Glycoproteins and Glycolipids in
Disease Processes
Earl F. Walborg, Jr., EDITOR The University of Texas System Cancer Center
A symposium sponsored by the ACS Division of Carbohydrate Chemistry at the 175th Meeting of the American Chemical Society, Anaheim, March 14-15, 1978.
ACS SYMPOSIUM SERIES 80
AMERICAN
CHEMICAL
SOCIETY
WASHINGTON, D. C. 1978
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Data
Library of Congress
Glycoproteins and glycolipids in disease processes. (ACS symposium series; 80 ISSN 0097-6156) Includes bibliographies and index. 1. Glycoproteins—Congresses. 2. Glycolipids—Congresses. 3. Tumor antigens—Congresses. 4. Carbohydrate metabolism disorders—Genetic aspects—Congresses. I. Walborg, Earl F. II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series : American Chemical Society. ACS symposium series; 80. QP552.G59G6 ISBN 0-8412-0452-7
616.3'99 ASCMC8
78-11920 80 1-480 1978
Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ACS Symposium Series Robert F. Gould, Editor
Advisory Board Kenneth B. Bischoff
Nina I. McClelland
Donald G. Crosby
John B. Pfeiffer
Jeremiah P. Freeman
Joseph V. Rodricks
E. Desmond Goddard
F. Sherwood Rowland
Jack Halpern
Alan C. Sartorelli
Robert A. Hofstader
Raymond B. Seymour
James P. Lodge
Roy L. Whistler
John L. Margrave
Aaron Wold
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide
a medium for publishin format of the SERIE parallels that of the continuing I N CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PREFACE T t is fitting that the proceedings of this symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the life and scientific contributions of my friend and fellow scientist, Ward W. Pigman. Much of Wards professional effort was devoted to the interchange of scientific information, both by his editorial skills and his support of national and international symposia on glycoconjugate chemistry. This symposium was born in Ward's mind in 1976 during deliberations of the Division of Carbohydrat Society concerning symposia topics for the coming years. Ward felt that sufficient progress had been made concerning the relationship of glycoconjugates to a variety of disease processes to warrant a symposium devoted to this topic. At that early date, the Carbohydrate Chemistry Division made a commitment to sponsor a symposium on some aspect of glycoprotein research and designated Ward as chairman with responsibility to formulate the program. Early in 1977, Ward informed me of his plans for the symposium and asked if I would serve as co-chairman. I gladly agreed and we set about the task of preparing a tentative program and contacting prospective speakers. On September 28, 1977, during the Fourth International Symposium on Glycoconjugates being held at Woods Hole, Ward and I finalized the symposium program. Upon completion of this task Ward expressed to me his personal satisfaction that the years of basic research on glycoconjugates wasfindingincreasing relevance to understanding a variety of disease processes. Ward's sudden death on September 30, 1977, was a shock to the many participants of the Wood's Hole meeting, many of whom were his long-time friends. In a way it was fitting that his last days found him engaged in an international meeting on glycoconjugates, an endeavor he had vigorously promoted and fostered for many years. Many of the participants in the symposium on "Glycoproteins and Glycolipids in Disease Processes" were present at the Wood's Hole meeting and by common consent it was decided that the planned symposium and this monograph should be dedicated to Ward Pigman's life and contributions to glycoconjugate research. The efforts of many persons and agencies were responsible for the success of the symposium and the publication of this monograph. First, I should like to express my appreciation to all of the participants for their contributions to the symposium and to the monograph. The supix In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
port of the Division of Carbohydrate Chemistry, particularly the efforts of Derek H. Ball and Gary D. McGinnis, is gratefully acknowledged. Grants from the National Institute of General Medical Sciences (GM 25226) and the National Cancer Institute (CA 24513), which provided travel support for participants in the symposium, are gratefully acknowl edged. Particular gratitude is extended to Edward Hampp (N.I.G.M.S.) and Mary A. Fink (N.C.I.) for their efforts in making this symposium a reality. On numerous occasions I have been encouraged by Gladys Pigman. Able assistance was obtained from Beverly Sea, Carey Osborn, Reita Maughan, and Pat McKirahan, staff at the Research Division— Science Park, The University of Texas System Cancer Center. Science Park—Research Division
E A R L F. WALBORG JR
The University of Texas July 20, 1978
χ
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
To Ward W . Pigman
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
DEDICATION TO WARD W. PIGMAN It is appropriate that this Symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the memory of my friend and colleague, Ward W. Pigman, who passed away on September 30, 1977, during his attendance at the 4th International Meeting on Glycoconjugates being held at Woods Hole, Massachusetts. Ward convinced the Carbohydrate Division of the American Chemical Society that a symposium on this subject warranted consideration for the spring meeting of the ACS to b efforts and those of Earl F. Walborg, Jr., this symposium has become a reality. Barely two hours before Ward's death, I had the opportunity to discuss his plans for this symposium, and I realized how deeply he felt about it. In a way, this symposium represents the ultimate goal of the long scientific road he and quite a few others among us have traveled, a road through carbohydrate chemistry that has led us from the use of analytical and physical chemistry in the study of reaction mechanisms to synthetic chemistry andfinallyto the elucidation of the structure of more and more complex carbohydrates, including glycosaminoglycans of connective tissue and glycoproteins from mucous secretions and the surface of the cancer cell. I feel that I have a deep understanding of Ward's research interests because both of us followed quite similar pathways, becoming interested in connective tissue polysaccharides in the late forties. In 1930 at the age of 20, he joined the National Bureau of Standards, where he was active until 1943. During this time, he obtained his B.A. and MA. degrees at George Washington University and his Ph.D. degree at the University of Maryland. At the Bureau he was associated with Horace Isbell, one of the leading American carbohydrate chemists. A series of papers reflecting Isbell's interests in the physical and organic chemistry of carbohydrates resulted from this collaboration. Soon a change in Ward's interests was noticeable, a turn to more biological problems with a study of glycosidases. This turning point was marked by a paper published in collaboration with H. Isbell and B. Helferich and by Ward's obtaining a Lalor Fellowship to spend one year in Leipzig, a stay that was unfortunately interrupted by the start of World War II. He returned to the National Bureau of Standards, and then during the last two years of the war he became a group leader at Corn Products, where most of his efforts were directed toward starch chemistry. Following his brief assoxiii In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ciation with Corn Products, he moved to the Institute for Paper Chemistry at Appleton, Wisconsin, an institute for basic research supported by the industry, where he also served as a group leader. During the Wisconsin period, Ward's interests included pectins, cellulose, carbohydrate-degrading enzymes, and tannins. In 1949 Ward moved into the academic community, accepting a position at the University of Alabama Dental School where he was Associate Professor until 1960, Professor and then Dean of the Graduate School from 1963 to 1968. He remained associated with this institution as a Visiting Professor until his death. In 1960 Ward assumed the additional burden of a second academic position as Professor and Chairman of the Department of Biological Chemistry at New York Medical College. These moves to academic positions in a dental and medical school correspond to a final shift in series of papers on the reaction between amino acids and carbohydrates, the so-called Browning reaction, initiated at Appleton, papers appeared on dental plaque and saliva, and then the beginning of the work for which Ward is now best known:first,investigations on hyaluronic acid degradation and then an outstanding series of papers on submaxillary mucins. Before this work had been started, the contribution of Ward to carbohydrate chemistry had already been recognized by his selection as the recipient of the Hudson Award by the Carbohydrate Chemistry Division of the American Chemical Society in 1959. Other awards followed, such as medals of the Société de Biochimie de France and of the University of Milan. Enumerated below are a few of Ward's contributions to glycoconjugate chemistry. (a) Elucidation of the mechanism of degradation of hyaluronic acid by ascorbic acid and free radicals. (b) Purification of submaxillary glycoproteins and elucidation of many aspects of their peptide and saccharide structure. (c) Elucidation of the ^-elimination mechanism for cleavage of the glycopeptide bond between 2-acetamido-2-deoxy-D-galactose and serine or threonine, the predominant glycopeptide linkage of the mucin glycoproteins. (d) Suggestion that the peptide moiety of the mucin glycoproteins is composed of repeating sequences of 28 amino acids. Although present evidence suggests a more complex arrangement, this hypothesis will continue to deserve attention.
xiv In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
This symposium and the resulting monograph bear the mark and vision of Ward W. Pigman, and therefore are fitting memorials to his life and scientific contributions to glycoconjugate chemistry. Harvard Medical School
and Massachusetts General Hospital Boston, Massachusetts August 21, 1978
ROGER JEANLOZ
XV In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Introduction ROGER W. JEANLOZ Laboratory for Carbohydrate Research, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114
To some chemist member Chemistry of the American Chemical Society, which is organizing this symposium, it may be unexpected that, for the next few days, we w i l l be discussing compounds that encompass lipids and proteins, in addition to carbohydrates, and their relation to disease processes. It is within the life span of many of us that natural product chemists have realized that nature is not compartmentalized into such nice and clean-cut categories as carbohydrates, proteins, and l i p i d s ; in fact, very few carbohydrates exist that are devoid of a peptide or l i p i d component, as well as there are few proteins devoid of a carbohydrate component. The roles of the carbohydrate component, of glycoproteins and glycolipids that are located at the surface of the animal c e l l or present in the connective tissue that links the c e l l s , have received increased recognition in the past decade and w i l l be discussed during the next few days. The subjects of the f i r s t session of this symposium w i l l be the methods of structure identification presently being developed, as well as the biosynthesis and degradation of glycoproteins and glycolipids. Because glycoproteins and glycolipids of biological interest are generally available only in minute amounts, the methods of structure identification, which w i l l be discussed by Dr. Walborg in his general presentation of current concepts of glycoprotein structure, are being developed at the micro- and even nano-gram l e v e l . Dr. Schachter and associates w i l l entertain us with the very active development of glycoprotein biosynthesis presently taking place. For many years, the concept of glycoprotein and glycolipid biosynthesis was dominated by the concept of elongation of the chains by one carbohydrate unit at a time, through sugar nucleotides; this concept, f i r s t developed for such homoglycans as glycogen and starch, was very ably demonstrated for glycoproteins and glycolipids by Roseman's group. Nearly twenty years ago, this unified concept of chain elongation was shown to be invalid for bacterial polysaccharides, and the importance of isoprenoid 3 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
sugar
phosphate
recognized. work
on
sugar have in
sugar
phosphates
the
intermediates
years in
the
D-glucose
etc.
It
with
the
glycoprotein
is
of
of
more
the
than
polysaccharide group,
established
in
the
the
role
role
component
the
passing
of of
transfer interest
processes:
One
^-glycoproteins
and
involves
rather
where
the
carbohydrate
takes
place
through
transfer at
l i p i d
elongation, are
The which
of
their
these
be
to
units
very
simple
components
two
the we
the
are
faced
processes
animal
and
mechanism, backbone
synthesized
degraded
coexist
now
in
peptide
before
where
sugar
the
chain,
complex
process
from
years
peptide
active
to
few
intermediates
^-glycoproteins,
oligosaccharides
and p a r t i a l l y
a
past
new
sugar
units
nucleotides.
or
are
Time
merely
the
of
blood-group
those
structures
are
found
length
at
the
to
of
eukaryotic
glycoglycerolipids
their
chain
as
b
solely
variation or
large
restricted
composition
process
is
single
residues
their
complex
in
w i l l
diversity of
of
its
inadequat
encompass
pyranosyl and
as
glycolipids
associates The
second
show w h e t h e r
expression
stage
intermediates
the
transferred
w i l l
of
the
a
is
was
for
isoprenoid
The
that
two
plant
of
known
these
to
of
synthesis
already
biosynthesis.
of
core
residues
existence
in
L e l o i r ' s
development
biosynthesis
of
ago,
nucleotides,
witnessed
role
as
Ten
the
up
to
surface
typing,
of
have
for
50
of
the
a-D-galacto-
chain
of
carbohydrate
units
the
organisms,
glycosphingolipids.
example
within
(evidence
having
result
(for
only
and
has
cells
been
of
reported),
during
the
specific
genetic
defects,
a l l
reasons
for
been
glycolipids), chains
oncogenic their
active
role
investiga
tion. Finally, the
most
microgram
specific chains
the
by
ago,
Dr.
of
we
by
were
Maley
Eylar of
D.
not
Dr.
degradation
methods the
identification of
this
his
been
are
we
and to
of
It
able
the the
more
progresses
glycoproteins these
to
my
the
I
laboratory structure such
that
present
addition of established and
structures
of
α -acid an
glycolipids disease
endo-
found
extract
that
biochemical
chemical i n
glyco
x
of
the
25
exo-
enzyme, in
be
when,
with
lack
such
recently
with
carbohydrate
remember
time
made
off
at
the
and which w i l l
the
was
elucidations
without
s p l i t
linkage
deploring
group,
is
the
in
on
were
his
structure possible
associates.
working
pneumoniae
great
correlation of
subject
was
manipulating.
allowed
chemical
have
that
r e a l i z i n g at
Kobata
has the
not
and
human p l a s m a ,
glycosidase later
recent
carbohydrate-peptide
Dr.
glycosidases protein
the would
endo-glycosidases
near
discussed years
of
level
methods
that
structural
and
made
possible
processes,
Symposium.
RECEIVED August 2, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
the
2 Biosynthesis and Catabolism of Glycoproteins HARRY SCHACHTER, SAROJA NARASIMHAN, and JAMES R. WILSON Research Institute, Hospital for Sick Children, Toronto, Canada
Glycoproteins are biosynthesis is, not unexpectedly Although much has been learned about this process in the past ten years, there are many aspects which we do not understand. In the limited space permitted for this article, it will be possible only to outline the major steps in the biosynthesis of some of the gly coprotein types; more detailed reviews are available (Schachter and Rodén, 1973; Montreuil, 1975; Schachter, 1974a,b, 1977, 1978; Waechter and Lennarz, 1976). I. Initiation of oligosaccharides of the asparagine-N-acetyl-Dglucosamine linkage type. The largest group of animal glycoproteins are those which contain only or predominantly oligosaccharides that are linked to the polypeptide back-bone by an N-glycosidic linkage between N-acetyl-D-glucosamine (GlcNAc) and asparagine (Asn). Many secreted glycoproteins (α1-acid glycoprotein and other plasma globulins, immunoglobulins, various gonadotrophins, ovalbumin, various enzymes, etc.) and membrane-bound glycoproteins (red cell membrane glycophorin, rhodopsin, the envelope glycoproteins of certain viruses such as vesicular stomatitis virus and Sindbis virus) contain Asn-GlcNAc linkage type oligosaccharides. It has become clear that the synthesis of the sugar-amino acid linkage pre-determines the general nature of the oligosaccharide that is subsequently assembled, i.e., that the synthesis of this linkage is an important control point. It is therefore important to understand the initiation process. In recent years it has become apparent that many (but not all) Asn-GlcNAc oligosaccharides share a common "core" structure (Montreuil, 1975) : Man-α1,3 Man-al,6
Man-31,4-GlcNAc-31,4-GlcNAc-Asn
0-8412-0452-7/78/47-080-021$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Further,
the
Hemming,
Heath,
1976,
and
strong
work
is
evidence
in
Soon
after
suggested cells. that
It
tissues
of
of
mammalian
core
is
scheme
Jeanloz,
and
has
Lennarz,
provided
pre-assembled prior
for
role
of
the
to
as
a
incorporation
initiation
p o l y p r e n o l - l i n k e d sugars
polysaccharide
a
process
similar however,
the
1);
be
pioneering
evidence and
nucleotides
(Fig.
might
assembly,
reaction
the
to
work
for
the
of
had
in
Leloir s
group
1
transfer
i n
mammalian
N-acetylglucosamine
from a
phosphate,
phosphate
workers
higher
dolichol
dolichol
been
various
occuring
their
phosphoryla-
sugars
formed
are,
Man-3-monophos-
GlcNAc-a-pyro-phosphate-dolichol, Glc-3
and
intermediates tissues
thyroid,
hen
ha
(rat
and p i g
oviduct,
l i v e r ,
human
mouse
myeloma
lymphocytes,
calf
tumor,
pancreas,
. Another
covery that
of
rat
glucose
liver
to
thought
to
water,
in
but
the
be
protein.
This
of
study
in
this of
the
product
product and
an
which
was
in
to
dolichol
the
was
of
radioactive
was
at
f i r s t
subsequently
shown
w h i c h was
et
aJU ,
pyrophosphate
ac to
insoluble
chloroform-methanol
(Behrens
dis
noted
endogenous
chloroform-methanol-water
solvent
was It
transfer
glucose
oligosaccharide acid
group
1
effect
acid-insoluble
soluble
The
length
of
the
tissue
and w i t h
reports
from
contain
about
mannose
and
mouse
20
residues
η
assembly
esting
to
formation
the
led
be
in
(2:1,
(10:10:3,
1971)
v/v)
the
oligosaccharides
units
residues. for
oligosaccharide of
GlcNAc
and
attached
conditions
laboratory
microsomes,
;
way in
5-7
used
indicated
to
for
the of
With
tissues
other
appeared
the to
residues
of
varies The
oligosaccharide
consisting example),
dolichol
synthesis.
to
N-acetylglucosamine, (hen
oviduct
predominant
lack
glucose
mannose,
and
as
and
dolichol contain
follows:
-Man-31,4-GlcNAc-31,4-GlcNAc-pyrophosphate-dolichol of
note of
oligosaccharide
glycose
glucose
myeloma
(Man-α-)
the
Leloir's
pyrophosphate
from
could
L e l o i r s
oligosaccharide.
tissues.
with
The
an
from
monophosphate
trichloroacetic was
intense
two
microsomes
pyrophosphate
discovery
many
contribution pyrophosphate
dolichol
form
dolichol
v/v)
major
dolichol
from
ceptor
to
reviews)
intermediate
general
mannose
l i p i d
(Leloir,
Waechter
1.
the
sugar
phate-dolichol
a
The
see
recent
common
glucose,
polyprenol
etc)
this
strong
respectively,
bovine
for
provided
of
laboratories others,
bacterial
was,
respective ted
in
that
first
1978,
that
Figure
established
and
oligosaccharide
polypeptide.
shown
several
Schachter,
lipid-linked into
of
Lennarz
this the
structure
inversions
3-linked
mannose
is of
shown
in
anomeric
from
Fig.
1.
It
is
configuration
GDP-a-Man and
inter
in
α-linked
the mannose
Man-3-monophosphate-dolichol. M.J.
carried
Spiro
out
containing
et
careful dolichol
a l .
(1976a,b)
and
investigations pyrophosphate
on
R.G. the
Spiro
et
formation
oligosaccharides
al of by
(1976)
have
glucoseseveral
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
tissues.
Working
with
these
workers
phate
oligosaccharide
Biosynthesis
thyroid
demonstrated with
slices
and
rather
than a
23
Catabolism with
the
formation
of
the
tentative
structure:
microsomes,
dolichol
pyrophos
(Man-α-) -(Glc) _ ~(Man-α-) -Man-GlcNAc-GlcNAcβ
1
2
4
PY r o p h o Evidence and
was a l s o
thymus
pyrophosphate preparations these
large
molecules lack
slices calf
dolichol
which
Parodi
for the of
pyrophosphate only
However, et
It
of
dolichol
pyrophosphat
glucose
believed
to
is
so
that
dolichol The
final
asparagine work and the to
i n i t i a t i o n process
and Lennarz, labelled
that
membrane the
that
that
is
of the
unglycosylated presence
of
by
retory
The
is
It
always
of
attached
pathway
transfer
However
to
the presence
pyrophosphate
of
of
of
further
forms
of
pathway. for
from
that
any amino sequence
the
dolichol
three
which is
sec
under
oligosaccharide
acid
does
A. effects
not
i n the tripeptide
almost this
time
i n
sec
and ribonuclease
polypeptide
some
a
glycoprotein
the transferase
for
rates
evidence
oligosaccharide
into
synthesis
ovalbumin,
the dolichol
denatured
an asparagine X can be
that
i n secretory
α-lactalbumin
recognized to
by
obtained
specificity
(Thr)-where
dolichol
has
tunicamycin,
normal
oligosaccharide
has been
i n h i b i t e d by
almost
appears
synthesis
however,
continued
oligosaccharide
substrate
Asn-X-Ser 1974).
the
suggested
since
i t
to
proteins
i n the
work,
endo
bound
secretory
only
and
The
1977);
at
been
asparagine
i n i t i a l l y
of
cells
intermediate
a l l tightly
recent is
l i p i d
an
catalyzed
reactions.
known
synthesis
have
the d o l i c h o l
showing
More
myeloma
has not yet
between
involved
glycosylated
proteins-ovalbumin,
incorporation stood.
is
(1977)
i t
of
to
Earlier
and Lennarz,
tunicamycin,
glycoprotein,
synthesis
the
from
findings
be
transfer
preparations
were as
ovalbumin
ovalbumin
and Lennarz
pyrophosphate
of
(Struck
retory
of
these
might
the
(Fig. 1).
i n these
identified
blocks
Pless
involvement
linkage
glycoproteins.
which
glucose-
configuration.
is
rat l i v e r ,
however,
experiments
study;
glycosylation
antibiotic
with
microsome
synthesized
pathway
N-acetylglucosamine
and
et a l . ,
oligosaccharide
chain
oligosaccharide
n o t be
under
dolichol
membrane-bound
shown an
tissues
the
1976) that
acceptors;
i n these
and could
smaller
(Herscovics
large
anomeric
pyrophosphate
an N - g l y c o s i d i c
acceptors
of
i n the polypeptide
N-acetylglucosamine
of
dolichol
protein
but
glucose-3-monophosphate-
from
i n the
of
microsome
produce
(Behrens
pancreas
step
transfer
genous
calf
synthesizing
glucose
established
established
of
of
hen oviduct endogenous
rat liver
inversion
residue
(Waechter
from
single
from
that
do n o t
and N-acetylglucosamine
a
oligosaccharide
kidney dolichol
b
transfer
involves
interesting
oligosaccharides
and from
capable
containing cules
is
mannose
microsomes
a l . , 1972) were
by o v i d u c t ,
glucose-containing
t h y r o i d and hen oviduct
contain
a l . , 1977a,b)
formation
similar
oligosaccharides. from
glucose.
1971; et
obtained
tissue
sphate-do1icho1
sequence(Marshall,
not
necessarily
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
result a
i n
glycosylation
protein
may,
cosylated (Thr)The
forms
site
work
but
that
A l l
may
be
completely
lactalbumin sequence
the
three and
ineffective
fully
protein A)
must
the
required tripeptide
and
the
Although serve
smaller This
into
observation transfer that
GlcNAc
contain
structures clear by
such
and
oligosaccha-
have
not
as yet
which
lack
and
since
1975)
o fa l l
glucose
stomatitis stages in
1978).
involve The
t othis
the
the
The
viral
class
Work
and
Sindbis
assembly
leads
e ta l . , envelope
plasmacytoma processing
Asn-GlcNAc
oligosaccharides
oligosaccharide i n
2.
F i g . may
f o l -
mannose
evidence has
i n d i -
envelope
subsequent 1977;
glyco-
rough
processing
o f mannose Robbins,
glycoproteins processing since
c e l l
(Tabas
line
GlcNAc,
Man a n d
endoplasmic
are
reprehowever,
biosynthesis o f was
e tal_.,
initiation
(and
1977;
i s , also
shown
1978) .
and
I t i spostulated
undergo
containing the
be
enveloped
virus)
t o removal
glycoproteins;
in
containing
must
the
o f the
type
type
residues, i t
reported with
o fAsn-GlcNAc oligosaccharides
summarized
occurs
Asn-
a n d o f some
recently
o fglycoprotein,
a mouse
are
process
o f the
o f mannose
residues;
(Hunt
processing large
large
N-
protein-bound oligosaccharide i s
mannose
oligosaccharide
initiation
have
virus
in
viruses,
rich
by
the
contain
3 mannose
residues
processing".
o fmembrane-bound IgG
oligosaccharides. primarily
oligosaccharide
the
residues
confined
oligo-
the
Asn-GlcNAc linkage
only
11 r e s i d u e s
glucose)
secretory
i n
than
N-acetyllactosamine
and
laboratories
a tearly
e ta l . ,
effective
acceptors
which
o f the the
contain
probably sentative
of
type.
o f glucose
and
more
involves
incorporation o fa large
o f these
moieties
glucose.
glycoproteins
This
accept
incorporation
pyrophosphate
envelope
Tabas
the
protein
pathway
these
a
not
were
proteins
oligosaccharide
9 times
oligosaccharides
(vesicular
large
i n
oligosaccharides
glucose
Several
that
proteins quite
vivo
"oligosaccharide
viruses
to
denatured
o fp r o t e i n - b o u n d o l i g o s a c c h a r i d e s
removal
residues.
cated
dolichol
(Montreuil,
that
residues
lowed
a
tripeptide
sequence
oligosaccharides
small
donors
about
mannose
linkage Since
not
are
pyrophosphate
Processing
not
glycosylation t o
tha
i n
acetylglucosamine, II.
relatively
t o endogenous
glucose-free
suggests
dolichol
of
site.
protein
oligosaccharides
saccharide
for
this
did
such
acceptors
(ovalbumin, a -
glycosylation
pyrophosphate
carry
asoligosaccharide
important phate
1-2
for
for
ungly-
-Asn-X-Ser
exogenous
several
and
a tanother
required
without
sequence
requirements
dolichol
and which
oligosaccharide
is
a tone
proteins
1977);
Lennarz,
resolved.
glucose
do
with
the
and
glycosylated
unfolded
however,
ride
can
be
acceptor
with been
(1977)
proteins
asacceptors;
Pless
both
glycosylated
contained
several
exact
i n
unglycosylated
Lennarz
effective
RNase
whereas
be
exist
or
and
1974;
(Marshall,
example,
o fPless
indicates occur.
for
by Glc
reticulum
subsequent that
a l l
transfer (Fig. (see
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
of
2). next
2.
Glycoprotein
SCHACHTER ET AL.
Biosynthesis
25
and Catabolism
UOP-GicNAc Dd-P *UMP G l c N Acc*-PP- - P - D o l U D P ^ G l c N A c J-
(GOP^Mon) (GOP^M •
n 1
n
γυΟΡ
(Dol-P)4
(
n
GlcNAc GlcNAc*P-P-Dol IcNAc^P-P-Dol GDP-Man *GDP
ί I " ^Man^P-Dol)
£
Mon-GlcNAc^GlcNAc^P-P-Dol—^(Man) *Man*GlcNAc*GlcNAc*P-P-Dol n
"
l U 0 1
P
Acceptor
)
Nooi-p-p ( Man\,
Man - GlcN Ac - GlcN Ac * A S N
Figure 1. Initiation of Asn-GlcNAc-type gosaccharides are preassembled tions: Dol,
oligosaccharides.
Oli
Peptide ^
(Glc) -(Man) -(GlcNAc) -P-P-Dolichol 2
n
(Glc) -(Man) -(GlcNAc) -Asn2
2
n
2
Oligosaccharide processing
V M-
Gn-Tr I
Gn-3l,2-M-cyl,3\ Μ-β1,4-Gn-Gn-Asn-
<
(MM)
(MGn) Gn-M^ M-Gn-Gn-AsnGn-pl,2-M-cyl,3
N
M-Gn-Gn-AsnGn-pl,2-M-ol,6
x
(GnGn)| Gn-My M-Gn-Gn-AsnGn-M^ Fuc
V
α
à2,6 G G βΙΛ pl,4 Gn Gn Pl,2 pi,2 M M 1,6\ /ol,3 M
6n
G-pl,4-Gn-M
Gn-pl,2-M-cyl,3, G-pl,4-Gn-M
x
1
/
Gn-(Fuc) -Âsnn-Çn-As (Fuc)
(GS)
G - p l , 4 - G n - p l , 2^-0*1,6' (GG) (GGn)
Figure 2. Biosynthesis of Asn-GlcNAc oligosaccharides of the N-acetyllactosamine type. Initial transfer of oligosaccharide to peptide can involve a glucose-containing intermediate. Processing to the Man GlcNAc Asn core (MM) is then believed to occur, followed by the Golgi-located elongation reactions. 3
Abbreviations:
M,
Ό-mannose;
2
Gn,
N-acetyl-O-glucosamine;
Asn,
asparagine;
Fuc,
-L-fucosc;
G,
Ό-gahctose; S, sialic acid; Tr, transferase. The glycopeptides are named according to the sugars present at the nonreducing ends; the sugar on the Man-al,6-branch is named first.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
26
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
section). type of
Oligosaccharides
(Montreuil,
glucose
1975)
residues destined
(Montreuil,
1975)
residues
t o become
undergo
Man3GlcNAc2Asn
undergoes
elongation
acid,
section
not
noside
type
while
interesting primitive
others
suggestion
the processing structures
Subcellular Fig.
membrane-bound for
secretory
these
to
site
3
of
when
now c o n s i d e r a b l e
secretory
the
nascent
Blobel
peptide
across
this
theory
nascent
peptide
binding
of
reticulum nascent
a
peptide
to
a
a
r
the more
e
of
oligo-
biogenesis
has been 1977)
proposed
site
creates
a
membrane.
moieties
Toneguzzo
the
which
"signal
the
causes
endoplasmic f o r passage
of
of
hypo-
end o f
somehow
on the
channel
and
membrane,
terminal
the i n t r a v e s i c u l a r space
of
the transfer
reticulum
sequence"
that
but are
ribosomes
To e x p l a i n
t h e amino
a
except
the plasma
the peptide
of
suggested
membrane
a l . , 1977;
receptor
and thereby
into
with
have
"signal
to the
structures.
of
to
the endoplasmic that
i s
organisms
conversion
1974a,b,
that et
made
structures
type
path
1978).
(1975a,b)
the ribosome ( F i g . 3)
fuse
Wirth
proposes
carries
have
on membrane-bound
1974a,b,
It
the oligoman-
extensively
scheme
attached
evidence
core
initiation.
similar
vesicles
the then
apparatus.
higher
enable
(Schachter
1975;
Schachter
and Dobberstein
thesis" ;
a
are translated
and Lodish,
1975;
to
Asn-GlcNAc
do n o t r e m a i n
secreted
(Morrison
more
and that
of a l l
form
become
(1978)
the subcellular
There
Ghosh,
scale
apparatus
glycoproteins
glycoproteins
et. a l .
to
F u c , G a l and s i a l i c
the oligomannoside
glycoprotein;
glycoproteins i s
are processed Tabas
type
(removal
Golgi
N-acetyllactosamine
shows
Oligo-
structure
GlcNAc,
oligosaccharides
that
developed
core
i n the c e l l ' s
i n the evolutionary
mannoside I l l .
This
(loss
residues).
residues)
(the a d d i t i o n o f
structure.
3
mannose
processing
(Fig. 2).
why some
oligomannoside
the N-acetyllactosamine
extensive
IV below)
yet understood
Man GlcNAc2Asn
some
the
limited processing
a n d a l l b u t 3 mannose
structure see
t o become
undergo
and possibly
saccharides glucose
destined
probably
the
of
endoplasmic
reticulum. It
has been
shown
for several
acetylglucosamine
i s
s t i l l
the ribosome
attached
Molnar
and Sy,
1970). be
to
If
Sherr
the dolichol
concluded
peptide.
1967;
incorporated
that
Kiely
et
a l .
a n d mannose
were
bound
to
ribosomes.
It
ments
whether
peptide of has
stage
peptide been
poration
o r whether
from
of
should
have
present
while
Jamieson
Cowan
has
shown these
takes
also
that
that
(1977)
i t can nascent
both
glu-
chains
various
place
occurs
found
i s
into
ovalbumin
on the nascent
and Lodish
1,2),
incorporated
from
core
i t
and Robinson,
(Figs.
at
experi-
the
after
C o n f l i c t i n g evidence (1977)
occurs
Rothman
1966;
on nascent
process
the ribosome.
carbohydrate
while
i n fact
was n o t c l e a r this
be
some N -
and Schachter,
operative
also
a l l incorporation of
obtained;
glycoprotein
is
that
peptide
and U h r , 1969;
(1976)
cosamine
tissues
nascent
(Lawford
pathway
mannose
into
on this
l i t t l e
peptide
suggest
nascent
release point
incor-
of
that
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
oxacid the
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and Catabolism
UOS R I B O S O M A L S U B U N I T mRNA
60S
RIBOSOMAL
SUBUNIT
HYDROPHOBIC SEQUENCE
- mRNA
CYTOPLASMIC ^ F A C E LIPID B I L A Y E R O F R O U G H ENDOPLASMIC
RETICULUM
TTTTTTTTT Hiiiliii
ÎÎÎÎÎÎÎÎ--" UUUU- -" * \
RIBOSOME BINDING PROTEIN '
INTRAVESICULAR FACE
mRNA
GLYCOSYLTRANSFERASE
EXTRACELLULAR INTRACELLULAR _
FACE
FACE
Figure 3. A scheme depicting biosynthesis of a membrane-bound glycoprotein. Translation probably starts on a free ribosome and the nascent peptide carries some sort of "signal" near its amino-terminal end. This signal organizes the appearance of a ribosome-binding protein in the endoplasmic reticulum and thereby creates a channel for passage of nascent peptide through the membrane. Oligosaccharide is incorporated into peptide within the channels of the rough endophsmic reticulum (stages 5 and 6). The signal sequence is probably cleaved off at some stage in assembly although there can be exceptions. The glycoprotein migrates to the Golgi apparatus for the elongation reactions (stage 7) and then to the plasma membrane (stages 8 and 9). An analogous scheme can be drawn for secretory glycoproteins except that the glycoprotein would not be firmly anchored to the membrane during the assembly process.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
27
28
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
entire
oligosaccharide
during
assembly
t i t i s
virus.
that,
i n their
through ation; and
of
In
fact,
both
processes
the ribosome.
data
of
Jamieson
out
before
report
involved phate
worked
endoplasmic vitro
as
(Vargas
reticulum.
a l .
these
peptide
(1978)
at
locus
liver. The
shown
different;
they
contained
mole,
levels
process
this
mannose
for
7
some
type.
not
i n
nascent
to
of
Thus,
of
compatible
acid
of
with
containing mannose.
oligosaccharide endoplasmic
oligosaccharide
the low
plasma
plasma
and the
a n d showed
to
t h e ZZ that
but
of
per
2 residues
residues In
into
individuals
ZZ l i v e r s
completion.
of
suggest
incorporated normal
i t
carried
levels
strongly
mannose
whereas
of
isolated
or galactose
normally
has
significantly
(1978)
reticulum; of
by the
acid)
protein
not
results
ZZ
1978).
on the average
i s
from
ejt a ^ . ,
and increased
These
by removal
the protein
liver
a^-antitrypsin
3 oligosaccharides
per oligosaccharide)
process
of
of
i n
and lung
i n secretion
liver
II
(protease
oligosaccharides
ejt a l .
s i a l i c
kidney
(Jeppsson
human
i n section
for glutamic
3-4
i s
subsequently
a-^-antitrypsin
studies
however,
carry
Hercz
by
interesting
t h e P-^
l i v e r ,
the normal were,
an
and
at
retention
N-acetylglucosamine
i n the rough
reason,
of
specimen
glucose,
residues
residues
to
microscopic
plasma
appeared
were
a mannose-rich
polypeptide 3
of
low l e v e l s
are prone
i n t h e ZZ p r o t e i n
oligosaccharide
and
f i r s t
similar
described
(lysine
an autopsy
the data
each
GlcNAc that
both
as
substitution
from
no f u c o s e ,
decreased mannose;
the
glycoprotein
reticulum
a blockage
N-acetyllactosamine from
rough
deficiency
secretory
substantial
compositions
isolated
protein
i n a
provided
a r e due t o
reported
carbohydrate
a
have
reticulum vesicles.
acid
ZZ p r o t e i n the
a
protein
An amino been
enzymes pyrophos
i n the
however,
for the Ζ allele
relatively
and electron
endoplasmic
recently
were,
processing"
individuals
have
this
l i v e r ,
homozygous have
such
Light
of
the
providing
acceptors
endoplasmic
"oligosaccharide
rough
latter
carried
dolichol
enriched
Institute
i n human
Persons
in
the
be
preliminary
that
hen oviduct,
acceptors
this
(above) .
levels
A recent
the
are not
1977) .
undergoes
individuals
peptide
while
must
c a r r i e d out on α^-antitrypsin
i n the rough
disease.
protein
from
of
between
(1977)
studies
glycosyl
Ra
that,
sera;
were
from
to
insertion
release
indicated
oligosaccharide
confirmation
inhibitor)
secretory
oligosaccharide
glycosylated
their
a
to protein
and Carminatti,
et
coupled
membrane
before
evidence
peptide
f o r the
Studies Hercz
is
stage stoma
strong
nascent
and Lodish
(1977)
prepared
endogenous
system;
of
peptide
vesicular
for the discrepancy
c a n be made.
and Lennarz
fraction
well
of
provide
without
but further
generalizations
evidence
served
with
protein
oligosaccharide
direct
not occur
a n d Rothman
i n transferring
microsome
workers
r e t i c u l u m membrane
does
(1977)
by C z i c h i
the nascent
insertion
The reasons
a membrane
at
glycoprotein
are completed
the former
studied
added
system,
the endoplasmic
from
i s
the latter
i n vitro
glycosylation
known;
core
the envelope
(to cannot,
normal
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
livers,
the protein
(addition below;
added
normally,
that
interferes
out
of
where
the
complete
the
rough
since
with
some
apparatus
for
elongation
residues,
see
section
contain
is
IV.
Elongation within
which
reticulum to
The blockage
a
the
protein
the
pathway
sugars
must
a^-antitryp-
of
processed,
external
liver
subsequent
of
region
i n this is
and i t
or
movement
ZZ
processed
i n ZZ
glycosylation
IV
is
i n the
are
not defective
ZZ a ^ - a n t i t r y p s i n
the
fucose
The d e f e c t
or prevents
for addition of
into
c e l l
i s
not
moves
and i s
to
the
secreted
plasma.
oligosaccharides As curtail
apparatus
acid
substitution
normal
endoplasmic occurs.
acid
29
Catabolism
glycoproteins
apparatus
amino
and
Golgi
apparatus). plasma
processing,
processing
Golgi
Golgi other
either
oligosaccharide
the
n o r ZZ p r o t e i n s
the processing
postulated
sin
normal since
to
Gal and s i a l i c
i n the
n o t known;
be
travels
GlcNAc,
neither
usually is
of
Biosynthesis
pointed
processing
oligomannoside will
not be
fate
of
the
out of
type
Golgi
apparatus
of
Asn-GlcNAc
o i n section
II
(above),
oligosaccharides
are n o t known;
considered
further.
oligosaccharides
which
this
In
the
destined type
this
of
which
become
the
oligosaccharide
section,
are processed
factors
to
to
we d e a l
the
with
the
core
structure : Man-al,3 Man-31,4-GlcNAc-31,4-GlcNAc-Asn
^ Man-al,6 Fig.
2
shows
process acid
subsequent addition
i n the
end o f
Golgi
this
The fied
p r i m a r i l y by
the
toxic
proteins has tain
et
on the
at
least
the use o f
of c e l l
the
structure II.
i n wild
be
of
G a l ,
these
discussed
a
sugar,
a
GlcNAc
f o r nomenclature)
This
s i a l i c
sugars
briefly
are at
residue,
has been
I,
Evidence type
cells
(Stanley c e l l
ejt a l . ,
line
because
was
a l . ,
incompletely
type
Chinese
ovary
MM ( s e e
F i g . 2)
F i g . 2) acting
and the is
one
summarized
of
con-
acting
on
desig-
glycopeptides
w h i c h we d e s i g n a t e absence
It
cells
w h i c h we on
to
glyco-
glycosylated.
hamster
i n d i c a t i n g the presence
cells
the
1975;
resistant
lectin-binding
were
and the other
cells
to
c l a r i -
phytohemagglutinin-resistant
lectins
wild
MGn ( s e e
i n lectin-resistant et
f i r s t
structure
GlcNAc-transferase
I
structure.
GlcNAc,
two N - a c e t y l g l u c o s a m i n y l t r a n s f e r a s e s , with
the
Narasimhan
this
addition
will
The mutant
surface
that
nate
ferases
as
ovary
several
with
transferase
the
F i g . 2
hamster
shown
glycopeptides
catalyzing
a l . , 1977).
action
now b e e n
of
(MM, s e e
Chinese
Narasimhan
of
sugars:
section.
structure of
four
apparatus,
addition
core
mutant
elongation
of
and F u c ; t h e enzymes
located the
the
involves
of
GlcNAc-
both
trans-
GlcNAc-transferase
i n Table
I
(data
1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
from
30
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES The
were
derived
Fig.
2).
glycopeptides from
Glycopeptide
myeloma
structure
on
was
glycosidase by
A.
to
and
important
Strecker
MM.
The
et
was
of
of
We
ted
can
that
enzyme
into
thus
state
1977). than
controls
aid
31,2
data
linkage t o
enzyme
3-N-
t o
reaction
the
also
I t is,
add
Man-al,3absolute
shown
action
(Ito
ejt
adhered
than
into
Schachter, likely
in
Fig.
Both
depend
the
2),
sialic
residues. mutant and
however,
in
resistance
we
have
i t i s not
certain
the
Man-al,6-
terminus.
was
determined
on
by
subsequent
I i s MGn
GlcNAc-
above
leads
only had
yet
on any
linkage above The
for
addition
action
o fGal
and
surprising that
the
Chinese
hamster glycoprotein
lectins. MGn
(Narasimhan
GnM a v a i l a b l e
e t a l . ,
for
testing
I I i s specific
synthesized
i s 31/2;
GlcNAc-transferase
product
o f the
reaction
for this
I i s there-
2).
relationship
described
above
petition)
studies
between
i spresently have
the
under
indicated
two
study.
that
I
o f the
o fGlcNAc-
t o incomplete
i fGlcNAc-transferase
1977).
the
addition
not
t o several
I I acts
The
GlcNAc-transferase
lectin-resistant
not
asdescribed
e ta l . ,
(Fig. The
the
described
and
(Narasimhan
a s does
residue
I t i s therefore
GlcNAc-transferase
that
(Narasimhan
process.
cell
preparation).
a preferred
elongation
glycosylation
in
path.
o f a GlcNAc
enzyme
Man-al,6-
the
2 i s thus
entire
o fthis
action
incorpora-
available
the
acid
been
t oa Man-al,6-
residue i s not
product
1975); w e t o the
o f GlcNAc-transferase
addition
(Fig.
prepara-
o f C I . p e r f r i -
a l . ,
GlcNAc has
in
o f the
resistant
however,
a GlcNAc
and o f Fuc
GnGn
CI
and
terminus
pathway
Schachter,
t o the
rather
product
GnM.
and
susceptibility
the
Narasimhan
residue
fore
the
a l l
terminus
that
The
an
that
I
1977);
the
in
The GlcNAc
ovary
o f NMR
i s completely
transferase absence
with
o f the
I reaction
product
than
I can
i f the
a l . ,
next
residue
was
susceptibility
Wilson
studied
indicating
rather
terminus rather
the
Man-al,3-
the
transferase et
product
Narasimhan,
recently
o f MM ( W i l s o n ,
2)
(Fig.
Genetics, acid
t ob
1975;
have
found
terminus We
The
GlcNAc-transferase
this
sialic
and
carried
branch point
a GlcNAc by
endo-3-N-acetylglucosaminidase
ngens have
linkage
ajL. ,
ejt
the
spectra
t oConcanavali
GlcNAc-Man tion) .
I adds
determined
acetylglucosaminidase.
Ogata
220
and by
(1977a).
GlcNAc-transferase
strongly
c a r r i e d out
I t s
sequential
o fMedical
o f the
I
multiple
columns.
1977)
Department
Table (see
subsequent
resonance
Man-al,3-
a t the
and
analysis,
e t a l . ,
magnetic
in GS
a human
filtration
assignment
a n d was
a l .
3-linkage
from
digestion
gel
shown
structure
carbohydrate
Carver, The
N-acetyllactosamine
from
and
by
assays
the
obtained
proton
J . P .
the
with
(Narasimhan
resolution
o fToronto.
especially
for
pronase
exchange
established
Grey
University
ion
Gby
digestions
360 MHz h i g h out
GS w a s
immunoglobulin
purification
used
a glycopeptide
GlcNAc-transferases Mixed
substrate
MM a n d M G n c o m p e t e
for
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
(coma
2.
SCHACHTER ET AL.
common this
enzyme
finding
catalytic I
and
I
would
is
active does
have
(Narasimhan
disprove
a common t o have
i nthe
aJL.
biosynthesis
(1977)
in
wild
type
and
cells.
I nw i l d
type
cells,
virus
carries
(3 m a n n o s e
virus
i nthe
acetyllactosamine from
the
mannose
I
residues
per
processing
absence
one
possibility
As can
2).
(Fig. buted
The
usually enzyme
assayed was
using
into
the
rather
than
(Wilson
and
transferase (see
F i g .
Kessel with
adding
Fuc
because This
not
Thus,
i t cannot
scheme
saccharides
occur
t o the
acetyllactosamine-type others
donot
but
the
fucosyltransferase Perhaps ferase
i s the
to
terminal
the
related, lactose
this
i f not
More shown
can
act
on
Fuc
residue
t o the a l . ,
GnGn
near
the
process
I has
acted.
Asn-GlcNAc a Fuc
fucosyl-
MGn a n d
i s added
o f the
work
the
oligo
why
some N -
residue
while
appropriate
possibility.
thoroughly
et.
recent
know
with
cleavage
that
both
donot
the
residue
migrated
Golgi-localized
have
a^-acid that
product by
Fuc
has
o r absence
GlcNAc o f GnGn. (Brew
the
and
and i s
GlcNAc
endoglycosidase
we s t i l l
a likely
identical,
d i s t r i
Munro
1977)
internal
analyzing
oligosaccharides
most
1975;
showed
GlcNAc-transferase
galactosyltransferase
synthetase
most
Fuc
residue
i s widely
(1976)
radioactive
after
presence
seems
the
and CI;
Fuc;
2),
(Fig.
Chou e t a l . ,
oligomannoside-type
contain
subunit o f
Asn
e t a l . ,
e t a l .
i s a late
until
known;
β-galactosidase-treated
o n MM b u t
why
the
oligosaccharide.
addition
explains never
5
between
i s not
t o MM
reaction
Munro
i np r e p a r a t i o n )
although
connection
regulatory
nearest
1977;
acceptors
act
The
added
this
Wilson
uncharged
chain,
virus
(about
processing.
been
sialidase-,
Schachter, w i l l
expected
i n removing
1971;
Asn-GlcNAc moiety with
absence o f mature
during oligosaccharide
e t a l . ,
as acceptor.
2).
polypeptide
cells.
i s somehow
catalyzing
glycopeptide
charged
the the
I and p r o c e s s i n g
GlcNAc
Schachter,
i nfact
than
However,
s i a l y l - N -
involved
endo-3-N-acetylglucosaminidase the
mannose
postulated
above)
residues
enzyme and
from
Surprisingly,
more
a s a GlcNAc has
1973;
glycoprotein
the
(see
mannose
(Jabbal
Schachter,
by
I
be incorporated
oligo
lacks
bepredicted
I-deficient
i s that
soon
line
ovary
o f the
oligosaccharide).
2).
F i g .
o f GlcNAc-transferase
two
hamster
glycoprotein
c e l l
studied glyco
(se
the
last
have
envelope
Chinese
which
oligosaccharide)
GlcNAc-transferase
the
per
as would
carries
the
GlcNAc-transferase
virus
N-acetyllactosamine-type
(see
cells
subunit above.
(1978)
e ta l .
envelope
residues
arms,
mutant
saccharide
the
GlcNAc-transferases GlcNAc-transferase
described
stomatitis
lectin-resistant
GlcNAc-transferase
that
different
regulatory
mutant
Although
o f two
subunit;
lectin-resistant
only
saccharides grown
suggests
and Tabas
o f vesicular
1977) .
e t a l . ,
catalytic
31
and Catabolism
existence
an additional
protein mature
the
lectin-resistant
Hunt e t the
Biosynthesis
i t nevertheless
share
then
absent
site
not
sites,
I I may
Glycoprotein
characterized which
This
adds
enzyme
A protein 1968) .
The
Gal
glycosyltransi n 31/4
i s believed component A protein
linkage
t o be
o f milk has
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
a
very
32
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
low
affinity
for glucose;
lactalbumin),
affinity
lactose
is
mammary
gland,
A
synthesized.
protein
can
lactose
(or a very
incorporate
terminal
i n the presence
for glucose Since
Gal into
glycoproteins glycoprotein
absence
fetuin,
linkage
of
Asn-GlcNAc oligosaccharides.
α-lactalbumin and r a t serum
homogeneity
by c l a s s i c a l
by
affinity
chromatography
or
UDP-hexanolamine-Sepharose
Andrews, and
1970;
Brew,
active and
1974;
Fraser
bovine
forms
with
that
smaller
due t o
1976). enzyme
of
molecular
be
41,000 b y been
to
from
milk
enzyme
is
the
the
enzyme
elongation
milk,
et
a l . , 1970)
et
and H i l l ,
to and
1971;
a l . , 1976;
51,000
potent
Powell
larger
that
a
be
protease molecule
is
form
milk
single
by
form
lactating
X-100
of
of
enzyme
weight has
sheep
colostrum
also mammary
fat
gland
enzyme
and, on p u r i f i c a t i o n ,
showed
molecular
molecular
t h e mammary It
the
bovine
this
t h e mammary
e_t a l . ,
of
molecular
and from bovine
proteo
from
inhibitors;
of
inactibelieved
(Magee
isolated
with
t h e two
heat
Galactosyltransferase
enzyme.
produced
the
could
component of
55,000-59,000
and i t
i n bovine
et_ a l . , 1 9 7 7 ) ;
component
be
substrate,
reagents
membranes
1977)
to
et. a l . , 1 9 7 4 ) ;
for
found
1% T r i t o n
the milk
M
from
trypsin.
properties of
of
purified
estimated
protease
smaller
a major
and a minor
K
derived
(Powell
components,
properties
a^-acid
a n d human
(Trayer
(Magee
to
(1974)
and Brew,
two
enzymic
a of
solubilized with
69,000
weights
the Golgi
was
The
is
contains
membranes
GlcNAc-
a-lactalbumin-Sepharose
Geren
sulfhydryl
weight
the action
(Smith
globule
columns
regard
and Brew
which
isolated
glands
form
degraded
GlcNAc,
been
(Fitzgerald either
The
and GlcNAc-terminal
certainly
have
i n
d i s t r i b u t e d and
The f u n c t i o n
The bovine
respectively
molecular
colostrum could
free
enzymes
using
a plasmin-like
Powell
to
almost
a l . , 1974;
and i n h i b i t i o n by
the
lysis
widely
only
organ.
an
are similar with
vation
present
i n this
(a-
and
milk
42,000-44,000,
forms
is
methods
Khatra et
The
is
only
3-galactosidase-treated
the
colostrum
increased
is
for example).
in
bovine
of
the Β protein
and glycopeptides,
(sialidase-, or
occurs
protein)
81/4
oligosaccharides
of
greatly
α-lactalbumin
synthesis
similar
is
was
by p r o t e o l y s i s
weight
weight
gland of
53,000-55,000.
enzyme
suggested the
65,000-
resembled
that
the
the
soluble
Golgi-bound
transferase. The
various
glycoproteins 1977;
Lehman e t of
this
addition
of
Mn2+,
et
studied et
enzyme
the galactosyltransferase
about
have
Bell
a l . , 1974;
1973; et
10-15%
Trayer been
UDP-galactose,
(Ebner,
a l . , 1975a,b;
Khatra
of
a l . , 1975;
kinetics been
forms
and contain
carbohydrate
are
(Geren
and H i l l ,
1971).
published
and the order
α-lactalbumin,
Tsopanakis
a l . , 1976;
Powell
of
and acceptor
and Herries,
K i t c h e n and Andrews,
et a l . ,
Detailed
1976;
and Brew,
have
Geren
1975;
1974a,b).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL. The
a-1,6There
scheme
terminus is
no
in
F i g .
prior
direct
to
evidence
preferntial
both
bovine
immunoglobulin
myeloma
immunoglobulin
communication) GnG.
Also,
transferase
that
specificity.
contain
Rao
depicts
addition
such ple
2
Biosynthesis
e_t
the
a l .
purified
ferred
Gal
more
rapidly
G than
to
Elongation residue
to
various
Asn-GlcNAc
primarily reports have
GG a2,3
of
and
other
reported
glycoprotein
is
(Fig.
a
tissues acid
into
Roden,
interesting,
G
(Tai
et
al_.,
G
(Baenziger have
swine
(Montreuil,
Isemura shown of
assumed
that
measuring
(Bartholomew
further, were the
et
of
rat
1977b)showed
which
makes
sialic is
acid
evidence
may
be
incorporation (see
section i n
s i a l i c
acid
into
to
transferase two
forms
by
and of
a l . ,
to
be
occasional
a2,3 into
of
acid
et_ a l _ . from
1972). et
preparations,
and
1972)
Support
rat,
pork,
catalyze
two
the
However,
probably
1977;
not
of
sialidase-
Van
den
the
Eijnden
was
a2,6;
transferase
involved
in
oligosaccharides.
this
i n
analysis
on
synthesized Thus,
There
3 -sialyltransferase 1
incorporation transferases
activities
sialyltransferases
action
a l . ,
Asn-GlcNAc that
and
recently
into
which i n
Ser(Thr)incorporate
linkages
other
discovered. (1977a) of
have
recently
CMP-sialic acid:
bovine
affinity
equal
until
colostrum
these
colostrum.
chromatography
on
above,
specific
this
reported enzyme
with
p u r i f i -
was
a2-6
purified
CDP-hexanolamineLike
the
sialyltransferase
activity,
the
3-D-galactoside The
CDP-ethanolamine-Sepharose.
discussed
CMP-sialic
(Schachter
separate
linkage.
V below)
s i a l y l -
sialyl-a2,6-lactose;
linkage
is
a
Various
from
1977b)
and
two
only
The
be
homogeneity
sialyltransferase Sepharose
crude
Asn-GlcNAc oligosaccharides
remain
440,000-fold
reported
membrane
Schachter,
(Stoffyn
the
had
crude
et
and
that
sialyl-a2,3-lactose
Paulson
acid
i n
suggested
acid
It
that
Schachter,
indication of
involved
to
been
have
sialyltransferase
that
sialic
cation
immuno-
sialyltransferase.
development
glycoprotein
oligosaccharides.
a2,6
p-
trans-
s i a l i c
protein.
s i a l i c
one
Paulson
and
GalNAc than
a
Golgi-localized
using
(Hudgin
liver
oxacid no
of
glycoprotein
finding
indicated
treated
was
a
sialyl-a2,3-lactose
et_ a l . , there
by
linkages
however,
same
1978).
than
the
1973;
(Hudgin
of
than
galactosyl-
porcine
have
(1971)
the
a^-acid
assays,
liver
liver
involved product
for
contain
more
differential
rat
Schmid
1974a,b,
from
a l . ,
both
the
embryonic
to
these
came
a n d human
synthesis
a
glycopeptides.
1975);
incorporating
Schachter,
i n
bovine
and
linkage
sialidase-treated
idea
personal
nodes
addition
oligosaccharides
been
this
from
that multi-
rather
that
acid-galactose
type
were for
2)
lymph
shows
human
chromatography
GnGn p r e p a r e d the
and
(Fig.
reported
by
Man-
however,
and K o r n f e l d ,
mesentary
Sialic
the
linkage
been
fact
1975)
GGn
affinity
completed
to
terminus.
galactosyltransferase
a2,6
capable
1973;
Gal
sialyl-a2,4-galactos
and
have
of
Man-a-1,3-
GlcNAc-terminal
2).
sialyl-a2,2-galactose transferase
the
structure
to
other
the
is
aminophenyl-8-GlcNAc-Sepharose globulin
addition
to
33
Catabolism
It
(1976)
from
and
molecular
galactosylwas
found
weights
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
in of
34
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
56,000 that
and 43,000
the
larger
smaller
enzyme.
might
as
observed
crude
enzyme
enzymes
were
with
1973).
Both
ot^-acid
glycoprotein,
sialic
acid-free
termini
whereas
purified as
the latter
above,
t h e a2,6
both We
have
crude
shown
described
above
and pork
liver
of
tissues
Although
organelles the
that
appears i n which
into
the
fuses
a
with
applied
on the site
cells
of
membrane
is
with
within
towards
F i g . 3). membrane by
evidence
comes
radioactivity o r by
auto
and Leblond,
the Golgi
1977).
apparatus,
the plasma
membrane
The v e s i c l e
membrane
glycoproteins
glycoproteins
a
,
Golgi
radioactive
techniques
Bennett
cells)
the
incorporation of
1978;
membrane
1974a,b,
subcellular
certain
Supportive
and secretory
whereas
lateral
are
probably
diffusion
process.
(threonine)-N-acetyl-D-galactosamine
similar
to
on the synthesis
studies
into
to
those
Almost
glycoproteins
two o v e r - l a p p i n g of
mucins of
with
blood
discuss
glycoprotein
the highly
active
specialized
(Watkins,
1978).
A brief
maxillary
mucin biosynthesis involved
1974;
discussion
i n mucin
the
mucins,
will
of
serve
has
since ovine to
synthesis glands tracts,
of
the
and
i n particular
various
will
be group
detailed
and Roden,
and porcine
illustrate
(Fig.
dealt
human b l o o d
Schachter of
synthesis
been
classified
No a t t e m p t
field
and g l y c o l i p i d biosynthesis
are available
(i)
a n d mucous
antigenicity.
Schachter principles
cells
work
and g e n i t o - u r i n a r y
group
human A B O - L e w i s
have
and can be
categories:
(ii)
above
Ser(Thr)-GalNAc-linkage
"mucins"
by goblet
respiratory
the synthesis
described of
a l l the reported
or
gastro-intestinal,
reviews
of
Approaches
secretion
to
of
elongation
are pulsed
migrates
serine
secretory
made
surfaces of
completed (see
Schachter
oligosaccharides.
oligosaccharides.
mucins
liver
sialyltransferase
i n other
by biochemical
is
the plasma
type
and
and
a l . , 1974; may o c c u r
1974a,b,
the c e l l
with
broadly
et
vesicle
the plasma of
and
sialyl-lactose.
N-acetylglucosaminyltransferases,
a r e made
either
glycoprotein
Biosynthesis type
the
of
conclusive.
intact
elongation
from
become p a r t V.
highly
transport
extruded
The
colostrum
enzymes
(Schachter
finished
within
termini.
from
isomers
(Letts
and measurements
Once
Gal-$1,3-GalNAc
(Munr
glycoproteins
radiography
various
Gal-βΐ,4-GlcNAc
preparations
the major
from
sugars
acid-free
sialyl-a2,6-lactose;
that
these
apparatus work
been
and Roden,
only
(in particular,
evidence
$-
had previously sialic
contain
for
with G a l -
are highl
and
1978).
former
galactosyltransferase
rat
other
with
shown, the
synthesizes
a n d a2,3
fucosyltransferase,
and other
(Schachter
active
of
specific
IgG and IgM but not w i t h
have
sialyltransferase
mentioned
make
highly
fetuin, the
highly
specificities
preparations
but not
product
low a c t i v i t y
Gal-31,4-Glc
substrate
mucins;
were
and showed
Gal-$l,6-GlcNAc, similar
a degradation
enzymes
an acceptor
galactosides;
i t was p o s t u l a t e d ,
be
Both p u r i f i e d
Gal-$l,4-GlcNAc 31,3-GlcNAc,
respectively; enzyme
the
4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1973; sub general
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
35
and Catabolism
TABLE I N-acetylglucosaminyltransferase l e v e l s of w i l d type and l e c t i n r e s i s t a n t Chinese hamster ovary c e l l s using as acceptors glycopeptides prepared from human IgG; see F i g u r e 2 for glycopeptide nomenclature. IgG glycopeptide acceptor
N-acetylglucosaminyltransferase activity (nmol/mg/hour) Wild type cells
MM
Lectin-resistant cells < 0.2
3.7
MGn GnGn
R
-0H
<0.2
<0.2
U D p
-
G o l N A c
, R-0-GalNAc
CMP-SIOUC
Acid
(POLYPEPTIDE CORE)
,
R
.
Q
.
G
a
L
N
A
c
.
S
A
(OSM) UOP-Gal
R - Ο GalNAc I Gal
CMP-SA (?)
-R-O-GalNAc-SA Gal ι
ODP-Fuc I
CMP-SA (?)
R-0-GalNAc I Gal-Fuc
•R-O-GalNAc-SA Gal—Fuc (A")-PSM ι
UDP-GalNAc
R-0-GalNAc
I
I (?)
CMP-SA (?)
Gal— Fuc I GalNAc
i -R-O-GalNAc-SA Gal—Fuc I GalNAc (A*)-PSM
Figure 4.
Assembly of ovine (OSM) and porcine (PSM) submaxilhry mucins
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
36
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
The f i r s t important p o i n t i s t h a t i n i t i a t i o n does not i n volve pre-assembly o f o l i g o s a c c h a r i d e as a l i p i d intermediate. I t has been reasonably e s t a b l i s h e d i n s e v e r a l systems t h a t the Ser(Thr)-GalNAc l i n k a g e i s synthesized by the i n c o r p o r a t i o n o f a GalNAc from UDP-GalNAc i n t o a high molecular weight acceptor. The t r a n s f e r a s e has a high s p e c i f i c i t y f o r acceptor; f o r example, the enzyme from s a l i v a r y gland w i l l donate GalNAc t o mucin t r e a t e d with g l y c o s i d a s e s t o remove a l l carbohydrate but not t o a l a r g e number o f other high and low molecular weight acceptors. An i n t e r e s t i n g exception i s the t r a n s f e r o f GalNAc t o a b a s i c p r o t e i n i s o l a t e d from bovine myelin (Hagopian e t al_., 1971) ; t h i s p r o t e i n i s not normally a g l y c o p r o t e i n and the s i g n i f i c a n c e o f i t s acceptor a c t i v i t y i s not known. Although pronase treatment of carbohydrate-free mucin acceptor destroys acceptor a c t i v i t y , a recent r e p o r t has shown t h a t the enzyme w i l l t r a n s f e r GalNAc to t r y p t i c peptides o f carbohydrate-free ovine submaxillary mucin ( H i l l e t a l . , 1977) s e r i n e and threonine residue saccharides have i n d i c a t e d no homologies t o d e f i n e the substrate s p e c i f i c i t y o f the t r a n s f e r a s e (Marshall, 1974; H i l l e t a l . , 1977). The enzyme r e q u i r e s high molecular weight acceptors but i t i s not c l e a r why some hydroxyamino a c i d s are g l y c o s y l a t e d but others are not. I t i s i n t e r e s t i n g t h a t the porcine submaxillary gland UDP-GalNAc:polypeptide N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e does not adhere t o UDP-hexanolamine-Sepharose and has thus f a r been p u r i f i e d only 3 0 - f o l d ; however, t h i s p r e p a r a t i o n was r e l a t i v e l y f r e e o f other mucin g l y c o s y l t r a n s f e r a s e s ( H i l l e t aJL., 1977) . There i s an important branch p o i n t immediately a f t e r GalNAc i n c o r p o r a t i o n . I f s i a l i c a c i d i n c o r p o r a t i o n occurs t o form the sialyl-a2,6-GalNAc d i s a c c h a r i d e , f u r t h e r carbohydrate i n c o r p o r a t i o n ceases and the predominant form i n ovine sub m a x i l l a r y mucin r e s u l t s ( F i g . 4 ) . I f , however, Gal i s i n c o r p o r a ted to form the Gal-βΐ,3-GalNAc d i s a c c h a r i d e , the various o l i g o saccharides present i n p o r c i n e submaxillary mucin r e s u l t (Fig. 4 ) . The r e l a t i v e p r o p o r t i o n s o f the s i a l y l - and g a l a c t o s y l t r a n s f e r a s e s c o n t r o l t h i s branch p o i n t (Schachter et_ al^. , 1971; McGuire, 1970). The C M P - s i a l i c acid:GalNAc-mucin a2-6 s i a l y l t r a n s f e r a s e has been p a r t i a l l y p u r i f i e d from sheep submaxillary gland (Carlson e t a l . , 1973 ) and porcine submaxillary gland (Sadler e t a l . , 1977). The b e s t substrate f o r t h i s enzyme i s s i a l i d a s e - t r e a t e d ovine submaxillary mucin (GalNAc-mucin) and other s i a l i c a c i d - f r e e mucins. The enzyme does not a c t on s i a l i d a s e - t r e a t e d αι-acid g l y c o p r o t e i n nor on l a c t o s e and i s t h e r e f o r e d i f f e r e n t from the s i a l y l t r a n s f e r a s e s d i s c u s s e d i n the previous s e c t i o n ; a v a r i e t y o f low molecular weight compounds both with and without t e r m i n a l GalNAc were i n e f f e c t i v e as acceptors although Gal-81/3-GalNAc had low acceptor a c t i v i t y . A n t i f r e e z e g l y c o p r o t e i n , which contains many Gal-81r3-GalNAc u n i t s , i s an e x c e l l e n t acceptor (Sadler e t a l . , 1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and
Catabolism
37
The key enzyme i n c o n t r o l l i n g s y n t h e s i s appears t o be the UDP-Gal:GalNAc-mucin 81-3 g a l a c t o s y l t r a n s f e r a s e (Schachter et a l . , 1971). This enzyme i s t i g h t l y bound t o membrane and a l l attempts a t p u r i f i c a t i o n have been u n s u c c e s s f u l . The enzyme w i l l not a c t i f GalNAc i s s u b s t i t u t e d with a s i a l i c a c i d residue (Fig. 4 ) . However, i f Gal i s i n c o r p o r a t e d , other sugar residues can be added. Thus, s i a l i c a c i d can then be added t o the i n t e r n a l GalNAc. F u r t h e r , Fuc can be i n c o r p o r a t e d i n a l , 2 linkage t o the Gal r e s i d u e ; t h i s enzyme i s s i m i l a r t o the human blood group H f u c o s y l t r a n s f e r a s e and can t r a n s f e r Fuc t o a v a r i e t y o f high and low molecular weight compounds with a t e r m i n a l 8-galactoside residue (McGuire, 1970) . The f u c o s y l t r a n s f e r a s e has r e c e n t l y been p u r i f i e d to near homogeneity from porcine submaxillary glands using GDP-hexanolamine-Sepharose (Beyer e t aJL. , 1977) . I t i s i n t e r e s t i n g t h a t although the f u c o s y l t r a n s f e r a s e w i l l a c t on a v a r i e t y o f 8 - g a l a c t o s i d e s i t p r e f e r s Gal-81,3-GalNAc Beyer e t a l . , 1977). The f i n a l enzyme i n the porcine submaxillary mucin syn t h e s i s scheme i s UDP-GalNAc:Gal-mUcin N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e . This enzyme i s analogous t o the human blood group Α-dependent N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e and occurs only i n those p i g s g e n e t i c a l l y disposed towards making A - p o s i t i v e mucin. The enzyme i n c o r p o r a t e s GalNAc i n a l , 3 linkage t o the Gal residue o f F u c - a l , 2 - G a l - t e r m i n a l acceptors. The p o r c i n e submaxillary gland enzyme was p u r i f i e d 38,000-fold by a f f i n i t y chromatography on UDP-hexanolamine-Sepharose (Schwyzer and H i l l , 1977a) and d e t a i l e d k i n e t i c s t u d i e s have been c a r r i e d out (Schwyzer and H i l l , 1977b). The pure enzyme has a molecular weight o f about 100,000 and may c o n t a i n 2 subunits. I t i s c l e a r from the above t h a t mucins are assembled by step-by-step a d d i t i o n o f monosaccharides; no pre-assembly seems to occur. The s u b c e l l u l a r path o f b i o s y n t h e s i s has not been worked out i n as great d e t a i l as f o r Asn-GlcNAc o l i g o s a c c h a r i d e s but passage through the G o l g i apparatus probably occurs (Bennett and Leblond, 1977; Schauer e t a l . , 1974). Glycophorin and other g l y c o p r o t e i n s have been reported to c a r r y a sialyl-a2,3-Gal-81,3-(sialyl-a2,6)-GalNAc t e t r a s a c c h a r i d e s t r u c t u r e (Marchesi e t a l . , 1976). The s i a l y l t r a n s f e r a s e c a t a l y z i n g t h i s a2,3 linkage t o galactose i s probably the same enzyme t h a t c a t a l y z e s s y n t h e s i s o f s i a l y l 0.2,3-lactose ; the s y n t h e s i s o f the a2,3 and a2,6 isomers o f s i a l y l - l a c t o s e by colostrum and l i v e r were d i s c u s s e d i n the p r e vious s e c t i o n . Sadler e t a l . (1977) p u r i f i e d two d i s t i n c t s i a l y l t r a n s f e r a s e s from porcine submaxillary gland by the use o f CDP-hexanolamine-Sepharose a f f i n i t y chromatography; one enzyme was the C M P - s i a l i c acid:GalNAc-mucin a2-6 s i a l y l t r a n s f e r a s e d i s cussed above, the other was an enzyme a c t i n g on a v a r i e t y o f acceptors to make a s i a l y l - a 2 , 3 - G a l l i n k a g e . Among the acceptors found a c t i v e with the l a t t e r enzyme were l a c t o s e , G a l - 8 1 , 3 -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
38
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
9?
S-a2,6-G-Bl,4-Gn-Bl , 4 . . * S-a2,6-G-Bl,4-Gn-Bl,2-M-o
^y-el,4-Gn-el> S-a2,6-G-Bl,4-Gn-Bl,2-M-al,6 Gn
|n-Asn
61,4> S-a2,6-G-Bl ,4-Gn-Bl ,4*'
<sf 0 Θ DEFICIENT ENZYME
IN-BORN ERROR
4-L-aspartylglycosylamlne amldohydrolase (enzyme 1)
Aspartylglycosylamlnurla (AGU)
a-mannosldase (enzyme 3) 8-N-acetylhexosamlnldases A and Β (enzyme 4)
STRUCTURES ACCUMULATING
Gn-Asn M-cl,6-M-el,4-Gn-el,4-Gn-Asn
M-ol,3-M-el,4-Gn
GL,,-gangl1os1dos1s variant 0 (SandhoffJatzkewltz disease)
Gn-Bl,2-M-al,3-Μ-βΙ,4-Gn
n c
Gn-6l,4-M-al,3-M-el ,4-Gn
Pollltt and Pretty (1974) Pollltt and Jenner (1969) Lundblad et a l . (1976) AkasaM et aTT (1976) Norden et al.. (1974)
Strecker et a l . (1977b) Ng Ylng KTn and Wolfe (1974)
Gn-Bl,2-M-al,3 v Gn-βΐ,4-Μ-βΙ,4-Gn Gn-Bl ,2-M-al , β ' Gn-Bl ,4 y Gn-βΐ,2-M-al,3 or 6 Gn-Bl,2-M-ol ,6 or 3
B-galactos1dase (enzyme 5)
(L,,-gangliosidosis ™
G-Bl,4-Gn-el,4^
Ng Y1ng K1n and Wolfe (1975)
G-Bl,4-Gn-Bl,2-M-ol,3 or 6
Wolfe et ai. (1974)
* M-βΙ,4-Gn G-Bl,4-Gn-el,2-M-al ,6 or 3 S1al1dase
Mucolipidosis I
(enzyme 6)
Mucolipidosis II (I Cell Disease)
S-a2,6-G-Bl,4-Gn-Bl,2-M-al,3
Michel ski et a l . (1977) Strecker et al_. (1976)
S-a2,6-G-Bl,4-Gn-Bl,2-M-al,6
Figure 5. Scheme depicting accumulation of glycopeptides and oligosaccharides derived from Asn-GlcNAc oligosaccharides in the urine and organs of patients with in-born errors of metabolism involving the absence of glycosidases. A typical Asn-GlcNAc oligosac charide of the N-acetyuactosamine type is shown at the top of the figure. Abbreviations: S, sialic acid; G, Ό-galactose; Gn, N-acetyl-O-glucosamine; M, Ό-mannose, F, \,-fucose; Asn, asparagine.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
Glycoprotein
SCHACHTER ET AL.
GalNAc,
antifreeze
submaxillary an
enzyme
glycoprotein
mucin.
capable
Pork
to
a sialyltransferase
GalNAc-Ser(Thr) least
saccharides. as
these
These
Many
organisms, variety
It
i s
will
porcine contain
(Hudgin and
has also
been
shown
sialyl-a2,3-Gal-81,3-
making
Therefore,
a c t on Ser(Thr)-GalNAc type act not only
on secretory
o n membrane-bound
glyco-
glycoproteins
therefore
reasonable
be widely
distributed.
at
oligo-
to predict
such
that
tides
limitations
plants
have
and both
which
t o a group
storage
of
appear
to be derived 5
Fig.
summarizes
the
exoglycosidases
end
one a t a time.
prevents
the action
apparent
that
known.
of
this
i n micro-
and vertebrate
animals
i n organs
and urine
from Asn-GlcNAc
normally
remove
The absence
briefly
of
sugars
I t
from
next
of action
process
o f these i n the
It
i s
(enzyme
also No.2,
are required
backbone.
diseases
that
exoglycosidase
N o . l , F i g . 5)
from polypeptide
located
i s evident
i n line.
o f the exo- and endo-
studies i s
(enzyme
d i s -
structures
the non-reducing
of a particular
o f t h e enzyme
of
glycopepsome
oligosaccharides.
data.
endo-8-N-acetylglucosaminidases
Pathological
degradation
consider
some o f t h i s
oligosaccharide
sequence
mucopolysaccharides,
We w i l l
and an asparaginase
separate
exact
discussion
the presence
o
glycolipids,
involving accumulation
5)
extensive
shown
invertebrate
andoligosaccharides.
orders
Fig.
preclude
laboratories
o f e x o - and endo
relevant
involving
to
I V above)
to
Glycoprotein Catabolism. Space
ly
enzymes
but also
two enzymes
topic. a
shown
( V a n d e n E i j n d e n e t a l . , 1977a)..
(mucins)
glycophorin.
VI.
section
two s i a l y l t r a n s f e r a s e s
proteins
previously
39
Catabolism
sialyl-a2,3-lactose
o f making
1972;
see also
and
and s i a l i d a s e - t r e a t e d
liver,
Schachter, contain
Biosynthesis
enzymes indicate
The i s not that
lysosomes.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
the
40
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
LITERATURE CITED Akasaki, Μ., Sugahara, K., Funakoshi, I., Aula, P. and Yamashina, I. (1976). FEBS Letters 69, 191-194. Andrews, P. (1970). FEBS Lett. 9, 297-300. Bartholomew, B.A., Jourdian, G.W., and Roseman, S. (1973). J. Biol. Chem. 248, 5751-5762. Behrens, N.H., Parodi, A.J. and Leloir, L.F. (1971). Proc. Nat. Acad. Sci. USA 68, 2857-2860. Bell, J.E., Beyer, T.A. and Hill, R.L. (1976). J. Biol. Chem. 251, 3003-3013. Bennett, G. and Leblond, CP. (1977). Histochemical J. 9, 393417. Beyer, T.A., Prieels, J.P. and Hill, R.L. (1977). Proc. Inter national Symp. Glycoconjugates, Woods Hole, Mass., in press Blobel, G. and Dobberstein, B. (1975a). J. Cell Biol. 67, 835-851. Blobel, G. and Dobberstein, B. (1975b). J. Cell Biol. 67, 852-862. Brew, K., Vanaman, T.C. and Hill, R.L. (1968). Proc. Nat. Acad. Sci. USA 59, 491-497. Carlson, D.M., McGuire, E.J., Jourdian, G.W., and Roseman, S. (1973). J. Biol. Chem. 248, 5763-5773. Chou, T.H., Murphy, C. and Kessel, D. (1977). Biochem. Biophys. Res. Communs. 74, 1001-1006. Cowan, N.J. and Robinson, G.B. (1970). FEBS Lett. 8, 6-8. Czichi, U. and Lennarz, W.J. (1977). J. Biol. Chem. 252, 7901-7904. Ebner, K.E. (1973). In "The Enzymes" (P.D. Boyer, editor), Edit 3, Vol. 9, Part B, pp. 363-377, Academic Press, New York.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and Catabolism
41
Fitzgerald, D.K., Brodbeck, U., Kiyosawa, I., Mawal, R., Colvin, B., and Ebner, Κ.Ε. (1970). J. Biol. Chem. 245, 2103-2108. Fraser, I.H. and Mookerjea, S. (1976). Biochem. J. 156, 347-355. Geren, C.R., Geren, L.M. and Ebner, K.E. (1975a). Biochem. Biophys. Res. Communs. 66, 139. Geren, C.R., Magee, S.C. and Ebner, K.E. (1975b). Biochemistry 14, 1461-1463. Geren, C.R., Magee, S.C. and Ebner, K.E. (1976). Arch. Biochem. Biophys. 172, 149-155. Geren, C.R., Geren, L.M. Biochim. Biophys. Acta 497, 128-132. Hagopian, Α., Westall, F.C., Whitehead, J.S., and Eylar, E.H. (1971). J. Biol. Chem. 246, 2519-2523. Hercz, Α., Katona, Ε., Cutz, Ε., Wilson, J.R., and Barton, M. (1978). Submitted for publication. Herscovics, Α., Bugge, B. and Jeanloz, R.W. (1977a). J. Biol. Chem. 252, 2271-2277. Herscovics, Α., Golovtchenko, A.M., Warren, CD., Bugge, B. and Jeanloz, R.W. (1977b). J. Biol. Chem. 252, 224-234. Hill, H.D. Jr., Schwyzer, Μ., Steinman, H.M. and Hill, R.L. (1977). J. Biol. Chem. 252, 3799-3804. Hudgin, R.L. and Schachter, H. (1972). Can. J. Biochem. 50, 1024-1028. Hunt, L.M., Etchison, J., Robertson, J., Robertson, M. and Summers, P.F. (1977). Abstracts 174th. National Meeting of the Am. Chem. Soc, Chicago, 111. Isemura, M. and Schmid, K. (1971). Biochem. J. 124, 591-604. Ito, S., Muramatsu, T., and Kobata, A. (1975). Arch. Biochem. Biophys. 171, 78-86. Jabbal, I. and Schachter, H. (1971). J. Biol. Chem. 246, 51545161. Jamieson, J.C. (1977). Can. J. Biochem. 55, 408-414.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
42
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Jeppsson, J-0, Laurell, C-B., and Fagerhol, M. (1978). Eur. J. Biochem. 83, 143-153. Kessel, D., Sykes, E. and Henderson, M. (1977). J. Natl. Cancer Inst. 59, 29-32. Khatra, B.S., Herries, D.G. and Brew, K. (1974). Eur. J. Biochem. 44, 537-560. Kiely, M.L., McKnight, G.S., and Schimke, R.T. (1976). J. Biol. Chem. 251, 5490-5495. Kitchen, B.J. and Andrews, P. (1974a). Biochem. J. 141, 173-178. Kitchen, B.J. and Andrews 587-590. Lawford, G.R. and Schachter, H. (1966). J. Biol. Chem. 241, 5408-5418. Lehman, E.D., Hudson, B.G. and Ebner, K.E. (1975). FEBS Lett. 54, 65-69. Letts, P.J., Pinteric, L. and Schachter, H. (1974). Biochim. Biophys. Acta 372, 304-320. Lundblad, Α., Masson, P.K., Norden, N.E., Svensson, S. Ockerman, P.Α., and Palo, J. (1976). Eur. J. Biochem. 67, 209-214. Magee, S.C., Mawal, R. and Ebner, K.E. (1974). Biochemistry 12, 99-102. Magee, S.C., Geren,C.R.and Ebner, K.E. (1976). Biochim. Biophys. Acta 420, 187-194. Marchesi, V.T., Furthmayr, H. and Tomita, M. (1976). Ann. Rev. Biochem. 45, 667-698. Marshall, R.D. (1974). Biochem. Soc. Symp.40,17-26. McGuire, E.J. (1970). In "Blood and Tissue Antigens" (D. Aminoff, editor), pp. 461-478, Academic Press, New York. Michalski, J.C., Strecker, G., Fournet, B., Cantz, M. and Spranger, J. (1977). FEBS Lett 79,' 101-104.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and Catabolism
43
Molnar, J. and Sy, D. (1967). Biochemistry 6, 1941-1947. Montreuil, J. (1975). Pure and Applied Chemistry42,431-477. Morrison, T.G. and Lodish, H.F. (1975). J. Biol. Chem. 250, 6955-6962. Munro, J.R. and Schachter, H. (1973). Arch. Biochem. Biophys. 156, 534-542. Munro, J.R., Narasimhan, S., Wetmore, S., Riordan, J., and Schachter, H. (1975). Arch. Biochem. Biophys. 169, 269-277. Narasimhan, S., Stanley P J. Biol. Chem. 252
d Schachter H (1977)
Ng Ying Kin, N.M.K. and Wolfe, L.S. (1974). Biochem. Biophys. Res. Communs.59,837-844. Ng Ying Kin, N.M.K. and Wolfe, L.S. (1975). Biochem. Biophys. Res. Communs.66,123-130. Norden, N.E., Lundblad, Α., Svensson, S. and Autio, S. (1974). Biochemistry 13, 871-874. Ogata, S., Muramatsu, T. and Kobata, A. (1975). J. Biochem. (Tokyo) 78, 687-696. Parodi, A.J., Behrens,N.H.,Leloir, L.F. and Dankert, M. (1972) Biochim. Biophys. Acta 270, 529-536. Paulson, J.C., Beranek, W.E., and Hill, R.L. (1977a). J. Biol. Chem. 252, 2356-2362. Paulson, J.C., Rearick, J.I. and Hill, R.L. (1977b). J. Biol. Chem. 252, 2362-2371. Pless, D.D. and Lennarz, W.J. (1977). Proc. Nat. Acad. Sci. USA 74, 134-138. Pollitt, R.J. and Jenner, F.A. (1969). Clin. Chim. Acta 25, 413-416. Pollitt, R.J. and Pretty, K.M. (1974). Biochem. J. 141, 141-146. Powell, J.T. and Brew, K. (1974). Eur. J. Biochem. 413, 217-228.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
44
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Powell, J.T. and Brew, K. (1975). J. Biol. Chem. 250, 6337-6343. Powell, J.T., Jarlfors, U. and Brew, K. (1977). J. Cell Biol. 72, 617-627. Rao, A.K., Garver, F., and Mendicino, J. (1976). Biochemistry 15, 5001-5009. Robbins, P.W. (1977). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Rothman, J.E. and Lodish, H.F. (1977). Nature 269, 775-780. Sadler, J.E., Rearick, J.I., Paulson, J.C. and Hill, R.L. (1977). Proc. International Symp Glycoconjugates Wood Hole Mass., in press Sato, T., Yosizawa, Z., Masubuchi, M. and Yamauchi, F. (1967). Carbohyd. Res.5,387-398. Schachter, H. (1974a). Adv. Cytopharmacol. 2, 207-218. Schachter, H. (1974b). Biochem. Soc. Symp. 40, 57-71. Schachter, H. (1977). In "Mucus in Health and Disease" (M. Elstein and D.V. Parke, editors), pp. 103-129, Plenum Publish. Co. Schachter, H. (1978). In "The Glycoconjugates" (M. Horowitz and W. Pigman, editors), Vol. 2, Academic Press, New York, in press. Schachter, H. and Roden, L. (1973). In "Metabolic Conjugation and Metablic Hydrolysis" (W.H. Fishman, editor), Vol. 3, pp. 1-149, Academic Press, New York. Schachter, H., Jabbal, I., Hudgin, R.L., Pinteric, L., McGuire, E.J., and Roseman, S. (1970). J. Biol. Chem. 245, 1090-1100. Schachter, H., McGuire, E.J. and Roseman, S. (1971). J. Biol. Chem. 246, 5321-5328. Schauer, R., Buscher, H.-P., and Casals-Stenzel, J. (1974). Biochem. Soc. Symp. 40, 87-116. Schwyzer, M. and Hill, R.L. (1977a). J. Biol. Chem. 252, 2338-2345.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2.
SCHACHTER ET AL.
Glycoprotein
Biosynthesis
and Cataholism
45
Schwyzer, M. and Hill, R.L. (1977b). J. Biol. Chem. 252, 2346-2355. Sherr, C.J. and Uhr, J.W. (1969). Proc. Nat. Acad. Sci. USA 64, 381-387. Smith, C.A. and Brew, K. (1977). J. Biol. Chem. 252, 7294-7299. Spiro, M.J., Spiro, R.G. and Bhoyroo, V.D. (1976a). J. Biol. Chem. 251, 6400-6408. Spiro, M.J., Spiro, R.G. and Bhoyroo, V.D. (1976b). J. Biol. Chem. 251, 6420-6425. Spiro, R.G., Spiro, M.J d Bhoyroo V.D (1976) J Biol Chem. 251, 6409-6419 Stanley, P., Narasimhan, S., Siminovitch, L., and Schachter, H. (1975). Proc. Nat. Acad. Sci. USA72,3323-3327. Stoffyn, P., Van den Eijnden, D. and Stoffyn, A. (1977). Fed. Proc.36,744. Strecker, G., Hondi-Assah, T., Fournet, B., Spik, G., Montreuil, J., MarOteaux, P., Durand, P., and Farriaux, J-P. (1976). Biochim. Biophys. Acta 444, 349-358. Strecker, G., Fournet, Β., Spik, G., Montreuil, J., Dorland, L., Haverkamp, J., Schut, B.L. and Vliegenthart, J.F.G. (1977a). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Strecker,G.,Herlant-Peers, M-C, Fournet, Β., Montreuil, J., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., and Farriaux, J-P. (1977b). Eur. J. Biochem. 81, 165-171. Struck, D.K. and Lennarz, W.J. (1977). J. Biol. Chem. 252, 1007-1013. Tabas, I., Schlesinger, S. and Kornfeld, S. (1978). J. Biol. Chem 253, 716-722. Tai, T., Ito, S., Yamashita, K., Muramatsu, T. and Kobata, A. (1975). Biochem. Biophys. Res. Communs.65,968-974. Tonneguzzo, F. and Ghosh, H.P. (1975). FEBS Lett.50,369-373. Trayer, I.P. and Hill, R.L. (1971). J. Biol. Chem. 246, 6666-6675.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
46
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Tsopanakis, A.D. and Herries, D.G. (1976). Biochem. Biophys. Res. Communs.68,1102. Turco, S.J., Stetson, B. and Robbiris, P.W. (1977). Proc. Nat. Acad. Sci. USA 74, 4411-4414. Van den Eijnden, D.H., Dieleman, B. and Schiphorst, W.C.M. (1977a). Proc. Fourth International Symp. Glycoconjugates, Woods Hole, Mass., in press. Van den Eijnden, D.H., Stoffyn, P., Stoffyn, A. and Schiphorst, W.E.C.M. (1977b). Eur. J. Biochem. 81, 1-7. Vargas, V.I. and Carminatti, H. (1977). Mol. Cell. Biochem. 16, 171-176. Waechter, C.J. and Lennarz, 45, 95-112.
(1976).
Watkins, W.M. (1974). In "The Red Cell" (D.M. Surgenor, editor), 2nd edition, Vol. 1, pp. 293-360, Academic Press, New York; Biochem. Soc. Symp.40,125. Wilson, J.R., Williams, D. and Schachter, H. (1976). Biochem. Biophys. Res. Communs.72,909-916. Wirth, D.F., Katz, F., Small, B. and Lodish, H.F. (1977). Cell 10, 253-263. Wolfe, L.S., Senior, R.G. and Ng Ying Kin, N.M.K. (1974). J. Biol. Chem. 249, 1828-1838. RECEIVED April 7, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3 Structure and Metabolism of Glycolipids C. C. SWEELEY, Y.-K. FUNG, B. A. MACHER, J. R. MOSKAL, and H. A. NUNEZ Department of Biochemistry, Michigan State University, East Lansing, MI 48824
Glycolipids have bee some important biologica as contact inhibition, intercellular adhesion, immunochemical tissue specificity, hormone receptor function, and internalization of various external macromolecular and supramolecular materials. Close to a hundred different glycolipids have been isolated and characterized from various terrestrial and marine organisms to date, providing sufficient structural diversity for these proposed functional roles. Interest in the chemistry of these substances was stimulated by the discovery of a family of inherited abnormalities in the metabolism of some of the glycolipids (13). Subsequently they became a subject of intense study by cell biologists and immunologists when it was generally recognized that they are membrane constituents of most tissues; current areas of research interest are the immunochemical specificity of glycolipids, their localization and mobility in bilayer membranes, lectin-binding properties, and viral transformation-specific changes in their composition. The aim of this review is to provide an introduction into the chemistry, methods of analysis and metabolism of the major classes of glycolipids that occur in eukaryotic organisms. Structure Unlike the complex lipopolysaccharides of bacterial membranes or the glycosides of hydroxylated fatty acids in yeasts, the glycolipids of eukaryotic organisms are of two major types, called glycosphingolipids and glycoglycerolipids. Glycosphingolipids are composed of a family of longchain aliphatic bases (sphingosines), fatty acids and carbohydrates, whereas the glycoglycerolipids consist of glycerol, fatty acids (or fatty ethers) and carbohydrates. The glycosphingolipids can be subdivided into several classes on the basis of gross molecular features. Thus, there are neutral glycosphingolipids, gangliosides (glycosphingolipids containing sialic acid), sulfoglycosphingolipids (containing sulfate ester groups on the carbohydrate moiety) and phosphoglycosphingolipids. There are also neutral and sulfoglycoglycerolipids. Complete review of the structures and occurrence of the glycosphingolipids and glycoglycerolipids is not possible in this brief presentation. Recent reviews with more extensive 0-8412-0452-7/78/47-080-047$09.75/0 © 1978 American Chemical Society American Chemical Society Library In Glycoproteins and Glycolipids in Disease 1 1 5 5 1 6 t h St. N.Processes; W. Walborg,DC,E.;1978. ACS Symposium Series;W American ashingtChemical on, D. Society: C. 2CWashington, 036
48
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
information about specific topics are cited whenever possible to assist the reader with a need for more than a casual introduction to the field. An excellent text (4) on the isolation, structure analysis and metabolism of glycolipids, with an emphasis on details of methods and procedures, should also be consulted. Ceramide is the hydrophobic portion of a glycosphingolipid that is anchored in a bilayer membrane. It consists of a mixture of molecular species in which the long-chain bases such as sphing-4-enine are attached through amide linkages to several fatty acids. The most common longchain bases are shown below, but a variety of other chain-length homologs, branched sphingosines and multiply unsaturated types are known to exist (5). They are all derived from sphinganine, previously known as dihydrosphingosine, which is D-erythro-2-aminooctadecane-1,3-diol or 2S,3R-2-aminooctadecane-l,3-diol (6). Methods have been described for their analysis as aldehydes after periodate oxidation (7), N-acetyltrimethylsilyl derivatives (8), dinitrophenyl derivatives (9), and as the alcohols obtained from intac ozonolysis and reduction (10>) CHUOH
CH9OH
I
HC-NH
I
HC - OH
I
(CH9)..
I
2
CH-
1
*
3
I
CH9OH
I
HC-NH
9
I
I
9
HC-NH
I
I
9
1
H C - OH
H C - OH
HC
HC
II
II
I
I
CH
(ÇH2)12 CH^ Sphinganine
CH9OH
I
4-Sphingenine
CH
(ÇH2)W
HC— N H 9
I
HC -
I
OH
HC-OH
I
(CH9)n
I CH
3
CH^ 4-Eicosasphingenine
4-D-Hydroxysphinganine The fatty acids occurring in the ceramide moiety are a variable mixture of saturated and monounsaturated homologs from C ^ to C 2 , and the α-hydroxy derivatives of these fatty acids. Lignoceric (24:uf and nervonic (24:1, Δ ) acids are generally major components, along with substantial amounts of palmitic (16:0), stearic (18:0), arachidic (20:0), behenic (22:0) and tricosanoic (23:0) acids. In some cases the α - h y d r o x y fatty acids are the dominant species. Comparisons of the fatty acid composition of a wide variety of glycosphingolipids have been reported
01).
In the glycosphingolipids, ceramide is attached to a mono- or oligosaccharide by a glycosidic linkage with the primary hydroxyl group of the long-chain base. Although it has only been proved conclusively in a few instances by nmr spectroscopy or chemical synthesis, it is assumed that the glycosidic linkage between ceramide and the carbohydrate moiety
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
49
has the 3 configuration, and that the sugar residues exist in the pyranose ring form. An example is shown in Figure 1, galactosyl-(gl->-4)-glucosyl31+D-ceramide (lactosylceramide). A class of phosphoglycosphingolipids occurs in higher plants and fungi; these structures consist of carbohydrate groups with an inositol residue linked to ceramide through a phosphodiester bridge. A substance of this type from corn was proposed to be GlcNal^4GlcUAal^6inositol(2^1aMan)-l-0-phosphorylceramide (169). A related compound from tobacco leaves (170) has been shown to be GlcNAcal+4GlcUAal^2myo-inositol-l-Q-phosphorylceramide (171). In the glycoglycerolipids studied thus far, the carbohydrate residues are attached to C-3 of sn-glycerol by either a- or 3-glycosidic linkages. Three important types of glycoglycerolipids are 3-3-galactosyl-l,2-diacylsn-glycerol (12), 3'-sulfo-3-3-galactosyl-l-alkyl-2-acyl-sn-glycerol (seminolipid) (13,14), and a sulfotriglycosyldiglyceride of human gastric secretions, 3-ot-(6'-sulfoglucosyl-(a l+6)-glucosyl-(al+6)-glucosyl)-l-alkyl2-acyl-sn-glycerol (15). Structural variatio glycosphingolipids is so diverse that a special nomenclature has become necessary in referring to these substances. A semisystematic nomencla ture has been recommended by the IUPAC-IUB Commission on Biochemi cal Nomenclature (^6) and has been reviewed in some detail (6,17,18). Compounds containing a single monosaccharide unit were historically called cerebrosides. Now they are identified by the sugar they contain; examples are galactosylceramide (GalCer) and glucosylceramide (GlcCer). Similarly, the diglycosylceramide containing a lactose unit is called lactosylceramide (LacCer). More complex glycosphingolipids are given names that identify a particular portion of the carbohydrate moiety, indicating the family to which it belongs and the number of glycose units in the chain. Prefixes (given below) for the various families imply a specific structure, including the sequential arrangement of the glycose units as well as the positions and anomeric configuration of the glycosidic linkages. Thus, the glycosphingolipid of the globo type that contains all four sugar residues is referred to as globotetraglycosylceramide and is abbreviated GbOse^Cer. Similarly, the glycosphingolipid of the ganglio type with three sugar units is called gangliotriglycosylceramide (GgOse^Cer); this substance is G a l NAc3l->4Gal31^4GlcCer. Substituents that are not part of the root oligosaccharide are given at the beginning of the name, using a Roman numeral to indicate the monosaccharide unit (counting from ceramide) on which the substituent is located and a superscript Arabic numeral to indicate the position of the glycosidic linkage. According to this system, Forssman hapten (Gal Ν Ac al-* 3GalNAc31+3Galal+4Gal3 l+4GlcCer) should he called IV -a-N-acetylgalactosaminyl-globotetraglycosylceramide (I\raGalNAc-GbOse^Cer). Methods of Analysis of Glycolipids Determination of the complete chemical structure of a glycolipid usually requires a combination of several techniques. Some of the methods can be used with nanomolar amounts of sample, whereas others
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
50
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 1. Lactosylceramide (LacCer), galactopyranosyl-(βl -» 4)-glucopyranosyl—(1 -» r)-ceramide
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Prefix
Abbreviation
lacto lactoneo muco gala globo globoiso ganglio
Lc Lcn Mc Ga Gb Gbi Gg
Structure
and Metabolism
51
Structure GaKg l+3)GlcNAc(e l+3)Gal(B l-*4)Glc Gal(Bl+4)GlcNAc(ei+3)Gaiei+4)Glc Gal( β 1 +3)Gal( β 1 +4)Gal( β 1+ 4)Glc GalNAc( l+3)Gal( l+4)Gal(al+4)Gal* GalNAc(ei+3)Gal(al-*4)Gal(31+4)Glc GalNAc( Bl+3)Gal(al+3)Gal(6 l-*4)Glc Gal( β ΐ +3)GalNAc( ei+4)Gal(6 l+4)Glc
^Substances more complex than Galal->-4GalCer in this family have not so far been isolated from biological material. require about one micromole; carbon-13 nmr spectroscopy is applicable at present only if a large amount (ca. 150 mg) of sample is available. Most of the structural studies chromatographic separation of the glycolipid (column and thin-layer chromatography), followed by several commonly used analytical proce dures: fatty acid, long-chain base and carbohydrate composition by gasliquid chromatography after acid-catalyzed methanolysis; stepwise degra dation of the carbohydrate moiety with specific glycosidases; and analysis of the partially methylated alditol acetates derived from permethylated glycolipid. Frequent use of mass spectrometry has been made in recent studies to validate the structures of the partially methylated alditol acetates and to determine some structural features of the intact permethylated glycolipid. Some of the newest techniques are reviewed briefly in the following section. Excellent separations of glycolipids according to fatty acid differ ences have been obtained by high pressure liquid chromatography of perbenzoylated or O-acetyl-N-p-nitrobenzoyl derivatives (19-21), requir ing only nanomolar amounts of samples for detection at 254 nm with a suitable flow-through detector. Mixtures of natural glycolipids from GlcCer to GbOse. Cer can be separated by chromatography on Sorbax Sil (22). Though the technique appears to be ideally suited for the separation of substances with different molecular weights, sufficient studies have not been made to determine whether isomeric glycolipids (different sugar arrangement, different positions and/or configuration of glycosidic link ages, different epimeric components) can be separated effectively. An alternative chromatographic approach involves the careful analysis by thin-layer chromatography of the effluent material from a column. This technique appears to be most effective when use is made of an ion exchange material such as DEAE-cellulose or DEAE-Sephadex and when a very large number of samples from the column are spotted adjacent to each other on the thin-layer plates. The resulting map looks like a two-dimensional chromatogram with excellent resolution of components that would overlap if analyzed together on the plate. This procedure has been exploited in the study of blood group glycolipids from human erythrocytes (23) and for analyses of the ganglioside composition of brain (24,25). Additional fractionation of gangliosides has been obtained
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
52
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
on a preparative scale by DEAE-Sephadex column chromatography and subsequent separation on a new spherical silica, called Iatrobeads (26-29). Analysis of carbohydrate composition by gas-liquid chromatography is common practice. Trimethylsilyl derivatives of methyl glycosides (obtained by acid-catalyzed methanolysis) or alditol acetates (obtained by acetolysis and hydrolysis) can be used with suitable internal standards to obtain molar ratios of the sugar components with about 100 nanomoles of sample ( 3 0 - 3 2 ) · These conditions cannot be used to distinguish between N acetyl and N-glycolyl derivatives of neuraminic acid in the intact glycolipids. Mild acid-catalyzed methanolysis (0.05 Ν methanolic HC1 at 8 0 ° for 1 hr) has been used instead, giving high yields of the β - m e t h y l ketosides of N-acetyl and N-glycolylneuraminic acid, the trimethylsilyl derivatives of which separate readily by gas chromatography (33). The sequential arrangement of the sugar residues in complex glycolipids can be deduced enzymatically or by mild acid hydrolysis or methanolysis. The enzymatic procedure involves the stepwise hydrolysis of the glycolipid with specifi evidence for the nature of the terminal glycose residue at each step as well as the anomeric configuration of the glycosidic linkage (34). It is customary to first remove sialic residues from gangliosides by 1 M formic acid hydrolysis for 1 hr at 1 0 0 ° (35), a procedure which also liberates fucose (36). Negative results in the enzymatic analysis need to be interpreted with some care since they might be caused by improper detergent in the reaction mixture or a glycosidase specificity that gives poor K m or maximal velocity values with a particular structure (34). It is important to realize that the procedure requires a set of highly pure glycosidases. The other method involves partial methanolysis with 0.3 M HC1 in chloroform/methanol at 6 0 ° for 40 min; it has been used effectively for analyses of a heptaglycosylceramide (37) and various blood group Α - a c t i v e glycolipids from gastric mucosa (36). Two methods make use of permethylation for the analysis of glycolipids and can be used together to obtain information about the sequential arrangement of carbohydrate residues and the positions of glycosidic linkages. Permethylation is generally carried out in dimethylsulfoxide with a mixture of sodium dimethylsulfinylcarbanion and methyl iodide (38), a mild procedure (room temperature) which gives high yields of completely methylated product. Precautions have to be taken to exclude plasticizers from the starting materials, particularly dimethylsulfoxide. Mass spectra of the permethylated intact glycolipid reveal several kinds of structural detail, including the composition of the ceramide moiety, the nature of the terminal glycose unit, the total number of glycose units and, to some extent, the sequence of sugars (3945). Spectra of the product obtained by reduction of the N-methyl amide linkage with lithium aluminum hydride are somewhat superior as ions at high mass are stabilized and the volatility of the derivative increases to some extent (39-45). Useful information results from the cleavage reactions (at glycosidic linkages) that result from random charge localization on ring oxygen atoms. Unfortunately these ions are obscured to some extent and often represent weak contributions to the total mass spectrum, due to the presence of the ceramide moiety.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
53
An alternative approach involves conversion of the glycolipid to an oligosaccharide by ozonolysis (46) or treatment with osmium tetroxide (47), both of which attack at the double bond of the sphingosine moiety, yielding a product that is readily hydrolyzed to the free oligosaccharide. The permethylated oligosaccharide has excellent mass spectral character istics, as illustrated in the case of lacto-N-tetraose and difucohexaose I in Figures 2 and 3. The molecular ion was not observed in either case upon electron impact ionization but may be present in chemical ionization mass spectra. The ion at highest mass in the case of the lacto-N-tetraose derivative is at m/e 858; it results from the loss of a C-6 substituent (· CH^OMe) from whichever ring bears the charge (on oxygen) in the molecular ion. Three ions (below) of approximately equal intensity, at m/e 219, 464 and 668 in Figure 2, are predicted for the sequence hexoseN-acetylhexosamine-hexose-R by arithmetic calculations; they result from the cleavage of different glycosidic linkages, with charge retention on the ring oxygen and loss of the remainder of the chain as a free radical.
Ν ME AC Further loss of methanol from J^and a disaccharide from 3 both give m/e 187 (4), whereas loss of the terminal hexose residue from 2^ gives an intense ion at m/e 228 (5). The presence of m/e 464 (a hexosaminecontaining disaccharide moiety) and the absence of m/e 260 (a terminal N acetylhexosamine unit) are strong evidence for the hexose-hexosamine
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
71
100
ΙΟΙ
150
668
159
650 100
M
I 'Ί 1
\
150
300
1
1
I
\
1
I
1
I
1
I
J I 'Ί I I ' I
850
1
858
/
MeOÇHs Ν
'Se/' /423
8CO
I 11
/66Θ
/
\ MtOÇHg
668\
350
r
2
T
1
Γ
τ
450
432
Χ3
1
950
I M 'Ί ' I ' 1
1000
1
500
464
M Ί I I I I I I M ' I
MW 903
Τ ' I
900
r
9
400
LACTO-N-TETRAOSE
464 \
I M M ι I ' I
2I9\
250
228
1
HO lb
Figure 2. Mass spectrum of permethyhted hcto-N-tetraose, obtained by electron impact ionization at 70 eV with a Varian MAT CH-5 double-focusing mass spectrometer (direct probe)
600
200
187
V-i • f • |-»|t f Ι Γ Ι | + | Λ * ι ' Λ Γ f i
50
100 η
ο
η
S
S!
S 3
ο ο ο ο
>
3
8
3.
SWEELEY ET AL.
55
Glycolipid Structure and Metabolism
tUC
S •S Se
1 ο ο ο
1 Ο
.s
Ë a.
a.
I CUD
s
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
56
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
arrangement. The presence of a strong fragment ion at m/e 228 is good evidence for a 1,3 linkage of the terminal hexose unit to the hexosamine, since this product is stabilized by the pair of electrons on the nitrogen. The product from loss of terminal hexose in a 1,4 linked disaccharide would not be similarly stabilized and would likely lose methanol to give an intense ion at m/e 196. In fact, this is the case with the doubly substituted hexosamine moiety in the difucohexaose (Fig. 3); further conversion of the ion at m/e 812 to m/e 606 by loss of the C-4 substituent results in a very weak ion, compared with that obtained by loss of the C-3 substituent from m/e 812 to give m/e 402. The ion at m/e 196, for loss of both of C-3 and C-4 substituents from m/e 812, is strong, as expected on the basis of these arguments. More precise information about the positions of glycosidic linkages can be obtained by the analysis of the monosaccharides liberated from the permethylated glycolipids by acid-catalyzed methanolysis (48) or by hydrolysis and reduction to partially methylated alditols (49-51). In either case free hydroxyl groups derivatives are analyzed b metry (50,52-54). A large number of model compounds have been analyzed and their retention behavior and mass spectral characteristics reported (55). Tfie mass spectra are similar on casual inspection but generally have at least one ion that is uniquely derived from a particular structure. Epimeric structures are differentiated on the basis of their retention behavior rather than their mass spectra. Use of this technique to distinguish between terminal, 1,3- and 1,4substituted N-acetylhexosamines is illustrated in Figures 4-6. Similarities in the mass spectra result from cleavage between C-2 and C-3 in all three cases to give an intense ion at m/e 158, which loses ketene to give another intense ion at m/e 116. As a rule strong ions are formed when cleavage can occur between two carbons substituted with O C H ^ groups, while virtually no ionization occurs by cleavage between two carbons bearing acetoxy groups. The three kinds of hexosamine are most readily distinguished by the presence of m/e 202 and 205 in the case of the product from terminal G a l N A c , m/e 233 from 1,4-linked G a l N A c and m/e 318, which is strong only in the product of 1,3-linked G a l N A c . Mass chromatography is a powerful tool that can be used to advantage for the analysis of the partially methylated alditol acetates from permethylated glycolipids, using a computerized gas chromatography-mass spectrometry system. Quick and accurate analysis of the products can be obtained without careful inspection of the total mass spectra. For example, Forssman pentaglycosylceramide (GalNAcal-*3GalNAcBl-*3Galal+4Gal31+4GlcCer) would be converted by permethylation analysis to equimolar amounts of N-methyl-N-acetyl-l,5-di-0-acetyl3,4,6-tri-O-methylgalactosaminitol from the terminal G a l N A c and N methyl-N-acetyl-1,3,5-tri-0-acetyl-4,6-di-0-methylgalactosaminitol from the internal G a l N A c residue. Mass chromatograms of m/e 205, 233 and 318 (Fig. 7) clearly show that the terminal and 1,3-substituted G a l N A c products are present in the mixture and that the 1,4-substituted G a l N A c product is not. Previous discussion of this technique by Laine (private communication, ref. 171 and Abstracts, Society for Complex Carbohydrates, New Orleans, October 8, 1976) suggests that it might be used with
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
57
Glycolipid Structure and Metabolism CH2OCOCH3 158
145
-
a
a' - S U » -
ÇN^COCH-.
"205
202
205/
H
5
CH.O CH i _ L C H 3 0 CH
161
HCOCOCFL
I
3
CH2OCH3 Figure 4.
Mass spectrum of
N-methyl-N-acetyU3,4,6-tri-0-methyl-l,5-di-0-acetylgalactosamintol
43
L
Λ. 158 /
116
J
MLL
Α..
I
~
233
5
^ C H
3
J ^ ^ C O Œ L
233 CH.O CH
1/ '
.1
Λ
I 158
I
3
-
i t fI Li t i a i eI- 2 * T J.
I M
.L - « ·
-·
·
-
a J i a l e
-
·
CH.COO CH
I
3
H C O C O C H 3 I CH2OCH3
Figure 5.
Mass spectrum of N-methyl-N-acetyl-3fi-di-0-methyl-14,5-tri-0acetylgalactosaminitol CH2OCOCH3 43
•
Τ.,.,,.,Ιμ.,.Α
I ~ "cTÇ
116
158
y!
H
" "
318
9 ^cocH3 N
CH.COOCH 3
I
CH30-CH
H C O C O C H 3 I
CH2OCH3 Figure 6.
Mass spectrum of
N-methyl-N-acetyl^-di-O-methyl-l^^-tri-O-acetylgalâctosaminitol
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 7. Mass chromatography of m/e 205, 233, 272, and 318 along with total ion intensity (Til) for gas chromatography—mass spectrometry analysis of partially methylated alditol acetates from Forssman pentaglycosylceramide. Ion peaks of m/e 272 and 318 at scan 176 are specific for 1,3-linked GalNAc (see Figure 6), and the peak of m/e 205 at scan 149 is specific for a terminal GalNAc (see Figure 4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
59
nanomolar amounts of glycolipid. Carbon-13 nuclear magnetic resonance spectroscopy offers some unique capabilities in the structural analysis of glycolipids, not the least of which may be the possibility to analyze interactions at the cell surface. Large are required to obtain a natural abundance Fouriertransform C nmr spectrum, using a high field instrument to resolve the complex of resonances for ring carbons not involved in glycosidic bonds or otherwise derivatized. Assignments have recently been made by Sillerud et^l. (56) for the resonances of every carbon nucleus of G . , . ganglioside JÛ α - Ν - a c e t y l n e u r a m i n o s y l - g a n g l i o t e t r a g l y c o s y l c e r a m i d e ) , Based on care ful comparisons of the spectrum of this ganglioside with those of neuraminosyllactose, ceramide and several related glycolipids. We have used the assignments proposed by Sillerud et al. (56) for ^ ganglioside to analyze the C nmr spectrum of a complex neutral glycosphingolipid, globotetraglycosylceramide, and present some of the assignments in Figure 8. A higher resolution spectrum and data for a few model compounds are necessary fo In the tetrasaccharide moiety the glycosidic carbons resonate in the range typical of α and β glycosidic linkages at 100 to 106 ppm. The hydroxymethyl carbons appear at 61 to 63 ppm, the sugar ring carbons of a glycone linkage appear at 79 to 83 ppm and the ring sugar carbons not involved in inter-sugar linkages fall in the 72 to 77 ppm region, with the exception of the C-2 of N-acetyl-D-galactosamine and C-4 of the galactosyl moieties. The ceramide moiety gives a set of resonances that resemble very closely those observed for ganglioside G . . . micelles (56). The alkyl chain shows a large and broad peak for the bulk methylene carbons and sharper peaks for the terminal methyl and neighboring meth ylene carbons in the region of 14 to 40 ppm. The rest of the carbons show resonances typical of the carbonyl group (175 ppm), of a carbon-carbon double bond (132 ppm), of a hydroxymethyl carbon linked to a glycosidic oxygen (69 ppm), of a secondary alcohol (72 ppm), and of a nitrogen-linked methinyl carbon (55 ppm).
s a m e p s l
Glycosphingolipid Biosynthesis The biosynthesis of glycosphingolipids should begin with studies dealing with the synthesis of ceramide (57-59). However, from a biological standpoint, it is the composition and sequence of the glycose units that is the most interesting and most thoroughly investigated. A number of investigators have studied the biosynthesis of gluco-and galactosylceramide (60-64) and to date it is still unclear whether these compounds are made in vivo by conversion of sphingosine to psychosine and then to cerebroside, or by acylation of sphingosine to yield ceramide, from which cerebroside is derived. However, the synthesis of lactosylcer amide from UDP-galactose and glucosylceramide has been established in embryonic chicken brain by Basu et al. (64) and confirmed by Hauser and coworkers (65) in rat spleen. It should be noted here that it was primarily the finding that embryonic chicken brain homogenates are rich in various glycosyltransferases and that detergents (such as Triton X-100) greatly facilitated their measurement (66) that gave the means and impetus to
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 6
PPM
80
Figure 8. Carbon-13 NMR spectrum of globotetraglucosylceramide from porcine erythrocytes. The 15.08 MHz, proton-decoupled spectrum was obtained in a Bruker WP-60 at 35°C, using 100 mg sample in 1.5 mL pyridine-d (TMS internal standard). Transients (25,000) were accumulated at a sweep width of 3000 Hz and a 45°C pulse (8 psec). (Sweeley, Barker, and Nunez, manuscript in preparation)
100
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
61
further study glycosphingolipid biosynthesis. Lactosylceramide (LacCer) appears to be the precursor for the various classes of more complex glycosphingolipids (see Figure 9). Glycosylation of this molecule by a galactosyltransferase (65) gives globotriglycosylceramide, the precursor of globotetraglycosylceramide and globopentaglycosylceramide (Forssman hapten). Sialylation of LacCer by sialyltransferase gives II - α - Ν - a c e t y l n e u r a m i n o s y l lactosylceramide (66-68) and N-acetylgalactosaminyltransferase converts LacCer to gangliotriglycosylceramide (69), a precursor of the gangliosides. Finally, the addition of N-acetylglucosamine to LacCer by the appropriate transferase gives lactoneotriglycosylceramide, and subsequent reactions lead to the blood group-active glycosphingolipids (70). As mentioned above, studies by Basu, Steigerwald, Kaufman and Roseman (71-75) historically were the first to be reported on the in vitro biosynthesis of glycosphingolipids, in particular the gangliosides. From their work the pathway in Figure 9 was proposed. Later this pathway was corroborated in frog brai neuroblastoma clones (80). Even further support for the ganglioside pathway in Figure 9 was obtained using an in vivo strategy consisting of incubating cultured murine neuroblastoma cells with radiolabeled precur sors, followed by isolation of the glycolipid products (81). The synthesis of globotriglycosylceramide, the precursor of globote traglycosylceramide, has been established in rat spleen (65). Heat inactivation patterns and inhibition by different glycosphingolipids showed that this galactosyltransferase was different from that which catalyzes the transfer of UDP-galactose to glucosylceramide. Chien et al. (82) demonstrated a β - Ν - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e that would catalyze the synthesis of globotetraglycosylceramide from globotriglycosylcer amide and U D P - G a l N A c . More recently Kijimoto et al. (70) and Ishibashi et al. (83) have reported an α - Ν - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e activity in guinea pig tissues that can convert globotetraglycosylceramide to globopentaglycosylceramide (Forssman hapten) by addition of N-acetylgalactosamine via U D P - G a l N A c . Yeung et al (84) demonstrated the synthesis in vitro of both globoside and Forssman hapten in a clone of cultured adrenal tumor (Y-l) cells. Dawson and Sweeley (85) concluded that the synthesis of globotetraglycosylceramide of porcine erythrocytes occurrs in bone marrow cells. Studies on the biosynthesis of the blood group-active glycosphingo lipids have also been made (see Figure 10). Basu and coworkers have reported the synthesis of lactoneotetraglycosylceramide from lactotriglycosylceramide (the core blood group structure) and a blood group B-active pentaglycosylceramide from lactoneotetraglycosylceramide by 3-galactosyltransf erase (86) and an α - g a l a c t o s y l t r a n s f e r a s e (87) isolated from rabbit bone marrow cells, respectively. They also reported the presence of galactosyltransferase activities involved in the synthesis of this blood group B-active glycolipid in cultured mouse neuroblastoma cells (80). GDP-fucose:glycolipid fucosyltransferase activities related to blood group structures have also recently been demonstrated. The biosynthesis in vitro of blood group H . Gal(2 -*4a Fuc) gl-*~4GlcNAc6 1-*· 3Gal e i + 4 G l c C e r {88^9j~and human B-type A Gala l + 3 G a l ( 2 « - l a F u c ) β1+ 4GlcNAcBl+ 3Galgl+^GlcCer (90) glycosphingolipids have been described in a Golgi-enriched
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Galactosyltransferose
Figure 9.
N-Acetylgalactosaminyltransferase
Sialyltransferase
4Glctfl^lCei
N-Acetylgalactosaminyl transferase
CMP-NeuAc^
Sialyltransferase
PAPS
UDP-Gal NAc^j
G A N G L I O
T Y P E
Sialyltransferase
3 Ga I Ν A c £ l - 4 G a ! ( 3 - 2rXNeu Ac )β 1 - 4 GI cB I - 1
Galactosy I transferase
T Y P E
glycosphingolipids
NeuAca2~3Gaim-3GalNAcâl-4Gal(3~20NeuAc)£1^4Glci31-
Ga l â 1 -
UDP-Gal-
Ce r
N- Acetyl galactosaminyl transferase
3
» 3-S0 -GalaH4Gal/?WCer
GalNAcfll^4Gal(3-2aNeuAc)51—4Glc£l~lCer
G A L A
Sulfo^onsferose
Gal^l-3GalNAc01^4Gal(3^2CrNeuAc8^2aNeuAclffl-4Glc81-lCer
Galactosyltransferose
Ga INAc#l - 4 G a l ( 3 - 2KNeuAc 8~20CNeuAc)/31- 4 G l c â l - ICer
UDP-GalNAc-
1
Ga1al-4Gol/a-lCer
NeuAciC2 — 8 N e u A c # 2 — 3 G a l # l - 4 G l c , S l — ICer
—
Sulfotransferase
Pathways for the biosynthesis of sulfo-, neutral (Forssman hapten), and acidic (gangliosides)
T Y P E
a 1 - 4 Gal/31 - 4 G l c / S l - 1 Cer
N
G L O B O
G a l N A c a l - 3 G a l N A c β\-3Gal
UDP-GalNAc
CMP-NeuAc
NeuAcOC2^3Galâl
CMP - NeuAc -
Sialyltransferase
Galactosyltransferose
Gal/01-4Glc/Sl-lCer
UDP-Gal
GlC/Gl - I C e r
• Gal/91-lCer UDP-GalJ 1 Galactosyltransferose
NeuAcC* 2-lGQ\fi\*~5Q>Q\mce\ - 4 G a l ( 3 - 2 C t N e u A c 8 ~ 2 û C N e u A c ) / 3 l - 4 G l c / ? W C e r
N-Acetylgalactosaminyltronsferase
GalNAc/Sl-3Galai-4Gal/Sl-4Glc/3l-lCer
UDP-GalNAc >
Galal-4Gal/31~4Glc/3l-lCe-
w,N
~ j
UDP-Gal Gclactosyltransferase
UDP-GlcΊ Glucosyltransferase
Ceramide
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. jGalactosyltransferose
Fucosyltransferase UDP-Gal
JGalactosyltransferose
Figure 10.
TYPE
N j
Galoctosyltransf erase
LACTO
TYPE
Pathways for the biosynthesis of blood group-related
LACTONEO
Blood Group B - l
l
Fucosyltransferase
Blood Group Β
Blood Group A
u
UC
N
Gal(2-la F )/S 1 -3GlcNAc β l-3Gal 3 l-4Glc/31-lCer
GDP-Fuc
Gakxl-3Gal(2-laF c)/8l-4GlcNAc/91-3Gal/?l-4Glc/3l-lCer
|N-Acetylgolactosaminyltransferase
Gal(2MaFuc)tfl-4acNAcfll-3Gol<3l-4Glc/3l-«lCer
GDP-Fuc Ν
jGaloctosyltransf erase Galtfl-3GlcNActfl-3Gal/3l-4Glc/3l--lCer Galoctosyltransferase
• Galcri-3GaU?l-3GlcNAc51-3Gal/31— 4 G l c â l — lCer
glycosphingolipids
Gala 1 -3Gal(2-la Fuc) β l-3GlcN Ac/? 1 -3Gal β 1 -4Glc β 1 -1 Cer
- GlcNAc9l~3Gal#i-4Glc/3HCer
Gal(3l-4GlcNAc/9l-3Gol/Sl-4Glc/91-lCer
Fucosyl transferase
UDP-GlcNAc.l . . , \N-Acetylglucosaminyltransf erase
GalNAcal-3Gal(2-laFuc)/Sl-4GlcNAc/i3l-3Gol Sl*4GlQSl-lCer
UDP-GalNAc χ
Galactosyl transferase
GlcNAc(0CFuc)-flt-3Gol^l- 4Glc51-tCer -
GDP^Fuc
GalSl-4Glc£l-lCer
S" ο
Ci
a,
"S
Ci Ο
> t-1
w w
i
GO
64
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
fraction isolated from bovine spleen. Pacuzka and Koscielak (91) also demonstrated the synthesis of these glycolipids with enzymes from human serum. Moreover, Presper et al (92) found a fucosyltransferase in human (IMR-32) neuroblastoma cells that is involved in the synthesis of types H - l and B - l glycolipids as well as a tetraglycosylceramide precursor of a novel L e a blood group glycosphingolipid. Many other glycosphingolipids have been purified and structurally characterized but their biosynthesis in tissues or cultured cells have not yet been determined. Two particularly interesting groups are the branched blood group-active glycolipids such as those reported by Watanabe et al. (93) and Slomiany and Slomiany (94) and the "macroglycolipids" such as those characterized by Gardas and Koscielak (95). The above data establish that glycosphingolipids are synthesized by the stepwise addition of activated sugars directly to the appropriate glycolipid acceptor. It will be interesting to see if this holds true for the "macro-glycolipids" as well, since they appear to contain a number of repeating units. Perhaps intermediat those described in glycoprotein biosynthesis. In conclusion, increasing numbers of reports are appearing in which glycolipid biosynthetic pathways are demonstrated in various cell culture systems. Using such model systems in concert with the characterization of endogenous and cell surface glycoconjugates should be very helpful in furthering our understanding of the role of glycosphingolipids in cellular processes and tumorigenesis. Acceptor specificity of
glycosyltransferases
The synthesis of the carbohydrate moiety of glycosphingolipids apparently takes place in a sequential manner and is catalyzed by glycosyltransferases with specificity for both nucleotide sugar donor and glycolipid acceptor. Until recently (97), purified glycosyltransferases have not been available to test for acceptor specificity with glycolipid substrates. Therefore, judgments have been made on the basis of indirect evidence (Table I) Hildebrand and Hauser (65) compared the heat lability and metal ion requirements of galactosyltransferases from rat spleen. A galactosyl transferase utilizing glucosylceramide as an acceptor molecule was inactivated by heat treatment ( 5 0 ° C for 5 minutes), while lactosylceramide galactosyltransferase activity was only slightly affected (20% reduction). Although both transferase activities were stimulated by manganese, only glucosylceramide galactosyltransferase was enhanced by magnesium. A third difference between the two transferase activities was their response to the addition of various sphingolipids. Lactosylceramide synthesis was inhibited by sphingosine, galactosylsphingosine, lactosylsphingosine and ceramide, whereas globotriglycosylceramide synthesis was inhibited by lactosylsphingosine, ceramide and galactosylceramide. Substrate competition experiments have shown that rabbit bone marrow contains two galactosyltransferase activities, 3-galactosyltransferase synthesizing lactotetraglycosylceramide and an a-galactosyltransferase synthesizing lactopentaglycosylceramide (820. When comparisons
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. P a c u s z k a and K o s c i e l a k (1976) K a u f m a n et a[. (1963) F i s h m a n et a l . (1972) S c h w y z e r and H i l l (1977)
Heat l a b i l i t y Viral t r a n s f o r m a t i o n Pure enzyme
LacCer,II3aNeuAc-GgOse^Cer
LacCer,II3aNeuAc-GgOse^Cer
Bloodj^roup g l y c o l i p i d s , A , Le , H - m e g a and H
N e u A c - t r a n s f erase
N e u A c - t r a n s f erase
GalNAc-transferase
Fuc-transferase
Gene expression
LcOse3Cer,LcOse^Cer
G a l - t r a n s f erase
LcnOse^Cer asialoganglioside
GlcCer,LacCer
G a l - t r a n s f erase
Basu and Basu (1973)
Reference
Substrate c o m p e t i t i o n
Evidence for Acceptor Specificity
H i l d e b r a n d and Hauser (1969)
Acceptors
Glycosyltransferases
1. M e t a l ion 2. Heat l a b i l i t y 3. Inhibition by sphingolipids
Enzymes
A c c e p t o r S p e c i f i c i t y of
TABLE I
66
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
were made of C-galactose incorporation (from UDP-galactose) in the presence of each acceptor individually and when both acceptors were present together, an additive relationship was found. The authors therefore concluded that two galactosyltransferases were present. Fucosyltransferase activities have been measured in human serum, with lactoneotetraglycosylceramide and "asialoganglioside" as acceptors (91). Serum of A B O and Lewis blood group contained fucosyltransferase activity with both acceptors, while serum preparations from two Bombay blood donors (O, ) lacked asialogangliofucosyltransferase activity. Thus, the two fucosyltransferase activities appear to be products of different genes. Reports by Kaufman et al. (73) and Fishman et al. (96) demonstrated that glycosyltransferases with glycolipid products of the same anomerity may be different proteins. They differentiated similar sialyltransferase activities (with lactosylceramide and N-acetylneuraminosylgangliotetraglycosylceramide as acceptors) on the basis of in vitro (heat lability) and in vivo (viral transformation lactosylceramideisialyltransferas II -a-N-acetylneuraminosylgangliotetraglycosylceramide:sialyltransferase. Fishman et al. (96) demonstrated that Swiss 3T3 cells contain these same sialyltransferase activities. Following transformation by SV-40 virus, N acetylneuraminylgangliotetraglycosylceramide sialyltransferase was re duced, whereas lactosylceramideisialyltransferase activity did not change. In all of the cases cited above, enzyme activities were measured in homogenates or membrane preparations. Recently Schwyzer and Hill (97) reported on the acceptor specificity of a homogeneous preparation of N acetylgalactosaminyltransferase from porcine submaxillary glands. The enzyme specifically transferred N-acetylgalactosamine from U D P - G a l NAc to substrates (both glycoprotein and glycolipid) with a blood group H structure. Fucod+2Gal Bl->4GlcNAc β1-> 3Gal 61->4Glc Cer and H-megg glycolipid were both acceptors for the enzyme, while A and Le glycolipids did not serve as substrates. The studies discussed above certainly suggest that glycosyltransfer ases display a specificity for glycolipid acceptors; however, some references can be cited which argue for nonspecificity. Chien et al. (82) found a single N-acetylgalactosaminyltransferase from embryonic chicken br^in that could utilize lactosylceramide, globotriglycosylceramide and II -N-acetylneuraminosyl-lactosylceramide as glycolipid acceptors, based on the results of substrate competition experiments. The same workers (88) have used a similar experimental approach to demonstrate multiple acceptor recognition with a fucosyltransferase from bovine spleen. Although several studies have dealt with the question of acceptor specificity, little is known concerning the possible role of protein modifiers (e.g., " A protein" in lactose synthase) and chemical alteration (i.e., phosphorylation, adenylation) in glycosyltransferase reactions. In the reports discussed above, the acceptor specificity (or lack of) may be a consequence of the presence or absence of such modifications or alterations. These ideas will be discussed further in the section dealing with the regulation of glycosyltransferases.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
67
Glycosyltransferase localization Roseman has proposed that glycosyltransferases on the surface of mammalian plasma membranes may be involved in intercellular glycosylation, i.e., glycosylation of surface glycoproteins and glycolipids of one cell by glycosyltransferases on the surface of an adjacent cell, and that enzyme-substrate complexes of these enzymes could be the key mechanism governing intercellular adhesion and thus, possibly, tumorigenesis (75). Since that time a large body of data has emerged, both in support of this idea (see review by Shur and Roth (HO)) as well as against it (see review by Keenan and Morre (111)). In order to establish the presence of cell surface glycosyltransferases, a number of criteria must be fulfilled. The purity of isolated membranes must be very carefully established by both membrane marker enzymes and electron microscopy. When using whole cells, one must control for possible hydrolysis of sugar nucleotides and transport of free sugars into the cell, and enzym carefully monitored. Further, glycosyltransferase assays must be optimized under all conditions examined. A number of investigators arguing for the presence of cell surface glycosyltransferases have controlled for sugar nucleotide breakdown (112-114). Others have controlled for intracellular glycosyltransferase "leakage" (112,115-117). Upon closer inspection of the experimental design of these various studies, it can be seen that none has included all of the appropriate controls, as discussed above. Keenan and Morre and their coworkers have presented the strongest evidence against cell surface glycosyltransferases. Figure 11 depicts the membrane flow hypothesis put forth by Morre (118,119). In essence, membrane biosynthesis includes the actual transfer of membranous material from one subcellular site to another, finally reaching (via fusion) the cell surface. Endoplasmic reticulum membranes are transferred to the Golgi-apparatus where they undergo modification to become plasma membrane-like. Completed membrane material is then incorporated into secretory vesicles which move to and fuse with the plasma membrane, discharging their intravesicular contents into the extracellular milieu. Morre goes on to postulate such a route for cell surface glycolipids and glycoproteins. Initial data to support the ideas above came from electron microscopic autoradiography (120) where it was shown that glycosylation of glycoproteins takes place in the Golgi-apparatus. Studies with highly purified and well characterized membrane fractions shortly followed. High specific activities for several glycoprotein (121) and glycolipid (122) glycosyltransferases in Golgi-apparatus preparations were established. More recently, lactose synthase (123) and glycosyltransferases for ganglioside biosynthesis (124) have been demonstrated in mammary gland and liver Golgi, respectively. Glycosyltransferase activities concentrated in the Golgi-apparatus of small intestines (125), pancreas (126), thyroid (127), and many other tissues have also been reported. The strength of this work relies on (1) the careful demonstration of purified membranes, (2) the significantly higher specific activity of glycosyltransferases in
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
j
VACUOLE
NUCLEUS Figure 11. Synthesis of cell surface glycoconjugates according to the "membrane-flow" hypothesis
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
69
Golgi-membranes compared to plasma membranes or homogenatess and (3) autoradiographic localization of radiolabeled glycose moieties in Golgi and endoplasmic reticulum. To date no conclusive evidence has been given for intercellular glycosylation of lipids or proteins by surface glycosyltransferases. However, many reports have appeared which implicate cell surface glycosyltransferases in a wide varity of recognition phenomena (116,128133). In conclusion, it seems quite reasonable to assume that there are glycosyltransferases on the cell surface, based on the number and diversity of reports, even if all of the appropriate controls have not been adequately considered. However, conclusions as to their function are tentative at best. The fact that glycosylation enzymes are found in the Golgi-apparatus seems to be well established. Moreover, the idea that some glycosyltransferases appear on the cell surface via "membrane flow" and function only as membrane repair systems cannot be ruled out. Clearly, this area of researc further studies will provid complex glycoconjugates, irrespective of whether the functionally most important glycosyltransferases are on the cell surface or in the Golgiapparatus. Regulation of
glycosyltransferases
Several studies (98-101) with glycoprotein glycosyltransferases have demonstrated an in vitro effect of nucleotide phosphates. Both inhibitory and stimulatory effects have been observed. Enzymes utilizing glycolipid acceptors are also affected by adenine- and uridine-containing compounds (Table II). In a study by Ishibashi et al. (102) both positive and negative modulating effects on N-acetylgalactosaminyltransferases from guinea pig kidney were noted with uridine nucleotides. Interestingly, the a transferase which catalyzes the synthesis of globopentaglycosylceramide was markedly inhibited by U D P while the β - t r a n s f e r a s e (globotetraglycosylceramide synthesis) was activated. UDP-glucose was similar to U D P in its effects on the transferases, whereas several other uridine compounds suppressed both activities. Other information has been presented on the possible regulatory effects of substrates and products of glycosyltransfer ases and is referred to in Table II. Lactose synthase provides an interesting example of glycosyltrans ferase regulation (103). In vitro, α - l a c t a l b u m i n causes galactosyltransfer ase to become a lactose synthase. In the absence of a-lactalbumin, glucose is a very poor acceptor (Km > 1M) and the enzyme serves as a glycoprotein galactosyltransferase. The possibility that protein modifiers exist for the regulation of glycolipid synthesis can now be examined. The availability of techniques to obtain purified glycosyltransferases, along with cell systems, in which viral transformation (see section on Acceptor Specificity) appears to result in a specific loss of a glycosyltransferase, should provide the means to test such a possibility. The regulation of glycosyltransferase activity has been demonstrat ed in intact animal and cell culture systems (Table II). The level of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
70
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES TABLE II REGULATION O F G L Y C O S Y L T R A N S F E R A S E S GLYCOSYLTRANSFERASE(S)
EFFECTOR
EFFECT (+ or -)
REFERENCE
In Vivo UDP-Gal:Ceramide Galactosyltransferase
Testosterone
ω
Gray ( 197l) a
U D P - G a l : G M l Galactosyltransferase, UDP-Gal:Glucosylceramide Galactosyltransferase, UDP-Gal:Lactotriosylceramide Galactosyltransferase
dBcAMP
ω
Basu etal.(1976) b
CMP-NAN:Lactosylceramide Sialyltransferase
Butyrate
ω
Simmons et al. (1975).c M â c h e r , et al. ( 1 9 7 7 Γ
GDP-Fucose:neolactotetraosylceramide Fucosyltransferase
6-mercaptoguanosine
ω
Presper é t a l . (1978)e
In Vitro Lactose synthase
Morrison, 3.F. and Ebner K . E . (1971)8
substrate inhibition
Porcine blood group A:N-acetylgalactosaminyltransf erase
UDP Fucosyllactose
(-) (-) (-)
UDP-Gal:Glucosylceramide and Lactosylceramide Galactosyltransf erases UDP-Gal Ν A c : Tr igl ycosylcer am ide N-Acetylgalactosaminyltransf erase
Uridine UDP UDP-Glucose UDP-Galactose
UDP-GalNAc:Globoside N-Acetylgalactosaminyltransf erase
Uridine UDP UDP-Glucose UDP-Galactose
Schwyzer and Hill (1977)h
Chandrabose, K . A . , and. MacPherson, L A . (1976)1 Ishibashi et al. (1976)j
* G . M . Gray (1971) Biochem. Biophys. Acta, 239, <»9<>-500. Basu, S., Moskal, 3.R. and Gardner, D . A . , (1976) "Ganglioside Function:Biochemical and Pharmacological Implications , (Porcellati, G . , ed.) Plenum Publishing Corp., N . Y . p. *5-63. ^Simmons, 3.L., Fishman, P . H . , Freese, E., and Brady, R.O., (1975) 3. Cell Biol., 66, 414-424. d Macher, Β . Α . , Lockney, M . , Moskal, 3.R., Fung, Y - K . and Sweeley, C.C.,tl978) Exp.. Cell Res., unpublished, f Presper, K . A . , Basu, M . , and Basu, S., (1978) Proc. Nat. Acad. Sri. U.S.A., 75, 289-293. f B e l l , J.E., Beyer, T . A . and Hill, R.L., (1976) 3. Biol. Chem., 251, 3003-3013. ^Morrison, 3.F. and Ebner, K . E . , (1971) 3. Biol. Chem., 246, 3992-3998. "Chandrabose, K . A . and MacPherson, I . A . t 7 Ï 9 7 6 T B ï ô c h i m . Biophys. Acta, 429, 96-111. !Schwyzer, M.and Hill, R . L . , (1977) 3. Biol. Chem., 252, 23*6-2355. J Ishibashi, T., Atsuta, T. and Makita, Λ Τ Π 9 7 6 ) Biochim. Biophys. Acta, » 2 9 , 759-767. b
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
71
digalactosylceramide in the kidneys of female C 3 H / H e and C57/BL mice was considerably lower than in male mice of the same strains (104). Administration of testosterone to the female mice resulted in a remarkable increase in digalactosylceramide levels. Enzymatic studies demonstrated a regulatory effect by testosterone on UDP-galactose:ceramide galactosyltransferase in these mice. Further information concern ing steroid regulation of glycosyltransferases has not been published. Basu et al. (105) assayed a number of glycosyltransferase activities in clones (NIE-115, adrenergic and NS-20, cholinergic both derived from C-1300 tumors of A/3 mice) of mouse neuroblastoma cells and found that three enzymes involved in glycolipid synthesis were stimulated by Ν , Ο dibutyryladenosine cyclic 3,,5'-monophosphate (dBcAMP) (Table II). Lactoneotetraglycosylceramide synthesis was increased 5-fold in NIE-115 cells and 2-fold in NS-20 cells. UDP-galactose:glucosylceramide galacto syltransferase activity was increased 3-fold in NS-20 cells and remained unchanged in NIE-115 cells, while the synthesis of II a-N-acetylneuraminosylgangliotetraglycosylceramid treated NS-20 cells. It is possible that d B c A M P may exert its stimulatory effect on glycosyltransferases via phosphorylation. Alternatively, these changes may reflect alterations in cell cycle distributions of the cell populations. This suggestion would be in line with findings presented by Wolf and Robbins (106) and Chatterjee et al. (107) that glycolipid synthesis is greatest during certain portions of the cell cycle (M, G j ) . Our own studies (Ref. 108 and Fig. 12) and those of Simmons et al. (109) on the effect of butyrate in cultured human cells demonstrated that glycosyltransferase levels can be adjusted within hours of drug treatment. Simmons et al. (109) originally showed that butyrate treatment of HeLa cells for 18-24 hr resulted in a 15-20 fold increase in CMP-sialic acid:lactosyltransferase activity and that this enzyme activity returned to control levels when the butyrate was removed. M â c h e r et al. (108) have studied the same enzyme in K B cells and found a similar induction. They have also demonstrated that K B cells apparently are synchronized by butyrate treatment (Fig. 13). Furthermore, release from butyrate treatment and addition of fresh medium resulted in a remarkable induction of UDP-Gal:lactosylceramide galactosyltransferase (Fig. 14). Therefore, it seems that butyrate treatment and release results in differential activation of sialyltransferase and galactosyltransferase activities in human cells. Glycosphingolipid Catabolism Virtually all the enzymes necessary for the stepwise catabolism of glycolipids have been studied in vitro and in many cases they have been partially or completely purified. These enzymes, which are exoglycosidases, have received much attention because of their involvement in the hereditary lipidoses. Recently, the potential of using enzyme replacement therapy for the treatment of these diseases has been examined. Another effort in the field has been directed toward an understanding of the kinetic properties and specificity of these enzymes. A brief discussion of the exoglycosidases involved in glycolipid metabolism is given below.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
0
4
8
f-
12
(+) Butyrate
16
20
& 4·
4
8
12
16
20)
24
(-) Butyrate
Figure 12. C-thymidine uptake after release from butyrate and double thymidine block in KB cells. Redrawn from Simmons et al., J. Cell Biol. (1975) 66,414. 14
Figure 13. The effect of butyrate on CMP-NAN:hctosyheramide sialyltransferase activity in HeLa cells. ( O — O ) Release from butyrate; (·—·) release from thymidine block.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
73
Occurrence and inducibility Glycosidases are widely distributed in microorganisms, animals and plants. Neuraminidase, for example, was first discovered in a virus (134) and later found to occur in a variety of microorganisms, avian and mammalian tissues. The induction of specific bacterial glycosidases in the presence of the corresponding substrate is well documented. For example, α - g a l a c t o s i d a s e and β - g a l a c t o s i d a s e are not constitutive but are inducible in E . coli (135). With regard to tissue and cellular distribution, the glycosidases occur widely in organs (brain, kidney, liver and spleen), bone marrow, plasma, spermatozoa, platelets and erythrocytes. Although some glycosidases have been found in mitochondria (136), plasma membranes (137) and chloroplasts (138), the lysosome is the only organelle that contains the full complement of hydrolase activities necessary for the complete degradation of glycolipids and glycoproteins. Purification and physical propertie Virtually all glycosidases purified to date are glycoproteins; several kinds of purification procedures are available that are based on affinity methods. In re'cent years, immobilized substrate analogs have been used to facilitate the purification of several glycosidases (139). Their purification can also be accomplished by the use of immobilized plant lectins specific for certain carbohydrate units on the enzymes. Concanavalan A-Sepharose, for example, has been used for the isolation of glycosidases such as a-N-acetylgalactosaminidase (140) and the β - Ν acetylhexosaminidases. Variation in the composition of the carbohydrate moiety leads to microheterogeneity, for example molecular weight and charge, in an otherwise homogeneous glycosidase preparation. Variation in sialic acid content has often been cited as a cause of multiple isoelectric forms of these enzymes. Multiple forms of L-fucosidase , for example, have been shown (141) to be convertible to the neutral forms by neuraminidase treatment. An excellent discussion on the issue of homogeneity of glycoproteins has appeared elsewhere (142). Another problem of the multiple forms, which we wish to emphasize, has to do with substrate specificity. Often, the presence of a contaminating glycosidase cannot be readily detected unless the incubation time is sufficiently long. This is especially important to realize when glycosid ases are used for structural eludication of glycoconjugates. L i and L i , for example, have shown (34) in studies of the core glycopeptide of ovalbumin that the liberation of one of the mannosyl units was due to the presence of 0.06% β -mannosidase in an apparently homogeneous α -mannosidase preparation. Substrate specificity The substrate specificities of glycosidases are much more complica ted than their names imply. Figure 15 shows the^structures of several glycosphingolipids (globopentaglycosylceramide, II ,IV - α , α - d i - N - a c e t y l neuraminosyl-gangliotetraglycosylceramide, and fucolipid A a ) and the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
74
Figure 14. The effect of butyrate on UDPgal'Mctosylceramide a-galactosyltransferase activity in KB cells
°
2 f
4
6
- (^Butyrate
2
*
2
4
•+· Time (hrs)
6
Globopentaglycosylceramid A Β C D Ε G a l N A c ( a l +3)GalNAc(B 1+ 3)Gal(a 1-*)Gal( 81 -4)Glc( β 1 1 OCer
II-,IV--a,g-Di-N-acetylneurarriinosyl-RanRliotetraRlycosylceramide G H I Κ Galtë 1-»· 3)GalNAc( Bl+*)Gal(0 l+4)Glc(B 1* 1 OCer (a2-3)F
(a2-3) 3 NeuAc
NeuAc
Fucolipid A L Ν Ο Q R G a l N A c ( al+3)Gal(B l - ^ ) G l c N A c ( Bl+3)Gal(3 l ^ ) G l c ( B 1 + l')Cer |(al-2)M Fuc
LINKAGE A, L Β, Η, Ο C D, G , I, N , Q Ε =Κ = R F, 3 Μ, Ρ
2
(-) Butyrate
|(al->3)P Fuc
ΕΝΖΥΜΕ a-N-Acetylgalactosaminidases Β -Hexosaminidases α-Galactosidases β-Galactosidases β-Glucosidases Neuraminidases α-Fucosidases
Figure 15. Structures of three glycosphinpolipids and the exoglycosidases which catalyze the sequential hydrolysis of glyco lipid glycosidic linkages (A to R)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4
-I
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
75
corresponding exoglycosidases involved in their metabolism. Several points regarding the mode of action of these enzymes follow. Glycosidases from different sources often have different specificities. Several kinds of neuraminidase have been isolated, each of which has unique properties. Degradation of G n . begins with the removal of the sialic acid linked to the galactose aT trie non-reducing end (Figure 15, linkage F), using neuraminidase from Vibrio cholerae. The other sialic acid of G ^ j is r é s i s t e n t to this enzyme (as is the product) (143). Removal o i frie vicinal N-acetylgalactosamine unit irdm G . . . is necessary before this neuraminidase can hydrolyse the ketosidic linKage shown in (Figure 15, linkage J). It has been shown, however, that neuraminidase from Clostridium perfringens (144) can remove this sialic acid (linkage 3) from GJ^J directly; thus the specificity of a given enzyme often varies depending upon its source. While each glycosidase has an absolute specificity toward the anomeric linkage, absolut This is shown by compariso acetylhexosaminidase. α - Ν - a c e t y l g a l a c t o s a m i n i d a s e lacks specificity for the C-2 substituent as well as its configuration on the carbohydrate position at the C-2 (145,146). As shown in Figure 16, the enzyme can hydrolyze all three substrates (at different rates) which differ in the substituent and/or absolute configuration (shaded portions) at C-2. The enzyme, however, does show absolute specificity at the C-4 position. In contrast, β - Ν - a c e t y l h e x o s a m i n i d a s e has an absolute requirement for an equatorial acetamido moiety at C-2 but the configuration at C-4 is not important, as shown in Figure 17. Moreover, α - g a l a c t o s i d a s e A has been shown (147) to have an absolute requirement at both C-2 and C-4 positions and is thus distinguished from the Β form of α - g a l a c t o s i d a s e (15,16). Dean et al. (147) and Schram et al. (148) have shown that α - g a l a c t o s i d a s e Β is an o-N-acetylgalactosaminidase. Multiple forms of a given enzyme may exist which differ in their ability to hydrolyze glycolipids but not artificial water soluble substrates. Two forms of human β - g a l a c t o s i d a s e have been shown to hydrolyze the artificial substrate, 4-methylumbelliferyl- β - D - g a l a c t o p y r a n o s i d e (149,150). One of these forms ( G M .-ganglioside: β - g a l a c t o s i d a s e ) was more specific for G . , , - and asialo υ M 1 -ganglioside, while the other form (galactosylceramidase) was more specific for galactosyl- and lactosyl ceramide. Similarly, two forms of β - Ν - a c e t y l h e x o s a m i n i d a s e have been found which catalyze the hydrolysis of globotetraglycosylceramide and 4methylumbelliferyl-B-N-acetylhexosaminide. Only οτψ ( β - Ν - a c e t y l h e x o s aminidase A) of these forms was able to hydrolyze II -N-acetylneuraminosyl-gangliotriglycosylceramide. Two forms of human β - g l u c o s i d a s e have been identified. Both hydrolyze 4 - m e t h y l u m b e l l i f e r y l - β - D - g l u c o p y r a n o side but only glucocerebroside: g-glucosidase is capable of hydrolyzing glucocerebroside (151,152). This form of the enzyme appears to be lysosomal in origin.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
76
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Journal of Cell Biology
Figure 16. Three-dimensional of terminal carbohydrate residues that bind to a-N-acetylgahctosaminidase. R represents the remaining portion of the sphingolipid. Substituents that the en zyme cannot distinguish are shaded (109).
Journal of Cell Biology
Figure 17. Three-dimensional projections of ter minal carbohydrate residues which bind to β-Νacetylhexosaminidases. R represents the remaining portion of the sphingolipid. Substituents that the enzyme cannot distinguish are shaded (109).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
77
Lipid-protein interactions Detergents are essential for the dispersion of most glycolipid substrates in glycosidase assay mixtures. In addition, it has been shown that detergents can stimulate or inhibit glycosidases directly. Presum ably, detergents mimic the effect of naturally occurring membrane amphiphilic lipids. β - G l u c o c e r e b r o s i d a s e : β - g l u c o s i d a s e , for example, has been shown to be activated by bile salts (151,153,154) such as deoxycholate, glycocholate and taurocholate; acidic phospholipids such as phosphatidylserine, phosphatidylinositol and phosphatide acid are also active (152,155). Interest ingly, the water-soluble β - g l u c o s i d a s e , which does not have activity toward lipid substrates, is inhibited by these amphiphiles. On the other hand, the β - g l u c o c e r e b r o s i d a s e : β - g l u c o s i d a s e , which hydrolyzes lipid substrates was inhibited by choline-containing lipids. Ho (156,157) has proposed a glucocerebrosidase enzyme system composed of glucocerebro sidase (factor C), a glycoprotei phospholipid. The acidic phospholipid appears to be necessary for the association of the activator and the enzyme. The activator-enzyme complex hydrolyzes artificial water-soluble substrates and G l c C e r . Sodi um taurocholate can substitute for the activator. Recently, Hechtman (158)Jias reported the isolation of an activator required for the hydrolysis of II -a-N-acetylneuraminosyl-gangliotriglycosylceramide by β - h e x o s a m i n i d a s e A . This activator does not affect the rate of hydrolysis of synthetic substrates or gangliotriglycosylceramide. Modulation of other glycosidase activities has also been reported. L i and L i (159) have isolated an activator which stimulates the hydrolysis of a number of glycolipids. However, this activator may be interacting with substrate rather than enzyme, judging from the fact that a 1:1 molar ratio of activator and substrate is required. , Synthetic lipoidal activators have also been reported (160). Figure 18 shows the structures of some synthetic fatty acyl amides which resemble fatty acyl sphingosine. The hydrolysis of galactosylceramide by β - g a l a c t o s i d a s e was stimulated by fatty acyl derivatives of 2-amino-2methyl-l-propanol, with the N-decanoyl derivative being the most active. In general, the V but not the Κ was changed. Omission of the branched methyl group resulted in inhilfftion instead of stimulation. The reports cited above suggest that protein activators stimulate the activity of glycosidases toward glycolipid substrates but do not modify the enzymatic activity toward water-soluble substrates. Our recent studies on the interaction of sodium taurocholate with a-N-acetylgalactosaminidase from canine liver (146,161) indicate that amphiphiles may stimulate the hydrolytic activities of glycosidases toward both artificial water-soluble substrates and glycolipid substrates. Low concentrations of sodium taurocholate stimulate the activity of the enzyme toward the artificial substrate Ρ Ν Ρ - α - G a l N A c . However, as the concentration of taurocholate is increased, inhibition results. A Lineweaver-Burk plot indicates that sodium taurocholate, at all concentrations, lowers the V m a x and the Κ for the hydrolysis of the artificial substrate. The percent maximal inhibition levels off at the critical micelle concentration of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES H I C — I H
Figure 18. Fatty acid amides which affect hydrolytic activity of rat brain β-galactosidase toward galactosylceramide. The N-decanoyl derivative was the most effective (160).
^
X
«
ChU
H I I C — C I I NH H I C-O I
—
6 , 8, 10, 12, 14,
OH
16
GLYCOSIDASES
φ ®
RELEASE OF G L Y C O S I D A S E S RECOGNITION OF ENZYME BY RECEPTOR
© ©
UPTAKE OF ENZYME PY P I N 0 C Y T 0 S I S FUSION OF PINOCYTIC VESICLE WITH P R I M A R Y
LYS0S0ME
Figure 19. Pictorial model of exocytosis and endocytosis of lysosomal glycosidases
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3.
SWEELEY ET AL.
Glycolipid
Structure
and
Metabolism
79
taurocholate (7 mM). Moreover, sodium taurocholate at very low concentrations significantly quenches the intrinsic fluorescence of the enzyme. Our data (161) indicate that the enzyme activity is modulated by the interaction with the monomeric but not the micellar form of taurocholate. These studies may shed light on the mode of action of glycosidases in their natural membrane environment. Physiological significance of glycosidases Besides the well publicized lipidoses resulting from glycosidase deficiencies, attention has shifted toward the release and uptake of these enzymes from cells. An abundance of evidence indicates that tumors, studied in vivo, release large quantities of lysosomal enzymes into their environment (162-164). Presumably this release of degradative enzymes is associated with tumor invasiveness and maintainance of the neoplastic state. Other studies (dealing with I-cell disease) have shown that specific recognition molecules on lysosomal glycosidases. A diagram of these processes is depicted in Figure 19. The uptake depends upon both the carbohydrate moiety of the glycosidases (165,166) and the presence of specific receptors on the plasma membrane (167,168). Clearly, more detailed studies are needed, results of which should be significant in the design of enzyme replacement therapy. Acknowledgements The authors would like to especially acknowledge the secretarial work of Ms. Dorothy Byrne. Mr. Kenneth J. Dean and Dr. S.-S.J. Sung are also gratefully acknowledged for providing data dealing with a - N acetylgalactosaminidase and α - g a l a c t o s i d a s e . This work was supported by NIH research grants (AM 12434 and H L 17462).
Literature Cited 1. 2. 3. 4. 5. 6. 7.
Lysosomes and Storage Diseases, ed. H.G. Hers and F. van Hoof, Academic Press, New York, 1973. The Metabolic Basis of Inherited Disease, 4th Ed., ed. J.B. Stanbury, J.B. Wyngaarden and D.S. Fredrickson, McGraw-Hill, New York, 1978. Practical Enzymology of the Sphingolipidoses, ed. R.H. Glew and S.P. Peters, Alan R. Liss, Inc., New York, 1977. Glycolipid Methodology, ed. L.A. Witting, Amer. Oil Chemists' Society, Champaigne, 1976. Karlsson, K.A., in Chemistry and Metabolism of Sphingolipids, ed. C.C. Sweeley, North-Holland Publishing Co., Amsterdam, 1970, pp. 6-43. Sweeley, C.C. and Siddiqui, B., in The Glycoconjugates. Vol. I. Mammalian Glycoproteins and Glycolipids, ed. M.I. Horowitz and W. Pigman, Academic Press, New York, 1977, pp. 459-540. Sweeley, C.C. and Moscatelli, E.A., J. Lipid Res., 1, 40 (1959).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
80
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
8.
Gaver, R.C. and Sweeley, C.C., 3. Am. Oil Chem. Soc, 42, 294 (1965). 9. Karlsson, K.A., Nature,188,312 (1960). 10. Kawamura, N. and Taketomi, T., 3. Biochem. (Tokyo),81,1217 (1977). 11. Martensson, E., Biochim. Biophys. Acta, 116, 296 (1966). 12. Norton, W.T. and Brotz, M., Biochem. Biophys. Res. Commun., 12, 198 (1963). 13. Kornblatt,M.J.,Schachter, H. and Murray, R.K., Biochem. Biophys. Res. Commun., 48, 1489 (1972). 14. Ishizuka, I., Suzuki, M. and Yamakawa, T.,J.Biochem. (Tokyo), 73, 77 (1973). 15. Slomiany, B.L., Slomiany, A. and Glass, G.B.J., Eur. 3. Biochem., 78, 33 (1977). 16. IUPAC-IUB Comission on Biochemical Nomenclature, Lipids,12,455 (1977). 17. Burton, R.M., in Glycolipi Oil Chemists' Society, Champaign, 1976, pp. 1-11. 18. Macher, B.A. and Sweeley,C.C.,in Methods in Enzymology, ed. V. Ginsburg, Academic Press, New York, in press. 19. Iwamori, M., Moser, H.W., McCluer, R.H. and Kishimoto, Y., Biochim. Biophys. Acta, 380, 308 (1975). 20. McCluer, R.H. and Evans,J.E.,J.Lipid Res., 17, 412 (1976). 21. Suzuki, Α., Handa, S. and Yamakawa, T., 3. Biochem. (Tokyo), 80, 1181 (1976). 22. Suzuki, Α., Handa, S. and Yamakawa, T., Abstr.Japan-U.S.Seminar on Structure and Functional Significance of Membrane Glycolipids, Honolulu, October 5, 1977. 23. Hakomori, S. and Watanabe, K., in Glycolipid Methodology, ed. L.A. Witting, Amer. Oil Chemists'Soc.,Champaign, 1976, pp. 13-47. 24. Iwamori, M. and Nagai, Y., 3. Neurochem., in press. 25. Iwamori, M. and Nagai, Y., Proc. Japanese 3. Biochem. Lipids, 19, 257 (1977). 26. Ando, S. and Nagai, Y., Biochim. Biophys. Acta, 424, 98 (1976). 27. Momoi, T., Ando, S. and Nagai, Y., Biochim. Biophys. Acta, 441, 488 (1976). 28. Ueno, K., Ando, S. and Yu, R.K.,J.Lipid Res., in press. 29. Ohashi, M. and Yamakawa, T.,J.Biochem. (Tokyo), 81, 1675 (1977). 30. Vance, D.E. and Sweeley,C.C.,J.Lipid Res., 8, 621 (1967). 31. Chambers, R.E. and Clamp, J.R., Biochem.J.,125, 1009 (1971). 32. Wong, C.G., Sung, S.-S.3. and Sweeley, C.C., in Methods in Carbohydrate Chem., ed. R. Whistler, Academic Press, in press. 33. Yu, R. and Ledeen, R.W., 3. Lipid Res.,11,506 (1970). 34. Li, Y.T. and Li,S.C.,in The Glycoconjugates. Vol. I. Mammalian Glycoproteins and Glycolipids, ed. M.I. Horowitz and W. Pigman, Academic Press, 1977, pp. 52-68. 35. Svennerholm, L., Mansson,J.E.and Li, Y.T.,J.Biol. Chem., 248, 740 (1973). 36. Slomiany, A. and Slomiany, B.L., Eur.J.Biochem., 76, 491 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3. SWEELEY ET AL.
Glycolipid Structure and Metabolism
37. Slomiany, Α., Slomiany, B.L. and Horowitz, M.I., in Glycolipid Methodology, ed. L.A. Witting, Amer. OilChemists'Soc.,Cham paign, 1976, pp. 49-75. 38. Hakomori, S.,J.Biochem. (Tokyo), 55, 205 (1964). 39. Karlsson, K.A., Biochemistry, 13, 3643 (1973). 40. Karlsson, K.A., Pascher, I. and Samuelsson, B.E., Chem. Phys. Lipids, 12, 271 (1974). 41. Smith, E.L., McKibbin,J.M.,Karlsson, K.A., Pascher, I. and Samuelsson, B.E., Biochim. Biophys. Acta, 388, 171 (1975). 42. Holm. M., Pascher, I. and Samuelsson, B.E., Biomed. Mass Spect., 4, 77 (1977). 43. Ledeen, R.W., Kundu, S.K., Price, H.C. and Fong, J.W., Chem. Phys. Lipids, 13, 429 (1974). 44. Hanfland, P. and Egge, H., Chem. Phys. Lipids, 16, 201 (1976). 45. Karlsson, K.A., in Glycolipid Methodology, ed. L.A. Witting, Amer. Oil Chemists'Soc.,Champaign 1976 pp 97-122 46. Wiegandt, H. and Baschang (1965). 47. Hakomori, S.,J.Lipid Res., 7, 789 (1966). 48. Kundo, S.K., Ledeen, R.W. and Gorin, P.A.J., Carbohyd. Res. 39, 179 (1975). 49. Bjorndal, H., Lindberg, B. and Svensson, S., Acta Chem. Scand., 21, 1801 (1967). 50. Bjorndal, H., Lindberg, B. and Svensson, S., Carbohyd. Res., 5, 433 (1967). 51. Bjorndal, H., Hellerqvist, C.G., Lindberg, B. and Svensson, S., Angen. Chem. Int. Ed., 9, 610 (1970). 52. Bjorndal, H., Lindberg, B., Pilotti, A. and Svensson, S., Carbohyd. Res.,15,339 (1970). 53. Sung, S.-3.3., Esselman,W.J.and Sweeley, C.C.,J.Biol. Chem., 248, 6528 (1973). 54. Stellner, K., Saito, H. and Hakomori, S., Arch. Biochem. Biophys., 155, 464 (1973). 55. Jansson, P.E., Kenne, L., Liedgren, H., Lindberg, B. and Lonngren, J., Chem. Commun. (Univ. of Stockholm), 1976 (No. 8), pp. 1-75. 56. Sillerud, L.O., Prestegard, J.H., Yu, R.K., Schafer, D.E. and Konigsberg, W.H., Biochemistry, in press. 57. Braun, P.E. and Snell, E.E.,J.Biol. Chem., 243, 3775 (1968). 58. Stoffel, W., LeKim, D. and Sticht, G., Z. Physiol. Chem., 349, 1637 (1968). 59. Scribney, M., Biochim. Biophys. Acta, 125, 542 (1966). 60. Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 243, 5802 (1968). 61. Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 248, 1388 (1973). 62. Cleland, W.W. and Kennedy, E.P.,J.Biol. Chem., 235, 45 (1960). 63. Morell, P. and Radin, N.S., Biochemistry, 8, 506 (1969). 64. Basu, S., Schultz, Α., Basu, M. and Roseman, S., 3. Biol. Chem., 246, 4272 (1971). 65. Hildebrand, J. and Hauser, G.,J.Biol. Chem., 244, 5170 (1969).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
81
82
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
66. Basu, S., Ph.D. Thesis, Univ. of Michigan, Ann Arbor, Michigan, 1966. 67. Basu, S. and Kaufman, B., Fed. Proc, 24, 479 (1965). 68. Kaufman, B., Basu, S. and Roseman, S., Methods Enzymol., 8, 365 (1966). 69. Basu, M., Chien,J.L.and Basu, S., Biochem. Biophys. Res. Commun., 60, 1097 (1974). 70. Kijimoto, S., Ishibashi, T. and Makita, Α., Biochem. Biophys. Res. Commun., 56, 177 (1974). 71. Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 240, PC4115 (1965). 72. Steigerwald, J.C., Basu, S., Kaufman, B. and Roseman, S.,J.Biol. Chem., 250, 6727 (1975). 73. Kaufman, B., Basu, S. and Roseman, S., Proc. of the Third Int. Symposium on the Cerebral Sphingolipidoses, ed. by S.M. Aronson and B.W. Volk, Pergamo 74. Kaufman, B., Basu (1968). 75. Roseman, S., Chem. Phys. Lipids, 5, 270 (1970). 76. Yip, M.C.M. and Dain, J.A., Biochem. 3.,118,247(1970). 77. Yip, M.C.M. and Dain, J.A., Biochim. Biophys. Acta, 206, 252 (1970). 78. Hildebrand,J.,Stoffyn, P. and Hauser, G.,J.Neurochem.,17,403 (1970). 79. Arce, Α., Maccioni,H.J.and Caputto, R., Fed. Proc, 29, 925 (1970). 80. Moskal, J.R., Gardner, D.A. and Basu, S., Biochem. Biophys. Res. Commun.,61,751 (1974). 81. Kempf, S.F. and Stoolmiller, A.C.,J.Biol. Chem., 251, 7626 (1976). 82. Chien, J.L., Williams, T. and Basu, S., 3. Biol. Chem., 248, 1778 (1973). 83. Ishibashi, T., Kijimoto, S., and Makita, Α., Biochim. Biophys. Acta, 337, 92 (1974). 84. Yeung, K.K., Moskal, J.R., Chien, J.L., Gardner, D.A. and Basu, S., Biochem. Biophys. Res. Commun., 59, 252 (1974). 85. Dawson, G. and Sweeley,CC.,J.Biol. Chem., 247, 410 (1972). 86. Basu, M. and Basu, S.,J.Biol. Chem., 247, 1489 (1972). 87. Basu, M. and Basu, S.,J.Biol. Chem., 248, 1700 (1973). 88. Basu, S., Basu, M. and Chien,J.L.,J.Biol. Chem., 250, 2956 (1975). 89. Basu, S., Basu, M., Moskal, J.R., Chien,J.L.and Gardner, D.A., in Glycolipid Methodology, ed. L.A. Whittig, 1976, p. 123-139. 90. Presper, K.A., Basu, M. and Basu, S., Fed. Proc, 35, 1441 (1976). 91. Pacuszka, T. and Koscielak,J.,Eur.J.Biochem., 64, 499 (1976). 92. Presper, K.A., Basu, M. and Basu, S., Proc. Natl. Acad. Sci., U.S.A., 75, 789 (1978). 93. Watanabe, K., Laine, R.A. and Hakomori, S., Biochemistry, 14, 2725 (1975). 94. Slomiany, B.L. and Slomiany, Α., Biochim. Biophys. Acta, 486, 531 (1977). 95. Gardas, A. and Koscielak,J.,FEBS Lett., 42, 101 (1974).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3. SWEELEY ET AL.
Glycolipid Structure and Metabolism 83
96. Fishman, P.H. and Brady, R.O., in Biological Roles of Sialic Acid ed. A. Rosenberg and C.L. Schengrund, Plenum Publishing Corp., New York, 1976. 97. Schwyzer, M. and Hill, R.L.,J.Biol. Chem., 252, 2346 (1977). 98. Letoublon, R., Richard, M., Louisot, R. and Got, R., Eur. J. Biochem., 18, 194 (1971). 99. Ko, G.K.W. and Raghupathy, E., Biochim. Biophys. Acta, 313, 277 (1973). 100. Eylar, E.H. and Cook, G.M.W., Proc. Natl. Acad. Sci. U.S., 54, 1678 (1965). 101. Testas, M., Chao, H. and Molnar,J.,Arch. Biochem. Biophys., 138, 135 (1970). 102. Ishibashi, T., Atsuta, T. and Makita, Α., Biochem. Biophys. Acta, 429, 759 (1976). 103. Bell, J.E., Beyer, T.A. and Hill, R.L., J. Biol. Chem., 251, 3003 (1976). 104. Gray, G.M., Biochim 105. Basu, S., Moskal,J.R. , , Gangliosid Biochemical and Pharmacological Implications, ed. Porcellati, G., Plenum Publishing Corp., New York, 1976, pp. 45-63. 106. Wolf, A. and Robbins, P.W.,J.Cell Biol., 61, 676 (1974). 107. Chatterjee, S., Sweeley, C.C. and Velicer, L.F., Biochem. Biophys. Res. Commun., 54, 585 (1973). 108. Macher, B.A., Lockney, M., Moskal, J.R., Fung, Y-K. and Sweeley, C.C., Expt. Cell Res., in preparation. 109. Simmons, J.L., Fishman, P.H., Freese, E. and Brady, R.O.,J.Cell Biol., 66, 414 (1975). 110. Shur, B.D. and Roth, S., Biochim. Biophys. Acta, 415, 473 (1975). 111. Keenan, T.W. and Morre, D.J., FEBS Lett., 55, 7 (Î975). 112. Roth, S., McGuire, E.J. and Roseman, S.,J.Cell. Biol.,51,536 (1971). 113. Datta, P., Biochemistry, 13, 3987 (1974). 114. Patt, L.M. and Grimes,W.J.,J.Biol. Chem., 249, 4157 (1974). 115. Weiser, M.M.,J.Biol. Chem., 248, 2542 (1973) 116. Podolsky, D.K., Weiser, M.M., LaMont,J.T.and Isselbacher, K.J., Proc. Natl. Acad. Sci., U.S.A.,71,904 (1974). 117. Roth, S. and White, D., Proc. Natl. Acad. Sci., U.S.A., 69, 485 (1972). 118. Franke, W.W., Morre,D.J.,Deumling, B., Cheetham, R.D., Kartenbeck,J.,Jarasch,E. and Zentgraf, H., Z. Naturforsch., 26b, 1031 (1971). 119. Morre, D.J., Keenan, T.W. and Huang, C.M., in Advances in Cytopharmacology ed. B. Cecarelli, F. Clementi andJ.Meldolesi, Vol. 2, Raven Press, New York, 1974, pp. 107-126. 120. Bennett, G., LeBlond,C.P.and Haddad, Α.,J.Cell Biol., 60, 258 (1974). 121. Schacter, H.,Jabbal,I., Hudgin, R.L., Pinteric, L., McGuire, E.J. and Roseman, S.,J.Biol. Chem., 245, 1090 (1970). 122. Morre,D.J.,Merlin, L.M. and Keenan, T.W., Biochem. Biophys. Res. Commun., 37, 813 (1969). 123. Brodbeck, U. and Ebner, K.,J.Biol. Chem., 241, 762 (1966).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
84
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
124. Keenan, T.W., Morre, D.J. and Basu, S., J. Biol. Chem., 249, 310 (1974). 125. Mahley, R.W., Bennett, B.D., Morre, D.J., Gray, M.E., Thistlewaithe, W. and LeQuire, V.S., Lab Invest., 25, 435 (1971). 126. Ronzio, R.A., Arch. Biochem. Biophys., 159, 777 (1973). 127. Chabaud, O., Bouchilloux, S., Ronin, C. and Ferrand, M., Biochimie, 56, 119 (1974). 128. LaMont, J.T., Perrotto, J.L., Weiser, M.M. and Isselbacher, K.J., Proc. Natl. Acad. Sci., U.S.A., 71, 3726 (1974). 129. Podolsky, D.K. and Weiser, M.M., Biochem. J., 146, 213 (1975). 130. Bosmann, H.B., Biochem. Biophys. Res. Commun., 48, 523 (1972). 131. Bosmann, H.B., Beiber, G.F., Brown, A.E., Case, K.R., Gersten, D.M., Klimmerer, T.W. and Lione, Α., Nature, 246, 487 (1973). 132. McClean, R.J. and Bosmann, H.B., Proc. Natl. Acad. Sci., U.S.A., 72, 310 (1975). 133. Shur, B. and Roth, S.,Am Zool. 13 1129 (1973) 134. Burnet, F.M., McCrea 27, 228(1946). 135. Koppel, J.L., Porter, C.J. and Crocker, B.F., J. Gen. Physiol., 36, 703 (1953). 136. Subba, Rao, K. and Pieringer, R.A., J. Neurochem.,17,483 (1970). 137. Fleischer, B. and Fleischer, S., Biochim. Biophys. Acta, 183, 265 (1969). 138. Gatt, S. and Baker, E.A., Biochim. Biophys. Acta, 185, 402 (1969). 139. Opheim, D.J. and Touster, O., J. Biol. Chem., 252, 739 (1977). 140. Sung, S.-S.J. and Sweeley,C.C.,in Current Trends in Sphingolipidoses and Allied Disorders, Vol. 68, ed. B.W. Volk and L. Schneck, 1976, pp. 323-337. 141. Alhadeff, J.A. and O'Brien, J.S. in Practical Enzymology of the Sphingolipidoses, ed. R.H. Glew, S.P. Peters, A.R. Liss Inc., New York, 1977, pp. 247-281. 142. Martin, I. and Horowitz, M.I., The Glycoconjugates, Vol. 1, 1977, Academic Press ed. M.I. Horowitz and W. Pigman, New York, p. 1534. 143. Kuhn R. and Wiegandt, H., Chem. Ber., 89, 2013 (1963). 144. Rauvala, H., FEBS Letts., 65, 229 (1976). 145. Dean, K.J., Sung, S.-S.J. and Sweeley,C.C.,in Enzymes of Lipid Metabolism, eds. P. Mandel, L. Freyst and S. Gatt, Plenum Publishing Corp., New York, in press. 146. Fung, Y.-K. and Sweeley,C.C.,Fed. Proc, in press. 147. Dean, K.J., Sung, S.-S.J. and Sweeley,C.C.,Biochem. Biophys. Res. Commun., 77, 1411 (1977). 148. Scham, Α., Hamers, M.N. and Tager, J.M., Biochim. Biophys. Acta, 482, 138 (1977). 149. Suzuki, K., in Practical Enzymology of the Sphingolipidoses, eds. R.H. Glew and S.P. Peters, Alan R. Liss, Inc., New York, 1977, pp. 102-132. 150. Tanaka, H., and Suzuki, K., Brain Res.,122,325 (1977). 151. Ho, M.W., Biochem. J.,136,721 (1973). 152. Peters, S.P., Coyle, P. and Glew, R.H., Arch. Biochem. Biophys. 175, 589 (1976). 153. Gatt, S., Biochem., J., 101, 687 (1966).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3. SWEELEY ET AL.
Glycolipid Structure and Metabolism 85
154. Gatt, S. and Rapport, M.M., Biochim. Biophys. Acta,113,567 (1966). 155. Blonder, E., Klibansky, C. and DeFries, Α., Biochim. Biophys. Acta 431, 45 (1976). 156. Ho, M.W. and Light, D., Biochem.J.,136, 821 (1973). 157. Ho, M.W., in Enzyme Therapy in Lysosomal Storage Disease eds. 3.M. Tager, G.J.M. Hooghwinkel and W. Th Daems, Amsterdam, North Holland, 1974, pp. 239-246. 158. Hechtman, P., Can.J.Biochem., 55, 315 (1977). 159. Li, S. and Li, Y.-T.,J.Biol. Chem., 251, 1159 (1976). 160. Arora, R.C. and Radin, N.S., Lipids, 7, 56 (1971). 161. Fung, Y.-K. and Sweeley,C.C.,in preparation. 162. Bosmann, H.B. and Hall,T.C.,Proc. Natl. Acad. Sci., U.S.A., , 71, 1833 (1974). 163. Sylven, B., Eur. 3. Cancer, 4, 463 (1968). 164. Koono, M., Ushijima, K. and Hayashi, H., Int. 3. Cancer,13,105 (1974). 165. Stahl, P., Schlesinger 264, 86 (1976). 166. Kaplan, Α., Fischer, D. and Sly, W.S., J. Biol. Chem., 253, 647 (1978). 167. Hickman, S., Shapiro, L.J. and Neufeld, E.F., Biochem. Biophys. Res. Commun., 57, 55 (1974). 168. Kawasaki, T. and Ashwell, G.,J.Biol. Chem., 252, 6536 (1977). 169. Carter, H.E., Strobach, D.R. and Hawthorne, 3.N., Biochemistry, 8, 383 (1969). 170. Kaul, K. and Lester, R.L., Plant Physiol., 55, 120 (1975). 171. Hsieh, T.C.Y., Kaul, K., Laine, R.A. and R.L. Lester (private communication). RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4 Endo-β-acetylglucosaminidases — Their Metabolic Role in Disease Processes and Their Use in the Study of Glycoprotein Structure and Function FRANK MALEY, ANTHONY L. TARENTINO, and ROBERT B. TRIMBLE New York State Department of Health, Division of Laboratories and Research, Albany, NY 12201
In the course of studie tides from RNase Β an tinase extracts (1, 2) capable of hydrolyzing the di-N-acetyl chitobiosyl linkage of oligosaccharides to yield the following products: R-GlcNAc + GlcNAc(Man)(GlcNAc) where R = Asn; Asnpeptide, Asn-protein, and x = 5 or greater, while y = 0 to 4. This enzyme was called endo-ß-N-acetylglucosaminidase-H (endo-H) because its specificity appeared to be directed towards substrates composed of at least five mannose residues. Further investigation of these initial crude preparations from Streptomyces plicatus revealed them to contain two enzymes, one with a specificity for long chain mannosyl oligosaccharides, and the other with a primary specificity for short chain oligosac charides such as (Man)1(GlcNAc)Asn (2). The latter enzyme, which can be separated from endo-H by gel filtration (Figure 1), has been designated endo-L and will be discussed later. We realize now that it was the presence of this enzyme in our early endo-H preparations that enabled us to affect the isolation of Manβ1-4GlcNAc and to clearly demonstrate for the first time that the core sequence of most Asn-associated glycopeptides is composed of this disaccharide (1, 3, 4). With the former enzyme, designated endo-H, it was shown (1, 5) that the ovalbumin oligosaccharide chain is linked to the distal GlcNAc of the di-N-acetyl chitobiosyl unit and not to the primary GlcNAc as originally believed (6). These high mannosyl or simple oligosaccharides, such as those associated with ovalbumin, ribonuclease B, deoxyribonuclease, carboxypeptidase Y, mungbean nuclease, invertase, immunoglobulin M, and thyroglobulin, were shown by us (6, 7) to be substrates for endo-H and stand in contrast to the resistant "complex" oligosaccharides associated with most immunoglobulins (8, 9, 10) and viral glyco proteins (11) which are composed of a core sequence of Manal "Man 31+4G1 cNAc 31+4G1 cNAc-Asn Manal x
y
2
0-8412-0452-7/78/47-080-086$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
Endo-β-acetylglucosaminidases
87
where fucose i s u s u a l l y l i n k e d to the f i r s t GlcNAc. The manner i n which t h i s u n i t i s synthesized remains to be determined, but i t appears to r e s u l t from the p r o c e s s i n g of l a r g e r molecular weight o l i g o s a c c h a r i d e chains that are r i c h i n mannose (12, 13). I t has become apparent that enzymes such as endo-H and a r e l a t e d endoglycosidase from Diplococcus pneumoniae, endo-D (14), w i l l provide v a l u a b l e a s s i s t a n c e i n a s s e s s i n g both the s t r u c t u r e and f u n c t i o n of g l y c o p r o t e i n s , t h e i r mechanism of b i o s y n t h e s i s and t h e i r p o t e n t i a l r o l e i n disease processes. But, because of the i n c r e a s i n g i n d i s c r i m i n a t e use of these enzymes without regard to t h e i r s p e c i f i c i t y of a c t i o n and t h e i r attendant contaminants, erroneous c o n c l u s i o n s may r e s u l t . I t i s f o r t h i s reason that I would f i r s t l i k e to review some of the p r o p e r t i e s of these enzymes which are o f t e n overlooked, and to i n c l u d e some of our recent f i n d i n g s which would appear to extend the range of a c t i v i t y of endo-H. As shown i n F i g u r e H r e l a t i v e to endo-D, i t i s c l e a r that endo-H hydrolyzes longer mannosyl o l i g o s a c c h a r i d e s than endo-D, an enzyme which has a much greater s p e c i f i c i t y f o r the branched complex o l i g o s a c c h a r i d e core (Man)3(GlcNAc)2Asn (15). In c o n t r a s t to endo-D, endo-H can e f f e c t i v e l y hydrolyze o l i g o s a c c h a r i d e chains w i t h as many as 50 or more mannosyl r e s i d u e s (j6) and even the complex core r e g i o n , but at a r a t e that i s s e v e r a l orders of magnitude lower than t h a t a f f e c t e d by endo-D, p r o v i d i n g fucose i s not present on the proximal N-acetylglucosamine of the core r e g i o n (Figure 3 ) . As i n d i c a t e d , the presence of fucose completely impairs endo-H s a l r e a d y low a c t i v i t y against the complex core o l i g o s a c c h a r i d e , but has no e f f e c t on endo-D a c t i v i t y . Because of endo-H s c a p a c i t y to r e l i e v e s p e c i f i c g l y c o p r o t e i n s of t h e i r o l i g o s a c c h a r i d e u n i t s , i t can be a v a l u a b l e asset i n e s t i m a t i n g molecular weights of g l y c o p r o t e i n s by SDS-acrylamide g e l e l e c t r o p h o r e s i s , a technique that o f t e n y i e l d s i n a c c u r a t e results with glycoproteins. Thus, by c a r e f u l l y u t i l i z i n g endo-H i n the removal of o l i g o s a c c h a r i d e s from i n v e r t a s e (16), a p r o t e i n w i t h a mass that i s 50% carbohydrate, i t could be shown that t h i s enzyme c o n s i s t s of two i d e n t i c a l s u b u n i t s , each w i t h a molecular weight of 61,000 (Figure 4) a f t e r s u b t r a c t i o n of the remaining N-acetylglucosamines. Incomplete removal of the carbohydrate, however, g i v e s the appearance of two nonequivalent c h a i n s , one w i t h a molecular weight of 65,000 and the other of 68,000 (Figure 5). But s i n c e endo-H i s s t a b l e i n 0.5% SDS, the o l i g o s a c c h a r i d e chains of the i n v e r t a s e molecule can be made completely a c c e s s i b l e to endo-H and almost a l l of i t s 18 o l i g o s a c c h a r i d e chains removed (Table I ) . In the case of CPase Y , four o l i g o s a c c h a r i d e chains were found to be a s s o c i a t e d w i t h a 51,000 d a l t o n monomer (Table I I ) . ?
1
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
88
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
40
60
100
80 FRACTION NUMBER
Figure 1. Separation of S. plicatus endo-L (O) and endo-H (±) on a Sephadex G-100 column (1.5 X 220 cm). For further details see Ref. 2. (ao)„-Asn (GlcNAd2(Man)3
100 Asn ( G l c N A c ) 2 (Man) 5
Asn ( G l c N A c ^ (Mon) 6
Asn ( G l c N A c ) 4 (Man) 6
TMan) 5 GlcNAc-GlcNAc-ol
(aa) x -Asn(GlcNAc) 2 (Man) 3
3
0
I
HOURS Biochemical and Biophysical Research
Communications
Figure 2. A comparison of the rates of hydrolysis of various oligosaccharides by endo-H and endo-D. The described substrates were incubated for the indicated times with (A) 2.5 ng of pure endo-H or (B) 0.5 fig of protein containing endo-D. Adapted from Tarentino and Maley (15).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
89
Endo-β-acetyl^ucosaminidases
(αα) χ -Asn(6lcNAc)2 ( Μ α η ) 3
Biochemical and Biophysical Research Communications
Figure 3. The rate of hydrolysis of ( Fuc)t( Man ) ( GlcNAc ) Asn-(aa )x and its defucosylated derivative by endo-H. The' [ C]-N-acetylated derivatives were incubated with 10 times more endo-H than those in Figure 1. Adapted from Tarentino and Maley (15). 3
2
u
HOURS
Journal of Biological Chemistry
Figure 4. Gel electrophoresis of native (lanes 1-5) and S-carboxymethyhted (SCM) invertase (lanes 7—11) after incubation with endo-H. Endo-H (0.5 μ-g, lane 6) was used to treat 2 mg of native or S-carboxymethylated invertase in a 1 mL reaction mixture. Aliquots were electrophoresed in a flatplate discontinuous SDS-pohjacrijlamide el system. Adapted from Trimble and ialey (16,), which should be referred to for further details.
f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
90
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
TABLE I Carbohydrate content of i n v e r t a s e
Preparation
Native
invertase
(CM)-invertase
Enzyme treatment
preparations
GlcN
Chains/ holoenzyme
Man
mol/120,000 g p r o t e i n 18 35.8 570 4 89 22.3 21.9 18 4
None Endoglycosidase Endoglycosidase + a-mannosidase None Endoglycosidas
35.9 18.9
1 cm A l l values are based on an A:280 of molecular weight of 120,000.
565 16
18 1
2.25 and a holoenzyme
Journal of Biological Chemistry
TABLE I I Carbohydrate content of carboxypeptidase Y f r a c t i o n s before and a f t e r endoglycosidase treatment
Enzyme p r e p a r a t i o n
Native Endoglycosidase-treated Denatured and endoglycosidase-treated
CPase Y molar r a t i o Mannosyl GlcN Man chains 7.90 4.95 3.72
55 15 0
4 1 0
Based on a peptide molecular weight of 51,000.
Biochemical and Biophysical Research
Communications
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
Endo-β-acetylglucosaminidases
91
A l t e r n a t i v e l y , molecular weights of the r e l e a s e d o l i g o s a c charides can be estimated by B i o g e l P-4 chromatography p r o v i d i n g appropriate standards are used ( Γ 7 ) . The s i g n i f i c a n c e of the r o l e of carbohydrate attached to p r o t e i n has been of great concern for some time and while removal of the carbohydrate has not g r e a t l y i n f l u e n c e d the enzymic and p h y s i c a l p r o p e r t i e s of most of the g l y c o p r o t e i n s we and others have s t u d i e d , an a l t e r a t i o n i n such p h y s i c a l parameters as s o l u b i l i t y has been noted, p a r t i c u l a r l y i n the case of i n v e r t a s e which possesses such a high mass of t h i s m a t e r i a l . Recently i t has been shown by Wang and H i r s (18) that carbohydrate i n f l u e n c e s the spec t r a l p r o p e r t i e s of ovine RNase B. Even more r e c e n t l y , we have found the s t a b i l i t y of carbohydrate depleted i n v e r t a s e to be markedly a l t e r e d at pH 4.0 r e l a t i v e to n a t i v e i n v e r t a s e (Figure 6). This parameter i s a l s o r e f l e c t e d i n the decreased c a p a c i t y of the carbohydrate depleted enzyme to renature when denatured by g u a n i dine h y d r o c h l o r i d e , suggestin i n f l u e n c e s the c a p a c i t y Because of the r a t h e r high content of carbohydrate i n t h i s enzyme, i t i s d i f f i c u l t to extrapolate these s t u d i e s to other g l y c o p r o t e i n s with considerably lower amounts of carbohydrate. Caution must be e x e r c i s e d i n i n t e r p r e t i n g these f i n d i n g s however, and the p o t e n t i a l for contaminants a s s o c i a t e d with even h i g h l y p u r i f i e d endoglycosidases makes t h i s even r i s k i e r . Thus, even the presence of small q u a n t i t i e s of exoglycosidases or p r o teases can g r e a t l y i n f l u e n c e the r e s u l t s obtained c o n s i d e r i n g the lengthy incubations often used by i n v e s t i g a t o r s . Witness the case of endo-D which was i n i t i a l l y b e l i e v e d to r e l e a s e complex o l i g o saccharides (19) u n t i l i t was r e a l i z e d on further p u r i f i c a t i o n that p e r i p h e r a l sugars as s i a l i c a c i d , g a l a c t o s e , and N - a c e t y l g l u cosamine must f i r s t be r e l e a s e d by contaminating exoglycosidases before the (Man^GlcNAc c h a i n could be r e l e a s e d (14). Even when p u r i f i e d about 4,000 f o l d to apparent homogeneity by the procedure shown i n Table I I I , endo-H contains t r a c e s of p r o t e o l y t i c a c t i v i t y which are not r e a d i l y apparent unless more s e n s i t i v e techniques than o r d i n a r i l y used are a p p l i e d , such as measuring the r e l e a s e of r a d i o a c t i v i t y from hemoglobin l a b e l e d with l ^ C - l a b e l e d g l y c i n e e t h y l e s t e r . Thus, i t i s shown i n F i g u r e 7, which presents a G-75 e l u t i o n p r o f i l e , the l a s t step i n the p u r i f i c a t i o n of endo-H, that small t r a c e s of p r o t e o l y t i c a c t i v i t y are s t i l l present i n the enzyme peak. Denatured CPase Y i s par t i c u l a r l y s e n s i t i v e to t h i s p r o t e o l y t i c a c t i v i t y and i t i s shown i n F i g u r e 8 that t h i s p r o t e i n i s hydrolyzed to v a r y i n g degrees by d i f f e r e n t f r a c t i o n s from the endo-H peak, even though the enzyme appears to be homogenous at t h i s stage. However, treatment of those f r a c t i o n s c o n t a i n i n g p r o t e o l y t i c a c t i v i t y with phenylmethane s u l f o n y l f l u o r i d e almost completely e l i m i n a t e s t h i s contaminant. One must be cautious i n a s s e s s i n g the molecular weight of l a b e l e d o l i g o s a c c h a r i d e s , that glycopeptides are not i n r e a l i t y being considered.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 5. Electrophoresis of native invertase treated with endo-H. The systems used for digestion and electrophoresis were simifor to those in Figure 4. The two forms of the enzyme represent incompletely removed oligosaccharide chains associated with the subunits.
HOURS Figure 6. Stability of native and carbohydrate (CHÔ)-depleted invertase maintained at pH = 4.0. Each enzyme form (1 fig) was incubated in 1 mh of 0.01M sodium citrate pH = 4.0 at 4°C for the indicated times and then assayed (F. K. Chu and F. Maley, in preparation).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
III
50
37
19.7
67
5.5
0.13
72
100
<%)
Recovery
T h i s procedure represents a m o d i f i c a t i o n from that p r e v i o u s l y d e s c r i b e d (2), and w i l l appear i n a future volume of Methods i n Enzymology.
a S t e p 1 m a t e r i a l was prepared i n 1 0 - l i t e r batches. Steps 2 through 4 were performed on the zinc paste from 30 l i t e r s of c u l t u r a l f i l t r a t e . Step 5 was performed by combining two preparations from the preceeding s t e p .
69
Sephadex G-75, pH 8.45
5.
3.5
94
17
404
SP-Sephadex C-25, pH 4.6
4. 29
126
996
680
DE-52 C e l l u l o s e , pH 8.45
3.
0.02
136
6,750
1,300
Ammonium s u l f a t e , 0.4-0.9
189
34,430
60,300
2.
0.0053
Activity (units)
Protein (mg)
Specific activity (units/mg)
plicatus
Volume (ml)
C u l t u r a l f i l t r a t e and zinc p r e c i p i t a t i o n
step
1.
Purification
P u r i f i c a t i o n of endo-3-N-acetylglucosaminidase H from Streptomyces
TABLE
94
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES 1.51
Figure 7. Assessment of residual proteo lytic activity present in the Sephadex G-75 elution profile from Step 5 of Table III. The substrate used to measure proteolysis was [ C]-glycine ethylester hemoglobin (13,000 cpm/mg protein). The peak of enzyme activity was associated with a single band of protein on polyacrylamide gel electroprhoresis. ( Ο — θ ) Endo-H ac tivity (cpm χ 10 ) or (A—^protease ac tivity (cpm X JO ). 14
4
2
20
40
60
80
FRACTION NUMBER (3 ml)
Figure 8. Assay for proteolytic activity in various fractions from the elution profile of Figure 7. Carboxypeptiaase Y (1 mf>) was incubated with 0.2 unit of endo-H from the indicated fractions in a 1 mh of reaction mixture for 4 hr at 37°C. Equal aliquots from these reactions were denatured with sodium dodecul sulfate and electrophoresed on SDS-polyacrylamide Eel as described in Trimble and Maley (7). The migration distances of untreated (-\-CHO) ana depleted (-CHO) carboxypeptiaase Y and endo-H are indicated in the first two lanes at the left.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
Endo-β-acetylglucosaminidases
95
From what was i n d i c a t e d i n Figure 2, endo-H appears most a c t i v e with substrates such as (Man)5(GlcNAc)2Asn, but because of the u n a v a i l a b i l i t y u n t i l now of compounds such as (Man)4(GlcNAc)2~ Asn and (Man)2(GlcNAc)2Asn, i t was not p o s s i b l e to determine the lower l i m i t s of the enzyme's s p e c i f i c i t y . R e c e n t l y , however, T a i et a l . (20) i s o l a t e d (Man)4(GlcNAc)2Asn from an ovalbumin d i g e s t by use of the Montgomery column (6^) and found that t h i s compound l i k e (Man)5(GlcNAc)2Asn was an e x c e l l e n t substrate for endo-H. By t r e a t i n g (Man)5(GlcNAc)2Asn with α - m a n n o s i d a s e for l i m i t e d time p e r i o d s , we have been able to i s o l a t e (Man)4(GlcNAc)2Asn and (Man)β(GlcNAc)2Asn through the use of B i o g e l P-4 chromatography. It was i n i t i a l l y b e l i e v e d , from the B i o g e l P-4 e l u t i o n p r o f i l e , that the l a t t e r compound was (Man)2(GlcNAc)2Asn, but by comparing i t s e l u t i o n time with other g l y c o s y l - A s n d e r i v a t i v e s on the amino a c i d analyzer (Figure 9 ) , i t was e s t a b l i s h e d that t h i s o l i g o s a c charide was composed of (Man)3(GlcNAc)2Asn (Figure 9A, b ) . The i n s e r t to Figure 9A e s t a b l i s h e graphic procedure for c h a r a c t e r i z i n up to s i x mannosyl r e s i d u e s . M e t h y l a t i o n of (Man)4(GlcNAc)2Asn revealed t h i s compound to d i f f e r from that i s o l a t e d by T a i et a l . (19) i n that the p e r i p h e r a l mannosyl linkage was al+6 (Figure 10 and Table IV) instead of al->3 as shown by these i n v e s t i g a t o r s . In contrast to the branched s t r u c t u r e of (Man)3(GlcNAc)2Asn, a s s o c i a t e d with the core of the complex o l i g o s a c c h a r i d e s , that i s o l a t e d from the α - m a n n o s i d a s e d i g e s t i o n of (Man)5(GlcNAc)2Asn, was determined by methylation (Table IV) and periodate o x i d a t i o n to be l i n e a r and to possess the p e r i p h e r a l s t r u c t u r e shown i n F i g u r e 10. Both (Man)4(GlcNAc)2Asn and l i n e a r (Man)3(GlcNAc)2Asn appear to be hydrolyzed as r a p i d l y by endo-H as i t s most a c t i v e s u b s t r a t e , which to date i s (Man)5(GlcNAc)2Asn, and c o n t r a s t s sharply w i t h the rather slow h y d r o l y s i s by t h i s enzyme of branched (Man)3(GlcNAc)2Asn (Figures 2 and 3). By u t i l i z i n g a modified Smith degradation on (Man)5(GlcNAc)2Asn (21, 22), (Man)2(GlcNAc)2Asn could be i s o l a t e d and v e r i f i e d by compositional a n a l y s i s , methyla t i o n (Table I V ) , and chromatography (Figure 9B). T h i s compound was hydrolyzed at only h a l f the r a t e of branched (Man)3(GlcNAc)2~ Asn, while (Man)1(GlcNAc)2Asn i s probably not hydrolyzed at a l l by endo-H. Thus, the statement i n our a b s t r a c t that (Man)2(GlcNAc)2Asn i s as good a substrate as (Man)5(GlcNAc)2Asn i s i n e r r o r and should be corrected by r e p l a c i n g the former compound with l i n e a r (Man)3(GlcNAc)2Asn. (Man)1(GlcNAc)2Asn, however, could be very e f f e c t i v e l y hydrolyzed to Man31->4GlcNAc + GlcNAc-Asn by endo-L, the enzyme r e f e r r e d to e a r l i e r as enabling us to i s o l a t e and to c h a r a c t e r i z e t h i s d i s a c c h a r i d e (1). That t h i s enzyme i n not c h i t o b i a s e i s i n d i c a t e d by i t s i n a b i l i t y to hydrolyze (GlcNAc) 2 Asn. Endo-L has now been p u r i f i e d about 300 f o l d and possesses proper t i e s d i s t i n c t l y d i f f e r e n t from endo-H, not the l e a s t of which i s t h e i r substrate s p e c i f i c i t i e s . Other d i f f e r e n c e s are a s s o c i a t e d with the higher molecular weight of endo-L and i t s sharper pH optimum (23). A comparison of these enzymes and t h e i r capacity to
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
96
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
175
35
52.5
70
0.1 M Ν α - C I T R A T E (pH 2.60), ml Figure 9.
Chromatographic separation of glycosyl asparagine derivatives on the amino acid analyzer using AG-50 X-4 resin.
The various stands used (prepared from ovalbumin) are indicated by the dashed line and repre sent (Man) (GlcNAc) Asn, 1-2-6; (Man) (GlcNAc) Asn, 1-2-5; (Man) (GlcNAc) Asn 1-2-1; (GlcNAc) Asn, 1-2; GlcN Ac-Asn, 1-1. Peaks a and b resulted from a limited a-mannosidase digestion of 1-2-5 as shown in Figure 10; peak c in Β was prepared by the periodate oxidation of 1-2-5; peak d in C was obtained by treating 1-2-2 with periodate. The elution times (t), measured at peaks of various glycosyl-Asn derivatives, were plotted as shown in the insert vs. the number of mannose residues in the compounds. As indicated, compounds a, b , and c fall on the line at 4, 3, and 2 mannose residues respectively, which is consistent with their mannosyl content (Table IV). The proposed mannosyl linkages shown in Figure 10 were obtained from the methylation data in Table IV. More specific details on the methodology used are in prepa ration. 6
2
5
2
t
2
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 1.00
(Man) 2 (GlcNAc) 2 A s n b
endo-H
1.0
0.84
2.2
1.4
1.1
1.3 1.0
1.0
Methylated mannitol products (2,3,4,6) (2,3,4) (2,4,6) (2,4)
s u b s t r a t e s for
'Periodate o x i d a t i o n product from (Man)r (GlcNAc)οAsn.
Prepared from IgM (9) ; R = p e p t i d e .
1.96
3.00
2.00
1.55
(Man) 3 (GlcNAc) 2 - R a 1.80
2.84
1.82
1.00
4.06
1.85
(Man) 3 (GlcNAc) 2 A s n
Man
Analysis GlcNAc
1.00
Asn
of compounds tested as
(Man) 4 (GlcNAc) 2 A s n
Isolated compound
Composition
TABLE IV
98
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
hydrolyze s p e c i f i c dansylated substrates prepared from ovalbumin (Figure 11) r e v e a l s that endo-L i s not very e f f e c t i v e against sub s t r a t e s c o n t a i n i n g more than two r e s i d u e s of mannose. In a s i m i l a r v e i n , endo-H i s not e f f e c t i v e against compounds c o n t a i n i n g two mannosyl residues or l e s s . We cannot be c e r t a i n at present whether the small amount of a c t i v i t y seen with (Man)3(GlcNAc)2Asn as s u b s t r a t e i s n a t i v e to endo-L and i s not due to traces of endo-H. There i s , however, a greater degree of confidence that homogenous endo-H decreases i n a c t i v i t y by 10,000 f o l d i n going from l i n e a r (Man)3(GlcNAc)2Asn to (Man)2(GlcNAc)2Asn. These recent s p e c i f i c i t y s t u d i e s suggest the need for c a u t i o n i n e s t i m a t i n g o l i g o s a c c h a r i d e s i z e based on the s e n s i t i v i t y or r e s i s t a n c e of glycopeptides to endo-H. Following our c h a r a c t e r i z a t i o n of the p r o p e r t i e s of endo-H (2, 6, 15) and that of endo-D by Koide and Muramatsu (14) a number of other u s e f u l endoglycosidases have been i d e n t i f i e d . Thus, an endo-3-N-acetyl-galactosaminidas f i l t r a t e s of Diplococcu from E s c h e r i c h i a f r e u n d i i (25), two endo-3-N-acetylglucosaminidases from C l o s t r i d i u m perfringens (26), one with s l i g h t l y d i f f e r ent s p e c i f i c i t y than endo-H, and more r e c e n t l y an amidase from almond emulsin (27). The amidase, although not p u r i f i e d to a s i g n i f i c a n t extent, possesses the i n t r i g u i n g property of r e l e a s i n g o l i g o s a c c h a r i d e s from a stem bromelein glycopeptide by h y d r o l y z i n g the glucosaminyl asparagine bond. If t h i s enzyme i s more gener a l l y a p p l i c a b l e , i t should be extremely u s e f u l i n the study of glycopeptides and p o s s i b l y g l y o c p r o t e i n s , but not enough i n known about i t s s p e c i f i c i t y at the present time. As far as the existence of an animal endoglycosidase with p r o p e r t i e s s i m i l a r to those of endo-H or endo-D, i t s presence can be i n f e r r e d from the s t r u c t u r e of o l i g o s a c c h a r i d e s excreted i n the u r i n e and present i n the t i s s u e s of animals with c e r t a i n lysosomal storage d i s e a s e s . Thus, mannosyl o l i g o s a c c h a r i d e s with a s i n g l e N-acetylglucosamine on the reducing end were found i n the u r i n e and t i s s u e of b l a c k angus c a t t l e (28) and humans (29) a f f l i c t e d with α - m a n n o s i d o s i s . O l i g o s a c c h a r i d e s c o n s i s t i n g of 7 to 10 sugar r e s i d u e s with the complex t e t r a s a c c h a r i d e core of (Man^GlcNAc have a l s o been found i n p a t i e n t s with GM]^ or GM2 g a n g l i o s i d o s i s (30, 31, 32), and f i b r o b l a s t s from p a t i e n t s with α - f u c o s i d o s i s (32) apparently accumulate the d i s a c c h a r i d e Fucal-*6GlcNAc, a d i s a c c h a r i d e found at the reducing end of many "complex" type oligosaccharides. Whether these products accumulate due to the above s p e c i f i c exoglycosidase d e f i c i e n c i e s combined with cleavages a f f e c t e d by a s i n g l e endoglycosidase i s not known, but we have described an enzyme i n animal t i s s u e s p a r t i a l l y capable of e x p l a i n i n g these f i n d i n g s (33). The enzyme was p u r i f i e d over 1,500 f o l d from hen-oviduct and as shown i n F i g u r e 12 hydrolyzed [ l ^ C ] - N - a c e t y l a t e d g l y c o s y l - A s n d e r i v a t i v e s derived from ovalbumin i n a manner s i m i l a r to that described for endo-H. As an added bonus, t h i s enzyme p r e p a r a t i o n
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
MALEY ET AL.
Endo-β-acetylglucosaminidases
Manal - ^ 6 Μ β η β 1 ^ » 4 G 1 c N A c 0 1
•>
4G1 c N A c - A s n
10.
b) H
+
Manal , ^ 6 Manal
^
Manal ^
ManÊl Manal
Manal
4G1 cNAcS1
4G1 c N A c - A s n
^
r
6Manal
a-Mannosidase
Man 31 <
M a n a l ^ » 3Manal
-•6ManBl.
Manal
Figure 10. Proposed structures of compounds prepared from (Man) (GlcNAc) Asn by periodate oxidation and limited α-mannosidase digestion. The structures proposed are based on compositional analyses, in addition to the methylation and periodate oxidation data presented in Figure 9 and Table IV. R = (GlcN Ac) -Asn. 5
2
2
CLEAVAGE OF OV-OLIGOSACCHARIDES BY E N D O - L AND -H Asn(GlcNAc) Man 2
AsniGlcNAcfeMai^
AsnfêlcNAcfeMons Asn(GlcNAc)4Mûn
6
OR ENDO-L
~
"
ENDO-H
-
+
+ -
-
- + +
-
-
- +
-
-
+
-
+
-
+
-
-
Figure 11. Studies on the substrate specificity of endo-H and endo-L. The corresponding dansyl derivatives of the compounds indicated were treated with endo-L and endo-H as shown, and aliquots were chromatographed ascendingly on Whatman 3-MM with butanol-ethanol-water (2:1:1).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Journal of Biological Chemistry
Figure 12. Substrate specificity of hen oviduct endoglycosidase. The indicated [ C]-N-acetylated derivatives were etectrophoresed on paper before and after treatment with this enzyme preparation, followed by the preparation of a radioautogram. Adapted from Trimble et al. (23) which should be consulted for further details. 14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4.
MALEY ET AL.
Endo-β-acetylglucosaminidases
101
a l s o hydrolyzed the complex core glycopeptide c o n t a i n i n g fucose, a property of endo-D but not of endo-H. A l s o c h a r a c t e r i s t i c of endo-D i s the f i n d i n g that the complex branched g l y c o p e p t i d e , (Man)3(GlcNAc)2Asn, i s hydrolyzed at a f a s t e r r a t e than (Man) 6 (GlcNAc)4Asn (33), the reverse of that obtained with endo-H (Figure 2 ) . I f an enzyme s i m i l a r to t h i s one i s present i n animal t i s s u e s from other s p e c i e s , the type of o l i g o s a c c h a r i d e s a s s o c i ated with the above mentioned genetic d i s o r d e r s can e a s i l y be rationalized. Through the use of a new assay we have developed where the mannosyl non-reducing end of g l y c o s y l - A s n d e r i v a t i v e s , such as (Man)5(GlcNAc)2Asn-dansyl, are l i g h t l y o x i d i z e d with p e r i odate and then reduced with sodium b o r o t r i t i d e , endoglycosidase a c t i v i t y has been found i n s e v e r a l r a t t i s s u e s , as w e l l as i n Tetrahymena p y r i f o r m i s (34). An advantage of t h i s s u b s t r a t e i s the r e s i s t a n c e of the l a b e l e d d i o l to α - m a n n o s i d a s e , an enzyme which could y i e l d a f a l s e p o s i t i v e endoglycosidase a c t i v i t y i n crude t i s s u e e x t r a c t s . Although an animal genetic d i s o r d e r i m p l i c a t i n g endo-3-Nacetylglucosaminidase has not been reported y e t , the question a r i s e s as to what might be expected i f an organism i s d e f i c i e n t i n t h i s enzyme. In a l l p r o b a b i l i t y t h i s defect would be d i f f i c u l t i f not impossible to assess s i n c e the numerous types of e x o g l y c o s i dases present i n animal t i s s u e s would l i m i t the accumulation of long chain o l i g o s a c c h a r i d e s a s s o c i a t e d with glycopeptides i n v o l v ing glucosaminyl-Asn l i n k a g e groups. That i s unless α - m a n n o s i d a s e was a l s o d e f i c i e n t , i n which case o l i g o s a c c h a r i d e s with GlcNAc on the reducing end would not be found and glycopeptides with mannosy 1 - r i c h o l i g o s a c c h a r i d e s should accumulate. At present, the r o l e of endo-H and other s i m i l a r l y r e l a t e d enzymes appears to be r e s t r i c t e d to that of degrading g l y c o p r o t e i n s and g l y c o p e p t i d e s , but t h e i r p o t e n t i a l c o n t r i b u t i o n to the b i o s y n t h e s i s and processing of g l y c o p r o e t i n s should not be ignored. In any event, the d i s c o v e r y of the endoglycosidases has added a new dimension to the study of g l y c o p r o t e i n s , which h o p e f u l l y w i l l continue to provide new and important information on t h e i r s t r u c t u r e and f u n c t i o n .
Acknowledgements We would l i k e to express our a p p r e c i a t i o n to D r . Thomas H. Plummer, J r . and Dr. F r e d e r i c k K. Chu for t h e i r a i d with some aspects of t h i s study.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
102
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Literature Cited 1. Tarentino, A. L., Plummer, T. H., Jr., and Maley, F., J. Biol. Chem. (1972) 247, 2629. 2. Tarentino, A. L., and Maley, F., J. Biol. Chem. (1974) 249, 811. 3. Sukeno, T., Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., Biochem. Biophys. Res. Commun. (1971) 45, 219. 4. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., J. Biol. Chem. (1973) 248, 5547. 5. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., J. Biol. Chem. (1974) 249, 818. 6. Huang, C.-C., Mayer, H.-E., Jr., and Montgomery, R., Carbohydrate Res. (1970) 13, 127. 7. Trimble, R. B., and Maley, F., Biochem. Biophys. Res. Commun. (1977) 78, 935. 8. Baeziger, J., and Kornfeld 7260. 9. Tarentino, A. L., Plummer, Τ. Η., Jr., and Maley, F., Biochemistry (1975) 14, 5516. 10. Kornfeld, R., and Kornfeld, S., Annu. Rev. Biochem. (1976) 45, 217. 11. Burke, D. J., and Keegstra, K., J. Virol. (1976) 20, 676. 12. Robbins, P. W., Hubbard, S.C.,Turco, S. J., and Wirth, D. F., Cell (1977) 12, 893. 13. Tabas, I., Schlesinger, S., and Kornfeld, S., J. Biol. Chem. (1978) 253, 716. 14. Koide, Ν., and Muramatsu, T., J. Biol. Chem. (1974) 249, 4897. 15. Tarentino, A. L., and Maley, F., Biochem. Biophys. Res. Commun. (1975) 67, 455. 16. Trimble, R. B., and Maley, F., J. Biol. Chem. (1977) 252, 4409. 17. Etchinson, J. R., Robertson, J. S., and Summers, D. F., Virology (1977) 78, 375. 18. Wang, F.-F.C.,Hirs, C. H. W., J. Biol. Chem. (1977) 252, 8358. 19. Muramatsu, T., Atkinson, P. H., Nathenson, S. G., and Ciccarini, C., J. Mol. Biol. (1973) 80, 781. 20. Tai, T., Yamashita, Κ., and Kobata, Α., Biochem. Biophys. Res. Commun. (1977)78,434. 21. Montgomery, R., Wu, Y.-C, and Lee, Y. C., Biochemistry (1965) 4, 578. 22. Garner, E. F., Goldstein, I. J., Montgomery, R., and Smith, F., J. Am. Chem. Soc. (1958) 80, 207. 23. Trimble, R. B., Tarentino, A. L., and Maley, F., in preparation. 24. Umenoto, J. Bhavanandan, V. P., and Davidson, Ε. Α., J. Biol. Chem. (1977) 252, 8609. 25. Fukuda, Μ. Ν., and Matsumura, G., J. Biol. Chem. (1976) 251, 6218.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
4. MALEY ET AL.
Endo-β-acetylglucosaminidases
103
26. Tai, T., Yamashita, Κ., Ito, S., and Kobata, Α., J. Biol. Chem. (1977) 252, 6687. 27. Takashi, N., Biochem. Biophys. Res. Commun. (1977) 76, 1194. 28. Lundblad, Α., Nilsson, B., Nordén, Ν. Ε., Svensson, S., Ôckerman, P.-Α., and Jolly, R. D., Eur. J. Biochem. (1975) 59, 601. 29. Nordén, Ν., Lundblad, Α., Svensson, S., and Autio, S., Biochemistry (1974) 15, 871. 30. Wolfe, L. S., Senior, R. G., and Kin, Ν., J. Biol. Chem. (1974) 249, 1828. 31. Kin. Ν., and Wolfe, L. S., Biochem. Biophys. Res. Commun. (1974) 59, 837. 32. Tsay, G.C.,and Dawson, G., Biochem. Biophys. Res. Commun. (1975) 63, 807. 33. Tarentino, A. L. and Maley F. J Biol Chem (1976) 251 6537. 34. Tarentino, A. L., Maley, , (1977) , 185. RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5 Mucous Glycoproteins in Cystic Fibrosis THOMAS F. BOAT and PIWAN CHENG Department of Pediatrics, Rainbow Babies and Childrens Hospital, Case Western Reserve University, School of Medicine, Cleveland, OH 44106 Cystic fibrosis (CF) Caucasians, causes muc progressiv g children and young adults. It also is responsible for intestinal obstruction at birth (meconium ileus), pancreatic insufficiency with maldigestion and failure to grow, and obstructive liver disease in this age group. Furthermore, obliteration of the vas deferens causes aspermia and infertility in most males with CF. Individuals with CF secrete sweat which contains excessive amounts of chloride and sodium. Diagnostic criteria include detection of elevated chloride levels in sweat of individuals with typical pul monary, gastrointestinal, or genital manifestations of the disease (1). The pathophysiology of CF has been the subject of extensive investigation (2). However, the basic defect responsible for the disease has not been identified. Claims have been made by several investigators that circulating factors which can be detected by inhibition of oyster gill (3) or rabbit tracheal (4) ciliary motil ity, or secreted factors which inhibit sodium reabsorption by ductal epithelium in sweat and salivary glands (5), may be related closely to the gene defect. Alteration of short circuit current across rat intestinal epithelium, inhibition of sugar and amino acid transport by intestinal epithelium, augmentation of leukocyte degranulation, inhibition of phagocytosis by alveolar macrophages, and inhibition of debranching enzyme activity have also been as cribed to circulating or secreted "factors" associated with CF. Polyamines, their condensation products (6) and small polycationic peptides (7) have been implicated as "factors" but a substance has not been isolated and characterized which is clearly responsible for any of these effects. Cell membrane properties with the ex ception of increased resistance of fibroblasts to oubain toxic ity (8) appear unaltered. Recent evidence suggests a deficiency of proteolytic activity in CF serum (9) and abnormal interaction of circulatingα -macroglobulinwithproteolyticenzymes(10). Pursuit of initial observations in many of these studies has led to contradictory results, making most studies difficult to inter2
0-8412-0452-7/78/47-080-108$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous Glycoproteins
in Cystic
Fibrosis
109
p r ê t or f i t i n t o an o v e r a l l p a t h o p h y s i o l o g i c scheme. One poss i b l e e x p l a n a t i o n f o r i n c o n s i s t e n c i e s i n s t u d i e s c a r r i e d out to date i s that CF may be due to genetic abberations at more than one l o c u s . On the other hand the pathogenesis of most c l i n i c a l manif e s t a t i o n s i n CF i s r e l a t i v e l y w e l l understood and appears to be r e l a t e d to two anomalies: 1) abnormal behavior of the mucuss e c r e t i o n s , g i v i n g r i s e to o b s t r u c t i o n , and i n some cases i n f e c t i o n w i t h i n mucus-secreting organs ( l u n g , i n t e s t i n e , pancreas, b i l i a r y t r a c t , s a l i v a r y g l a n d s , and g e n i t o u r i n a r y t r a c t ) , and 2) abnormality of the e l e c t r o l y t e composition of e c c r i n e sweat glands, o c c a s i o n a l l y r e s u l t i n g i n heat p r o s t r a t i o n due to massive s a l t l o s s (2). T h i s review w i l l examine the abnormality of mucous s e c r e t i o n s i n d e t a i l and present current t h i n k i n g about i t s biochemical b a s i s . Emphasis w i l l be given to lung mucus because a i r f l o w o b s t r u c t i o n and lung i n f e c t i o n are r e s p o n s i b l e for most of the m o r b i d i t CF. Mucus from i n d i v i d u a l s with CF g e n e r a l l y i s d e s c r i b e d as abundant and e x c e s s i v e l y s t i c k y . R h é o l o g i e s t u d i e s of lung mucus have been c a r r i e d out l a r g e l y on sputum (11) w i t h a high content of DNA (12) which masks the v i s c o e l a s t i c p r o p e r t i e s of the primary s e c r e t i o n s . While c a r e f u l r h é o l o g i e s t u d i e s of u n i n f e c t ed mucous s e c r e t i o n s with a low DNA content from CF subjects have not been r e p o r t e d , i t i s reasonable to assume that the o b s t r u c t i v e events l e a d i n g to the c l i n i c a l syndrome are the r e s u l t of abnormal p r o p e r t i e s of mucus, and perhaps of the mucous g l y c o p r o t e i n s themselves which seem to be the primary determinant of uninfected dog t r a c h e a l mucus (13). Three p o t e n t i a l causes f o r these abnormal p r o p e r t i e s can be proposed: 1) h y p e r s e c r e t i o n of normal mucus which overloads clearance mechanisms, r e s u l t i n g i n s t a s i s and o b s t r u c t i o n ; 2) inadequately hydrated mucus or mucus with an a l t e r e d i o n content such that g l y c o p r o t e i n complexes are formed which have abnormal r h é o l o g i e p r o p e r t i e s ; or 3) a l t e r a t i o n of chemical p r o p e r t i e s of mucous g l y c o p r o t e i n s r e s u l t i n g i n f o r mation of mucous g e l s with abnormal r h é o l o g i e c h a r a c t e r i s t i c s . Mucus H y p e r s e c r e t i o n : Morphologic examination of s e v e r a l organs from i n d i v i d u a l s with CF shows marked enlargement of the mucuss e c r e t i n g glands (hypertrophy) and a marked increase i n the number of mucus-secreting goblet c e l l s w i t h i n the surface e p i t h e l i u m (hyperplasia) (14). T h i s i s true not only of l u n g , but a l s o of i n t e s t i n e , s a l i v a r y glands, and u t e r i n e c e r v i x . In f a c t , goblet c e l l s appear e a r l y i n the course of the d i s e a s e i n the surface e p i t h e l i u m of v e r y small airways (bronchioles) where they normally do not r e s i d e i n s u b s t a n t i a l numbers. T h i s m e t a p l a s t i c change introduces mucus i n t o airways which are not equipped to handle g e l - l i k e s e c r e t i o n s . Mucus then impacts i n these small airways, producing the e a r l i e s t r e c o g n i z a b l e lung l e s i o n (15). Morphologic evidence f o r a hypersecretory s t a t e i n CF lungs at b i r t h i s c o n t r a d i c t o r y (16,17). However, i t i s c l e a r that duo-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1 1 0
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
denal mucus-secreting glands are enlarged and apparently hypera c t i v e at b i r t h (17). T h i s f i n d i n g suggests that increased s e c r e t i o n of mucus may be more than a secondary r e a c t i o n to c h r o n i c i n f e c t i o n or inflammation. A search f o r p a t h o p h y s i o l o g i c determinants of mucous hypers e c r e t i o n u n t i l r e c e n t l y turned up few l e a d s . In the l a s t s e v e r a l y e a r s , s t u d i e s of the e f f e c t of serum on c i l i a r y m o t i l i t y l e d to the observation that l a r g e amounts of "mucus" were r e l e a s ed from c i l i a t e d e p i t h e l i u m i n the presence of CF serum (18,19). Recently Czegledy-Nagy and Sturgess (20) studied t h i s response by scanning e l e c t r o n microscopy and observed that r a b b i t t r a c h e a l e p i t h e l i u m r e l e a s e d more s e c r e t o r y product i n the presence of CF than c o n t r o l serum. These observations are q u a l i t a t i v e and i n a d d i t i o n , products which were assumed to c o n t a i n mucous g l y c o p r o t e i n s were not i d e n t i f i e d . We have developed an assay to assess the e f f e c t of CF serum and other b i o l o g i c a l f l u i d s on the r e l e a s e of mucous g l y c o p r o t e i n G l y c o p r o t e i n s are l a b e l e glucosamine, and t h e i r r e l e a s e i n t o c u l t u r e medium i n the presence of 50% (v/v) CF serum, 50% (v/v) c o n t r o l serum, and no serum i s monitored. P r e l i m i n a r y r e s u l t s show that the presence of serum from any source i n the medium increases the r e l e a s e of l a b e l e d high molecular weight macromolecules which have a carbohydrate composition t y p i c a l of mucous g l y c o p r o t e i n s . Some CF sera t r a n s i e n t l y induce a higher s e c r e t o r y r a t e f o r these l a b e l e d macromolecules than i s achieved i n the presence of age and sexmatched c o n t r o l s e r a , but o v e r a l l we have not yet been able to demonstrate a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t f o r CF sera (Figure 1 ) . At longer exposure periods (1 hour or more) no suggestion of a d i f f e r e n t i a l CF serum e f f e c t on mucous g l y c o p r o t e i n r e l e a s e was found. The nature of the s t i m u l a t o r y e f f e c t i n some CF sera i s not known. Polyamines augment the r e l e a s e of g l y c o p r o t e i n s from canine t r a c h e a l expiants (22) but not human t r a c h e a l e p i t h e l i a l expiants (23). P o l y c a t i o n i c peptides a l s o increase the r e l e a s e of g l y c o p r o t e i n s i n v i t r o from dog t r a c h e a l e p i t h e l i u m (24). P o l y c a t i o n i c substances may i n f l u e n c e mucous g l y c o p r o t e i n synthesis by s t i m u l a t i n g the a c t i v i t y of g l y c o s y l t r a n s f erases at low Mn + ^ concentrations (25) . Whether t h i s occurs with i n t a c t t i s s u e s under p h y s i o l o g i c c o n d i t i o n s i s not known. Expiants of post-mortem t r a c h e o b r o n c h i a l e p i t h e l i u m from CF subjects s e c r e t e mucous g l y c o p r o t e i n s at a higher b a s e l i n e r a t e than do expiants from c h i l d r e n and a d u l t s who have d i e d of nonr e s p i r a t o r y c o n d i t i o n s (26). This observation i s c o n s i s t e n t w i t h an increased mass of mucus-secreting c e l l s i n t i s s u e s of subjects with severe b r o n c h i t i s as i t occurs i n CF, and does not r e f l e c t an increased i n t r i n s i c l e v e l of f u n c t i o n f o r CF s e c r e t i n g u n i t s . Further support f o r t h i s concept comes from autoradiographic s t u d i e s of Neutra (27) which show no d i f f e r e n c e i n the r a t e of movement of l a b e l e d g l y c o p r o t e i n precursors through goblet c e l l s of CF, compared with c o n t r o l , r e c t a l e p i t h e l i a l e x p i a n t s .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic
Fibrosis
Figure 1. Discharge of SO and Hlabeled glycoproteins by rabbit tracheal expiants in the presence of no serum (C), normal serum (N), and CF serum (CF). 35
/f
3
Expiants were cultured in the presence of 2 X 10 dpm of 3 H-6-D-glucosamine and 1 Χ 10 dpm of SO in 2 mh of medium for 24 hr (Fj), rinsed thoroughly with unlabeled me dium and then cultured for 10 min (PJ in 2 mL of unlabeled medium containing no serum or 50% (v/v) serum. Media harvested from P, and P culture periods, including washings, were treated with 5% trichloro acetic acid-1 % phosphotungstic acid and the washed precipitates were solubilized with hydroxide of hyamine-H 0 for scintillation counting (24). Data are expressed as a ratio of the rate of acid-precipitahle label released in P to that in P (56) for each experimental condition. Approximately 80% of labeled macromolecules released by rabbit tracheal explants were high molecular weight sub stances as assessed by BioGel A-5m chroma tography and had a carbohydrate composition typical for mucous glycoproteins (21). Con stant specific activity of discharged glycopro teins was achieved within 24 hr of exposure to medium containing labeled precursors. C vs. Ν; ρ < 0.01. C vs. CF; ρ < 0.01. Ν vs. CF; 0.05
8
35
i
2
2
2
2
r
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
111
112
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Sturgess and Reid observed w i t h autoradiographic techniques that the response of ^ s e c r e t o r y index ( p r o p o r t i o n of c e l l s show ing i n c o r p o r a t i o n of Η - g l u c o s e and movement of l a b e l to the lumenal surface) to cholinomimetic s t i m u l a t i o n was higher f o r hypertrophic glands of CF t r a c h e a l expiants than f o r glands from n o n - b r o n c h i t i c tracheas (28) . However, d i r e c t measurement of the s e c r e t o r y r a t e f o r mucous g l y c o p r o t e i n s from human t r a c h e a l exp l a n t s i n our hands has demonstrated a s i m i l a r s e c r e t o r y response to cholinomimetic agents f o r CF and n o n - b r o n c h i t i c t r a c h e a l exp l a n t s (26). T h e r e f o r e , a strong case cannot be made f o r i n creased i n t r i n s i c responsiveness to s t i m u l a t i o n of CF mucus s e c r e t i n g elements. D e f i n i t i v e data are not a v a i l a b l e to support the hypothesis that c h o l i n e r g i c tone i s increased i n C F , r e s u l t ing i n a chronic hypersecretory s t a t e ( 2 ) . Abnormal M i l i e u f o r Mucous G l y c o p r o t e i n s : Mucous s e c r e t i o n s of the CF t r a c h e o b r o n c h i a water content and highe s e c r e t i o n s (29). Meconium from meconium i l e u s p a t i e n t s i s a l s o r e l a t i v e l y dehydrated (29). The volume of p a n c r e a t i c s e c r e t i o n s i s diminished i n n e a r l y a l l CF s u b j e c t s , i n d i c a t i n g a decreased water output. The volume of s a l i v a r y s e c r e t i o n s i s not d i m i n i s h ed, but the p r o t e i n c o n c e n t r a t i o n of submaxillary s a l i v a i s higher than normal (29). In s h o r t , mucous-type exocrine s e c r e t i o n s are r e l a t i v e l y dehydrated i n c y s t i c f i b r o s i s . A paucity of water i n these s e c r e t i o n s may e x p l a i n the i n s p i s s a t i o n and d u c t a l or small airways o b s t r u c t i o n seen prominently i n C F . In the lungs, a l a c k of water could reduce the volume of p e r i c i l i a r y f l u i d and i n t e r f e r e with c i l i a r y m o t i l i t y ( 1 ) . In a d d i t i o n , r e l a t i v e l y " d r y " s e c r e t i o n s may have sub-optimal v i s c o e l a s t i c p r o p e r t i e s which would i n t e r f e r e w i t h normal c o u p l i n g of mucus to c i l i a r y movement and consequently w i t h mucus clearance from the lungs (30). The non-mucous s e c r e t i o n s ( p a r o t i d s a l i v a , sweat) of subjects with CF, however, are not reduced i n volume and are not concentrated w i t h respect to organic c o n s t i t u e n t s (29). T h e r e f o r e , a g e n e r a l i z e d defect i n water s e c r e t i o n i s not charac t e r i s t i c of t h i s d i s e a s e . Increased concentrations of calcium are found i n s e v e r a l s e c r e t i o n s of i n d i v i d u a l s w i t h CF, i n c l u d i n g submaxillary and p a r o t i d s a l i v a , t e a r s , and seminal plasma (29). Formation of complexes between d i v a l e n t c a t i o n s and the h i g h l y a c i d i c mucous g l y c o p r o t e i n s may c o n t r i b u t e s u b s t a n t i a l l y to the p h y s i c a l and r h é o l o g i e p r o p e r t i e s of mucous s e c r e t i o n s . For example, r a t goblet c e l l i n t e s t i n a l mucus becomes l e s s s o l u b l e i n the presence of i n c r e a s i n g concentrations of c a l c i u m (31). However, a s i m i l a r e f f e c t i n CF mucus s e c r e t i o n s has not been d e s c r i b e d . F u r t h e r more, calcium-mucin complex formation i s an u n l i k e l y pathogenetic f a c t o r because calcium l e v e l s are not elevated i n CF tracheob r o n c h i a l s e c r e t i o n s , c e r v i c a l s e c r e t i o n s , and meconium (29). The m i l k y appearance of CF submaxillary s a l i v a i n i t i a l l y was
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic
Fibrosis
113
considered to be evidence f o r c a l c i u m - g l y c o p r o t e i n i n t e r a c t i o n s (32) but the i n s o l u b l e m a t e r i a l has been shown to be a complex of calcium and a low molecular weight phosphoglycoprot e i n , u n r e l a t e d to the mucous g l y c o p r o t e i n s (33). I n t e r a c t i o n s of mucous g l y c o p r o t e i n s w i t h other c a t i o n i c compounds i n s e c r e t i o n s such as the polyamines and t h e i r metab o l i t e s (29), lysozyme, l a c t o f e r r i n , p r o l i n e - r i c h p o l y p e p t i d e s (34) and immunoglobulins (35) may w e l l o c c u r . However, the r o l e which complexes of mucous g l y c o p r o t e i n s and these substances p l a y i n determining the p r o p e r t i e s of mucus has not been f u l l y e x p l o r e d . The p o s s i b i l i t y that these complexes c o n t r i b u t e to a b n o r m a l i t i e s of mucus i n CF deserves c o n s i d e r a t i o n . A l t e r e d Physicochemical P r o p e r t i e s of Mucous G l y c o p r o t e i n s : Mucous g l y c o p r o t e i n s are r e s p o n s i b l e i n l a r g e part f o r the v i s c o e l a s t i c p r o p e r t i e s of t r a c h e o b r o n c h i a l s e c r e t i o n s . Determinants of these p r o p e r t i e s i n c l u d of non-covalent i n t e r a c t i o n gated g l y c o p r o t e i n molecules, r e s u l t i n g i n the formation of a gel matrix. P a r t i c i p a t i o n of other p r o t e i n s i n the formation of t h i s matrix has been suggested (36). Human r e s p i r a t o r y t r a c t mucous g l y c o p r o t e i n s c o n t a i n 75-80% carbohydrate, 20% or l e s s p e p t i d e , and a v a r i a b l e amount of s u l f a t e e s t e r (40), at l e a s t some of which i s present as D-galactose-6-S0^ (41). Fucose, g a l a c t o s e , glucosamine, galactosamine, and N-acetylneuraminic a c i d are the only i d e n t i f i a b l e sugar r e s i d u e s and are arranged i n a v e r y heterogeneous group (41) of galactosaminyl-threonine ( s e r i n e ) - l i n k e d o l i g o s a c c h a r i d e c h a i n s . Nearly one-half of the peptide amino a c i d s are threonine or s e r i n e r e s i d u e s , and at l e a s t 50% of these hydroxyamino a c i d s are g l y c o s y l a t e d (42). The molecular weight of reduced and carboxymethylated r e s p i r a t o r y g l y c o p r o t e i n prepared from lung washings of an i n d i v i d u a l with c y s t i c f i b r o s i s i s approximately 250,000 as determined by sedimentation e q u i l i b r i u m s t u d i e s (42), but these g l y c o p r o t e i n s behave as i f they are much l a r g e r molecules on agarose g e l f i l t r a t i o n (40). In the non-reduced s t a t e they p o l y merize to form complexes with estimated molecular weights of 600,000 to 3,000,000 (35,43). I t has been suggested that r e p u l s i v e forces between s i a l i c a c i d c a r b o x y l and s u l f a t e a n i o n i c s i t e s on adjacent o l i g o s a c c h a r i d e chains are r e s p o n s i b l e f o r the extended conformation of these g l y c o p r o t e i n s (44), but the cont r i b u t i o n of a n i o n i c groups to the r h é o l o g i e behavior of mucous g l y c o p r o t e i n s c u r r e n t l y i s a subject of debate (45,46). We have developed a system f o r p u r i f i c a t i o n of mucous g l y c o p r o t e i n s i n sputum or lung lavage f l u i d s and t h e i r f r a c t i o n a t i o n based on charge (40). Reduced and carboxymethylated g l y c o p r o t e i n s c o n t a i n i n g a l l of the blood group substance, are r e c o v ered at the v o i d volume of a B i o G e l A-5m column and separated i n t o a s e r i e s of components, ranging from s p a r s e l y a c i d i c to h i g h l y a c i d i c , on DEAE c e l l u l o s e (Figure 2 ) . These components have s i m i l a r peptide and carbohydrate compositions. The major
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
PHOSPHATE
PHOSPHATE
pH 8.0
pH 6.0
0 8 -
S
0 6
CVJ
.
0
M
N
o
C
I
Κα)
I.Or
1
2
0.3M N o C 0 2
3
HI
1
0.4
Ο
6
0.2
10
2C 20
30
F R A C T I O N
40
50
60
N U M B E R
Archives of Biochemistry and Biophysics Figure 2. Fractionation of reduced, carboxymethyhted mucous glycoproteins from a tracheostomized subject with no evidence for lower respiratory tract disease on DEAE cellulose (12 X 0.75 cm column). Glycoproteins were first purified by BioGel A-5m chromatography (40,). Glycoproteins were applied to the column in 0.005M phosphate buffer (pH = 8.0) and eluted stepwise as indicated. Five mL fractions were col lected. Less than 1% of material applied to the column was recovered in the run-through volume. Approximately 60% of material applied to the column was recovered in the four fractions. All four fractions con tained blood group-active substance and were typical mucous glycopro teins with respect to sugar and amino acid composition (40).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic
Fibrosis
115
s p a r s e l y a c i d i c component (I) i s a potent blood group a n t i g e n , has r e l a t i v e l y weak i n f l u e n z a v i r u s hemagglutination i n h i b i t i o n p r o p e r t i e s , and contains 1-2% s u l f a t e . The major a c i d i c com ponent (II) i s a l e s s potent blood group a n t i g e n , a stronger i n h i b i t o r of v i r u s hemagglutination, and i s h i g h l y s u l f a t e d (6-7% by w e i g h t ) . O l i g o s a c c h a r i d e chains of component I I have a s l i g h t l y longer mean c h a i n length than those of component I . However, both components d i s p l a y a wide range of o l i g o s a c c h a r i d e chain l e n g t h s , from 1-20 sugar r e s i d u e s / c h a i n . In general s u l fated sugars are found i n the long c h a i n s , and s i a l i c a c i d r e s i d e s i n chains c o n t a i n i n g 4-7 sugar residues (47). C y s t i c f i b r o s i s t r a c h e o b r o n c h i a l mucous g l y c o p r o t e i n s d i f f e r from those of subjects with other hypersecretory s t a t e s , e . g . , chronic b r o n c h i t i s , i n that they c o n t a i n predominently the h i g h l y a c i d i c components as shown i n F i g u r e 3. Consequently, CF t r a c h e o b r o n c h i a l s e c r e t i o n s as a whole are more h i g h l y s u l f a t e d and e x h i b i t stronger a c i d i been made i n our l a b o r a t o r y and by others (48) . CF s a l i v a r y g l y c o p r o t e i n s are more potent i n h i b i t o r s of i n f l u e n z a Β v i r u s hemagglutination than are s a l i v a r y g l y c o p r o t e i n s from c o n t r o l subjects (49), suggesting that they a l s o have increased a c i d i c properties. No unique mucous g l y c o p r o t e i n components have been found i n CF s e c r e t i o n s and none are m i s s i n g . There i s no evidence f o r a l t e r a t i o n s of the fucose and s i a l i c a c i d contents of CF t r a c h e o b r o n c h i a l mucous g l y c o p r o t e i n s , as postulated a number of years ago f o r i n t e s t i n a l g l y c o p r o t e i n s by Dische (50). Expiants of t r a c h e a l e p i t h e l i u m from subjects with CF a l s o s e c r e t e l a r g e amounts of the h i g h l y s u l f a t e d g l y c o p r o t e i n com ponents. T h i s p a t t e r n r e v e r t s to a non-CF type with predomin ance of s p a r s e l y s u l f a t e d g l y c o p r o t e i n (component I) when s e c r e t i o n by expiants i s stimulated by adding methacholine to the c u l t u r e medium during a 6 hour p e r i o d (Table I ) . These r e s u l t s i n d i c a t e that a l t e r a t i o n s i n the d i s t r i b u t i o n of mucous g l y c o p r o t e i n components from CF t r a c h e o b r o n c h i a l e p i t h e l i u m cannot be a t t r i b u t e d to e x c e s s i v e s t i m u l a t i o n through c h o l i n e r g i c mechanisms. The consequences of enhanced s u l f a t i o n of CF r e s p i r a t o r y t r a c t mucous g l y c o p r o t e i n s remain unclear . Evidence that s u l f a t i o n of macromolecules decreases the water of h y d r a t i o n of these substances (51) suggests that increased s u l f a t i o n and a r e l a t i v e p a u c i t y of water i n CF r e s p i r a t o r y t r a c t s e c r e t i o n s are r e l a t e d i n some way. A question of considerable importance i s the r e l a t i o n s h i p between extent of g l y c o p r o t e i n s u l f a t i o n and the genetic defect i n CF. I t has been suggested, based on r e s u l t s of h i s t o c h e m i c a l s t u d i e s , that increased amounts of s u l f a t e d g l y c o p r o t e i n may r e f l e c t long standing i r r i t a t i o n of the lung airways (52). Thus, the increased s u l f a t i o n of CF t r a c h e o b r o n c h i a l g l y c o p r o t e i n s may be secondary to i n f e c t i o u s b r o n c h i t i s . On the other hand, two pieces of information suggest that increased s e c r e t i o n of h i g h l y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
116
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Archives of Biochemistry and Biophysics
Figure 3. Ratios of the amounts of tracheobronchial glycoprotein in sparsely acidic components (I la) to amounts in highly acidic components (II, III) after fractionation on DEAE cellulose (see Figure 2). Secretions for these studies were obtained by bronchial lavage, performed by Robert E. Wood. Glycoproteins were quantitated by determining the total amount of fucose, galactose, galactosamine, glucosamine, and sialic acid in each fraction (40). f
TABLE I D i s t r i b u t i o n of Mucous G l y c o p r o t e i n Components Secreted by Tracheal Expiants of an O-secretor with CF, With and Without Methacholine S t i m u l a t i o n .
No Methacholine I
II
Methacholine'
III
I
II
III
34
49
16
57
32
11
Fucose^
3.3
4.0
—
1.2
0.9
—
Galactose^
6.1
6.3
—
2.7
3.1
—
1.0
1.0
—
1.0
1.0
—
2.6
5.1
—
2.3
3.2
—
0.8
0.3
—
0.3
0,3
—
% of T o t a l
Carbohydrate
2 Galactosamine • 2 Glucosamine N-acetylneuraminic 1. 2. 3.
acid"'"
Analyzed by gas l i q u i d chromatography (40); r e s i d u e s / 1 galactosamine. Analyzed by amino a c i d analyzer (55); r e s i d u e s / 1 galactosamine. 50 ug/ml methacholine added to the c u l t u r e medium for a 6 hour p e r i o d .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous Glycoproteins in Cystic Fibrosis
1.2H
no serum
control serum
S O j
CF serum
Figure 4. Ratio 5 of3 to H in glycopro teins released from rabbit tracheal expiants in the presence of no serum, 50% (v/v) normal serum, and 50% (v/v) CF serum. Data were generated and analyzed as outlined in legend for Figure 1. No serum vs. CF serum; ρ < 0.02. Control serum vs. CF serum; ρ < 0.02. 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
117
118
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
s u l f a t e d g l y c o p r o t e i n s by CF r e s p i r a t o r y e p i t h e l i a may have a c l o s e r r e l a t i o n s h i p to the gene d e f e c t . In double isotope s t u d i e s , the r a t i o of *^S0, to Η - g l u c o s a m i n e incorporated i n t o g l y c o p r o t e i n s secreted by CF r e s p i r a t o r y e p i t h e l i a l expiants i s s i g n i f i c a n t l y greater than the r a t i o f o r g l y c o p r o t e i n s secreted by age-matched c o n t r o l expiants (53). In a d d i t i o n , recent s t u d i e s i n our l a b o r a t o r y (Figure 4) suggest that b r i e f exposure of r a b b i t t r a c h e a l expiants to 50% CF serum e f f e c t s the r e l e a s e of g l y c o p r o t e i n s which are more h i g h l y s u l f a t e d than those r e l e a s e d i n the presence of 50% age and sex-matched c o n t r o l serum, CF t i s s u e s and/or body f l u i d s may possess a p r o p e r t y , independent of c h r o n i c i n f e c t i o n , which modulates the process of g l y c o p r o t e i n s u l f a t i o n , and which may p l a y a r o l e i n the pathogenesis of the lung d i s e a s e . I t i s u n l i k e l y that people with CF are missing a g l y c o p r o t e i n s u l f a t a s e a c t i v i t y , because we have been unable to detect t h i s a c t i v i t y i n both CF and nonCF t i s s u e homogenates an sputum. Substrate f o t o r y mucous g l y c o p r o t e i n , l a b e l e d and harvested i n v i t r o . Both i n t a c t and d e s i a l y l a t e d mucin were t e s t e d . Enzyme a c t i v i t y was assayed at s e v e r a l pHs, with and without d i v a l e n t c a t i o n s , and with and without T r i t o n X-100 (54). Summary: There are s e v e r a l , w e l l documented a l t e r a t i o n s i n the mucous s e c r e t i o n s of i n d i v i d u a l s with CF, any of which alone or i n combination, may i n t e r f e r e with mucous clearance from the lung or from passageways of other organs. I t seems c l e a r t h a t a d d i t i o n a l s t u d i e s to d e f i n e b a s i c aspects of normal mucous b i o c h e m i s t r y , physiology and pharmacology must precede or p a r a l l e l s t u d i e s of CF mucous s e c r e t i o n s i f data p e r t i n e n t to CF pathophysiology are to be generated. P a r t i c u l a r needs i n c l u d e an understanding of the chemical determinants of p h y s i c a l and r h e o l o g i c a l p r o p e r t i e s of these s e c r e t i o n s , and e l u c i d a t i o n of mechanisms f o r c o n t r o l of s u l f a t e d g l y c o p r o t e i n b i o synthesis. Acknowledgements T h i s work was supported i n part by Grants HL 14844 and HR 52957 from the United States P u b l i c H e a l t h S e r v i c e , and a Grant from the C y s t i c F i b r o s i s Foundation of C l e v e l a n d .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5. BOAT AND CHENG 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Mucous Glycoproteins in Cystic Fibrosis
Literature Cited Wood, R.E., Boat, T.F., and Doershuk, C.F. Am. Rev. Resp. Dis. 113:833 (1976). Davis, P., and di'Sant Agnese, P.A. New Eng. J. Med. 295:481 (1976). Mayo, B.J., Klebe, R.J., Lankford, B.J., Morris, N.R., Barnett, D.R., and Bowman, B.H. Texas Reports on Biology and Medicine 34:107 (1976). Conod, E.J., Conover, J., and Gaerlan, P. Pediat. Res. 11:45 (1977). Mangos, J.Α., and McSherry, N.R. Pediat. Res. 2:378 (1968). Dearborn, D.G. In: Advances in Polyamine Research, Vol. II, Eds. D. Morris, R. Campbell, D. Daves, D. Bartos, and F. Bartos, Raven Press, New York (1978), pp. 273-279. Bowman, B.H., Lankford B.J. Fuller G.M. Carson S.D. Kurosky, Α., and Commun. 64:1310 (1975) Epstein, J., and Breslow, J.L. Proc. Natl. Acad. Sci. USA, 74:1676 (1977). Rao, G.J.S., and Nadler, H.L. Pediat. Res. 9:739 (1975). Shapira, E., Martin, C.L., and Nadler, H.L. J. Biol. Chem. 252:7923 (1977). Davis, S.S. Bull. Physio-path. Resp. 9:47 (1973). Potter, J.L., Spector, S., Matthews, L., and Lemm, J. Am. Rev. Resp. Dis. 99:909 (1969). Khan, M.A., Wolf, D.P., and Litt, M. Biochim. Biophys. Acta 444:369 (1976). Reid, L., and deHaller, R. Mod. Probl. Pediat. 10:195 (1967). Zuelzer,W.W.,and Newton, W.A. Pediatrics 4:53 (1949). Reid, L. In: Ciba Foundation Study Group No. 32, Cystic Fibrosis, Eds. R. Porter and M. O'Connor, Little, Brown and Co., Boston (1968), pp.45-67. Oppenheimer, E.H., and Esterly, N.R. Arch. Path. 96:149 (1973). Bowman, B.H., Lockhart, L.H., et al. Science 164:325 (1969). Bogart, B.I., Conod, E.J., and Conover, J.H. Pediat. Res. 11:131 (1977). Czegledy-Nagy, E., and Sturgess, J.M. Lab. Invest. 35:58 (1976). Boat, T.F., Kleinerman, J.I., Carlson, D.M., Maloney, W.H., and Matthews, L.W. Am. Rev. Resp. Dis. 110:428 (1974). Baker, A.P. "GAP" Conference Reports, Cystic Fibrosis Foundation (1976). Boat, T.F., Cheng, P.W., and Wood, R.E. Mod. Probl. Pediat. 19:141 (1977). Baker, A.P., Hillegass, L.M., et al. Am. Rev. Resp. Dis. 115:811 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11
120
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
25. Baker, A.P., and Hillegass, L.M. Arch. Biochem. Biophys. 165:597 (1974). 26. Boat, T.F., and Cheng, P.W. In: Cystic Fibrosis, Projec tions into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Corp., New York, 1976, pp. 165-177. 27. Neutra, Μ., Grand, R.J., and Trier, J.S. Lab. Invest. 36:535 (1977). 28. Sturgess, J., and Reid, L. Clin. Sci. 43:533 (1972). 29. Dearborn, D.G. In: Cystic Fibrosis: Projections into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Co., New York, 1976, pp. 179-191. 30. Wood, R.E., Wanner, Α., Hirsch, J., and Farrell, P.M. Am. Rev. Resp. Dis. 111:233 (1975). 31. Forstner, J.F., and Forstner G.G Pediat Res 10:609 (1976). 32. Gugler, E., Pallavicini, , , , Agnese, P.A. J. Pediat. 71:585 (1967). 33. Boat, T.F., Wiesman, U.N., and Pallavicini, J.C. Pediat. Res. 8:531 (1974). 34. Bailleul, V., Richet, C., Hayem, Α., and Degand, P. Clin. Chim. Acta 74:115 (1977). 35. Degand, P., Roussel, P., Lamblin, G., Durand, G., and Havez, R. Bull. Physio-Path. Resp. 9:199 (1973). 36. Roberts, G.P. Arch. Biochim. Biophys. 173:528 (1976). 37. Shih, C.K., Litt, M., Khan, M.A., and Wolf, D.P. Am. Rev. Resp. Dis. 115:989 (1977). 38. Snary, D., Allen, Α., and Pain, P.H. Biochem. Biophys. Res. Commun. 40:844 (1970). 39. Hill, H.D., Reynolds, J.A., and Hill, R.L. J. Biol. Chem. 252:3791 (1977). 40. Boat, T.F., Cheng, P.W., Iyer, R., Carlson, D.M., and Polony, I. Arch. Biochem. Biophys. 177:95 (1977). 41. Roussel, P., Lamblin, G., Degand, P., Walker-Nasir, E., and Jeanloz, R.W. J. Biol. Chem. 250:2114 (1975). 42. Cheng, P.W., and Boat, T.F. Fed. Proc. 35:1444 (1976). 43. Lopez-Vidriero, M.T., Das, I., and Reid, L.M. In: Respira tory Defense Mechanisms, Part I, Eds., J.D. Brain, D.F. Proctor, and L.M. Reid. Marcel Dekker, New York, (1977), pp. 289-356. 44. Gottschalk, Α., and Thomas, M.A.W. Biochim. Biophys. Acta 46:91 (1961). 45. Meyer, F.A., King, M., and Gelman, R.A. Biochim. Biophys. Acta 392:223 (1975). 46. Deman, J., Mareel, Μ., and Brunyneel, E. Biochim. Biophys. Acta 297:486 (1973). 47. Cheng, P.W. Unpublished Observation. 48. Lamblin, G., Lhermitte, M., Lafitte, J.J., Filliat, M., Degand, P., and Roussel, P. Bull. Europ. Physiopath. Resp.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
5.
BOAT AND CHENG
Mucous
Glycoproteins
in Cystic Fibrosis
121
13:175 (1977). 49. Boat, T.F., Davis, J., Cheng, P.W., and Stern, R.C. Proceed ings of the Fourth International Symposium on Glycoconjugates, In press. 50. Dische, Z., di Sant'Agnese, P.Α., Pallavicini, C, and Youlos, J. Pediat. 24:74 (1959). 51. Ablett, S., Lillford, P.J., Baghdadi, S., and Derbyshire, W., In: Magnetic Resonance in Colloid and Interface Science. Eds., H. Resing, and C. Wade, A.C.S. Symposium Series 34 (1976), p. 344. 52. Jeffery, P.K., and Reid, L.M., In: Respiratory Defense Mechanisms, Part I, Eds., J.D. Brain, D.F. Proctor, and L.M. Reid, Marcel Dekker, New York, (1977), pp. 193-245. 53. Boat, T.F., and Cheng, P.W., In: Cystic Fibrosis: Projections into the Future, Eds., J.A. Mangos and R.C. Talamo, Stratton Intercontinental Medical Book Corporation New York (1976) pp. 165-177. 54. Jentoft, N., and Cheng, Unpublishe 55. Cheng, P.W., and Boat, T.F. Anal. Biochem. 85:276 (1978). 56. Boat, T.F., and Kleinerman, J.I. Chest 67:32 S (1975). RECEIVED April 17, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6 Glycoprotein Storage Disease J. NEVIN ISENBERG Pediatric Gastroenterology Division, Department of Pediatrics—UCLA, Center for the Health Sciences, Los Angeles, CA 90024
I. Alpha-1-antitrypsi attracted considerable attentio cause of its association with two divergent disease processes emphysema and cirrhosis. On routine serum protein electrophor esis, it is the predominant protein in the α globulin area. Iron ically (with regard to the current topic), the disease states are associated with a marked reduction in the protein appearing in this band. Thus, this routine clinical test can serve as a screening test for alpha-1-antitrypsin deficiency-type emphysema or cirrhosis. This subject has been reviewed in 1976 by Harvey L. Sharp, M.D., who first recognized the association between α1AT deficiency and childhood cirrhosis.1 In the subsequent paragraphs, we will utilize the material presented there and update the cur rent status with information published since then. 1
The protein has a molecular weight in the range of 50,000. It has approximately 12% carbohydrate which includes galactose, mannose, N-acetyl-glucosamine and sialic acid. The name, α1AT, comes from its electrophoretic position (-α1-) and from the fact that it is responsible for 90% of the serum trypsin inhibitory capacity. Since this potential may be less important physiologi cally than the inhibitory capacity for other proteases, it has also been referred to as α1 protease inhibitor as the study of protease inhibitors (P ) has increased in general. As it is an acute phase reactant, its serum levels and presumably synthesis increase during inflammation. i
Serum contains about 2ragof α 1AT per ml which has the capacity to inhibit 1.1 mg of trypsin. Both quantity (immuno diffusion methods) and function (trypsin inhibitory capacity) are used to evaluate serum from patients with emphysema or cir rhosis. The complexities begin when the deficient serum is typed. Using an acid starch gel electrophoresis,Fagerhol first appreciated the microheterogeneity of alAT which results in three major and five minor protein bands as products of each 0-8412-0452-7/78/47-080-122$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
i S E N B E R G
Glycoprotein
Storage
Disease
123
allele.2 When this separation was followed by antigen-antibody crossed electrophoresis, the f u l l range of 24 phenotypes for this genetic locus was appreciated. Since each a l l e l e i s expressed by a gene product (with microheterogeneity) the inheritance pattern i s regarded as autosomal codominant. P^ type "M" i s the predominant phenotype and has moderate ëLectrophoretic mobility. Phenotypes moving more rapidly are identified by letters which are before "M," while slower ones are designated by letters after "M." Reduced circulating alAT has been associated with a l l e l e s Ζ ( < 10%), Ρ (12.5%), S (30%) and with P j — i i which no gene product has been identified. The "M" a l l e l e i s 50% (MM=100%). Disease states are associated, then, with diploid phenotypes giving low a c t i v i t y such as PiZZ, PjSZ, Pj — , etc. Population genetic studies have shown the wide variation i n a l l e l e distribution. The highest gene frequency for P±Z i s .0262. i n Swedes vs. .011 pathologic a l l e l e most Pathologic P-^ types are very infrequent among non-whites. The Pj^ a l l e l e system seems closely linked to the locus (IgG heavy chain marker) with placement on chromosome 2,8 or 12 l i k e l y . — In Sveger's study2, 120 PjZZ infants and 48 PjSZ infants were de tected fran 200,000 l i v e births, indicating that one infant i n 1200 births had a potentially pathologic phenotype! The figure (proportionately lower) for the USA makes this inherited glyco protein disorder among the most conmon genetic disorders. Pre natal diagnosis i s not possible. The Lung Disease The postulated mechanism by which emphysema develops i n the deficient phenotypes i s thought to be the result of uninhibited proteolysis that destroj^s the fibrous network of the lungs. alAT does inhibit the leukocyte proteases including elastase and c o l lagenase and i s small enough to leave the vasculature to diffuse to a s i t e of inflanmation to function as a protease inhibitor. The susceptibility of hétérozygotes (e.g., PjMZ) to environmental influences which would provoke lung damage has been disputed.! In a recent study heterozygous children compared with normals fran the same families showed no differences except i n forced flow pulmonary functions analyzed by matched pairs .iL These abnormalities were of the type seen with <*1AT deficiency emphysema and suggest a predisposition for the hétérozygote. Only followup w i l l c l a r i f y the significance of this finding. The Liver Disease When Sharp f i r s t associated childhood cirrhosis with α 1AT deficiency (PjZZ), the hepatocytes contained eosinophilic cyto plasmic granules that were shown to be PAS positive after diastase
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
124
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
digestion of glycogen. These globules are most evident in peripor t a l hepatocytes and become more prominent with age i n those c h i l d ren affected. These granules are positive when sections are treated with fluorescein-labelled antibody to otlAT. These glob ules are found on close inspection i n individuals with PjZ pheno type whether or not cirrhosis i s present. A single report has identified globules i n the l i v e r of a 79-year-old man with PjMM phenotype that were PAS(+) and positive for alAT by irrmunofluorescent and iirrnunoperoxidase methodology.^ The significance of alAT globules i n the absence of a pathologic phenotype i s not clear at present. This material appears i n dilated portions of the endoplasmic reticulum (ER) and i n no other organelle when specimens are exam ined with the electron microscope. It i s usually amorphous with a fine granular or occasional f i b r i l l a r appearance Both smooth and rough endoplasmic reticulu apparatus. Cells are no have any material.Ζ These are concentrated i n periportal areas. The globules vary i n size from one to 40 microns i n diameter, with 15-20 microns diameter particles predominating, The l i v e r injury begins at b i r t h as evidenced by the f r e quency of cholestasis i n newborns with P^ZZ or PjSZ phenotypes. 1> 3 However, some children escape this phase but demonstrate cirrhosis when l i v e r biopsy i s done as part of the c l i n i c a l evaluation of hepatosplenomegaly. The cholestatic phase usually passes within six months. Although affected children then remain asymptomatic u n t i l cirrhosis becomes s u f f i c i e n t l y advanced to result i n por t a l hypertension with ascites and/or hematemesis, l i v e r function tests show continued evidence for hepatocellular injury without cholestasis. Interestingly, however, hyperbilirubinemia may pre cede enzyme elevation i n the newborn period! While originally the l i v e r disease presenting i n early c h i l d hood was thought to be almost uniformly f a t a l i n adolescence secondary to the consequences of advancing cirrhosis, others have had experiences to the contrary.§. The Toronto group reported four of 18 patients with neonatal cholestasis who have no l i v e r function or physical abnormality i n mid-adolescence.§ Early c i r rhosis with or without neonatal cholestasis was a bad prognostic factor.?. In Sveger's neonatal screening study, the PjZZ infants were c l a s s i f i e d into three groups according to the severity of hepatic involvement .-2. Fourteen of 122 had prolonged obstructive jaundice, eight had a suspicion of l i v e r disease by examination, and the remainder had no c l i n i c a l evidence for l i v e r disease. The PjSZ children were a l l i n the third category. Continued evalua tion of the infants over their f i r s t s i x months of l i f e showed that half of those i n the third category with P-^ZZ phenotypes had biochemical abnormalities indicating l i v e r injury. This was not
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
i S E N B E R G
Glycoprotein
Storage
Disease
125
found i n the SZ group. Sass-Kortask could draw no prognostic significance from b i opsies taken during the cholestatic phase. Ihey saw equivalent degrees of injury i n well survivors and early deaths Λ French workers have foimed a different conclusion on the basis of re viewing biopsies from fifteen P ZZ children with neonatal chole stasis . ϋ Those that shewed improvement i n l i v e r injury had chole stasis and c e l l u l a r injury, but only slight portal fibrosis. When fibrosis was extensive and b i l e ducts proliferated, early cirrho s i s occurred. A t h i r d group, i n which prolonged cholestasis was noted, had ductular hypoplasia. The follow-up period for most of the patients i n the series i s less than five years, which i s i n sufficient to make a f i n a l judgement; but those i n Groups II and III are progressing toward the complications of cirrhosis while those i n Group I seem to be inprovingJ* A l l had dPAS(+) globules whose size and nuirber di grouping.9 i
PAS(+) globules are also noted i n emphysematous adults of Pj^ZZ phenotype..1 Fibrosis, cirrhosis and hepatoma were noted. No alAT globules are seen i n the malignant c e l l s . — Adults with phenotypes resulting i n p a r t i a l deficiencies had no greater i n c i dence of l i v e r disease than PjMM c o n t r o l s . i l The Liver Storage Material Clearly, the accumulation (storage?) of alAT-like material in the l i v e r seems well documented and i s associated with cirrho s i s . The mechanism by which the hepatic injury occurs i s more d i f f i c u l t to envision than that proposed for the development of emphysema i n deficient phenotypes. The l i v e r disease i s not merely a product of deficient serum alAT since i t seems to be most easily related to the PjZ a l l e l e and not other deficient pheno types. Further, the PjZ protein retains f u l l protease inhibitor capacity proportionate to the amount present. An understanding of the mechanism for l i v e r injury w i l l very l i k e l y require a better understanding of how the α lAT-like mat e r i a l gets to the l i v e r i n the f i r s t place. It i s synthesized by the l i v e r and recently has been p a r t i a l l y characterized as a pro duct of human l i v e r c e l l monolayer cultures. PjM and Pj^Z types were cultured. Only the PjZ c e l l s shewed globules with alAT immunofluorescent p o s i t i v i t y . Ihe P.Ζ protein fran the c e l l s d i f fered from that released to the culture medium and extracted from the supernatant.— This may become an important laboratory ap proach to understanding more about the pathogenesis of l i v e r dis ease. In his review, Sharp l i s t e d four hypotheses that might ex plain the accumulation of alAT i n the livers of P-ZZ individuals:
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
126
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
1) A point mutation leading to an amino acid substitution i n the peptide which would interfere with subsequent transport from the l i v e r ; 2) A glycyl transferase deficiency resulting i n an inade quately glycosylated protein that could not be transported; 3) P^ZZ i s synthesized and secreted but more rapidly taken up by the l i v e r ; and 4) A pro- alAT i s the storage product because a cleaving enzyme i s missing. It could be added that the amino acid substitution (1) might result i n inadequate glycosylation (2) and secretory failure. Many groups are working on isolation of alAT from serum and l i v e r . No one has found a markedly larger alAT i n the l i v e r so that support for pro-alAT i s minimal. Increased hepatic uptake i s a known feature of asialoglycoproteins. Working with purified alAT, Gan's group shewed that removing s i a l i c acid an galactose residue, altere change binding to concanavilin A, trypsin, or chymotrypsin inhib itory activity or immunological reactivity .1!L The unaltered alAT had a h a l f - l i f e of 18 hours. After neuraminidase treatment re moving four of s i x s i a l i c acid residues, the h a l f - l i f e was re duced to 30 minutes. With subsequent treatment of the galactose residues with galactose oxidase, the h a l f - l i f e was restored to 18 hours .12 Similarly, isolated M and Ζ alAT were labelled with radio active iodine and injected into PjMM subjects. Simultaneous dis appearance rate measurement showed the h a l f - l i f e of M equal to seven days and for Ζ five days. Ihese differences are too small to explain the serum deficiency seen i n P^ZZ.!! Many investigators have suggested that the α 1AT of P^ZZ i s deficient i n s i a l i c acid, but the reported amount and composition of the carbohydrate on this variant protein shows many discrepan cies from group to group. In 1975, the Swedish group reported isolation and characterization of the alAT P^Z from the inclusion bodies i n a P^ZZ l i v e r . 15 It contained no s i a l i c acid, was v i r tually insoluble, f a i l e d to inhibit proteases but did shew thesanre irrinunoreactivity as serum alAT.l§_ Its amino acid composition was similar to PjM but the sugars were a l l reduced to the following percent of that found i n PjM: mannose - 50%, galactose - 25%, N-acetylglucosamine - 16%. These findings, i n the investigator's view, suggested a potential substitution i n the amino acid back bone leading to decreased s o l u b i l i t y , ineffective transport through the ER and secondarily inadequate glycosylation. 15. In the same p e r i o d , Sharp joined forces with Glew and co-workers to pursue the latter's o b s e r v a t i o n that sialyltransferase was deficient i n the serum
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
i S E N B E R G
Glycoprotein
Storage
Disease
127
of patients with the PjZZ phenotype. After more extensive studies, i t appeared that the decreased serum levels were i n general rela ted to advanced cirrhosis but not accompanied by decreased activ ity i n l i v e r homogenates. No kinetic differences i n the enzyme were noted between the sialyItransferase from PjMM and P±ZZ.± In 1976, two groups reported an amino acid substitution i n the protein backbone of the Ζ variant of alAT. 16,17 Both groups made peptide maps from digests of the isolated alAT made i n two different ways and found differences i n one or more peptides. Both agree that lysine i s substituted for glutamic acid i n the major variant peptide. The USA group believe that another glu tamic acid i n the M peptide i s replaced by glut amine i n the Ζ peptide. Similarly, an amino acid substitution was confirmed i n a histidine containing peptide of the S-variant.l§ Valine was substituted again for glutamic a c i d . T h e S and M variant had the same s i a l i c aci However, dissenting voices have been raised about the Ζ var iant findings. Owen and Carrell could find no peptide fran the Ζ variant in the expected part of the peptide map after t r y p t i c digestion of both M and Z.£0 A sequential analysis of an M pep tide not seen i n the Ζ map enabled prediction where the variant peptide should be. Thus far, sequential analyses of numerous peptides from the digests has f a i l e d to show any amino acid d i f ferences . Further information strengthening the argument for the po t e n t i a l accumulation of an a s i a l o - alAT comes from studies of Bnerson which imply that the l i v e r asialoglycoprotein binding protein (LABP) i s present i n the membranes of the Golgi apparatus and SER. Thus, P-^Z with exposed galactosyl residues secondary to incomplete s i a l y l a t i o n would bind to the SER and be no further t r a n s p o r t e d C o m p a r i n g binding to LABP i n vitro, native serum PiZ binds more avidly than native M but much less well than com pletely desialylated M. Ihis suggests that there i s circulating PjZ which has exposed galactosyl residues. Circular dichroism has been applied to the analysis of alAT fran normal serum. Sixteen to 20% i s α-helix, 30-50% i n the 3 pleated sheet conformation and the remainder i s aperiodic. This conformational p r o f i l e remained stable over a pH range fran 4.7 to 8.8, but was markedly changed by pH 2.5, guanidine hydrochlor ide and SDS. These findings implicate electrostatic interaction, hydrogen bonding and hydrophobic regions as v i t a l to the three dimensional structure of alAT..?? Obviously, the application of this technique to the variant α1AT with single amino acid sub stitutions may y i e l d information on conformational changes that could interfere with glycosylation. Its application to the problem of microheterogeneity w i l l likewise be interesting.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
128
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Microheterogeneity The factors responsible for the microheterogeneity i n the products of a single gene are not resolved. In some glycoproteins the microheterogeneity has been attributed to a variable degree of s i a l y l a t i o n . Using isoelectric focusing to analyze the DEAE c e l lulose - NaCl gradient column chromatographic separation of purif i e d P-^M, five pooled fractions each shaved up to three of the five bands present i n the otiAT preparation before DEAE fractionation. The f i r s t pool eluted with a low NaCl concentration had the more cathodal isoproteins, while the last pool (hi concentration NaCl) had the more anodal ones. If this was due to s i a l y l a tion differences, then Pool X should have been richer i n s i a l i c acid. However, a l l pools were homogenous by irnmunoelectrophores i s and had the same amino acid, hexose, hexosamine and s i a l i c acid concentrations! When the pools were enzymatically desialylated the bands i n each poo and relative positions o This study casts doubt on uneven s i a l y l a t i o n as an explanation for the microheterogeneity i n aiAT.i^L This a b i l i t y to separate the various forms of the gene product w i l l enable the ultimate identification of what i s responsible for the microheterogeneity. Allen has used isoelectric focusing to study the microheterogeneity i n P-^ZZ children with cirrhosis and compared i t to adults with emphysema and PjZZ i n d i viduals with no c l i n i c a l l y evident disease.?â A l l bands inhibit trypsin and were antigenically α 1AT by crossed irnmunoelectrophoresis. The c i r r h o t i c children had higher levels of alAT and increasing amounts with age of isoprotein i n the Z2 (anodal) band region (isoprotein point = 4.534). Ihus, i t appears that there are differences even i n the PiZ product which might be used i n conjunction with newborn screening to predict whether infants of PjZZ phenotype w i l l be at r i s k for l i v e r or lung disease. Using this Z2 band difference as their marker, family studies showed that penetrance of the Ζ a l l e l e was of the order of 70%.25 i f these observations hold true, then i t may be possible to better understand why seme P-^ZZ families have l i v e r disease without lung involvement and why the converse exists. Up to this point, en vironmental factors (which would stimulate the synthesis of acute phase reactant) were being sought to help explain the onset of l i v e r disease i n the newborn. The majority of investigators had been unable to identify any infectious or toxic agent present i n those children developing the cholestatic phase of the disease. 1. Furthermore, since the incidence of small for gestational age i n fants i s increased i n the P^ZZ phenotype, there i s some evidence that the disease may begin i n utero. Therapy No therapy exists for this glycoprotein storage disease.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
i S E N B E R G
Glycoprotein
Storage
Disease
129
Factors that increase serum levels of α 1AT i n P-^MM do not alter the level of P^ZZ. Steroids have not been helpful. Several pat ients have received l i v e r transplants with conversion of serum Pi phenotype from ZZ to MM. A l l died of transplantation compli cations. At autopsy, no PAS(+) globules were noted in the l i v e r which speaks strongly against a peripheral desialylation mechan ism for altering alAT with resultant storage.
11.
ASPARTYLGLUOOSAMINURIA
In the usual sense, storage disease implies accumulation of a macromolecule whose catabolism i s interrupted by a deficiency in one or more of the hydrolytic enzymes involved i n i t s break down. For glycoproteins, disturbances i n the breakdown of the peptide backbone or carbohydrat accumulation. However a l l proteins and have f a r reaching, perhaps lethal, effects. In addition, glycoprotein accumulation might be the consequence of disturbances i n the breakdown of the carbohydrate side chain. Yet, the carbohydrate side chains are shared by the glycolipids and abnormalities i n their cleavage are discussed elsewhere i n this symposium. The bond unique to the glycoproteins i s that between the f i r s t sugar moiety of the side chain and the amino acid residue in the peptide backbone. In man, the predominant bond i n serum glycoproteins seems to be the N-glycosidic linkage between N-acetylglucosamine and asparagine. The lysosomal enzyme respon sible for the hydrolysis of this bond i s [2-acetamido-l- ( β-Laspartamido) -l,2-dideoxy-3-D-glucose aspartamido hydrolase (EC 3.5.1.26)] AADGase. A deficiency i n this enzyme was f i r s t demonstrated i n two mentally retarded English siblings who had excreted large amounts of an unusual compound i n the urine which was identified as 2-acetamido-l-( 3 -L-aspartamido) -1,2-dideoxy- 3 -D-glucose (AADG).i Ibis was the i n i t i a l recognition of the disorder, asparty lglucosaminuria (AGU), which has subsequently been diagnosed in Finland? and the United States .2 The Finnish workers have demonstrated the prevalence of this disorder i n their country (1:26,000 l i v e births) by screening residents of national institutions for the mentally retarded.4The coarse features, visceromegaly and vacuolated lymphocytes re sult i n c l i n i c a l l y confusing this disease with the mucopolysac charidoses and mucolipidoses.5. Mental retardation characterized by impaired speech and motor clumsiness predominates .2* 4_ Connec tive tissue lesions including dyostosis multiplex, umbilical her nias and hypermobile joints are common.3,4 Systolic heart rauirnurs
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
130
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
are frequent. Our patient shaved mitral insufficiency on heart catheterization suggesting that this may be another metabolic disorder directly influencing valvular function.! Despite a pre disposition to recurrent infections, no d e f i c i t s i n leukocyte function have been demonstrated Extensive light and electron microscopic studies have been carried out on various tissues from affected patients.§^Z»-§- The major changes i n a l l tissues are lysosomal. In addition to the readily discernible lymphocyte vacuoles on the peripheral smear, electron dense filamentous material i s noted i n the enlarged lysoscmes of leukocytes .ϋ Virtually every lysosome i n the hepa t i c parenchymal c e l l s i s enlarged. Some PAS positive droplets are seen. Ultrastructurally, the lysosomes contain fine granular material, electron dense structures, and electron lucent l i p i d droplets.§^8. Kupffer c e l l lysoscmes are likewise involved.!L§_ Similar lysosomal change of proximal tubule c e l l jejunal enterocytes§, and i n fibroblasts fran a skin biopsy .8 Ihe most s t r i k i n g changes have occurred i n the neuronal cyto plasm from cerebral cortex, cerebellum and thalamic regions.Ζ The cytoplasm i s v i r t u a l l y f i l l e d with membrane bound vacuoles of either electron lucent granular material or electron dense membranous or granular material with occasional l i p i d droplets.No single feature i s pathognomonic for AGU although seldom i s such a variety of lysosomal changes seen i n other storage diseases. The reduced activity of AADGase was f i r s t noted i n a patient by assay of seminal fluid.,! Markedly decreased activity was ob served i n brain, l i v e r and spleen but not i n kidney biopsy mater i a l , â Leukocyte homogenates are also deficient i n AADGase JL Other lysosomal enzymes involved i n the catabolism of the carbohydrate side chain have normal activity levels except for N-acetyl-3-glucosaminidase which i s elevated.*Li* Measurement of the enzyme i n cultured fibroblasts peimits detection of hétérozygotes and enables prenatal diagnosis. 12 AADGase has been isolated from l i v e r of normals and AGU enabling mixing experiments that rule out inhibitors as a cause for the reduced activity. 11 > 1? Only one isoenzyme has been noted. 11»!? The pH optimum ( 7 . 7 ) and ΗΏ ( 0 . 1 6 mM) have been determined on a 1 7 , 5 0 0 f o l d purification of AADGase that was inhibited by N-acetylcysteine but not other SH agents.12 Even from the f i r s t description of AGU, compounds other than AADG were appreciated i n the urine. 1_ Characterization of these plus the nature of the principal tissue storage compound has taken place. The only quantitatively important compound i n t i s sue i s AADG. lâ Ihe higher molecular weight compounds containing asparagine but with additional carbohydrate residues have been termed glycoasparagines and f a l l into neutral and acidic groups when analyzed by high voltage electrophoresis at pH 2 . 0 and pH
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6.
i S E N B E R G
Glycoprotein
Storage
Disease
131
5.3.ϋ Five neutral and nine acidic glycoasparagines were sepa rated and analyzed. Neutral glycoasparagines (N-^ - N 5 ) differed in the number of neutral sugar residues attached to AADG. The acidic glycoasparagines f e l l into three groups with one or two s i a l i c acid residues; the sulphated compounds formed the t h i r d group. The neutral sugars identified were glucosamine > galac tose » mannose > glucose > fucose. Mannose was noted only i n the neutral group, glucose only i n acidic compounds and fucose in both.i^ Aula, working i n conjunction with several Japanese biochemists, has reported the sequence of several glycoasparagines as shown i n the table b e l o w l ^ ^ : GLYOOASPARAGINE
SEQUENCES IN THE URINE OF AGU AADG
a-NANA-(2+3)-3-Gal-(1+4)-AADG ot-NANA- ( 2+4 ) - 3-Gal- ( 1+4)-AADG a-NANA- ( 2+? ) -3-Gal- ( 1+? ) -3-GlcNAc- ( 1+? ) - 3-Gal- ( 1+4 ) -AADG α-Man- ( 1+6 ) - g-Man- ( l+4)-3-GlcNAc-( 1+4)-AADG That these compounds are present i n only small amounts i n the tissues thus f a r studied makes the source of urine glycoasparagines open to speculation. They may have their origin i n connective tissue. We have suggested that the AADG may serve as an accep tor compound for various transferases leading to synthesis of new glycoasparagines which are released from tissue sites after reaching some c r i t i c a l conformâtion.£-
ACKNOWLEDGEMENT To Harvey L. Sharp, M.D., for introducing me to both these diseases of glycoprotein metabolism.
LITERATURE CITED I. Alpha-1-Antitrypsin 1. Sharp HL: Gastroenterology 70: 611 (1976). 2. Fagerhol MK: Scand J Clin Lab Invest 23: 97 (1969).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
132
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
LITERATURE CITED (Cont.) I. Alpha-1-Antitrypsin (Cont.) 3. Sveger Τ: Ν Engl J Med 294: 1316 (1976). 4. Ihird International Workshop - Human Gene Mapping: Cytogenetics and Cell Genetics 16: (1976). 5. Vance JC, Hall WJ, Schwartz RH, Hyde RW, Roghmann KJ, and Mudholkar GC: Pediatrics 60 : 263 (1977). 6. Bradfield JWB and Blenkinsopp WK: J Clin Path 30: 263 (1977). 7. Yunis EJ, Agostini Jr RM, and Glew RH: Am J Pathol 82: 265 (1976). 8. Moro SP, Cutz E, CoxOT,Sass-Kortsak A: J Pediatr 88: 19 (1976). 9. Hadchouel M and Gautier M: J Pediatr 89: 211 (1976). 10. Blenkinsopp WK and Haffenden GP: J Clin Path 30: 132 (1977). 11. Fisher RL, Taylor L and Sherlock S: Gastro 71: 646 (1976). 12. Gautier M, Martin JP and Polim G: Biomedicine (Express) 27: 116 (1977). 13. Yu SD and Gan JC: Arch Biochem Biophys 179: 477 (1977). 14. Laurell CB, Nosslin Β and Jeppsson JO: Clin Sci Molec Med 52: 457 (1977). 15. Jeppsson JO, Larsson C and Eriksson S: Ν Engl J Med 293: 576 (1975). 16. Jeppsson JO: FEBS Letters 65: 195 (1976). 17. Yoshida A, Lieberman J, Gaidulus L and Ewing C: PNAS 73: 1324 (1976). 18. Ovens MC, Carrell RW and Brennan SO: BBA 453: 257 (1976). 19. Yoshida A, Ewing C, Wessels M, Lieberman J and Gaidulis L: Am J Hum Genet 29: 233 (1977). 20. Owens MC and Carrell RW: FEBS Letters 79 : 245 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
6. ISENBERG Glycoprotein Storage Disease LITERATURE CITED (Cont.) I. Alpha-1-Antitrypsin (Cont.) 21. Emerson WA: GAP Conference 1: 7 (1977). 22. Jirgensons Β: BBA 493 : 352 (1977). 23. Hercz A and Barton M: Can J Biochem 55: 661 (1977). 24. Allen RC and Hennigar GR: Am J Clin Path 67: 209 (1977). 25. Allen RC: GAP Conference 1: 16 (1977). II. ASPARIYLGLUOOSAMINURIA 1. Pollitt RJ, Jenne (1968). 2. Palo J and Mattsson K: J Ment Defic Res 14: 168 (1970). 3. Isenberg JN and Sharp HL: J Pediatr 86: 713 (1975). 4. Autio S: J Ment Defic Res (Monograph Series 1) 1972. 5. Fleisher TA, Isenberg JN and Sharp HL: J Pediatr 87: 833 (1975). 6. Arstila AU, Palo J, Haltia M, Riekkinen Ρ and Autio S: Acta Neuropath (Berl) 20 : 207 (1972). 7. Haltia M, Palo J and Autio S: Acta Neuropath (Berl) 31: 243 (1975). 8. Isenberg JN and Sharp HL: Human Pathol 7: 469 (1976). 9. Palo J, Riekkinen P, Arstila AU, Autio S and Kivimaki T: Acta Neuropath (Berl) &): 217 (1972). 10. Aula P, NäntöV, Laipio ML and Autio S: Clin Gen 4: 297 (1973). 11. Savolainen H: Biochem J 153: 749 (1976). 12. Dugal Β and Strømme J: Biochem J 165: 497 (1977). 13. Palo J, Pollitt RJ, Pretty KM and Savolainen H: Clin Chim Acta 47 : 69 (1973). 14. Pollitt RJ and Pretty KM: Biochem J 141: 141 (1974).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
133
134
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
LITERATURE CITED (Cont.) II. ASPARTYLGLUCOSAMINURIA (Cont.) 15. Sugahara K, Funakoshi S, Funakoshi I, Aula Ρ and Yamashina I: J Biochem 80: 195 (1976). 16. Akasaki M, Sugahara K, Funakoshi I, Aula Ρ and Yamashina I: FEBS Lett 69: 191 (1976). RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7 Lysosomal Enzyme Deficiency Diseases—Glycoprotein Catabolism in Brain Tissue ERIC G. BRUNNGRABER Missouri Institute of Psychiatry and the Departments of Psychiatry and Biochemistry, University of Missouri—Columbia, St. Louis, MO 63139 The heteropolysaccharid chain f glycoprotein i brain tissue provide th substrates for a number of lysosomal glycosidases. The genetically-induced absence of a glycosidase, or a mutation which causes one or more of these glycosidases to become non-functional, produces a block in the catabolism of these proteinlinked heteropolysaccharides. This pathological event, observed in the lysosomal enzyme deficiency diseases, causes an accumulation of undegraded glycopeptides or oligosaccharides, and an excessive urinary excretion of these substances. About 65 percent of the glycoprotein-carbohydrate of brain tissue is released in the form of sialoglycopeptides which contain NeuNAc, Fuc, GlcNAc, Gal, and Man (1) after treatment of the glycoproteins with proteolytic enzymes, usually papain or pronase. Approximately 20-40 percent of these sialoglycopeptides contain, in addition, sulfate ester groups. All of these sialoglycopeptides appear to contain 3 Man residues per glycopeptide molecule (Table I). Two, three, and possibly four chains consisting of -GlcNAc-Gal-NeuNAc (Fuc) are attached to this trimannoside core. The sialoglycopeptides were partially resolved by column electrophoresis. Fraction I contains the sialoglycopeptide with the highest molecular size and negative charge. The molecular size TABLE I SIALOGLYCOPEPTIDES DERIVED FROM BRAIN GLYCOPROTEINS Fraction: I II III IV V VI VII NeuNAc 4 4 3 2 1 1 3 Gal 4 4 4 3 3 2 2 Man 3 3 3 3 3 3 3 GlcNAc 6 6 6 6 5 4 4 Fuc 1 1 1 1 2 1-2 1-2 0-8412-0452-7/78/47-080-135$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
136
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
and charge decreased as the number and s i z e of the - G l c N A c - G a l NeuNAc (or Fuc) chains attached to the trimannoside core i s r e duced. The composition of f r a c t i o n s VI and VII i s of p a r t i c u l a r interest. These s i a l o g l y c o p e p t i d e s account for about 15-20 p e r cent of the glycopeptide-carbohydrate of whole r a t b r a i n . Two -GlcNAc-Gal-NeuNAc (or Fuc) chains are attached to the trimanno side core. The Man residues are s u f f i c i e n t l y exposed so that these glycopeptides show a greater a f f i n i t y to Con A-Sepharose columns (2) than the more a c i d i c p o l y s i a l o g l y c o p e p t i d e s of f r a c t i o n s I to IV. These glycopeptides a l s o have a greater a b i l i t y to i n h i b i t the p r e c i p i t a t i o n of glycogen by Concanavalin A than the more n e g a t i v e l y charged p o l y s i a l o g l y c o p e p t i d e s (3). I t was a l s o found (4) that these glycopeptides y i e l d m e t h y l a t i o n p r o ducts that resemble those obtained from a glycopeptide from transferrin:
( A)
Gal+GlcNAc (1+2 ) Man (1+6 )
^ Man+Gl cN Ac+Gl cN Ac-*As η Gal+GlcNAc (1+2 ) Man (1+3 ) '
S i a l o g l y c o p e p t i d e s of f r a c t i o n s I to V , c h a r a c t e r i z e d by higher values f o r the r a t i o s of NeuNAc/Man, Gal/Man, and GlcNAc/ Man may possess s t r u c t u r e s which resemble those of the g l y c o peptides d e r i v e d from f e t u i n ( 4 ) , as suggested some years ago (5) and f o r which Krusius et a l C4,6) have obtained some e v i d e n c e . : Gal-GlcNAc^ Man Gal-GlcNAc (B)
\
x
Gal-GlcNAc^ Gal-GlcNAc'"
Man'
/
Man-GlcNAc- Gl cN Ac -Agn
The b r a i n s i a l o g l y c o p e p t i d e s contained r e l a t i v e l y high amounts of t e r m i n a l nonsubstituted G a l and GlcNAc residues which suggested that the p e r i p h e r a l chains are often incomplete (6). As we s h a l l see, an accumulation of glycopeptides with s t r u c t u r e s corresponding to (A) have been shown to accumulate i n f u c o s i d o s i s , I - c e l l d i s e a s e , and the GM1- and GM2-gangliosidoses. An accumulation of o l i g o s a c c h a r i d e s or glycopeptides with 3 or 4 t e r m i n a l -GlcNAc-Gal-NeuNAc (or fucose) branches has not yet been r e p o r t e d . Does t h i s suggest the presence i n t i s s u e s of a h i t h e r t o undescribed endo-g-glucosaminidase which s p l i t s the bond between the GlcNAc r e s i d u e of the -GlcNAc-Gal-NeuNAc(or Fuc) branch chain l i n k e d to the trimannoside core? The existance of such an enzyme might e x p l a i n the r e l a t i v e l y l a r g e accumulation of o l i g o s a c c h a r i d e s and glycopeptides with s t r u c t u r e (A) i n the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
Lysosomal
Enzyme
Deficiency
Diseases
137
l i v e r , b r a i n and u r i n e i n s e v e r a l of the lysosomal enzyme d e f i ciency d i s e a s e s , s i n c e t h i s s t r u c t u r e would be the accumulated end product of the catabolism of a wide v a r i e t y of h e t e r o p o l y saccharide c h a i n s . Finne et a l (7) demonstrated the presence of d i s i a l o s y l (a-N-acetylneuraminyl-(2+8)N-acetylneuraminyl) groups i n the s i a l o g l y c o p e p t i d e s from b r a i n . Thus, glycopeptides of f r a c t i o n s I and I I , which contain 4 NeuNAc r e s i d u e s , may c o n t a i n d i s i a l o s y l groups, ensuring the a v a i l a b i l i t y of one or two of the 4 Gal residues as a t e r m i n a l sugar or as a sugar that i s p e n u l t i mate to a t e r m i n a l fucose or s u l f a t e r e s i d u e . Approximately 20-25 percent of the g l y c o p r o t e i n - c a r b o h y d r a t e of r a t b r a i n c o n s i s t e d of mannoglycopeptides which c o n t a i n 6 Man and 2 GlcNAc residues per glycopeptide molecule (8). Manno glycopeptides , r e a d i l y i s o l a t e d since they bind s t r o n g l y to Con A-Sepharose, contained t e r m i n a l Man residues and would not be a f f e c t e d i n the G M l - g a n g l i o s i d o s i would make a major c o n t r i b u t i o n to the accumulated o l i g o s a c c h a r i d e s i n mannosidosis. When p u r i f i e d l i v e r lysosomes were incubated i n the presence of f e t u i n or orosomucoid ( 9 ) , about one h a l f of the peptide bonds were cleaved and most of the t e r m i n a l NeuNAc residues were r e leased. Release of Gal and GlcNAc was slower and d i d not exceed 30 percent of the t h e o r e t i c a l y i e l d . Release of Man was not d e t e c t e d , and branch p o i n t s i n which Man r e s i d u e s were i n v o l v e d appeared to r e s i s t a t t a c k . Many s t u d i e s have shown that p u r i f i e d 3-galactosidase and β - Ν - a c e t y l g l u c o s a m i n i d a s e can cleave t e r m i n a l l y exposed c a r b o hydrate groups of i n t a c t g l y c o p r o t e i n s , and i t appears l i k e l y that t h i s process occurs i n the lysosomes of the i n t a c t organism. There may be an e x c e p t i o n , however. H i g h l y p u r i f i e d a-fucosidase from r a t l i v e r c a t a b o l i z e d the h y d r o l y s i s of 1-2, 1-3, and 1-4 f u c o s y l linkages i n g l y c o p e p t i d e s , but not i n i n t a c t g l y c o p r o t e i n s (10). Since fucose i s i n a t e r m i n a l l y l i n k e d p o s i t i o n , f a i l u r e to cleave i t w i l l prevent the f u r t h e r degradation of any chains terminated by t h i s sugar. An endo-3-N-acetylglucosaminidase which hydrolyzes the d i - N , N - a c e t y l c h i t o b i o s y l bond i n the l i n k a g e r e g i o n i n g l y c o peptides to produce o l i g o s a c c h a r i d e s w i t h GlcNAc i n a reducing p o s i t i o n and Asn-GlcNAc ( F i g . 2) has been demonstrated i n the hen oviduct (11) and methods f o r i t s assay have been described (12). Although most of the carbohydrate can be removed from n a t i v e g l y c o p r o t e i n s by the endo-glucosaminidase, denaturation of the g l y c o p r o t e i n s was necessary to e f f e c t complete renewal (13). The a c t i v i t y of t h i s enzyme accounts f o r the accumulation of o l i g o s a c c h a r i d e s i n some of the lysosomal enzyme d e f i c i e n c y diseases. O l i g o s a c c h a r i d e s may a l s o be produced by the a c t i o n of 4 - L - a s p a r t y l g l y c o s y l a m i n e amidohydrolase, which cleaves the bond between GlcNAc and asparagine (14). The s i z e and s t r u c t u r e of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
138
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
the carbohydrate group d i d not appear to be an important f a c t o r i n c o n t r o l l i n g a c t i v i t y , but the presence of amino a c i d s i n a d d i t i o n to the asparagine i n the peptide p o r t i o n of the g l y c o peptide molecule reduced the r a t e of t h i s r e a c t i o n . T h i s enzyme i s not a c t i v e on i n t a c t g l y c o p r o t e i n s (15). Treatment of the d e l i p i d a t e d b r a i n t i s s u e w i t h p r o t e o l y t i c enzymes r e l e a s e d a l l of the heteropolysaccharide chains of the g l y c o p r o t e i n s , along with glycosaminoglycans and n u c l e i c a c i d s . The l a t t e r substances are removed by p r e c i p i t a t i o n with c e t y l p y r i d i n i u m c h l o r i d e . D i a l y s i s of the glycopeptide p r e p a r a t i o n separates the s i a l o g l y c o p e p t i d e s , which are l a r g e l y n o n - d i a l y z a b l e , from the d i a l y z a b l e , Con-A-binding, mannoglycopeptides. In the storage d i s e a s e s , accumulated degradation products d e r i v e d from the s i a l o g l y c o p e p t i d e s w i l l appear i n the d i a l y z a b l e glycopeptide f r a c t i o n . Thus, an i n c r e a s e i n the carbohydrate content of t h i s f r a c t i o n does not n e c e s s a r i l y i n d i c a t e an ac cumulation of the norma i n d i c a t e the appearanc ( u s u a l l y present only i n t r a c e s ) d e r i v e d from the n o n d i a l y z a b l e sialoglycopeptides. Treatment of the d e l i p i d a t e d t i s s u e with p r o t e o l y t i c enzymes has the disadvantage of obscuring the evidence f o r the p o s s i b l e accumulation of glycopeptides or o l i g o s a c c h a r i d e s i n the t i s s u e . These substances can be recovered q u i t e simply by e x t r a c t i n g the t i s s u e with water or aqueous s o l v e n t s . As we s h a l l see, the l a t t e r procedure has the advantage of p r o v i d i n g the accumulated o l i g o s a c c h a r i d e s which have proved to be amenable f o r s e p a r a t i o n and s t r u c t u r a l a n a l y s i s . A thorough study would allow f o r the i s o l a t i o n of these substances p r i o r to s u b j e c t i n g the i n s o l u b l e t i s s u e r e s i d u e to p r o t e o l y s i s . GM2-gangliosidoses ( d e f i c i e n c y of β - h e x o s a m i n i d a s e ) . Tingey (16) reported that the hexosamine l e v e l i n the de l i p i d a t e d b r a i n t i s s u e from Tay-Sachs diseased p a t i e n t s was elevated. Hexosamine-containing substances c o u l d a l s o be ex t r a c t e d from the residue w i t h b o i l i n g water. While only 3-10 percent of the t o t a l b r a i n hexosamine could be e x t r a c t e d from normal b r a i n i n t h i s manner, the amount of the m a t e r i a l extracted from Tay-Sachs b r a i n was s u b s t a n t i a l l y e l e v a t e d . An e l e v a t i o n of protein-bound hexosamine i n Tay-Sachs disease was confirmed by others (17-21). Suzuki (20) found that the aqueous e x t r a c t prepared from the b r a i n of p a t i e n t s with Sandhoff's disease contained elevated l e v e l s of hexosamine while the i n c r e a s e i n hexosamine c o n c e n t r a t i o n of defatted Tay-Sachs b r a i n t i s s u e and i n aqueous e x t r a c t s prepared from the residue was moderate. Glycopeptides were recovered from the d e l i p i d a t e d c e r e b r a l c o r t i c a l t i s s u e by treatment of the m a t e r i a l w i t h papain (22-24). The NeuNAc content of the d i a l y z a b l e and n o n - d i a l y z a b l e g l y c o peptides obtained from Tay-Sachs b r a i n s was normal. Thus, catabolism of these heteropolysaccharide chains d i f f e r e d from that of the g a n g l i o s i d e s ( F i g . 1 ) : cleavage of the NeuNAc residues
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
Lysosomal
Enzyme
Deficiency
Diseases
139
was not dependent on the p r i o r removal of hexosamine. The conc e n t r a t i o n and carbohydrate composition of the n o n - d i a l y z a b l e s i a l o g l y c o p e p t i d e s d i d not d i f f e r from normal v a l u e s . On the other hand, a f o u r - f o l d e l e v a t i o n of hexosamine and mannose content i n the d i a l y z a b l e glycopeptides f r a c t i o n was observed i n both Tay-Sachs and Sandhoff's d i s e a s e s . The accumulated d i a l y z a b l e glycopeptides d i d not bind s t r o n g l y to Con A-Sepharose and presumably d i d not contain the t e r m i n a l Man linkages which i n t e r a c t with the l e c t i n . Thus, the accumulated m a t e r i a l d i d not correspond to the mannoglycopept i d e s , also present i n the d i a l y z a b l e glycopeptide p r e p a r a t i o n . Probably breakdown products of the s i a l o g l y c o p e p t i d e s , these accumulated glycopeptides presumably contained t e r m i n a l GlcNAc residues. Oligosaccharides which contained a trimannosyl core and GlcNAc at non-reducing t e r m i n i were i s o l a t e d from the l i v e r of a case with Sandhoff' GlcNAc-containing o l i g o s a c c h a r i d e , or the excessive e x c r e t i o n i n the u r i n e , was not observed i n Tay-Sachs disease (26). The o l i g o s a c c h a r i d e s were i s o l a t e d by homogenizing the l i v e r i n 10 volumes of chloroform-uiethanol-water ( 1 : 2 : 0 . 3 , v / v / v ) . The m a t e r i a l was then p a r t i t i o n e d i n t o an upper aqueous phase which was evaporated to dryness. Gangliosides were extracted from the d r i e d residue by repeated e x t r a c t i o n with dry chloroformmethanol. Glycopeptides were a l s o i s o l a t e d from d e l i p i d a t e d t i s s u e by means of p r o t e o l y s i s with papain. Tsay and Dawson (27) minced b r a i n t i s s u e i n water. The mixture was sonicated and c e n t r i f u g e d , and the water s o l u b l e m a t e r i a l was f r a c t i o n a t e d by g e l f i l t r a t i o n . The s t r u c t u r e of an i s o l a t e d o l i g o s a c c h a r i d e was determined by treatment with the appropriate g l y c o s i d a s e s , periodate o x i d a t i o n , and GLC analysis. Its s t r u c t u r e corresponded to that of the o l i g o saccharide p r e v i o u s l y i s o l a t e d from the l i v e r of Sandhoff p a t i e n t s (25). The amount i s o l a t e d from b r a i n was extremely small compared to the l e v e l s accumulated i n the l i v e r of these patients. GlcNAc (3,1+2) Man (a, 1+3). GlcNAc (3,1+2)Man (a, 1+6)
Man (3,1-^4) GlcNAc
Whether the o l i g o s a c c h a r i d e s i n b r a i n are a product of b r a i n metabolism i s not e n t i r e l y c l e a r . It i s p o s s i b l e that incompletely degraded m a t e r i a l s produced i n the l i v e r and organs can enter the c i r c u l a t i o n from which they may be taken up by brain c e l l s . On the other hand, b r a i n t i s s u e does appear to contain heteropolysaccharide chains (Table I) with a carbohydrate composition which corresponds to that of the accumulated product.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
140
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
The d e f i c i e n c y of hexosaminidase A i n Tay-Sachs d i s e a s e , and hexosaminidases A and Β i n Sandhoff f s disease r e s u l t s i n the accumulation of g a n g l i o s i d e GM2 ( F i g . 1) and o l i g o s a c c h a r i d e s ( F i g . 2, r e a c t i o n D) both of which c o n t a i n t e r m i n a l l y l i n k e d β-hexosamine r e s i d u e s . Both hexosaminidases A and Β have been shown to act on t e r m i n a l N - a c e t y l glucosamines of o l i g o s a c c h a r i d e s derived from g l y c o p r o t e i n s (28). G M l - g a n g l i o s i d o s i s , d e f i c i e n c y of F - g a l a c t o s i d a s e . An o l i g o s a c c h a r i d e c o n s i s t i n g of a degradation product d e r i v e d from g l y c o p r o t e i n s was i s o l a t e d from the l i v e r of p a t i e n t s with G M l - g a n g l i o s i d o s i s (29): Gal(β,1+4)GlcNAc(β, 1+2)Man(α,1+3)^ Gal (β, 1+4) GlcNAc (β, 1+2 )Man (α, 1+6 )
'
Man(β,1+4)GlcNAc
The n o n d i a l y z a b l e a f t e r d i g e s t i o n of the d e l i p i d a t e d residue from the c e r e b r a l gray matter showed an over two-fold e l e v a t i o n i n Gal content (31,32). Man and GlcNAc were a l s o e l e v a t e d . These r e s u l t s suggested the accumulation of glycopeptides with exposed Gal residues. Degradation can proceed no f u r t h e r a f t e r the removal of the t e r m i n a l NeuNAc and fucose r e s i d u e s . There was an es p e c i a l l y marked accumulation of glycopeptides which were d e r i v e d from the NeuNAc-rich, h i g h l y n e g a t i v e l y charged, glycopeptides the sugar content of which was c h a r a c t e r i z e d by h i g h values f o r the Gal/Man and GlcNAc/Man r a t i o s ( f r a c t i o n s I and I I , Table I ) . In a d d i t i o n to these changes, e l e c t r o p h o r e t i c a n a l y s i s of the n o n - d i a l y z a b l e glycopeptide p r e p a r a t i o n suggested that a l a r g e p r o p o r t i o n of the s i a l o g l y c o p e p t i d e s were d e f i c i e n t i n -GlcNAcGal-NeuNAc branches which are attached to the trimannoside c o r e . This change cannot be a t t r i b u t e d to the absence of β - g a l a c t o s i dase, and may represent secondary changes due to the e f f e c t s of the d i s e a s e . In a second case, the c o n c e n t r a t i o n of d i a l y z a b l e glycopeptides c o n t a i n i n g hexosamine, Gal and Man showed a 3-7 f o l d e l e v a t i o n which was dependent on the b r a i n area s t u d i e d . The accumulated glycopeptides d i d not bind to Concanavalin A Sepharose (24). A s i x y e a r - o l d boy was reported by P a t e l et a l (33) to have a 2.5 f o l d e l e v a t i o n i n g l y c o p r o t e i n - g a l a c t o s e i n brain. Tsay and Dawson (27) recovered an o l i g o s a c c h a r i d e from the b r a i n of G M l - g a n g l i o s i d o s i s p a t i e n t s the s t r u c t u r e of which was i d e n t i c a l to that recovered from l i v e r and u r i n e by Wolfe et a l (29,30,31). I t s s t r u c t u r e resembled that of the o l i g o s a c c h a r i d e s recovered from the b r a i n of Sandhoff p a t i e n t s , except for the two t e r m i n a l l y - l i n k e d Gal residues ( F i g . 2 ) . Only a r e l a t i v e l y small amount of t h i s m a t e r i a l was recovered from b r a i n ; the accumulation of g a n g l i o s i d e GM1 and glycopeptides released by p r o t e o l y s i s was c o n s i d e r a b l y g r e a t e r .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Figure
G M 3
GM2
GM1
^-glucosidase
galactosidase
1. Block in ganglioside
$
neuraminidase
^hexosaminidase
/ί-galactosidase
BLOCKED
+ Glc
GM2
Gal
GM1
BLOCKED
IN
DISEASE
GM2-gangliosidosis, and
GAUCHERS
>-Gal
+ Neu Ν A c
GANGLIOSIDOSIS
GANGLIOSIDOSIS
Gal
+ Gal Ν Ac
IN
+
IN
catabolism in GM-l-gangliosidosis, Gauchers Disease
Cer
Cer-GIc
Cer-Glc-Gal
NeuNAc
J 1 i— I
Cer-Glc-Gal
-BLOCKED
— I
-NeuNAc
I
Cer-Glc-Gal-GalNAc
•
2)NeuNAc
1
C e r ( 1 « — 1 . β ) G l c ( 4 « - 1 . β) G a l ( 4 « - 1 , ^ g ) G a l Ν A c ( 3 * - 1 . β)
S «s:
Ci
Ο > w Η
142
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Fuc F u c - G a l - GlcNAc - M a n
1
M a n - G l c N A c —GlcNAc - A s n Fuc-Gal - GlcNAc-Man A
^ (Endo-0-N-glucosaminidase)
^
Fuc-Gal-GlcNAc-Man
Fuc \ Man —GlcNAc
I GlcNAc-Asn
+
Fuc- Gal-GlcN Ac-Man Β Gal — G l c N A c —Man ΓM a n — G l c N A c
+
Fuc
Gal — G l c N A c — M a n ' C
\
G l c N A c - A s n + Fuc N-aspartyl-
^
glucosaminidase
( β-Galactosidase)
GlcNAc
+ Asn
GlcNAc — Man
\
G l c N A c —Man
/
Man —GlcNAc
+ Gal
( β-hexosaminidase)
i
Man, Man
>
Man
(Q£
1
-GlcNAc
Man
GlcNAc
mannosidase)
I an —GlcNAc M
(β
+
+ Man
mannosidase) +
GlcNAc
Figure 2. Probable catabolic pathway for the degradation of oligosaccharide chains derived from brain glycoproteins. Reactions B, C, D, and Ε are blocked in fucosidosis, GMl-gangliosidosis, GM2-gangliosidosis, and mannosidosis, respec tively.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
Lysosomal
Enzyme
Deficiency
Diseases
143
Gaucher*s Disease ( d e f i c i e n c y of glucocerebrosidase) Whether p a r t i a l degradation products derived from g l y c o p r o t e i n s accumulate i n the b r a i n of the neuropathic forms of Gaucher s disease i s not known. Kanfer et a l (34) t r e a t e d de f a t t e d l i v e r t i s s u e with p r o t e o l y t i c enzymes. Glycopeptides d e r i v e d from g l y c o p r o t e i n s were markedly elevated i n the Gaucher l i v e r ; a 20 f o l d e l e v a t i o n i n hexose, hexosamine, fucose and NeuNAc was recorded. The accumulated m a t e r i a l contained Gal and GlcNAc as the major sugars; Glc and Man were present i n t r a c e s . The disease i s caused by d e f i c i t i n glucocerebrosidase, a 3-glucosidase. Although glucose-containing g l y c o p r o t e i n s un doubtedly e x i s t (1), there i s no evidence f o r the accumulation of G l c - c o n t a i n i n g glycopeptides or o l i g o s a c c h a r i d e s . Gaucher spleen appears to produce an excess of a g l y c o p r o t e i n a c t i v a t i n g f a c t o r , capable of a c t i v a t i n g the glucocerebrosidase from normal spieen (35-37)· While the a c t i v a t o r from Gaucher spleen was a g l y c o p r o t e i n , that fro composition of the two of the g l y c o p r o t e i n a c t i v a t o r remains obscure. Metachromatic Leukodystrophy (MLD) and M u l t i p l e S u l f a t a s e D e f i c i e n c y (MSP). 1
The s i a l o g l y c o p e p t i d e p r e p a r a t i o n from b r a i n contains mole cules which c a r r y s u l f a t e - e s t e r groups. Consequently, i t ap peared l i k e l y that p a r t i a l degradation products derived from g l y c o p r o t e i n s which bear s u l f a t e d o l i g o s a c c h a r i d e chains might accumulate i n MLD, a disease caused by the absence of a f u n c t i o n ing s u l f a t a s e A. However, s u l f a t i d e s are the n a t u r a l substrate f o r t h i s enzyme. The hexosamine l e v e l s i n the d e l i p i d a t e d t i s s u e residue from cases of MLD, i n which i t was e s t a b l i s h e d that s u l f a t a s e A i s d e f i c i e n t , f e l l w i t h i n the normal range (39,40). The s i t u a t i o n d i f f e r s i n MSD, a v a r i a n t of MLD i n which the t i s s u e s of the p a t i e n t s l a c k s u l f a t a s e s A, B, and C. The non l i p i d hexosamine i n t i s s u e s from such p a t i e n t s show an e l e v a t i o n of protein-bound hexosamine (41), and i t has been shown that t h i s i s due, at l e a s t i n p a r t , by the e l e v a t i o n of glycosaminoglycans· I t i s not known whether glycopeptides or o l i g o s a c c h a r i d e s de r i v e d from g l y c o p r o t e i n s accumulate i n t h i s d i s e a s e . I t has been suggested (42) that O-sulfated glycosaminoglycans are the n a t u r a l substrate f o r s u l f a t a s e B, s i n c e t h i s enzyme i s d e f i c i e n t i n two diseases (MSD and Maroteaux-Lamy disease) i n which O-sulfated glycosaminoglycans accumulate (mainly dermatan s u l f a t e ) . However, c o - c u l t i v a t i o n of MSD f i b r o b l a s t s with S a n f i l i p p o A or Hunter c e l l s d i d not c o r r e c t f o r the degradation defect i n glycosaminoglycan-SO/j-S metabolism. S a n f i l i p p o and Hunter f i b r o b l a s t s l a c k heparan s u l f a t a s e and dermatan s u l f a t a s e , but a r y l s u l f a t a s e Β i n these c e l l s i s normal or elevated (42). These f i n d i n g s l e d to the suggestion that s u l f a t a s e Β may be i n v o l v e d i n degrading s u l f a t e d glycopeptides (43). 35
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
144
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
S u l f a t a s e Β appears to correspond to N-acetylgalactosamine 4 - s u l f a t a s e (44). The s u l f a t a s e r e s p o n s i b l e f o r the accumula t i o n of protein-bound hexosamine s t i l l remains to be i d e n t i f i e d . Nor i s i t yet e s t a b l i s h e d whether s u l f a t e d glycopeptides d e r i v e d from g l y c o p r o t e i n s accumulate i n MSD. The unknown s u l f a t a s e may be a component of s u l f a t a s e A or B, both of which e x i s t i n m u l t i p l e forms, or a s u l f a t a s e which does not respond to the s y n t h e t i c substrates commonly i n use to detect s u l f a t a s e a c t i " vity. The mucopolysaccharidoses. The defects i n H u r l e r , Hunter, and S a n f i l i p p o A and Β syndromes, those forms of the mucopolysaccharidoses i n v o l v i n g severe n e u r o l o g i c a l d e f i c i t s , are caused by nonfunctioning α - i d u r o n i d a s e , s u l f o i d u r o n a t e s u l f a t a s e , heparan-N-S-sulfatase, and Ofr-N-acetylglucosaminidase, r e s p e c t i v e l y . These linkages a r e , as f a r as i s known, absent i n g l y c o p r o t e i n s . Consequently, no a b e r r a t i o n i n g l y c o p r o t e i such a defect been r e p o r t e d b r a i n of a Hunter p a t i e n t showed no remarkable changes ( 4 5 ) · Diseases with a primary l e s i o n i n g l y c o p r o t e i n c a t a b o l i s m . Mannose-rich o l i g o s a c c h a r i d e s accumulate i n the b r a i n of mannosidosis p a t i e n t s (46-48): Man-Man-Gl cNAc? Man-Man-Man-Gl cNAc Man-Man
^Man-GlcNAc
Man' The disease occurs a l s o i n Angus c a l v e s , and the defatted b r a i n t i s s u e residue of the a f f l i c t e d animals showed a 4-6 f o l d e l e v a t i o n i n Man content (49). D e f i c i e n c y of α - m a n n o s i d a s e causes a b l o c k i n the catabolism of the h e t e r o p o l y s a c c h a r i d e c h a i n s : mannose-containing glycopeptides or o l i g o s a c c h a r i d e s are d e r i v e d from the s i a l o g l y c o p e p t i d e trimannoside c o r e , as w e l l as from the mannoglycopeptides· In p a t i e n t s with a d e f i c i e n c y of ot-fucosidase (fucosidosis), f u c o s e - c o n t a i n i n g substances were shown to accumulate i n the d e l i p i d a t e d t i s s u e , i n c l u d i n g that of the b r a i n (50). Aqueous e x t r a c t s of sonicated b r a i n t i s s u e y i e l d e d an o l i g o s a c c h a r i d e (27) : Fuc-Gal-GlcNAc-Man V
Man-GlcNAc Fuc-Gal-GfccNAc-Man^
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
Lysosomal
Enzyme
Deficiency
Diseases
145
A disaccharide, fucosylated N-acetylglucosamine, was also de tected i n the brains of fucosidosis patients (Fig. 2). Aspartylglucosaminuria i s caused by a d e f i c i t i n the enzyme l-aspartamido-3-N-acetylglucosamine amidohydrolase. Aspartylglucosaminylamine and aspartyloligosaccharides accumulate i n organs, including the brain (51,52). The mucolipidoses are caused by a deficiency of α-neuramini dase (53) resulting i n the excessive excretion of s i a l y l o l i g o saccharides. Most of the oligosaccharides isolated had a core consisting of three mannose residues to which two GlcNAc-GalNeuNAc chains, or incomplete portions of these chains, were attached (54). Although not a l l of the linkages i n the oligo saccharides of brain tissue have been definitively established, i t appears l i k e l y that their structure w i l l prove to be identi cal to that of one of the sialoglycopeptides isolated from urine (55): NeuNAc ( α , 2+6) Gal ( β , 1+4)GlcNA
/
Man(β,1+4)GlcNAc
NeuNAc ( α , 2+6 ) Gal ( 3,1+4 ) GlcNAc ( β , 1+2 )Man (α, 1+6 )' A sequence of the steps involved i n the catabolism of the heteropolysaccharides of brain glycoproteins has been postulated (Fig. 2) based on the structures of the oligosaccharides which accumulate i n the various lysosomal enzyme diseases discussed above. Niemann-Pick's Disease The primary lesion i n the i n f a n t i l e , type A, form of Nie mann Pick's disease i s a deficiency of the enzyme, sphingomye linase. While sphingomyelin i s the primary storage product, many cases which have been examined have demonstrated an increase i n gangliosides and cholesterol i n brain tissue. Tingey (16) and Norman et a l (56) have reported an elevation of proteinbound hexosamine as well. Brunngraber et a l (57) noted a two fold elevation of glycoproteins-carbohydrate which was recovered as sialoglycopeptides upon proteolytic digestion of the d e l i p i dated tissue residue from gray matter. Mannoglycopeptide levels remained within the normal range. The composition of the s i a l o glycopeptides was altered, since the number of -GlcNAc-GalNeuNAc chains attached to the internal trimannoside core was reduced. I t i s possible, but not demonstrated, that the accumu lated glycopeptides i n Niemann-Pick disease may correspond to the sialylated forms of the oligosaccharides which accumulate i n the GMl-gangliosidoses. I t was suggested that sphingomyelin catabolism may be an early step i n the degradation of plasma mem branes, and a lesion i n the catabolism of this phospholipid may have the secondary effect of impeding the degradation of other plasma membrane components.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
146
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Neuronal-Ceroid L i p o f u s c i n o s e s (NCF). A form of NCF may i n v o l v e g l y c o p r o t e i n storage. Adelman et a l (58) found a n e a r l y two f o l d i n c r e a s e i n the NeuNAc con tent of s i a l o g l y c o p e p t i d e p r e p a r a t i o n s obtained from t i s s u e sec t i o n s of the c e r e b r a l c o r t e x of a case with c l i n i c a l f e a t u r e s resembling those of Batten's d i s e a s e . C e l l r e c o g n i t i o n of lysosomal enzymes. Studies u t i l i z i n g c u l t u r e d f i b r o b l a s t s d e r i v e d from p a t i e n t s w i t h lysosomal enzyme d e f i c i e n c y diseases have shown that these c e l l s s e c r e t e lysosomal g l y c o s i d a s e s i n t o the medium, and are capable of r e - a s s i m i l a t i n g these enzymes. Hickman and Neuman (59,60) have proposed t h a t lysosomal enzymes are t r a n s p o r t e d from t h e i r s i t e of s y n t h e s i s and g l y c o s y l a t i o n i n the G o l g i apparatus to the lysosome by t h i s process of e x o c y t o s i s and subsequent p i n o c y t o t i c r e - i n c o r p o r a t i o n . Such a mechanism may be a p a r t of a membrane r e c y c l i n g mechanism s i n c e i n t e r n a l i z a t i o n of the enzyme i s patch of plasma membrane to fuse with the lysosome (61). F i b r o b l a s t s from p a t i e n t s a f f l i c t e d with one of the l y s o s o mal d e f i c i e n c y diseases are capable of a s s i m i l a t i n g the d e f i c i e n t enzyme which had been added to the c u l t u r e medium, thereby c o r r e c t i n g the metabolic d e f e c t . Uptake of lysosomal g l y c o s i dases i s dependent on the r e c o g n i t i o n of the h e t e r o p o l y s a c c h a r i d e c h a i n of the g l y c o p r o t e i n enzyme by a r e c e p t o r on the s u r f a c e of the f i b r o b l a s t . Thus, the t e r m i n a l Gal r e s i d u e appeared to be important f o r the r e c o g n i t i o n and uptake o f a-N-acetylglucosaminidase (62) by f i b r o b l a s t s of S a n f i l i p p o Β p a t i e n t s . Mannose residues appeared to be important f o r uptake of 3-galactosidase by f i b r o b l a s t s from p a t i e n t s with G M l - g a n g l i o s i d o s i s (63). Cultured f i b r o b l a s t s appear to recognize phosphohexosyl groups i n 3-glucuronidase, β-hexosaminidase, and 3-galactosidase (64,65) and a-L-iduronidase (66). The r e c o g n i t i o n of phospho hexosyl groups may be a general c h a r a c t e r i s t i c of p i n o c y t o s i s of lyosomal g l y c o s i d a s e s . These f i n d i n g s may be r e l a t e d to the e a r l i e r r e p o r t s on the i s o l a t i o n of a phosphoglycoprotein from b r a i n (67-69). Phosphorylated mannoglycopeptides were i s o l a t e d from the phosphoglycoprotein, and the phosphate r e s i d u e which i s attached to one of the h y d r o x y l groups of mannose, was removed by the a c t i o n of a l k a l i n e phosphatase (69). The r e l a t i o n of t h i s phosphoglycoprotein to the lysosomal g l y c o s i d a s e s remains to be e s t a b l i s h e d . I f the c a p a c i t y of the f i b r o b l a s t to s e c r e t e and r e - a s s i m i l a t e lysosomal enzymes i s a s p e c i f i c example of a more general phenomena, phosphoglycoproteins and/or g l y c o s i d a s e s may p l a y an important r o l e i n the turnover of the plasma membrane.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7.
BRUNNGRABER
1. 2. 3. 4. 5. 6. 7· 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Lysosomal
Enzyme
Deficiency
Diseases
147
Literature Cited Brunngraber, E. G.; Hof, H.; Susz, J.; Brown, B. D.; Aro, Α.; and Chang, I. Biochim Biophys Acta, 304; 781, 1973. Krusius, T. FEBS Lett, 66: 86, 1976. Hof, Η. I.; Susz, J. P.; Javaid, J. I.; and Brunngraber, E. G. Neurobiology, 5.: 347, 1975. Krusius, T.; Finne, J.; and Rauvala, H. FEBS Lett, 71: 117, 1976. Brunngraber, E. G. Adv Exptl Med and Biol, 25: 255, 1972. Krusius, T.; and Finne, J. Eur J Biochem, 78: 369, 1977. Finne, J.; Krusius, T.; and Rauvala, H. Biochem Biophys Res Communs, 74: 405, 1977. Javaid, J. I.; Hof, H. I.; and Brunngraber, E. G. Biochim Biophys Acta, 404: 74, 1975. Aronson, Ν. Ν., Jr.; and DeDuve C J Biol Chem 243: 4564, 1968. Opheim, D. J.; and Touster, 0. J Bio Chem, 252: 739, 1977. Tarentino, A. L.; and Maley, F. J Biol Chem, 251: 6537, 1976. Tarentino, A. L.; and Maley, F. Anal Biochem, 77: 185, 1976. Trimble, R. B.; and Maley, F. Biochem Biophys Res Communs, 78: 935, 1977. Kohno, M.; and Yamashina, I. Biochim Bipphys Acta, 258: 600, 1972. Fidler, I. J.; Montgomery, P.C.;and Cesarini, J. P. Cancer Res, 33: 1376, 1973. Tingey, A. J Neurochem, _3 230, 1959. Stefanko, St.; Guminska, M.; and Pietrzykowa, B. Schweiz Arch Neurol Neurochir Psychiat, 90: 295, 1962. Edgar, G. W. F. Proc of the Fifth Internatl Congr Neuro pathology, Zurich, 1965. Amsterdam, Excerpta Med Found, 1966, p. 328. Thieffry, S.; Bertrand, I.; Bargeton, E.; and Edgar, G.W.F. Rev Neurol, 102: 130, 1960. Suzuki, Y.; Jacob, J.C.;Suzuki,K.;Kutty, Κ.N.;and Suzuki, K. Neurology, 21: 313, 1971. Norman, R. M.; Tingey, A. H.; Newman, C. H. H.; and Ward, S. P. Arch Dis Child, 39: 634, 1964. Brunngraber, E.G.;Haberland,C.;and Brown, B. D. Brain Res, 38: 151, 1972. Brunngraber, E. G.; Brown, B. D.; and Aro, A. J Neurochem, 22: 125, 1974. Brunngraber, E. G.; Davis, L. G.; Javaid, J. I.; and Berra, B. Adv Exptl Med and Biol,68:31, 1976. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 59: 837, 1974. Wolfe, L. S.; and Ng Ying Kin, Ν. M. K. Adv Exptl Med and Biol, 68: 15, 1976. Tsay, G.C.;and Dawson, G. J Neurochem, 27: 733, 1976. American Chemicaf Society Library 1155 16th St. N. W. W a shiGlycolipids ngton, D.inC.Disease 200Processes; 36 In Glycoproteins and Walborg, E.; :
ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
148
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
28. Bearpark, T.; Bouquelet, S.; Fournet, B.; Montreuil, J; Spik, G.; Stirling, J.; Strecker, G. FEBS Lett, 84: 379, 1977. 29. Wolfe, L. S.; Senior, R. G.; and Ng Ying Kin, Ν. M. K. J Biol Chem, 249: 1828, 1974. 30. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 66: 123, 1975. 31. Brunngraber, E. G.; Berra, B.; and Zambotti, V. FEBS Lett, 34: 350, 1973. 32. Berra, B.; Di Palma, S.; and Brunngraber, E. G. Clin Chim Acta, 57: 301, 1974. 33. Patel, V.; Goebel, H. E.; Watanabe, I.; and Zeman, W. Acta Neuropathol, 30: 155, 1974. 34. Kanfer, J. N.; Stein, M.; and Spielvogel, C. Adv Exptl Med and Biol, 19: 225, 1972. 35. Ho, M. W.; and O'Brien J S Proc Natl Acad Sci US 68: 2810, 1971. 36. Ho, M. W.; O'Brien Biochem J, 131; 173, 1973. 37. Ho, M. W. Biochem J, 136: 721, 1973. 38. Peters, S. P.; Coyle, P.; Coffee, C. J.; Glew, R. H.; Kuhlenschmidt, M. S.; Rosenfeld, L.; and Lee, Y. C. J Biol Chem, 252: 563, 1977. 39. Haberland, C.; Brunngraber, E.G.;Witting, L. Α.; and Daniels, A. Acta Neuropathol, 26: 93, 1973. 40. Federico, Α.: Guazzi, G. C.; and Di Benedetta, C. Eur Neurol, 15: 163, 1977. 41. Eto, Y.; Meier, C.; and Herschkowitz, N. N. J Neurochem, 27;1071,1976. 42. Austin, J. Arch Neurol, 28: 258, 1973. 43. Eto, Y.; Wiesmann, U.; and Hierschkowitz, Ν. N. Arch Neurol, 30: 153, 1974. 44. Dorfman, Α.; Arbogast, B.; and Matalon, R. Adv Exptl Med and Biol, 68: 261, 1976. 45. Roukema, P. Α.; Overkerk, C. E. and Van Den Berg, G. Clin Chim Acta, 44: 277, 1973. 46. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; Ockerman, P. Α.; and Autio, S. J Biol Chem, 248: 6210, 1973. 47. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; and Autio, S. Biochemistry, 13: 871, 1974. 48. Tsay, G. C.; Dawson,G.;and Matalon, R. J Pediat, 84: 865, 1974. 49. Norden, Ν. E.; Lundblad, Α.; Ockerman, P. A. and Jolly, R. D. FEBS Lett, 35: 209, 1973. 50. Van Eoff, F.; and Eers, E. G. Eur J Biochem, 7: 34, 1968. 51. Palo, J.; and Savolainen, E. J Chromatog, 65: 447, 1972. 52. Palo, J. and Savolainen, E. Ann Clin Res, 5: 156, 1973. 53. Cantz, M.; Gehler, J.; and Spranger, J. W. Biochem Bipphys Res Communs, 74: 732, 1977.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
7. BRUNNGRABER
149 Lysosomal Enzyme Deficiency Diseases
54. Michalski, J. C.; Strecker, G.; and Fournet, B. FEBS Lett, 79: 101, 1977. 55. Strecker, G.; Peers, M. C.; Michalski, J. C.; Hondi-Assah, T.; Fournet, B.; Spik, G.; Montreuil, J.; Farriaux, J. P.; Maroteaux, P.; and Durand, P. Eur J Biochem, 75: 391, 1977. 56. Norman, R. M.; Tingey, A. H.; and Fowler, M. C. Proc of the Fifth Internatl Congr Neuropathol, Zurich, 1965, Amster dam, Excerpta Med Found, 1966, p. 143. 57. Brunngraber, E. G.; Berra, B.; and Zambotti, V. Clin Chim Acta, 48: 173, 1973. 58. Adelman, L. S.; Young, E.; and Bass, Ν. H. Neurology, 24: 1045, 1974. 59. Hickman, S.; and Neufeld, E. F. Biochem Biophys Res Communs, 49: 992, 1972. 60. Neufeld, E. F.; Sando, G. N.; Sarvin, A. J.; and Rome, L. H. J Supramol Struct 6: 95 1977 61. Brunngraber, E. G Springfield, Ill. , 62. Von Figura, Κ.; and Kresse, H. Prot Biol Fluids, 22: 275, 1975. 63. Hieber, V.; Distler, J.; Myerowitz, R.; Schmickel, R. D.; and Jourdian, G. W. Biochem Biophys Res Communs, 73: 710, 1966. 64. Kaplan, Α.; Fischer, D.; Achord, D.; and Sly, W. J Clin Invest, 60: 1088, 1977. 65. Kaplan, Α.; Achord, D. T.; and Sly, W. S. Proc Natl Acad Sci US, 74: 2026, 1977. 66. Sando, G. N.; and Neufeld, E. F. Cell, 12: 619, 1977. 67. Davis, L. G.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 30, 1976. 68. Davis, L. G.; Costello, A. J. R.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 35, 1976. 69. Davis, L. G.; Brettschneider, I.; and Brunngraber, E. G. Fed Proc, 36: 750, 1977. RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8 Incorporation of Exogenous Enzymes into Lysosomes A Theoretical and Practical Means for Correcting Lysosomal Blockage P. G. PENTCHEV, J.A. BARRANGER, A. E. GAL, F. S. FURBISH, and R. O. BRADY Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20014
The lysosomal storag types for delineating th inherited metabolic disorders. Progress has led to an identification of most primary enzymatic lesions in these disorders and to a better understanding of the cellular manifestations resulting from these enzymopathies. Specific enzymatic tests are now available for the unequivocal diagnosis of most of these storage diseases. Reliable heterozygote detection and prenatal diagnosis has enabled genetic counseling to effectively reduce the incidence of many of these crippling diseases (1). Investigators have also addressed considerable effort to the intriguing possibility of enzyme replacement as an effective therapeutic measure for the treatment of the lysosomal storage disorders. Although more than a decade of effort has been channelled in this direction, an unequivocal answer has yet to appear. An evaluation of the status of this research program can perhaps be best initiated by examining the cellular and biochemical bases for enzyme replacement. Remarkable insight into this phenomena was given in 1964, at a time when only one lysosomal storage disorder was recognized: "Both in our pathogic speculations and in our therapeutic attempts it may be well to keep in mind that any substance which is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction including replacement therapy."(2). The implication of the dynamic nature of lysosomal interactions is that one can, in theory, equate the ability of endogenous and exogenous enzymes to stimulate intralysosomal digestion (Figure I). It is now well documented that enzymes that are initially found in the extracellular environment can be incorporated into the lysosomal architecture of cells (Table I). It has 150 This chapter not subject to U.S. copyright Published 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PENTCHEV ET AL.
Lysosomal
PRIMARY L Y S O S O M E
Blockage
AUT0PHAG0S0ME
ENZ
RESIDUAL B O D Y
SECONDARY LYSOSOME
-Λ /
PHAGOSOME
ENDOCYTOSIS
PRIMARY L Y S O S O M E
AUTOPHAGOSOME
r
/
/ •
SECONDARY L Y S O S O M E ,
PHAGOSOME
^ENZ
RESIDUAL B O D Y
Λ
ENDOCYTOSIS
Figure 1. Representation of lysosomal interactions. (Top) Nor mal lysosomal digestion. (Bottom) Lysosomal digestion stimuhited by exogenous enzyme.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
152
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
also been shown that these "migrant" enzymes are functionally active in reversing previously blocked metabolic pathways and/or reversing abnormal morphology of lysosomes (Table II). In 1966, i t was predicted that Gaucher^ disease might prove to be the ideal model for enzyme replacement therapy (]_5). The accumulation of glycolipid occurs predominantly within the cells of reticuloendethelial system. Consequently intravenously admin istered enzyme preparations were expected to have ready access to major pathological depots of stored l i p i d . Our enzyme infusion studies with Gaucher patients began in 1973 with the successful isolation of human placental glucocerebrosidase (1_6). A brief summary of the results of these i n i t i a l t r i a l s is presented in Table III. Encouraged by these preliminary findings and having developed a more efficient and practical means of purifying large quantities of the enzyme (1_8), we began a series of infusions in patients using considerably more glucocerebrosidase than i n i t i a l l y employed. The results o ing. No significant changes in either the circulating or l i v e r levels of glycolipid were observed following the intravenous ad ministration of native glucocerebrosidase (Brady, R. et al_., un published observations). Concomittant c l i n i c a l observations such as regression of hepatomegaly also prove negative. On the other hand, Belchetz and collaborators (1_9) and Beutler's group (20) have reported a decrease in l i v e r size when glucocerebrosidase was administered over relatively long periods of time (13 and 3-1/2 months respectively). Furthermore, Belchetz, et al_., injected glucocerebrosidase that had been entrapped in liposomes while Beutler, et al^., used resealed erythrocyte ghosts to deliver the placental enzyme. A possible reason for the refractiveness of biochemical and c l i n i c a l responses to exogenous enzyme in our t r i a l s with the new glucocerebrosidase preparations may be an unfavorable cellular disposition of the enzyme. It has been shown that approximately 30% of the infused enzyme is taken up by the l i v e r in rats and monkeys (5). More recent studies have shown that approximately 90% of this uptake represents incorpora tion into hepatocytes ( 2 Π ) . The predominate uptake of enzyme by hepatocytes is an inefficient route for inducing l i p i d clearance since the accumulation of glycolipid occurs mainly within reticuloendothetial cells (22J. Modification and/or encapsulation of the enzymes will now have to be seriously considered in an attempt to specifically target the administered enzyme to these c e l l s . The eventual effectiveness of enzyme supplementation in the treatment of Gaucher's disease will also depend on several other parameters which unfortunately have not been experimentally evalu ated due to the lack of a suitable animal model (Table IV). To begin with, the inherent extent of enzymatically induced clearance will depend on the degree of accessibility of exogenous enzymes to stored l i p i d . Complete clearance of glucocerebroside can be effected when specimens of Gaucher l i v e r tissue are incubated with glucocerebrosidase in vitro (Figure II). However, the in situ
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8.
PENTCHEV ET AL. Table I.
Lysosomal
Blockage
Demonstrated Lysosomal Uptake of Exogenously Administered Enzymes
Enzyme
Reference
Tissue
3-Glucuronidase
Cultured
a-Glucosidase
Rat Liver
(4)
Glucocerebrosidase
Rhesus Monkey Liver
(5)
Hexosaminidase
Rat Liver
(6)
Table II.
Fibroblasts
(3)
Experimental Models for Correction of Lysosomal Accumulation with Exogenous Enzymes
Effect
Reference No.
Invertase and 3-glucosidase
Reversal of Sucrose and cellobiose accumulation in macrophases
7
Deficient lysosomal hydrolases:
Metabolic correction in patients' fibroblasts:
Mucopolysacchari dases
Mucopolysacchari doses
8
Arylsulfatase-A
Metachromatic leukodystrophy
9
Acid lipase
Wolman's Disease
10
ofr-Galactosidase
Fabry's Disease
11
3-N-Acetylhexosaminidase
Sandhoff Disease
Enzyme Addition
B. Invertase
Dextranase
12
Animals
Prevention of hepatic sucrose accumulation in rat
13
Reversal of hepatic dextran accumulation in rat
14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
154
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Table III.
Initial Results of Enzyme Replacement Trials in Gaucher's Disease (17)
1.
Intravenous infusions of highly purified human placental glucocerebrosidase were well tolerated by patients with no untoward immunological or pyrogenic response.
2.
Exogenous enzyme tion.
3.
A clearance of the elevated glycolipid levels was observed in the blood stream.
4.
Following enzyme infusion, lower levels of glycolipid were found in l i v e r biopsy samples.
Table IV.
Assessment of Lysosomal Responses in Evaluating the Effectiveness of Enzyme Replacement for the Treatment of Storage Disorders
1.
The extent of enzymatically induced clearance of stored material.
2.
The rate at which stored material
3.
The degree of lysosomal overloading in relationship to c l i n i c a l and pathological manifestations.
4.
The c l i n i c a l consequence of reversing intralysosomal accumulation.
accumulates.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PENTCHEV ET AL.
Lysosomal
Blockage
Figure 2. Clearance of glucocerebroside from Gaucher liver tissue by exogenous glucocerebrosidase in vitro. Fresh liver needle biopsy samples from a Gaucher patient were incubated in phosphate-buffered saline at pH = 6.0 for 12 hr at 37°C without and with 1000 units of highly purified glucocerebrosidase as indicated. Glucocerebroside in these preparations and in nonincubated samples was subsequently quantitated as previously described (23). Gl I = glucocerebroside; Cer-ceramide, PE = phosphatidyl ethanolamine.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Adult
Adult
Adult
F
F
M
M
F
F
F
F
F
3
4
5
6
7
8
9
10
Π
F&M
Adult
M
2
itrol
Adult
F
1
Adult
Adult
Adult
Adult
Juvenile
Adult
Sex
Patient
Type
Table V.
20-35
21
23
49
39
30
20
30
15
54
15
44
Age
100
29
25
23
23
23
0.03 - 0.07
22.90
17.90
17.80
16.50
16.40
15.40
11.20
-27
4.30
1.63
0.70
0.25
(mg/gram wet t i s s u e )
Liver Glucocerebroside
29
30
21
16
(% of Control)
Residual Glucocerebrosidase A c t i v i t y i n Leukocytes
Glycolipid Accumulation in Livers of Gaucher Patients
8.
PENTCHEV ET AL.
Lysosomal
Blockage
157
accessability of l i p i d may be more restrictive and perhaps a limiting factor. Preliminary studies with mouse l i v e r have shown that residual bodies have a lower tendency to fuse with lysosomes containing exogenous material (24). Secondly, the efficiency and course of enzyme replacement regimens w i l l depend on the degree to which an induced clearance can be sustained. Obviously, a rapid rate of accumulation relative to clearance w i l l limit the u t i l i t y of this form of treatment. It may be useful to attempt to stabilize the exogenous enzyme in order to prolong its intralysosomal digestive effect. The h a l f - l i f e of human glucocerebrosidase in the l i v e r of rats and Rhesus monkeys is approximately 8 hours (5). Generalized hypothermia (33°C) extends this halfl i f e to over thirty hours (5). Lipids have also been shown to stabilize the enzyme in vitro (Furbish, F. and Brady, R., unpublished observations). Stabilization and protection of the enzyme may also be achieved by cross-linking or binding to matrices (25). Thirdly, the progressio c l i n i c a l disease states remains obscure and speculative. The v a r i a b i l i t y of biochemical and c l i n i c a l manifestations in Gaucher's is i l l u s t r a t i v e of this gap in our knowledge. The range of elevated glucocerebroside in the l i v e r of patients with Gaucher's disease may vary over 100-fold (Table V). Often l i t t l e correlation was observed between the degree of glucocerebroside accumulation and the severity of c l i n i c a l manifestations. Accordingly, a given level of clearance induced by enzyme supplementation may show considerable variation in terms of c l i n i c a l benefits. F i n a l l y , i t has sometimes been t a c i t l y assumed that an enzymatically induced reversal of lysosomal blockage w i l l restore normality to the effected tissues. As already discussed, the reality of biochemically defined corrections has been well established with various models. However, descriptions of morphological correlates to biochemical correction are rather sparce in the literature. The classic experiments of Cohn et a l . {7) reporting a visual observation of the reversal of lysosomal swelling in the sucrose-loaded macrophages upon the uptake by the cells of invertase remains to date the most descriptive accounting of this phenomena. The complexities of biochemical, morphological and physiological relationships in organisms are enormous. The present status of our knowledge is not sufficient to evaluate potentially corrective or simply preventive roles for enzyme replacement. In conclusion, the r e a l i s t i c application of enzyme replacement as a therapeutic measure for the treatment of lysosomal storage disorders has yet to be established. Although the present state of the art is at a delicate embryonic stage of development, nature has extended a helpful hand in providing intracellular homing mechanisms. The continued creative and persistent efforts of investigators can l i k e l y complement this advantage towards a meaningful realization of this form of therapy.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
158
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
REFERENCES 1.
Brady, R., Metabolism 26: 329 (1977).
2. 3.
DeDuve,C.;Fed. Proc. 23, 1045 (1964). Lagunoff, D., Nicol, D., Prilzl, P., Lab. Invest. 29: 449 (1973).
4.
Huijing, F., Waltuck, B., Whelan, W., in Birth Defects Origi nal Article Series, Enzyme Therapy in Genetic Diseases, Vol. IX, No. 2, p. 191, (1973). Daniel Bergsma, Ed.
5.
Pentchev, P., Kusiak, J., Barranger, J., Furbish, F., Rapoport,S., Massey, J., Brady, R., in Enzymes of Lipid Metabolism, Plenum Press i Press 6. Schlesinger, P., Rodman, J., Frey, M., Lang, S., Stahl, P., Arch. Biochem. and Biophys. 172: 606 (1976). 7.
Cohn, Z., Ehrenreich, B., J. Exp. Med. 129: 201 (1969).
8.
Neufeld, L., Shapiro, L., Ann. Rev. Biochem. 44: 357 (1975).
9.
Porter, M., Fluharty, Α., Kihara, H., Science 172: 1263 (1971).
10. Kyriakides, E., Paul, B., Balint, J., J. Lab. Clin. Med. 80: 810 (1972). 11. Dawson, G., Matalon, R., Li, Y., Pediat. Res. 7: 684 (1973). 12. Cantz, M., Kresse, H., J. Biochem. 47: 581 (1974). 13. Jacques, P., Bruns, G., Abs. 2nd Meet. Fed. Eur. Biochem. Soc. A36 (1978) 14. Colley, M., Ryman, Β., Bioch. and Biophys. Acta. 451: 417 (1976). 15. Brady, R., N. Engl. J. Med. 275: 312 (1966). 16. Pentchev, P., Brady, R., Hibbert, S., Gal, Α., Shapiro, D., J. Biol. Chem. 248: 5256 (1973). 17. Brady, R., Pentchev, P., Gal, Α., Fed. Proc. 34: 1310 (1975). 18. Furbish, F., Blair, H., Shiloach, J., Pentchev, P., Brady, R. Proc. Natl. Acad. Sci. USA 74: 3560 (1977).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
8. PENTCHEV ET AL.
Lysosomal Blockage
159
19. Belchetz, P., Crawley, J., Braidman, I., Gregoriadis, G. Lancet ii, 116 (1977). 20. Beutler, E., Dale, G., Guinto, E., Kuhl, W. Proc. Natl. Acad. Sci. USA, 74: 4620 (1977). 21. Furbish, F., Steer, C., Barranger, J., Jones, J., Brady, R. Biochem. Biophys. Res. manuscript submitted. 22. Brady, R., King, F. In Lysosomes and Storage Diseases (1973) H. G. Hers and F. Van Hoof, Editors, Academic Press, New York, p. 381. 23. Brady, R., Pentchev, P., Gal, Α., Hibbert, S., Dekaban, Α., Ν. Engl. J. Med. 291: 989 (1974). 24. Daems, W., in Enzym p. 3, (1974), J. M. Tager, G. S. Hooghwinkel and W. Daems, Editors, North-Holland Publishing Company. 25. Wold, F., in Birth Defects, Original Article Series, Vol. IX, No. 2, Enzyme Therapy in Genetic Diseases, p. 46. (1973). Daniel Bergama, Editor, Williams and Wi1 king Publishers, Baltimore, Md. RECEIVED March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9 Blood Coagulation—Initiation and Regulation by Limited Proteolysis WALTER KISIEL Department of Biochemistry, University of Washington, Seattle, WA 98195
During the coagulation of blood, a number of plasma glycoprotein zymogens are converted to serine proteases in the presence of calcium ions and phospholipid. These activation reactions do not occur randomly but rather as a series of consecutive zymogen activation reactions where the product of one reaction is the catalyst for the next. The focal point of this cascade-like reaction is the formation of thrombin, the serine protease which converts fibrinogen to fibrin. In vivo, the formation of fibrin is thought to occur on the surface of platelets concurrent with their response to an injury in the vessel wall. Indeed, platelets constitute the primary defense in arresting the bleeding of ruptured blood vessels by their rapid adhesion to extravascular surfaces. The platelet adhesion reaction is followed by a morphological change in the platelet membrane which provides a catalytic phospholipid surface for optimal generation of thrombin. The ensuing fibrin serves to stabilize the hemostatic platelet thrombus by virtue of its mesh-like properties. Following tissue repair and vessel wall regeneration, the hemostatic plug is destabilized by the degradation of the fibrin network by plasmin arising from the fibrinolytic system. The common names, as well as the Roman numeral designations, of the glycoproteins participating in coagulation are listed in Table I. Prothrombin, factor VII, factor IX, factor X, factor XI, factor XII, and prekallikrein exist in plasma as precursors to serine proteases. Also listed in Table I are the coagulation disorders associated with the deficiency of a particular coagulation factor. In the past decade, enormous progress has been made in the isolation and characterization of most of these proteins. The availability of homogeneous preparations of each zymogen has made is possible to establish unambiguously the mechanism whereby it is activated, and its proteolytic specificity following activation. The results of these studies have proven invaluable in our understanding of the molecular events in coagulation, and 0-8412-0452-7/78/47-080-160$05.00/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
161
Coagulation
TABLE I The
P r o t e i n s o f Blood
Coagulation
Roman Numeral Des i g n a t i o n
Associated
Common Name
Disorder
Fibrinogen
Factor 1
Afibrinogenemia
Prothrombi n
F a c t o r 11
Prothrombin d e f i c i e n c y
Proaccelerin
Factor V
Parahemophi1ia
Proconvertin
F a c t o r VI1
ProSPCA d e f i c i e n c y
Factor
Hemophi1ia A
Tissue
Factor
Antihemophi1ic Christmas
Factor
F a c t o r IX
Christmas disease ( h e m o p h i l i a B)
Factor
Factor X
Stuart factor deficiem
Plasma T h r o m b o p l a s t i n A n t e c e d e n t (PTA)
F a c t o r XI
PTA
Hageman
F a c t o r XI1
Hageman
Factor
Factor XIII d e f i c i e n c y
Stuart
Factor
VIII
Factor
Fibrin-stabi1izing Factor Prekal1ikrein Factor)
XIII
deficiency trait
(Fletcher
HMW K i n i n o g e n (high molecular weight k i n i nogen, Fitzgerald factor)
Fletcher factor Fitzgerald
disease
trait
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
162
have g r e a t l y a s s i s t e d t h e r e s e a r c h e r i n f o r m u l a t i n g a more I n t e l l i g e n t approach t o the understanding o f the k i n e t i c s o f the overa l l c o a g u l a t i o n p r o c e s s in vivo. F u r t h e r m o r e , f r o m such s t u d i e s has emerged t h e c o n c e p t t h a t t h e p r i n c i p a l mechanism f o r t h e i n i t i a t i o n , p r o p a g a t i o n and r e g u l a t i o n o f t h e c o a g u l a t i o n process i n v o l v e s t h e s e l e c t i v e e n z y m a t i c c l e a v a g e o f u n i q u e p e p t i d e bonds. In t h i s a r t i c l e , t h e p r i m a r y e m p h a s i s w i l l be p l a c e d on more r e c e n t i n f o r m a t i o n r e g a r d i n g t h e mechanism o f a c t i v a t i o n o f e a c h c o a g u l a t i o n zymogen l e a d i n g t o f i b r i n f o r m a t i o n . In a d d i t i o n , c e r t a i n a c t i v a t e d c o a g u l a t i o n f a c t o r s have been o b s e r v e d t o c a r r y o u t s e c o n d a r y p r o t e o l y t i c r e a c t i o n s i n t h e t e s t t u b e and t h e s e w i l l be p r e s e n t e d and d i s c u s s e d i n r e l a t i o n t o t h e i r p o t e n t i a l r e g u l a t o r y r e l e v a n c e in vivo. I I.
M o l e c u l a r Events i n Blood
Coagulation
Two pathways a p p a r e n t l the s e r i n e p r o t e a s e w h i c pathways have been r e f e r r e d t o a s t h e i n t r i n s i c and e x t r i n s i c c o a g u l a t i o n p a t h w a y s . The i n t r i n s i c pathway i n c l u d e s t h o s e r e a c t i o n s t h a t l e a d t o f a c t o r Xa f o r m a t i o n u t i l i z i n g f a c t o r s p r e s e n t o n l y i n p l a s m a . These f a c t o r s i n c l u d e f a c t o r X I I , f a c t o r X I , f a c t o r IX and f a c t o r V I M . P r e s e n t l y , i t i s not c l e a r whether h i g h m o l e c u l a r w e i g h t k i n i n o g e n and p r e k a l 1 i k r e i n a r e a l s o r e q u i r e d f o r t h i s pathway inasmuch a s p l a s m a l a c k i n g t h e s e p r o t e i n s c l o t s p o o r l y upon e x p e r i m e n t a l l y i n i t i a t i n g t h e i n t r i n s i c s y s t e m . The e x t r i n s i c pathway o f f a c t o r Xa g e n e r a t i o n i n v o l v e s t h e p a r t i c i p a t i o n o f p l a s m a f a c t o r V I I and t i s s u e f a c t o r , a s p e c i f i c l i p o p r o t e i n r e l e a s e d f r o m t i s s u e s u r r o u n d i n g t h e damaged b l o o d vessel w a l l . These pathways c l e a r l y c a n be d i f f e r e n t i a t e d i n t h e t e s t t u b e d e p e n d i n g on t h e i n i t i a t i n g s t i m u l u s p r o v i d e d . Factor Xa formed by e i t h e r t h e i n t r i n s i c o r e x t r i n s i c pathway a p p e a r s biochemically indistinguishable. Following i t s formation, factor Xa t h e n t r i g g e r s a s e q u e n c e o f r e a c t i o n s w h i c h r e s u l t s i n t h e formation of insoluble f i b r i n . I t i s perhaps noteworthy t o m e n t i o n t h a t e a c h o f t h e s e pathways I s t h o u g h t t o be t r i g g e r e d by t h e i r i n i t i a l i n t e r a c t i o n w i t h e l e m e n t s o f t h e v a s c u l a r subendothelium. The r a t e and i n t e n s i t y o f f a c t o r Xa f o r m a t i o n in vivo c o n c e i v a b l y i s d i c t a t e d by t h e pathway f o l l o w e d w h i c h i n t u r n i s r e l a t e d t o t h e s e v e r i t y o f t h e v a s c u l a r trauma. Thus, v a s c u l a r trauma w h i c h i n v o l v e s a b r e a k i n t h e c o n t i n u i t y o f t h e v e s s e l with concomitant release o f t i s s u e f a c t o r necessitates immediate f o r m a t i o n o f f a c t o r Xa w h i c h p r e s u m a b l y o c c u r s v i a e x t r i n s i c clotting. On t h e o t h e r h a n d , r e l a t i v e l y m i l d v a s c u l a r trauma i n w h i c h t h e v e s s e l does n o t l o s e i t s c o n t i n u i t y , may c a l l f o r a r e l a t i v e l y s l o w , more c o n t r o l l e d f o r m a t i o n o f f a c t o r Xa. Under t h e s e c i r c u m s t a n c e s , f a c t o r Xa c o u l d be formed v i a t h e i n t r i n s i c pathway i n i t i a t e d by t h e i n t e r a c t i o n o f f a c t o r X I I w i t h a s u b endothelial collagen surface.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
A.
KISIEL
Blood
163
Coagulation
The A c t i v a t i o n o f F a c t o r X by t h e
I n t r i n s i c Pathway
The s e q u e n c e o f e v e n t s w h i c h l e a d t o t h e f o r m a t i o n o f f a c t o r Xa by t h e i n t r i n s i c pathway o f b l o o d c o a g u l a t i o n is schematically r e p r e s e n t e d i n F i g u r e 1, The f i r s t s t e p i n t h e s e c a s c a d i n g r e a c t i o n s i s the a c t i v a t i o n o f f a c t o r X I I . F a c t o r X I I has been i s o l a t e d f r o m human and b o v i n e p l a s m a and c h a r a c t e r i z e d i n r e g a r d t o i t s s i z e , s t r u c t u r e and c o m p o s i t i o n (J_,.2). F a c t o r XII i s o l a t e d f r o m e i t h e r s o u r c e a p p e a r s t o be a s i n g l e - c h a i n glycop r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 75,000. F a c t o r XI l a h a s a l s o been i s o l a t e d f r o m b o v i n e p l a s m a by F u j i k a w a e t a l . ( 3 ) . B o v i n e f a c t o r XI l a i s composed o f a heavy and a l i g h t c h a i n h e l d t o g e t h e r by d i s u l f i d e bonds. The l i g h t c h a i n h a s a m o l e c u l a r w e i g h t o f 28,000 and c o n t a i n s an a m i n o - t e r m i n a l s e q u e n c e o f Val-Val-Gly-Gly ( 3 ) , The a m i n o - t e r m i n a l s e q u e n c e o f the heavy c h a i n (mol wt 46,000) i s T h r - P r o - P r o - T r p - L y s - and i s i d e n t i c a l t o t h a t o b s e r v e d f o r the s i n g l e - c h a i n The c a r b o x y l - t e r m i n a l residu T h u s , the c o n v e r s i o n o f b o v i n e f a c t o r X I I t o f a c t o r XI l a i s due t o t h e c l e a v a g e o f a s i n g l e i n t e r n a l a r g i n y l - v a l i n e p e p t i d e bond. F a c t o r XI l a e x h i b i t e d a m i d a s e a c t i v i t y t o w a r d arginine-containing s u b s t r a t e s and i s r e a d i l y i n h i b i t e d by t h e s e r i n e p r o t e a s e i n h i b i t o r , d i i s o p r o p y l p h o s p h o f l u o r i d a t e (DFP). T h i s i n h i b i t o r i s bound e x c l u s i v e l y t o t h e l i g h t c h a i n o f f a c t o r XI l a i n d i c a t i n g t h a t the a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h i s c h a i n . Furthermore, the a c t i v e s i t e region i n t h e l i g h t c h a i n o f f a c t o r XI l a c o n t a i n s an A s p - A l a - C y s - G l n - G l y - A s p - S e r - G l y - G l y - s e q u e n c e w h i c h i s t y p i c a l o f the o t h e r p l a s m a s e r i n e p r o t e a s e s ( 2 ) . The mechanism whereby f a c t o r X I I i s a c t i v a t e d p h y s i o l o g i c a l l y i s p r e s e n t l y n o t c l e a r . M o r e o v e r , the p r o t e a s e ( s ) w h i c h c a r r i e s o u t t h i s r e a c t i o n has n o t been i d e n t i f i e d . The a c t i v a t i o n o f f a c t o r X I I h a s been t h o u g h t t o o c c u r in vivo f o l l o w i n g i t s i n t e r a c t i o n with a charged s u r f a c e which presumably i s s u b e n d o t h e l i a l collagen. The a c t i v a t i o n o f f a c t o r X I I has been i n v e s t i g a t e d i n the t e s t tube u t i l i z i n g g l a s s o r k a o l i n as the n e g a t i v e l y charged surface. I n c u b a t i o n o f s i n g l e - c h a i n f a c t o r XII w i t h k a o l i n prod u c e s a [ f a c t o r X l l - k a o l i n ] c o m p l e x w h i c h does n o t have e n z y m a t i c a c t i v i t y towards small s u b s t r a t e s . In a d d i t i o n , t h i s c o m p l e x i s not i n h i b i t e d by DFP s u g g e s t i n g t h a t f a c t o r X I I i s n o t c o n v e r t e d t o an a c t i v e enzyme s i m p l y by i t s i n t e r a c t i o n w i t h t h e k a o l i n surface. However, t h e [ f a c t o r X l l - k a o l i n ] c o m p l e x w i l l react w i t h f a c t o r XI and p r e k a l 1 i k r e i n c o n v e r t i n g t h e s e zymogens t o a c t i v e enzymes ( 3 ) . Whether f a c t o r X I I was c o n v e r t e d t o f a c t o r XI l a d u r i n g t h i s i n c u b a t i o n i s n o t known. G r i f f i n and C o c h r a n e (4) and Chan et^ aj_. (5) have r e p o r t e d t h a t human f a c t o r X I I i s a c t i v a t e d i n t h e p r e s e n c e o f k a i l i k r e i n , HMW k i n i n o g e n and k a o l i n . These s t u d i e s s u g g e s t t h a t f a c t o r X I I i n t e r a c t s s t o i c h i o m e t r i c a l l y w i t h HMW k i n i n o g e n on t h e k a o l i n s u r f a c e and i s s u b s e q u e n t l y a c t i v a t e d by k a l l i k r e i n . From t h i s i n f o r m a t i o n , i t i s c o n c e i v a b l e t h a t upon i n t e r a c t i o n w i t h a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
164
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
c h a r g e d s u r f a c e i n the p r e s e n c e o f HMW k i n i n o g e n , f a c t o r X\\ u n d e r g o e s a c o n f o r m a t i o n a l change w h i c h p e r m i t s i t s a c t i v e s i t e t o r e a c t s l o w l y and p r e f e r e n t i a l l y w i t h p r e k a l 1 i k r e i n . The r e l a t i v e l y s m a l l amount o f k a l l i k r e i n formed can r e c i p r o c a l l y a c t i v a t e s u b s t a n t i a l amounts o f f a c t o r X I f . Once formed i n s u f f i c i e n t l e v e l s , f a c t o r XI l a c a n * t h e n c a t a l y z e t h e a c t i v a t i o n o f t h e n e x t zymogen i n t h e c a s c a d e , namely f a c t o r X I . F a c t o r XI i s c o n v e r t e d t o f a c t o r X l a by f a c t o r XI l a , a r e a c t i o n a p p a r e n t l y augmented by HMW k i n i n o g e n and k a o l i n ( 6 ) . F a c t o r XI l a i s p r e s e n t l y t h e o n l y c o a g u l a t i o n p r o t e a s e known w h i c h can a c t i v a t e f a c t o r XI i n t h e t e s t t u b e . The a p p a r e n t o b l i g a t o r y r o l e o f f a c t o r XI l a i n the a c t i v a t i o n o f f a c t o r XI i s i n t e r e s t i n g i n v i e w o f the f a c t t h a t p a t i e n t s w i t h f a c t o r X I I d e f i c i e n c y have no d e m o n s t r a t i v e b l e e d i n g t e n d e n c i e s , y e t p a t i e n t s w i t h f a c t o r XI d e f i c i e n c y o f t e n b l e e d c o n s i d e r a b l y f o l l o w i n g i n j u r y or minor s u r g e r y . On possibl explanatio fo thi phe nomenon i s t h a t f a c t o r X I l - d e f i c i e n l e v e l s o f f a c t o r XII t coagulation g coagu l a t i o n p r o c e s s i s p r o p a g a t e d by an a m p l i f i c a t i o n mechanism, the s m a l l e s t l e v e l o f enzyme r e q u i r e d w o u l d be i n t h e i n i t i a l r e actions. An a l t e r n a t i v e e x p l a n a t i o n i s t h a t o t h e r h e r e t o f o r e unr e c o g n i z e d f a c t o r s may p l a y a m a j o r r o l e i n the p h y s i o l o g i c a l a c t i v a t i o n of f a c t o r XI, In t h i s c a s e , p r o t e o l y t i c c l e a v a g e o f f a c t o r XI may be a s e c o n d a r y r e a c t i o n c a t a l y z e d by f a c t o r XI l a and t h a t o t h e r p r o t e i n s s u c h as p r e k a 1 1 i k r e i n and p l a s m i n o g e n p r o a c t i v a t o r c o n s t i t u t e more s p e c i f i c p r i m a r y s u b s t r a t e s for f a c t o r XI l a . F a c t o r XI has been i s o l a t e d f r o m human and b o v i n e p l a s m a . Human f a c t o r XI i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 124,000. I t i s composed o f two i d e n t i c a l p o l y p e p t i d e c h a i n s (mol wt a b o u t 60,000) h e l d t o g e t h e r by d i s u l f i d e bonds. D u r i n g t h e a c t i v a t i o n o f f a c t o r XI an i n t e r n a l p e p t i d e bond i n e a c h o f the two c h a i n s i s c l e a v e d by f a c t o r XI l a ( 7 ) . T h i s g i v e s r i s e t o two heavy c h a i n s (mol wt 35,000) and two l i g h t c h a i n s (mol wt 25,000) in f a c t o r X l a . The a m i n o - t e r m i n a l amino a c i d s e q u e n c e o f the l i g h t chains in f a c t o r Xla i s I l e - V a l - G l y - G l y . T h i s sequence shows homology w i t h o t h e r p l a s m a s e r i n e p r o t e a s e s i n v o l v e d i n coagulation. The l i g h t c h a i n s a l s o c o n t a i n the a c t i v e s i t e s e r i n e w h i c h r e a c t s q u a n t i t a t i v e l y w i t h DFP. Thus, f a c t o r X l a i s a s e r i n e p r o t e a s e c o n t a i n i n g two a c t i v e s i t e s per m o l e c u l e o f enzyme, and r e p r e s e n t s t h e f i r s t c a s e o f a s e r i n e p r o t e a s e w i t h two c a t a l y t i c s i t e s ( 7 ) . The n e x t zymogen t o be a c t i v a t e d i n t h e i n t r i n s i c pathway i s f a c t o r IX. Human and b o v i n e f a c t o r IX have been i s o l a t e d and w e l l c h a r a c t e r i z e d i n t h e p a s t few y e a r s (8^,9). B o v i n e f a c t o r IX i s a s i n g l e - c h a i n g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000. Human f a c t o r IX i s a l s o a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 57,000 and p o s s e s s e s an a m i n o - t e r m i n a l amino a c i d s e q u e n c e n e a r l y i d e n t i c a l t o t h a t o f b o v i n e f a c t o r IX ( 9 ) .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
Coagulation
165
F a c t o r IX i s one o f t h e f o u r c o a g u l a t i o n f a c t o r s w h i c h r e q u i r e v i t a m i n K f o r i t s b i o s y n t h e s i s . The o t h e r f a c t o r s i n c l u d e p r o t h r o m b i n , f a c t o r X and f a c t o r Vitamin K p a r t i c i p a t e s i n the b i o s y n t h e s i s o f these p r o t e i n s i n t h e post-ribosoma1 carboxyl a t i o n o f s p e c i f i c g l u t a m i c a c i d r e s i d u e s p r e s e n t i n t h e aminoterminal p o r t i o n o f these p r o t e i n s (10). The new amino a c i d , y-carboxyglutamic a c i d , i s e s s e n t i a l f o r c a l c i u m i o n b i n d i n g by t h e s e p r o t e i n s and t h e i r s u b s e q u e n t I n t e r a c t i o n w i t h a c i d i c p h o s p h o l i p i d membranes. The y - c a r b o x y g l u t a m i c a c i d residues i n these p r o t e i n s tend t o o c c u r as a d j a c e n t r e s i d u e s w h i c h f a c i l i t a t e t h e c o o r d i n a t i o n o f one c a l c i u m i o n p e r p a i r o f y - c a r b o x y g l u t a m i c a c i d r e s i d u e s (JJ_). F a c t o r IX i s a c t i v a t e d by f a c t o r X l a i n t h e p r e s e n c e o f c a l cium ions i n a two-step process. In t h e f i r s t s t e p , s i n g l e - c h a i n f a c t o r IX i s c o n v e r t e d t o a t w o - c h a i n m o l e c u l e by t h e c l e a v a g e o f an i n t e r n a l A r g - A l a p e p t i d a c t i o n has no e n z y m a t i (mol wt 16,000) w i t h an a m i n o - t e r m i n a l sequence o f T y r - A s n - S e r G l y , and a heavy c h a i n (mol wt 38,000) w i t h an a m i n o - t e r m i n a l sequence o f A l a - G l y - T h r - I l e . T h i s i n t e r m e d i a t e i s then c o n v e r t e d t o f a c t o r IXa i n a r a t e - l i m i t i n g s t e p by l i m i t e d p r o t e o l y s i s . In t h i s r e a c t i o n , a s p e c i f i c A r g - V a l bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e h e a v y c h a i n i s c l e a v e d by f a c t o r X l a w i t h t h e c o n c o m i t a n t r e l e a s e o f an a c t i v a t i o n p e p t i d e . T h u s , t h e f a c t o r IXa formed i n t h i s r e a c t i o n i s composed o f two c h a i n s h e l d t o g e t h e r by d i s u l f i d e bonds. The heavy c h a i n (mol wt 27,300) c o n t a i n s t h e active s i t e serine residue and c o n t a i n s an a m i n o - t e r m i n a l s e quence o f V a l - V a l - G l y - G l y . The l i g h t c h a i n (mol wt 1 6 , 6 0 0 ) , w h i c h o r i g i n a t e s f r o m t h e a m i n o - t e r m i n u s o f t h e zymogen, c o n t a i n s the y-carboxyglutamic a c i d r e s i d u e s and t h e a m i n o - t e r m i n a l s e quence o f T y r - A s n - S e r - G l y ( l _ 2 ) . F a c t o r IXa i s t h e s e r i n e p r o t e a s e w h i c h c o n v e r t s f a c t o r X t o f a c t o r Xa by l i m i t e d p r o t e o l y s i s i n t h e i n t r i n s i c pathway. In t h i s r e a c t i o n , f a c t o r IXa f o r m s a m a c r o m o l e c u l a r c o m p l e x w i t h an a d d i t i o n a l p r o t e i n , f a c t o r V I I I , i n the presence o f p h o s p h o l i p i d and c a l c i u m i o n s . F a c t o r V I I I i s t h e plasma p r o t e i n t h a t i s a b s e n t i n i n d i v i d u a l s w i t h c l a s s i c h e m o p h i l i a , t h e most p r e v a l e n t c o a g u l a t i o n d i s o r d e r known. F a c t o r V I I I has been i s o l a t e d f r o m human and b o v i n e plasma and e x h i b i t s a m o l e c u l a r w e i g h t o f a b o u t one m i l l i o n (l_3,]_4). These p r e p a r a t i o n s p o s s e s s potent coagulant a c t i v i t y a s w e l l as von W i l l e b r a n d p l a t e l e t a g g r e g a t i n g a c t i v i t y . Treatment o f t h e bovine f a c t o r V I I I p r e p a r a t i o n w i t h c a t a l y t i c amounts o f b o v i n e t h r o m b i n r e s u l t s i n a 2 5 - f o l d i n c r e a s e i n f a c t o r V I I I procoagulant a c t i v i t y i n t h e presence o f CaCl2. Gel f i l t r a t i o n o f t h i s thrombin-treated preparation o f f a c t o r V I M r e s u l t s i n t h e s e p a r a t i o n o f f a c t o r V I M c o a g u l a n t a c t i v i t y and the p l a t e l e t a g g r e g a t i n g a c t i v i t y (l_5). However, no d e t e c t a b l e p r o t e i n was a s s o c i a t e d w i t h t h e f a c t o r V I M c o a g u l a n t a c t i v i t y . T h i s has l e d t o s p e c u l a t i o n t h a t t h e f a c t o r V I M r e p r e s e n t s o n l y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
166
GLYCOLIPIDS IN
DISEASE PROCESSES
a small f r a c t i o n o f the t o t a l p r o t e i n i n the f a c t o r V M t prepar a t i o n s d e s c r i b e d (15). The r o l e o f f a c t o r V I M in the a c t i v a t i o n o f f a c t o r X appears t o be one o f a r e g u l a t o r y n a t u r e . Conceivably, factor VMI could augment t h i s r e a c t i o n by p r o m o t i n g t h e f o r m a t i o n o f a more r e a c t i v e c o n f o r m a t i o n i n e i t h e r f a c t o r IXa o r i t s s u b s t r a t e , f a c t o r X. The f i n a l s t e p i n t h i s pathway i s t h e a c t i v a t i o n o f f a c t o r X, F a c t o r X i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000 and one o f t h e most w e l l c h a r a c t e r i z e d p r o t e t n s i n b l o o d coagulation. I n d e e d , t h e t o t a l amino a c i d s e q u e n c e o f b o v i n e f a c t o r X has been completed (16). i t c o n s i s t s o f a h e a v y c h a i n (mol wt 39,300) and a l i g h t c h a i n (mol wt 16,500) h e l d t o g e t h e r by d i s u l f i d e b o n d s . The h e a v y c h a i n c o n t a i n s 307 amino a c i d r e s i d u e s w i t h two g l y c o s y l m o i e t i e s l i n k e d t o Asn-35 and T h r - 3 0 0 ( 1 6 ) . The heavy c h a i n a l s o c o n t a i n s the a c t i v e s i t serin located I p o s i t i o 233 Th l i g h t c h a i n c o n t a i n s th acid residues. The c o n v e r s i o n o f b o v i n e f a c t o r X t o f a c t o r Xa by f a c t o r IXa o c c u r s as a r e s u l t o f t h e c l e a v a g e o f a s i n g l e p e p t i d e bond between Arg-51 and M e - 5 2 , T h i s r e s u l t s i n t h e r e l e a s e o f an a c t i v a t i o n p e p t i d e (mol wt 10,500) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f the heavy c h a i n a n a l o g o u s t o t h a t o b s e r v e d in the a c t i v a t i o n o f f a c t o r IX. F a c t o r Xa i s a s e r i n e p r o t e a s e w h i c h , i n t h e p r e s e n c e o f f a c t o r V, p h o s p h o l i p i d and c a l c i u m i o n s , c a t a l y z e s t h e c o n version of prothrombin to thrombin. B.
The
Activation
of
F a c t o r X by
the
Extrinsic
Pathway
The p r o p o s e d s e r i e s o f r e a c t i o n s w h i c h r e s u l t i n t h e a c t i v a t i o n o f f a c t o r X by t h e e x t r i n s i c pathway a r e d e p i c t e d i n F i g u r e 2. In t h e s e r e a c t i o n s , p l a s m a f a c t o r V I I , i n t h e p r e s e n c e o f c a l c i u m i o n s , f o r m s a c o m p l e x w i t h a l i p o p r o t e i n r e l e a s e d f r o m damaged tissue. T h i s c o m p l e x s u b s e q u e n t l y c o n v e r t s f a c t o r X t o f a c t o r Xa which appears i n d i s t i n g u i s h a b l e from t h a t observed f o l l o w i n g i n t r i n s i c a c t i v a t i o n o f f a c t o r X (T7). Present evidence suggests that f a c t o r VII i s c o n v e r t e d to f a c t o r V i l a in o r d e r to c a t a l y z e this reaction. The l i p o p r o t e i n s e r v e s as a h i g h m o l e c u l a r w e i g h t c o f a c t o r which i s a b s o l u t e l y e s s e n t i a l f o r the e x p r e s s i o n o f factor Vila proteolytic activity. F a c t o r VII i s a t r a c e plasma g l y c o p r o t e i n . I t s low c o n c e n t r a t i o n has v i r t u a l l y p r e c l u d e d t h e i s o l a t i o n o f t h i s p r o t e i n f r o m human p l a s m a . F a c t o r V I I has been i s o l a t e d f r o m b o v i n e plasma as a s i n g l e - c h a i n p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 45,500 (l_8,l_9). In the t e s t t u b e , s i n g l e - c h a i n f a c t o r V I I can be c o n v e r t e d t o a t w o - c h a i n p r o t e i n w h i c h e x h i b i t s 30-40 t i m e s t h e c o a g u l a n t a c t i v i t y o f t h e s i n g l e - c h a i n f o r m . The a c t i v a t i o n o f f a c t o r V I I , t h a t i s , t h e f o r m a t i o n o f t w o - c h a i n f a c t o r V I I , has been o b s e r v e d f o l l o w i n g t h e i n c u b a t i o n o f f a c t o r V I I w i t h f a c t o r XI l a ( 2 0 ) , f a c t o r X l a ( 2 0 , k a l l i k r e i n ( 2 J J , f a c t o r Xa ( 2 2 ) , and
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
167
Coagulation
t h r o m b i n (22). F a c t o r V M a i s composed o f a h e a v y c h g i n (mol wt 34,000) and a l i g h t c h a i n (mol wt 23,000) h e l d t o g e t h e r by d i s u l f i d e bonds. The a c t i v e s i t e s e r i n e i s l o c a t e d In t h e heavy c h a i n which c o n t a i n s t h e a m i n o - t e r m i n a l sequence o f I l e - V a l - G l y Gly (20). The l i g h t c h a i n c o n t a i n s t h e y - c a r b o x y g l u t a m i c a c i d r e s i d u e s and a r i s e s f r o m t h e a m i n o - t e r m i n a l p o r t i o n o f t h e s i n g l e c h a i n zymogen ( 2 0 ) , The c a r b o x y l - t e r m i n a l residue o f the l i g h t chain i s arginlne. Thus, f a c t o r V t l a Is a s e r i n e p r o t e a s e a r i s ing from t h e p r o t e o l y s i s o f a s i n g l e i n t e r n a l A r g - l l e p e p t i d e bond. F a c t o r V i l a has an a b s o l u t e r e q u i r e m e n t f o r t i s s u e f a c t o r i n o r d e r t o c l e a v e t h e A r g - 5 1 - M e - 5 2 p e p t i d e bond In t h e heavy c h a i n o f f a c t o r X, The p h y s i o l o g i c a l mechanism f o r t h e a c t i v a t i o n o f f a c t o r V I I i s p r e s e n t l y n o t known. W h i l e s e v e r a l s e r i n e p r o t e a s e s t h a t p a r t i c i p a t e i n t h e I n t r i n s i c pathway w i l l c o n v e r t f a c t o r V M t o f a c t o r V i l a in vitvo i t i c i p a t e i n theactivatio factor X i stheonly protei t h e p r o t e o l y t i c r a n g e o f f a c t o r V M a has n o t been Investigated, W h i l e c o n s i d e r a b l e i n f o r m a t i o n h a s a c c u m u l a t e d on t h e m o l e c u l a r p r o p e r t i e s o f f a c t o r V M , c o m p a r a t i v e l y l i t t l e i s known r e g a r d i n g i t s i n t e r a c t i o n w i t h p u r i f i e d t i s s u e f a c t o r and t h e i n t e r a c t i o n o f f a c t o r X w i t h t h i s [ f a c t o r V l l - t i s s u e f a c t o r ] complex. The t i s s u e f a c t o r a p o p r o t e i n , t h e p r o t e i n component o f t h e t i s s u e f a c t o r l i p o p r o t e i n , h a s been i s o l a t e d f r o m s e v e r a l t i s s u e s . The most h i g h l y c h a r a c t e r i z e d a p o p r o t e i n p r e p a r a t i o n a p p e a r s t o be o b t a i n e d f r o m human b r a f n m i c r o s o m e s ( 2 3 ) , T i s s u e f a c t o r apop r o t e i n prepared i n t h i s study i s a s i n g l e - c h a i n glycoprotein c o n t a i n i n g a b o u t 6% c a r b o h y d r a t e and e x h i b i t e d a m o l e c u l a r w e i g h t o f 52,000 by SDS g e l e l e c t r o p h o r e s i s . The t i s s u e f a c t o r a p o p r o t e i n e x h i b i t s no c o a g u l a n t a c t i v i t y i n t h e a b s e n c e o f phospholipid. A d d i t i o n o f mixed p h o s p h o l i p i d s t o t h e a p o p r o t e i n , however, p a r t i a l l y restores the tissue factor coagulant a c t i v i t y . The r o l e o f t i s s u e f a c t o r a p p e a r s t o be s i m i l a r t o t h a t o f f a c t o r V I M i n t h a t i t s e r v e s t o enhance t h e l i m i t e d p r o t e o l y s i s o f f a c t o r X by f a c t o r V i l a s e v e r a l o r d e r s o f m a g n i t u d e . No e n z y m a t i c a c t i v i t y has been o b s e r v e d a s s o c i a t e d w i t h homogeneous preparations o f tissue factor. Moreover, i n c u b a t i o n o f pure bovine f a c t o r VII w i t h c r u d e , h i g h l y a c t i v e p r e p a r a t i o n s o f t i s s u e f a c t o r does n o t r e s u l t i n t h e f o r m a t i o n o f t w o - c h a i n , a c t i v a t e d f a c t o r V I I (21_). y
C.
Prothrombin A c t i v a t i o n
by A c t i v a t e d
Factor X
P r o t h r o m b i n i s c o n v e r t e d t o t h r o m b i n by f a c t o r Xa i n t h e p r e s e n c e o f c a l c i u m i o n s , p h o s p h o l i p i d and f a c t o r Va ( F i g u r e 3 ) . P r o t h r o m b i n and i t s mechanism o f a c t i v a t i o n has been i n t e n s i v e l y i n v e s t i g a t e d o v e r t h e l a s t two d e c a d e s , and t h e d e t a i l s o f t h i s complex r e a c t i o n a r e r e a s o n a b l y c l e a r (24). Prothrombin i s a g l y c o p r o t e i n with a molecular weight o f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
168
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
SURFACE HMW KININOGEN PREKALLIKREIN
FACTOR X I I
FACTOR
XII
FACTOR
IX
FACTOR CA
[FACTOR
2 +
,
IX
A
PLj FACTOR
IX -FACTOR 3
VIII
A
2 +
VIII -CA A
-PL]
Figure 1. Abbreviated mechanisms for the activation of factor X by the intrinsic pathway
FACTOR
XII
FACTOR
XI
; ]
; L
KALLIKREIN FACTOR
X
A
THROMBIN
FACTOR V I I
FACTOR ?
+
CA-
Figure 2. Abbreviated mechanisms for the activation of factor X by the extrinsic pathway
[FACTOR V I I
; 1
VII
-CA
T ISSUE 2 +
FACTOR
-TISSUE
FACTOR X
FACTOR X
; L
|
FACTOR
FACTOR]
X
1
:L
C A , PL j FACTOR V 2 +
;L
[FACTOR X^-FACTOR V ,-CA -PL] ;
2+
FACTOR XIII
PROTHROMBIN
Figure 3. Mechanisms for the formation of cross-linked fibrin following the activation of factor X
FIBRINOGEN j
2+
FACTOR XII I
A
FIBRIN (CROSS-LINKED)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
169
Coagulation
70,000. I t i s o n e o f t h e f o u r v i t a m i n K-dependent c l o t t i n g f a c t o r s a n d c o n t a i n s 10 y - c a r b o x y g l u t a m f c acid residues, I t s c o n c e n t r a t i o n i n plasma f a r exceeds t h a t o f any o t h e r c o a g u l a t i o n zymogen. The amino a c i d s e q u e n c e o f b o v i n e p r o t h r o m b i n h a s been d e t e r m i n e d (25) and t h e s e q u e n c e o f human p r o t h r o m b i n Is n e a r i n g completion. B o v i n e p r o t h r o m b i n c o n t a i n s 582 amino a c i d r e s i d u e s w i t h 12 d i s u l f i d e b r i d g e s . The t h r e e c a r b o h y d r a t e c h a i n s o f bovine prothrombin a r e N - g l y c o s f d i c a l l y l i n k e d t o asparagine r e s i d u e s a t p o s i t i o n s 7 7 , 101 and 376 ( 2 5 ) , The c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n o c c u r s a s a t w o s t e p p r o c e s s on t h e s u r f a c e o f a m a c r o m o l e c u l a r a g g r e g a t e commonly r e f e r r e d t o a s t h e " p r o t h r o m b i n a s e c o m p l e x . " T h i s c o m p l e x c o n s i s t s o f f a c t o r X a , f a c t o r V a , p h o s p h o l i p i d and c a l c i u m i o n s . P r e - t r e a t m e n t o f f a c t o r Xa w i t h DFP o b v i a t e s t h e c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n i n d i c a t i n g t h a t f a c t o r Xa i s t h e a c t i v e p r i n c i p a l i n t h i s complex f a c t o r , appears t o f u n c t i o cofactor in this reaction. Its a b i l i t y to potentiate this r e a c t i o n i s i n c r e a s e d s e v e r a l f o l d by p r e - t r e a t m e n t w i t h f a c t o r Xa or thrombin (26). The f i r s t s t e p i n t h e a c t i v a t i o n o f p r o t h r o m b i n i sthe cleavage o f a p e p t i d e bond between A r g - 2 7 4 and T h r - 2 7 5 by f a c t o r X a . T h i s r e a c t i o n r e l e a s e s a l a r g e I n a c t i v e g l y c o p e p t i d e fragment (mol wt 3 3 , 5 0 0 ) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f p r o t h r o m b i n and y i e l d s a s i n g l e - c h a i n t h r o m b o g e n i c p r o t e i n c a l l e d i n t e r m e d i a t e 11, T h i s p r o t e i n h a s no c o a g u l a n t a c t i v i t y a n d h a s a m o l e c u l a r w e i g h t of 39,000. I n t e r m e d i a t e II i s s u b s e q u e n t l y c l e a v e d by f a c t o r Xa a t a p e p t i d e bond between A r g - 3 2 3 a n d l l e - 3 2 4 t o y i e l d two-chain thrombin. T h r o m b i n I s composed o f a heavy c h a i n (mol wt 3 2 , 0 0 0 ) and a l i g h t c h a i n (mol wt 5 , 7 0 0 ) h e l d t o g e t h e r by a d i s u l f i d e bond. The heavy c h a i n o f t h r o m b i n a l s o c o n t a i n s t h e a c t i v e s i t e serine residue. D.
Formation
of Cross-linked
Fibrin
The c o n v e r s i o n o f f i b r i n o g e n t o f i b r i n monomer by t h r o m b i n I s t h e p e n u l t i m a t e s t e p i n t h e b l o o d c o a g u l a t i o n scheme. Fibrinogen i s a l a r g e g l y c o p r o t e i n (mol wt 3 ^ 0 , 0 0 0 ) composed o f t h r e e p a i r s of non-identical polypeptide chains. These c h a i n s , d e s i g n a t e d a , 8 and y , a r e h e l d t o g e t h e r by s e v e r a l d i s u l f i d e bonds. The m o l e c u l a r w e i g h t s f o r t h e a , 8 and y c h a i n s o f human f i b r i n o g e n a r e 6 3 , 5 0 0 , 5 6 , 0 0 0 and 4 7 , 0 0 0 , r e s p e c t i v e l y (2]_). T h u s , t h e s u b u n i t s t r u c t u r e f o r f i b r i n o g e n may be e x p r e s s e d a s a282Y2. During t h econversion o f f i b r i n o g e n t o f i b r i n , four s p e c i f i c a r g i n y l - g l y c i n e p e p t i d e bonds i n t h e a and 8 c h a i n s a r e c l e a v e d by t h r o m b i n w i t h t h e c o n c o m i t a n t r e l e a s e o f f o u r f i b r i n o p e p t i d e s . Two f i b r i n o p e p t i d e s A a r e r e l e a s e d f r o m t h e a m i n o - t e r m l n u s o f t h e a c h a i n s a n d two f i b r i n o p e p t i d e s B a r e r e l e a s e d f r o m t h e amino-terminal end o f t h e 8 c h a i n s . Subsequent t o t h e r e l e a s e o f t h e s e f i b r i n o p e p t I d e s , t h e f i b r i n monomers r a p i d l y a g g r e g a t e
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
170
GLYCOLIPIDS IN DISEASE PROCESSES
i n an o r d e r l y p r o c e s s t o f o r m t h e " s o l u b l e " f i b r i n p o l y m e r . This f i b r i n polymer i s s o l u b l e i n d e n a t u r a n t s such as urea o r g u a n i d i n e - h y d r o c h l o r i d e , and i s r e a d i l y a t t a c k e d by t h e f i b r i n o l y t i c protease, plasmin. C o n v e r s i o n o f t h e " s o l u b l e " f i b r i n p o l y m e r t o an " I n s o l u b l e " f i b r i n c l o t i s t h e terminal r e a c t i o n i n blood c o a g u l a t i o n . This r e a c t i o n i s c a t a l y z e d by a c t i v a t e d f a c t o r X I I I i n t h e p r e s e n c e o f calcium ions. In t h i s r e a c t i o n , i n t e r m o l e c u l a r p e p t i d e c r o s s l i n k a g e s a r e formed by f a c t o r XI I l a between an amino g r o u p f r o m a s p e c i f i c l y s i n e r e s i d u e on o n e f i b r i n monomer and a g l u t a m i n y l r e s i d u e o f an a d j a c e n t f i b r i n monomer ( 2 8 ) . Two e ( y - g l u t a m y l ) l y s i n e p e p t i d e bonds a r e r a p i d l y formed between i n t e r m o l e c u l a r y chains. Upon l o n g e r i n c u b a t i o n , c r o s s l i n k a g e s a r e a l s o o b s e r v e d between i n t e r m o l e c u l a r a c h a i n s . The t o t a l number o f c r o s s l i n k a g e s a p p e a r s t o be a b o u t s i x p e r mole o f f i b r i n , two of which i n v o l v e cross 1inkage w i t chain wit the a c h a i n s . The p r e s e n c f i b r i n polymer which i g q u i t e r e s i s t a n t t o plasmin d i g e s t i o n . F a c t o r XI I l a , t h e t r a n s g l u t a m i n a s e w h i c h c a t a l y z e s t h e c r o s s l i n k i n g r e a c t i o n s i n f i b r i n , c i r c u l a t e s i n plasma i n a p r e c u r s o r form. In t h e p r e s e n c e o f c a l c i u m i o n s , f a c t o r X I I I i s c o n v e r t e d by t h r o m b i n t o f a c t o r XI I l a by l i m i t e d p r o t e o l y s i s . F a c t o r X I I I i s a g l y c o p r o t e i n composed o f two A c h a i n s (mol wt 7 5 , 0 0 0 ) and two B c h a i n s (mol wt 8 8 , 0 0 0 ) . The n a t i v e p r o t e i n i s a t e t r a mer w i t h a m o l e c u l a r w e i g h t o f 3 2 0 , 0 0 0 ( 2 9 ) . Factor XIII i s a c t i v a t e d by t h r o m b i n i n a t w o - s t e p p r o c e s s . In t h e f i r s t s t e p , t h r o m b i n c l e a v e s an a r g i n y l - g l y c i n e bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e A c h a i n s , g i v i n g r i s e t o an i n a c t i v e t e t r a m e r i c i n t e r m e d i a t e and two p e p t i d e s (mol wt 4 , 0 0 0 ) . In t h e n e x t s t e p , which r e q u i r e s c a l c i u m i o n s , t h e t e t r a m e r i c intermediate d i s s o c i a t e s i n t o a c a t a l y t i c d i m e r ( A ' 2 ) and an i n a c t i v e d i m e r o f B chains (30). A l t h o u g h t h e A' c h a i n s a r e p r e s u m a b l y i d e n t i c a l , t h e a c t i v e c y s t e i n e r e s i d u e i s f o u n d i n o n l y one o f t h e c a t a l y t i c subunits. T h u s , f a c t o r XI I l a a p p e a r s t o be an e x a m p l e o f t h e " h a l f - o f - t h e - s i t e r e a c t i v i t y " enzymes ( 3 1 ) . E.
Mechanism lation
Involving P r o t e o l y s i s i n theTermination
o f Coagu-
Under normal p h y s i o l o g i c a l c o n d i t i o n s , t h e a c t i v a t i o n o f a zymogen t h r o u g h p r o t e o l y s i s i s e s s e n t i a l l y an i r r e v e r s i b l e p r o cess. A c c o r d i n g l y , s e v e r a l c o n t r o l mechanisms a r e a v a i l a b l e t o r e g u l a t e and t e r m i n a t e c o a g u l a t i o n f r o m s p r e a d i n g t h r o u g h o u t t h e circulation. P e r h a p s t h e p r i n c i p a l mechanism f o r t h e r e g u l a t i o n o f t h e a c t i v a t e d c o a g u l a t i o n f a c t o r s i s t h e i r n e u t r a l i z a t i o n by s e r i n e protease i n h i b i t o r s found i n t h e blood. Indeed, about 10% o f t h e p r o t e i n i n p l a s m a i s s e r i n e p r o t e a s e i n h i b i t o r s ( 3 2 ) . A n o t h e r i m p o r t a n t mechanism f o r t h e c o n t r o l o f c o a g u l a t i o n i n v o l v e s t h e i n a c t i v a t i o n o f s p e c i f i c c l o t t i n g f a c t o r s by l i m i t e d
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
KISIEL
Blood
Coagulation
171
proteolysis. Although the d e t a i l s o f these reactions are j u s t b e g i n n i n g t o emerge, i t a p p e a r s t h a t t h r o m b i n i s c e n t r a l t o t h i s process. E x c e s s t h r o m b i n f o r m a t i o n i s c a r e f u l l y c o n t r o l l e d by several negative feedback r e a c t i o n s e i t h e r i n i t i a t e d o r d i r e c t l y c a t a l y z e d by t h r o m b i n . As p o i n t e d o u t e a r l i e r , p r o t e o l y t i c m o d i f i c a t i o n o f f a c t o r V and f a c t o r VI If by t h r o m b i n e n c h a n c e s t h e coagulant a c t i v i t y o f these p r o t e i n s several f o l d . Continued i n c u b a t i o n o f these p r o t e i n s w i t h t h r o m b i n , however, r e s u l t s i n t h e loss o f t h e i r coagulant a c t i v i t y (15,33). Thrombin a l s o c a t a l y z e s t h e r a p i d c l e a v a g e o f a p e p t i d e bond between A r g - 1 5 6 and Ser-157 i n prothrombin g i v i n g r i s e t o a non-thrombogenic fragment (mol wt 23,000) and a t h r o m b o g e n i c i n t e r m e d i a t e I (mol wt 5 5 , 0 0 0 ) . Intermediate I i s loosely associated with the "prothrombinase c o m p l e x " and c o n s e q u e n t l y c l e a v e d by f a c t o r Xa a t a much s l o w e r r a t e than p r o t h r o m b i n . Hence, t h e o v e r a l l e f f e c t i s t o reduce thrombin f o r m a t i o n as a R e c e n t l y , a new v i t a m i c o v e r e d and t e n t a t i v e l y i d e n t i f i e d a s P r o t e i n C ( J 4 J , Protein C i s a g l y c o p r o t e i n composed o f a heavy c h a i n (mol wt 40,000) and a l i g h t c h a i n (mol wt 22,000) h e l d t o g e t h e r by d i s u l f i d e bonds. Incubation o f P r o t e i n C with thrombin r e s u l t s i n the cleavage o f a s i n g l e p e p t i d e bond between A r g - 1 4 and l l e - 1 5 i n t h e heavy chain w i t h the concomitant formation o f a c t i v a t e d P r o t e i n C (35). A c t i v a t e d P r o t e i n C i s a s e r i n e p r o t e a s e and t h e a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h e heavy c h a i n . The amino a c i d sequence s u r r o u n d i n g t h e a c t i v e s i t e s e r i n e r e s i d u e i n a c t i v a t e d P r o t e i n C i s homologous t o t h e a c t i v e s i t e r e g i o n s o f t h e s e r i n e proteases involved i n blood coagulation. Activated Protein C m a r k e d l y p r o l o n g s t h e c l o t t i n g t i m e o f p l a s m a , and t h i s a n t i c o a g u l a n t e f f e c t was t o t a l l y o b v i a t e d by p r i o r i n c u b a t i o n o f t h e enzyme w i t h DFP. R e c e n t e v i d e n c e i n d i c a t e s t h a t a c t i v a t e d P r o t e i n C, i n t h e p r e s e n c e o f c a l c i u m i o n s and p h o s p h o l i p i d , i s a p o t e n t i n a c t i v a t o r o f f a c t o r V and f a c t o r V I M ( 3 5 , 3 6 ) » and t h a t t h e s e i n a c t i v a t i o n r e a c t i o n s were a b s o l u t e l y d e p e n d e n t on t h e e n z y m a t i c a c t i v i t y o f a c t i v a t e d P r o t e i n C. T h u s , i t a p p e a r s t h a t the l e v e l o f t h r o m b i n a c t i v i t y i s c a r e f u l l y r e g u l a t e d by n e g a t i v e feedback r e a c t i o n s which i n v o l v e the l i m i t e d p r o t e o l y s i s o f prot h r o m b i n , f a c t o r V and f a c t o r V I M e i t h e r d i r e c t l y c a t a l y z e d by thrombin o r i n d i r e c t l y through the formation o f a c t i v a t e d P r o t e i n C.
Literature Cited 1. 2. 3.
Griffin, J . H., and Cochrane, C. G. (1976) Methods Enzymol. 45, 56-65. Fujikawa, K., Walsh, K. A . , and Davie, E. W. (1977) Biochemistry 16, 2270-2278, Fujikawa, K., Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 4182-4188.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
172
4.
Griffin, J . H., and Cochrane, C. G. (1976) Proc. Natl. Acad. Sci.
5. 6. 7.
USA 73, 2554-2558.
Chan, J . Y. C., Habal, K. M., Burrowes, C. E . , and Movat, H. Z. (1976) Thromb. Res. 9, 423-430. Wiggins, R. C., Bouma, B. N . , Cochrane, C. G., and Griffin, J. H. (1977) Proc. Natl. Acad. Sci. USA 74, 4636-4640. Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 58315839.
8. 9. 10. 11.
DiScipio, R. G., Hermodson, M. A . , Yates, S. G., and Davie, E. W. (1977) Biochemistry 16, 698-706. Fujikawa, K., Thompson, A. R., Legaz, M. E . , Meyer, R. G., and Davie, E. W. (1973) Biochemistry 12, 4938-4945. Stenflo, J., Fernlund, P., Egan, W., and Roepstorff, P. (1974) Proc. Natl. Acad. Sci. USA 71, 2730-2733. Furie, B. C., Mann, K. G., and Furie, B. (1976) J. Biol. Chem. 251, 3235-3241
12. 13.
14. 15.
16.
Fujikawa, K., Legaz Biochemistry 13, 4508-4516 McKee, P. A . , Anderson, J . C., and Switzer, M. E. (1975) Ann. N.Y. Acad. Sci. 240, 8-33. Legaz, M. E . , Weinstein, M. J., Heldebrant, C. M., and Davie, E. W. (1975) Ann.N.Y.Acad. Sci. 240, 43-61. Vehar, G. A . , and Davie, E.W.(1977)Science 197, 374-376. Titani, K., Fujikawa, K., Enfield, D. L., Ericsson, L. H . , Walsh, K. A . , and Neurath, H. (1975) Proc. Natl. Acad. S c i . USA 72, 3082-3086.
17. 18.
Fujikawa, K., Coan, M. H . , Legaz, M. E . , and Davie, E. W. (1974) Biochemistry 13, 5290-5299. K i s i e l , W., and Davie, E. W. (1975) Biochemistry 14, 49284934.
19.
Radcliffe, R., and Nemerson, Y. (1975) J. Biol . Chem. 250, 388-395.
20.
21. 22.
K i s i e l , W., Fujikawa, K., and Davie, E. W. (1977) Biochemistry 16, 4189-4194. Kisiel, W., unpublished data. Radcliffe, R., and Nemerson, Y. (1976) J. Biol. Chem. 251 4797-4802.
23. 24.
Bjorklid, E . , and Storm, E. (1977) Biochem. J. 165., 89-96. Suttie, J . W., and Jackson, C. M. (1977) Physiol. Reviews 57,
25.
26.
1-70.
Magnusson, S., Peterson, T. E . , Sottrup-Jonsen, L . , and Claeys, H. (1975) in Proteases and Biological Control, eds. Reich, E . , Rifkin, D. B . , and Shaw, E. (Cold Spring Harbor Laboratory, New York), Vol. 2, pp. 123-149. Smith, C. M., and Hanahan, D. J. (1976) Biochemistry 15, 1830-1838.
27. 28.
McKee, P. A . , Rogers, L. A . , Marler, E . , and Hill, R. L. (1966) Arch. Biochem. Biophys. 116, 271-279. Lorand, L . , Downey, J., Gotoh, T., Jacobsen, A . , and Tokura, S. (1968) Biochem. Biophys. Res.Commun.31, 222-230.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
9.
29. 30. 31. 32.
33. 34.
35. 36.
KisiEL
Blood Coagulation
173
Schwartz, M. L., Pizzo, S, V., Hill, R, C., and McKee, P. A. (1973) J. Biol. Chem. 248, 1395-1407. Chung, S. I . , Lewis, M. C., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950. Levitzki, Α., Stallcup, W. Β., and Koshland, D. E. (1971) Biochemistry 10, 3371-3378. Heimburger, Ν., Haupt, Η., and Schwick, H. (1971) in Proc. Int. Res. Conf. Proteinase Inhibitors, eds. Fritz, H., and Tschesche, H. (W. de Gruyter, Berlin), pp. 1-21. Colman, R. W. (1969) Biochemistry 8, 1438-1445. Stenflo, J . (1976) J. Biol. Chem. 251, 355-363. Kisiel, W., Canfield, W. Μ., Ericsson, L. H., and Davie, E. W. (1977) Biochemistry 16, 5824-5831. Vehar, G., unpublished results.
RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10 Biosynthesis of Murine Leukemia Virus Membrane Proteins and their Assembly into Virus Particles R. B. ARLINGHAUS, E. C. MURPHY, JR., R. B. NASO, L. J. ARCEMENT, and W. L. KARSHIN Biology Department, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030
The cellular plasma membrane forms the surface layer of C-type RNA tumor virus particles. The c e l l ' s plasma membrane is also involved in one of the important phases of the l i f e cycle of RNA tumor viruses, that is the budding of the complete virus particles from the infected c e l l . C-type RNA tumor viruses contain a core structure surrounded by a l i p i d envelope through which spikes protrude. These spikes are believed to be composed, in mouse systems, of the v i r a l glycoprotein gp69/71 (1). Two non -glycosylated proteins termed p12(E) and p15(E) are also associated with the virion envelope. Their roles are not definitely known but recent studies suggest that gp69/71 and p15(E) exist as a complex and this complex forms the basic unit of the protruding spike (1, 2, 3). The core of these viruses is composed of four internal proteins termed p30, p15, pp12 (phosphoprotein) and p10 (4). The latter two have been shown to be associated with the v i r a l genomic RNA (5, 6). Viral p30 is thought to be the capsid protein of the core (4) whereas v i r a l protein p15 (a hydrophobic protein) could be involved in the association of the core with the l i p i d envelope. The virus also contains a DNA polymerase in its core. This polymerase can readily copy RNA into DNA with an appropriate p r i mer. It is present in virions in amounts estimated to be 50-100 molecules per virus particle (7) whereas the internal structural proteins are thought to be present in approximately 5,000 copies per virion (8). The envelope proteins [gp69/71, p15(E)] are also initially present in relatively large amounts but gp69/71 appears to be lost from the virion probably because of proteolytic degradation that releases the mature glycoprotein into the surrounding medium (9). 0-8412-0452-7/78/47-080-180$05.25/0
© 1978 American Chemical Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
181
These v i r a l p r o t e i n s are encoded by three d i f f e r e n t genes w i t h i n the 35S (-3 x 10^ daltons o r 10,000 bases) v i r a l genome. Each v i r a l p a r t i c l e i s thought to c o n t a i n two i d e n t i c a l copies of the RNA genome. The gene coding f o r the four i n t e r n a l core prot e i n s has been termed g a g o r group antigens; that coding f o r the v i r a l RNA-directed DNA polymerase i s termed p o l o r reverse t r a n s c r i p t a s e (RT); the envelope p r o t e i n s are coded w i t h i n the envelope gene or e n v (10). The purpose o f t h i s r e p o r t i s to summarize the events l e a d ing to the formation of the mature v i r a l envelope p r o t e i n s and t h e i r assembly i n t o v i r i o n s . We a l s o w i l l propose a model f o r v i r i o n assembly and budding of the v i r u s p a r t i c l e from the c e l l . f
f
f
f
f
f
The V i r i o n P r o t e i n s of Rauscher Murine Leukemia V i r u s Rauscher murine leukemia v i r u s (R-MuLV) coded p r o t e i n s can be r e s o l v e d by e l e c t r o p h o r e s i denaturing c o n d i t i o n s ( F i g can be seen are p30, p l 5 , pl2(E) and plO; minor amounts o f gp69/71 and pl5(E) are a l s o present i n the v i r i o n . A s u b s t a n t i a l amount o f an uncleaved precursor to the core p r o t e i n s , termed Pr65§ S, i s a l s o present i n v i r u s . The v i r a l phosphoprotein ppl2 i s not s o l u b i l i z e d by t h i s procedure but i t i s r e a d i l y seen i n v i r u s denatured w i t h 6 M quanidine-HCl and f r a c t i o n a t e d by molecu l a r s e i v e chromatography i n 6 M guanidine. The r a d i o a c t i v e v i r u s , whose p r o t e i n s are sljown i n F i g . 1, was prepared by i n c u b a t i n g i n f e c t e d c e l l s w i t h ^ C-arginine and l y s i n e f o r a 48 hr i n t e r v a l . Under these c o n d i t i o n s much of the v i r a l gp69/71 i s l o s t from the v i r u s p a r t i c l e (9). This l o s s of v i r a l gp69/71 may correspond to the low amount of pl5(E) that i s present i n mature v i r u s p a r t i c l e s (see below). Murine C-type v i r u s e s appear to c o n t a i n a complex made up of gp69/71 and pl5(E) (1, 2, 3). The gp69/71-pl5(E) complex can be d i s s o c i a t e d w i t h s u l f h y d r i l reagents such as mercaptoethanol (1, _2, 3) . This complex may be the b a s i c u n i t of the surface s t r u c ture of the v i r u s . V i r a l gp69/71 has been shown to be on the surface of the v i r u s by s e v e r a l methods. One i n v o l v e s l a c t o peroxidase-catalyzed i o d i n a t i o n of the v i r i o n s u r f a c e components (11, 12). Another i n v o l v e s sodium borohydride r e d u c t i o n f o l l o w ing galactose oxidase treatment (13). A t h i r d procedure concerns the use of immunoelectromicroscopy using monospecific a n t i s e r a r a i s e d against p u r i f i e d gp70 (14), and most important, anti-gp70 can n e u t r a l i z e i n f e c t i v i t y (15, 16). The surface l o c a t i o n o f pl5(E) i s i n d i c a t e d by the f i n d i n g that a n t i s e r a to pl5(E) can p r e c i p i t a t e i n t a c t v i r u s (17, 18, 19) u n l i k e a n t i s e r a prepared against v i r a l i n t e r n a l components. Thus, i t i s q u i t e evident that both pl5(E) and gp69/71 are present i n the same place i n the v i r i o n which i s c o n s i s t e n t w i t h t h e i r being complexed together. a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
182
Figure
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
1.
Preparative sodium dodecyl sulfate-polyacrylamide mature viral proteins
gel electrophoresis of
Rauscher murine leukemia virus (R-MuLV) was grown in a Balh/C spleen-thymus cell line (JLS-V5 cells) in the presence of C-arginine and lysine for 48 hr. Virus was purified as described (30), and viral proteins were separated by electrophoresis on a 11.25% polyacrylamide slab gel. The radioactive virus was distributed among the three sample wells after boiling in electrophoresis sample buffer (20). The radioactive bands were detected by fluorography using pre-flashed x-ray film so as to obtain a linear response to the amount of radiactivity (26). 14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
183
I n t r a c e l l u l a r V i r a l S p e c i f i c Glycoproteins V i r u s i n f e c t e d and u n i n f e c t e d NIH Swiss mouse embryo f i b r o b l a s t s (JLS-V16 c e l l s ) were incubated f o r 5-1/2 hr w i t h C glucosamine to detect v i r a l g l y c o p r o t e i n s . The cytoplasmic ext r a c t , t r e a t e d w i t h NP-40 and sodium deoxycholate detergents (20), was mixed w i t h an optimal c o n c e n t r a t i o n of antiserum r a i s e d i n r a b b i t s against p u r i f i e d R-MuLV that had been d i s r u p t e d by the above detergents (21). Figure 2 shows the r e s u l t s of f r a c t i o n a t i o n of the immunoprecipitates on a 10% sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l . Figure 2 lane A shows the r a d i o a c t i v e g l y c o p r o t e i n s were not observed i n u n i n f e c t e d c e l l s i n the area where two broad bands termed g P r 9 0 and gp69/71 were seen i n i n f e c t e d c e l l s ( F i g . 2, lane B). These r e s u l t s i n d i c a t e that two major g l y c o p r o t e i n s are present i n v i r a l i n f e c t e d c e l l s but not i n u n i n f e c t e d c e l l s . One of the g l y c o p r o t e i n s corresponds i n molecular weight to the other we have shown to b gp69/71 and the non-glycosylated p r o t e i n pl5(E) (see below and references 22, 23). Since both gp69/71 and g P r 9 0 r e a d i l y incorporate radioact i v e glucosamine, i t was o f i n t e r e s t to t e s t r a d i o a c t i v e l a b e l i n g w i t h other sugars. Of p a r t i c u l a r i n t e r e s t i s fucose s i n c e i t i s o f t e n a t e r m i n a l sugar i n g l y c o p r o t e i n s (24). Fucosyl tranferases are a l s o present i n plasma membranes o f the c e l l (25) thereby r e s t r i c t i n g f u c o s y l a t i o n to the l a t t e r stages of g l y c o p r o t e i n proc e s s i n g . With these p o i n t s i n mind, we then incubated i n f e c t e d c e l l s w i t h % - l a b e l e d fucose and p r e c i p i t a t e d the v i r a l glycoprot e i n s e i t h e r w i t h anti-RLV serum ( F i g . 2, lane C) or anti-gp69/71 ( F i g . 2, lane D). The r e s u l t s showed that g P r 9 0 d i d not i n corporate fucose whereas the mature v i r a l g l y c o p r o t e i n (gp69/71) d i d . Lanes G and H of F i g . 2 a l s o show that v i r i o n gp69/71 a l s o i n c o r p o r a t e s r a d i o a c t i v e fucose as w e l l as glucosamine. Thus, gPr90 appears to c o n t a i n glucosamine but l a c k s fucose and d i s t i n g u i s h e s i t c l e a r l y from the mature gp69/71. In a p a r a l l e l experiment v i r u s - i n f e c t e d c e l l s were incubated w i t h r a d i o a c t i v e glucosamine f o r 5-1/2 hr and the cytoplasmic ext r a c t was d i v i d e d i n t o two p a r t s . One p o r t i o n was immunoprecipit a t e d w i t h anti-gp69/71 ( F i g . 2, lane E) and the other p o r t i o n was i s o l a t e d by anti-R-MuLV immunoprecipitation ( F i g . 2, lane F ) . Again the major v i r a l glucosamine-containing g l y c o p r o t e i n s recogn i z e d bv e i t h e r sera were gp69/71 and g P r 9 0 . We note that n ti~R-MuLV sera a l s o p r e c i p i t a t e d a minor glucosamine-containing g l y c o p r o t e i n that migrates j u s t slower than g P r 9 0 ( F i g . 2, lanes B and F ) . This g l y c o p r o t e i n may be r e l a t e d to gag' gene products because i t i s not recognized by anti-gp69/71 but i t i s seen w i t h anti-RLV. Anti-RLV serum contains a n t i b o d i e s t o g a g and 'env' determinants but not to ' p o l determinants (26). I n f r e q u e n t l y another p r o t e i n was a l s o detected i n the anti-R-MuLV p r e c i p i t a t e from glucosamine l a b e l e d i n f e c t e d c e l l s . I t comi1 4
e n v
e n V
e n v
e n v
a
e n v
f
T
f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
f
184
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
a
grates w i t h Pr2008 S~P°l which i s the precursor to the v i r i o n r e verse t r a n s c r i p t a s e (26, 27, 28). Kabat and co-workers have r e ported t h a t F r i e n d leukemia v i r u s i n f e c t e d c e l l s c o n t a i n 'gag gene p r o d u c t - r e l a t e d g l y c o p r o t e i n s of 95,000 and 200,000 mol. wt. (29). The l a t t e r were found i n a d d i t i o n to the v i r a l 'env' gener e l a t e d g l y c o p r o t e i n s . The r o l e of the 'gag' gene-related g l y c o p r o t e i n s i n the v i r u s l i f e c y c l e i s unknown. To show the s p e c i f i c i t y of immunoprecipitation, R-MuLV i n f e c t e d c e l l s were p u l s e - l a b e l e d w i t h -^C-amino a c i d s f o r 10 min and p a r a l l e l c u l t u r e s were p u l s e - l a b e l e d f o r 10 min and then i n cubated i n complete growth medium f o r 60 min (chase). Cytoplasmic e x t r a c t s were p r e c i p i t a t e d w i t h monospecific antiserum d i r e c t e d a g a i n s t R-MuLV gp69/71 and p30 (22). Anti-gp69/71 p r e c i p i tated g P r 9 0 almost e x c l u s i v e l y i n p u l s e - l a b e l e d c e l l s ( F i g . 3, lane A) and both g P r 9 0 and gp69/71 i n chase incubated c e l l s ( F i g . 3, lane B). Anti-p30 serum, however, d i d not recognize gPr90 i n pulse-labele mainly precursors Pr80g A f t e r chase i n c u b a t i o n ( F i g . 3, lane D), a n a l y s i s of the anti-p30 p r e c i p i t a t e showed t h a t Pr80S § disappeared and P r 6 5 was r e duced i n i t s i n t e n s i t y w h i l e p30 appeared as a major band. No gp69/71 or g P r 9 0 were p r e c i p i t a t e d by a n t i s e r a d i r e c t e d a g a i n s t 30. The k i n e t i c s of v i r a l g l y c o p r o t e i n s y n t h e s i s were s t u d i e d by p u l s e - l a b e l i n g c e l l s w i t h -^C-amino a c i d s and chasing r e p l i c a t e c u l t u r e s f o r 2, 4 and 6 hr p e r i o d s . The r e s u l t s c l e a r l y showed that gp69/71 was not l a b e l e d i n a 10 min p u l s e - l a b e l i n g ( F i g . 4, lane B) but became r a d i o a c t i v e between 60 and 120 min of the chase i n c u b a t i o n ( F i g . 4, lane E) w h i l e a t the same time there was a s l i g h t decrease i n the i n t e n s i t y of g P r 9 0 . Chase incubat i o n s of up to 6 hr s t i l l showed a s i g n i f i c a n t amount of g P r 9 0 v but the r a t i o of gp69/71 to g P r 9 0 reached a value greater than one whereas a f t e r 2 hr of chase i t was l e s s than one. As p r e v i o u s l y r e p o r t e d , p r e c i p i t a t i o n of c e l l u l a r e x t r a c t s w i t h antiserum to d i s r u p t e d v i r u s revealed other v i r a l p r o t e i n s (e.g. p30 and p l 5 ) t h a t appeared upon chase at the expense of Pr80gag and Pr65§ 8 (20, 21, 2_2, 30) ( F i g . 4, lanes A, D, G, and J ) . V i r a l pl2(E) a l s o appeared during long chase periods (2 to 6 hr) whereas only pl5(E) was seen i n short chase periods (1 h r ) . 1
e n v
e n v
e n v
a
g a g
e n v
P
e n v
en
e n v
a
Presence of gp69/71 and p!5(E) Peptide Sequences i n g P r 9 Q e n V
e n V
We have p r e v i o u s l y shown t h a t g P r 9 0 and gp69/71 c o n t a i n many t r y p t i c peptides that comigrate on i o n exchange columns (22). However, the p a t t e r n s obtained were q u i t e complex, to s i m p l i f y the complexity of the t r y p t i c d i g e s t p r o f i l e s and the subsequent comparisons of the peptide sequences, we e l e c t e d to tag the v i r a l p r o t e i n s w i t h r a d i o a c t i v e t y r o s i n e (23, 30). F i g u r e 5 shows i o n exchange p r o f i l e s i n which l ^ C - t y r o s i n e - l a b e l e d g P r 9 0 tryptic peptides were mixed w i t h t r y p t i c peptides of % - t y r o s i n e l a b e l e d e n v
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
ARLINGHAUS E T A L .
Figure 2.
Murine
Leukemia
Virus Membrane
Pohjacrylamide gel electrophoresis of R-MuLV glycoproteins from infected cells and virus.
Proteins
185
specific
Cytoplasmic extracts from uninfected (lane A) and R-MuLV-infected NIH Swiss mouse embryo fibroblasts (JLS-V16 cells) (lane B) were labeled for 5V2 hr with C-glucosamine and the anti-R-MuLV immunoprecipitates were analyzed as in Figure 1. Cytoplasmic extracts from R-MuLV-infected JLS-V15 cells were labeled with H-fucose were immunoprecipitated with anti-gp69/71 serum (lane C) or anti-R-MuLV serum (lane D). R-MuLVinfected cells were labeled for 5 /2 hr with C-glucosamine and the antigp69/71 immunoprecipitate (lane E) or the anti-R-MuLV immunoprecipitate (lane F) also were analyzed. Columns G and H are H-fucose and C-glucosa7nine-labeled virus, respectively, obtained form growth medium from R-MuLV-infected JLS-V16 cells following a SV2 hr labeling period. 14
3
l
14
3
14
Figure 3. Analysis of intracellular RMuLV specific proteins precipitated by anti-gp69/71 and anti-p30 sera. Cytoplasmic extracts from R-MuLV-infected JLS-V16 cells labeled with C-labeled amino acids were prepared and immunoprecipitation was performed with either anti-gp69/71 or anti-p30 serum. Electrophoresis on 10% gels andfluorographywere as in Figure 1. Column A, anti-gp69/71 immune precipitate from a 10-min pulse labeling; column B, antigp69/71 immune precipitate from a 10-min pulse labeling with a 60 min chase; column C, anti-p30 immune precipitate from a 10min pulse labeling; column D, anti-p30 immune precipitate of a 10-min pulse labeling with a 60-min chase incubation. 14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 4. Formation chase experiment Cytoplasmic extracts from infected JLS-V16 cells were prepared, and immunoprecipitation was performed as described. Electrophoresis on 6-12% gradient gels and fluorography were carried out as in Figure 1. Columns Vi are C-labeled amino acid marker virus; columns A, B, and C, 10 min pulse with Clabeled amino acid protein hydrolysate; columns D, £, and F, 2-hr chase incubation of the 10-min pulse labeling; columns G, 77, and 7, 4-hr chase; columns J, K, and L, 6-hr chase; columns A, D, G, and J, anti-R-MuLV precipitated; columns B, E, 77, and K, anti-gp69/71 precipitated; and columns C, F, 7, and L, precipitated with anti-MuLV absorbed with excess R-MuLV proteins. 14
14
Figure 5. Ion exchange chromatography of tryptic digests of tyrosine-labeled gFr90 and gp69/71. The arrows show the two additional tyrosine-containing tryptic peptides in gPr90 that are not found in gp69/71. €nv
env
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
187
gp69/71. The r e s u l t s c l e a r l y show that g P r 9 0 shares many t y r o s i n e - c o n t a i n i n g t r y p t i c peptides w i t h gp69/71. Figure 5 a l s o shows that there are at l e a s t two a d d i t i o n a l t y r o s i n e - c o n t a i n i n g t r y p t i c peptides i n g P r 9 0 t h a t are not found i n gp69/71 (see arrows i n F i g . 5). An a n a l y s i s of a -*- C-tyrosine-labeled pl5(E) t r y p t i c d i g e s t mixed w i t h a ^ H - t y r o s i n e - l a b e l e d g P r 9 0 digest ( F i g . 6) c l e a r l y showed that these two a d d i t i o n a l t r y p t i c peptide f r a c t i o n s are d e r i v e d from p l 5 ( E ) . We conclude from these r e s u l t s that pl5(E) and gp69/71 are cleavage products o f g P r 9 0 . e n v
e n v
4
e n v
e n v
The Gene Order of the P r o t e i n s W i t h i n g P r 9 Q
e n V
e t l V
The order of gp69/71 and pl5(E) w i t h i n g P r 9 0 was examined by i n c u b a t i n g i n f e c t e d c e l l s w i t h r a d i o a c t i v e amino a c i d s i n the presence and absence of pactamycin, a drug that s e l e c t i v e l y i n h i b i t s the i n i t i a t i o n of t r a n s l a t i o n at 5 x 10""^ M but not e l o n g a t i o n (31, 32). In th t i a l l y l a b e l the C-termina In the case of g P r 9 0 , we could determine which p r o t e i n i s a t the C-terminus. R e s u l t s of such s t u d i e s (23) showed that pactamycin treatment f o r 30 to 60 seconds before and during p u l s e l a b e l i n g reduced the amount of r a d i o a c t i v e gp69/71 formation i n a chase i n c u b a t i o n , whereas r a d i o a c t i v e pl5(E) was increased r e l a t i v e to that found i n the absence of the drug. The r e s u l t s were q u a n t i t a t e d y i e l d i n g a r a t i o of pl5(E) to gp69/71 of 0.39 i n the c o n t r o l and 0.76 i n the presence of pactamycin. These r e s u l t s suggest that pl5(E) i s C-terminal i n g P r 9 0 . e n v
fenv
V i r a l P r o t e i n p!2(E) We have i d e n t i f i e d a new v i r a l p r o t e i n , termed p l 2 ( E ) , i n v i r u s p a r t i c l e s . I t can be d i s t i n g u i s h e d from phosphoprotein p l 2 (ppl2) on s e v e r a l grounds. F i r s t pl2(E) i s not phosphorylated (23), i t migrates i n the v o i d volume f r a c t i o n of a 6 M guanidineHC1 column whereas ppl2 e l u t e s i n the 12,000 mol. wt. range (23), and t h i r d l y i t has a peptide map much d i f f e r e n t from ppl2 (23); and l a s t l y , pl2(E) and ppl2 can be r e s o l v e d by 2-dimensional g e l s c o n s i s t i n g of i s o e l e c t r i c f o c u s i n g i n the f i r s t dimension and sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l e l e c t r o p h o r e s i s (PAGE) i n the second (23). We emphasize the p o i n t that i s o l a t i o n of pl2(E) from v i r u s by SDS-PAGE o f t e n y i e l d s p l 2 ( E ) contaminated w i t h fragments of p l 5 and p30. We have found i t necessary to use a two-step procedure i n v o l v i n g guanidine-HCl agarose column chromatography f o l l o w e d by SDS-PAGE i n order to o b t a i n a r e l a t i v e l y pure p r e p a r a t i o n of p l 2 ( E ) . The r o l e of pl2(E) i n v i r u s s t r u c t u r e i s not known. I t i s tempting to propose that i t i s a degradation product of pl5(E) and i s the r e s u l t of a p r o t e o l y t i c cleavage of the gp69/71-pl5(E) complex r e s u l t i n g i n the l o s s o f gp69/71 from the v i r i o n (4, 9) and the generation of an uncomplexed pl2(E) remaining i n the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
0--0
3
H-1yrosine labeled g P r 9 0
20
40
60
80
env
100
120
140
160
180
Fraction Number
Figure 6. Ion exchange chromatography of tryptic digests of tyrosine-labeled gPr90 and pl5(E). The pl5(E) used in this experiment was purified from virus by sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described in the legend to Figure 1. env
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
189
v i r u s envelope. We propose that t h i s p r o t e o l y t i c cleavage occurs a f t e r the v i r u s i s r e l e a s e d from the c e l l i n t o the c u l t u r e medium. We have shown t h a t as R-MuLV p a r t i c l e s age i n the c u l t u r e medium i n which they were r e l e a s e d , such p a r t i c l e s show a steady l o s s o f gp69/71 (Arlinghaus, unpublished r e s u l t s ) . C e l l - F r e e Synthesis of an Unglycosylated P15(E)
Precursor t o gp69/71 and
C e l l - f r e e t r a n s l a t i o n o f genomic RNAs from murine and avian RNA tumor v i r u s e s has shown that the f u l l - l e n g t h 35S RNA can code f o r the core p r o t e i n precursor P r 6 5 S 8 (28, 33, 34, 35. 36, 37) as w e l l as f o r a l a r g e r p o l y p r o t e i n c o n t a i n i n g both 'gag' and p o l gene products (_28, 34, 3 9 ) . The e n v gene product has never been observed to be coded f o r by f u l l l e n g t h v i r i o n genomic RNA although the i n f o r m a t i o n f o r t h i s gene product i s d e f i n i t e l y encoded i n th In order t o determin could be synthesized from i n t r a c e l l u l a r RNA from v i r a l i n f e c t e d c e l l s , we i s o l a t e d 18-25S RNA and 25-35S RNA from a t o t a l RNA ext r a c t by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . These RNA c l a s s e s were again f r a c t i o n a t e d on separate sucrose g r a d i e n t s and i n d i v i d u a l f r a c t i o n s were analyzed i n a mRNA-dependent c e l l - f r e e amino a c i d i n c o r p o r a t i o n system (28^, 34) . The r e s u l t s o f t h i s experiment, d i s p l a y e d i n F i g . 7 (lanes E and F) showed that only RNA of about 20-22S i n s i z e , can be t r a n s l a t e d i n t o a polypeptide prec i p i t a b l e by antiserum to gp69/71. This polypeptide had an apparent molecular weight of 68,000 to 70,000. I n some experiments t h i s polypeptide migrated as two bands (a d o u b l e t ) . Polypeptides both l a r g e r and s m a l l e r than the 68,000 t o 70,000 were a l s o recogn i z e d by anti-gp69/71 serum. However, the smaller polypeptides were found i n a l l RNA f r a c t i o n s and the l a r g e r ones were found i n c o n s i d e r a b l y s m a l l e r q u a n t i t i e s than the 68,000 t o 70,000 mol. wt. polypeptide. The i d e n t i t y o f these s m a l l e r and l a r g e r polypept i d e s i s unknown. Immunoprecipitation o f the c e l l - f r e e t r a n s l a t i o n product of the 25S t o 35S RNA c l a s s w i t h anti-gp69/71 i n d i cated t h a t the 68,000 t o 70,000 mol. wt. polypeptide was absent ( r e s u l t s not shown). Hence, a 20-22S RNA seems to be the s o l e mRNA f o r an envelope-related p r o t e i n i n R-MuLV i n f e c t e d c e l l s . The t r a n s l a t i o n product o f each o f the RNA f r a c t i o n s i n the 18-25S and 25-30S s i z e c l a s s e s was a l s o t e s t e d by immunoprecipit a t i o n w i t h anti-p30 serum. The r e s u l t s showed that the g a g o r core p r o t e i n precursor Pr658 § and the RT precursor Pr200§ 8"P are synthesized e x c l u s i v e l y from RNA which i s greater than 28S i n s i z e ( r e s u l t s not shown). T r y p t i c peptide a n a l y s i s o f t h i s 65,000 mol. wt. product showed that i t had methionine-containing t r y p t i c peptides c h r a c t e r i s t i c of a u t h e n t i c P r 6 5 ^ ^ . We have p r e v i o u s l y e s t a b l i s h e d that P r 6 5 8 8 i s the major product and Pr2008 8"" i s a minor product i n c e l l - f r e e p r o t e i n s y n t h e s i z i n g systems o f 35S R-MuLV genomic RNA t r a n s l a t e d (34). a
f
f
?
T
f
a
a
a
a
f
a
pO
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
o1
190
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 7. Sodium dodecyl sulfate slab gel electrophoresis of the cell-free product produced from 20-22S mRNA from R-MuLV infected JLS-V5 cells. Total cellular nucleic acid was extracted from RMuLV-infected JLS-V5 cells as described (28), and RNA was selectively obtained by washing the nucleic acid alcohol precipitate with 3M LiClSmM EDTA = (pH 6) which removes DNA.The RNA was fractionated by velocity sedimentation on a sucrose gradient, and the 18S-to-25S RNA fraction was pooled and again separated on a sucrose gradient. Aliquots of the fractions (1 ^g of RNA) were analyzed in a mRNA-dependent protein synthesizing system (28,34). The protein products were immunoprecipitated with anti-gp69/71 serum and analyzed on a 6-12% gradient slab-gel of polyacrylamide, as in Figure 1. Lane A is a pulse-chase experiment from R-MuLV-infected JLS-V16 cells showing authentic viral specific protein precursors obtained with anti-R-MuLV serum (20). Lanes B-G represent aliquots across a gradient in which 18S ribosomal RNA sediments to the middle of the centrifuge tube (lane D). Lane B represents the top two fractions, lane G represents the bottom two fractions, and fractions C, D, E, and F represent the in-between fractions.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
191
Peptide Maps o f the Envelope-related P r o t e i n Synthesized From I n t r a c e l l u l a r 20-22S mRNA I f the 68,000 t o 70,000 mol. wt. polypeptide coded f o r by 20-22S mRNA i s r e l a t e d t o R-MuLV envelope precursor g P r 9 0 , i t should possess t r y p t i c peptides c h a r a c t e r i s t i c o f gp69/71 and p l 5 ( E ) . Therefore, 3 5 - e t h i o n i n e - l a b e l e d 68,000 t o 70,000 mol. wt. p o l y p e p t i d e product of 20-22S mRNA was digested w i t h t r y p s i n and mixed w i t h a u t h e n t i c g P r 9 0 t r y p t i c peptides l a b e l e d w i t h 3 -methionine. The mixture was f r a c t i o n a t e d by i o n exchange chromatography ( F i g . 8 ) . A n e a r l y p e r f e c t c o - e l u t i o n of t r y p t i c methionine-containing peptide was observed. The one exception being a minor peak ( F r a c t i o n 45) i n the i n v i t r o synthesized product that was n o t observed i n the a u t h e n t i c g P r 9 0 . The pept i d e peak e l u t i n g a t F r a c t i o n 76 i s c h a r a c t e r i s t i c of pl5(E) (20, 22) as i s the peptide peak a t F r a c t i o n 83 i f the t r y p t i c peptides are not o x i d i z e d w i t h performi c h a r a c t e r i s t i c o f gp69/7 s u l t s ) . We conclude from these experiments that the 68,000 t o 70,000 mol. wt. p o l y p e p t i d e termed P r 6 8 , i s i d e n t i c a l t o gPr90 i n o v e r a l l peptide content and P r 6 8 probably r e p r e sents the u n g l y c o s y l a t e d form of that molecule. e n v
S
m
e n v
H
e n v
e n v
e n v
e n v
T r a n s l a t i o n of F r a c t i o n a t e d R-MuLV Genomic RNA e n v
Since a 20-22S i n t r a c e l l u l a r mRNA species codes f o r P r 6 8 , i t was o f i n t e r e s t to determine whether RNA from v i r u s p a r t i c l e s had a s i m i l a r coding c a p a c i t y . V i r a l RNA p r e p a r a t i o n s obtained from v i r u s , t h a t has remained 24 h r i n the c u l t u r e f l u i d , have i n a d d i t i o n t o 35S RNA a range o f fragments o f the f u l l l e n g t h genome (35S) whose sedimentation r a t e v a r i e s down to 4S. T r a n s l a t i o n of v a r i o u s s i z e c l a s s e s o f v i r i o n RNA f r a c t i o n a t e d on a sucrose grad i e n t i n d i c a t e d that a s i g n i f i c a n t amount o f a polypeptide o f the size of P r 6 8 and p r e c i p i t a b l e by anti-gp69/71 i s coded f o r by v i r a l RNA of l e s s than f u l l l e n g t h s i z e . I t i s necessary t o obt a i n the t r y p t i c peptide map o f t h i s p o l y p e p t i d e to determine that i t i s a e n v gene product and not a g a g gene r e l a t e d p r o t e i n that i s a r t i f a c t u a l l y seen i n the anti-gp69/71 p r e c i p i t a t e . e n v
f
f
f
T
Model f o r the Synthesis and P r o c e s s i n g o f R-MuLV P r o t e i n s Our r e s u l t s and those o f others (40, 41, 42, 43) i n d i c a t e that there a r e a t l e a s t two s i z e c l a s s e s of mRNAs that code f o r R-MuLV p r o t e i n s . The f u l l - l e n g t h genome s i z e 35S R-MuLV contains genes termed gag* o r core p r o t e i n s , p o l o r the DNA polymerase and e n v o r the envelope p r o t e i n s . However, only the g a g and p o l genes appear t o be t r a n s l a t e d from 35S genomic RNA (28^, 34, 39). Our f i n d i n g s i n d i c a t e that a s m a l l e r s i z e c l a s s of mRNA (-22S) codes f o r the envelope p r o t e i n s . Studies i n the a v i a n system i n d i c a t e the gene order w i t h i n the genome of non-defective ?
f
T
f
f
T
T
f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
T
192
GLYCOPROTEINS AND
GLYCOLIPIDS IN DISEASE PROCESSES
avian sarcoma v i r u s e s (another C-type RNA tumor v i r u s ) i s : 5'gag-pol-env-src -poly(A)-3 (44). Given the same gene order i n the R-MuLV genomic RNA (e.g. 'gag-pol-env'), our r e s u l t s i n d i c a t e t h a t only the f i r s t two genes are t r a n s l a t e d from v i r i o n RNA. Furthermore, our r e s u l t s suggest t h a t only one ribosome o f 15 t o 25, that has completed the t r a n s l a t i o n o f the 'gag gene, proceeds i n t o the ' p o l ' gene (26, 28, 34) ( F i g . 9 ) . This l a t t e r process we have termed read-through (28, 34) and the product o f t h i s read-through event i s a 200,000 mol. wt. p o l y p r o t e i n termed Pr200§ ° . I t contains peptide sequences and a n t i g e n i c determinants o f the 'gag' p r o t e i n and the v i r a l DNA polymerase (26, 27, 28, 34). We have shown i t to be the precursor o f the v i r i o n reverse t r a n s c r i p t a s e (26, 27). The d e t e c t i o n o f both R T - s p e c i f i c and core p r o t e i n - s p e c i f i c a n t i g e n i c determinants i n a s i n g l e precursor p o l y p r o t e i n ( P r 2 0 0 S " ) r a i s e d the f o l l o w i n g i s s u e concerning the t r a n s l a t i o n o f the v i r a l genome gene products proceeds b precursor o n l y , then t h i s would e n t a i l the s y n t h e s i s o f equimolar amounts o f g a g and ' p o l ' gene products. V i r u s p a r t i c l e s c o n t a i n much l e s s polymerase than s t r u c t u r a l p r o t e i n s 07, 8 ) . Moreover, measurement of the steady s t a t e amount o f R T - s p e c i f i c sequences i n v i r u s - i n f e c t e d c e l l s has i n d i c a t e d t h a t these sequences are present i n much s m a l l e r amounts than the v i r a l s t r u c t u r a l p r o t e i n s C7, 45). This i n d i c a t e s t h a t i f RT polypeptide sequences are i n deed made i n equimolar amounts compared t o the core p r o t e i n s , they must be q u i c k l y degraded. We have been able t o shed more l i g h t on t h i s p o i n t by comparing the amount o f RT precursors t o the amount of core p r o t e i n precursors a f t e r p u l s e - l a b e l i n g o f i n f e c t e d c e l l s (26, 27). These c o n d i t i o n s should more c l e a r l y approximate the r a t e o f s y n t h e s i s o f these p o l y p e p t i d e s . Our measurement revealed that 1/25 t o 1/10 as much R T - s p e c i f i c precursors are present i n the c e l l , a f t e r a p u l s e - l a b e l i n g , as core p r o t e i n precursors (26) . Moreover, intermediate RT precursors appear to be formed by c l e a v age of preformed Pr2008 8""P°l, w h i l e formation o f core p r o t e i n precursors i s only compatible w i t h t h i s mechanism i f very f a s t o r nascent chain cleavage o f Pr2008 S~P°l takes p l a c e , which i s not supported by our s t u d i e s w i t h amino a c i d analogs o r protease i n h i b i t o r s (26). This makes i t u n l i k e l y t h a t the pathways of synt h e s i s o f 'gag' and ' p o l ' gene products only i n v o l v e the cleavage of a common precursor. Three p o s s i b l i t i e s are t h e o r e t i c a l l y compatible w i t h our f i n d i n g s . F i r s t , the mRNA (35S i n s i z e ) coding f o r P r 2 0 0 8 S ~ P i s a minor RNA species compared t o a s m a l l e r s i z e mRNA i n v o l v e d i n the s y n t h e s i s o f 'gag' gene products. Second, there i s only one species o f mRNA coding f o r both 'gag' and ' p o l ' , but a t r a n s l a t i o n a l c o n t r o l mechanism a l l o w s the s y n t h e s i s on the mRNA of more 'gag' than ' p o l ' gene products. A t h i r d p o s s i b i l i t y i s that two c l a s s e s o f s i m i l a r s i z e mRNA (e.g. 35S) e x i s t i n the i n f e c t e d c e l l . One codes f o r 'gag' and the other codes f o r 'gag-pol'. A ,
?
T
1
ag P
a g
p O
f
T
a
a
a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
o1
10.
Murine
ARLINGHAUS E T A L .
Figure 8. gPrQQcnv
Leukemia
Virus
Membrane
Proteins
193
Ion exchange chromatography of tryptic digests of authentic i 68,000 mol wt polypeptide (pr68 ) made from ~ 22S mRNA from R-MuLV-infected JLS-V5 cells.
anc
t
n
cnv
e
Pr68 was labeled with S-methionine in the cell-free mRNA-dependent protein synthesizing system (as in Figure 7). gPr90 " was isolated from R-MuLV-infected JLS-V16 cells labeled with H-methionine. The proteins were purified by immunoprecipitation with anti-gp69/71 and sodium dodecyl sulfate gel electrophoresis. The bands were digested with TPCK-trypsin and fractionated on a cation exchange column (30). env
35
c
v
3
Translotionol control
t
35SI mole P r 8 0 Pr65
Pol
Q>
Poly (A) -vw\
T
0'
gPr90
fl0g
H—-1
20S ,nv
viral mRNA
— I mole
-
,afl
4
\
Pr 27
Pr 42
4
4 \
pi5
\
gp 69/71
iZT gag-pol
\
pl2E
pl2 p30 plO
1/8 - 1/15 mole Pr 200
pl5E
1 pol
- Pr 145
I = initiation T = termination
•
Polymerase
Figure 9.
A model illustrating the overall steps involved in the synthesis and processing of Rauscher murine leukemia viral proteins
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
194
GLYCOLIPIDS IN DISEASE PROCESSES
c o n s i d e r a t i o n o f other a v a i l a b l e i n f o r m a t i o n favors the second o r t h i r d p o s s i b i l i t y . Thus, the major species o f v i r a l mRNA i n murine and a v i a n RNA tumor v i r u s - i n f e c t e d c e l l s has a s i z e o f 35S, but species o f 21S and 16S have a l s o been detected (40, 41, ^ 2 , 43). Moreover, f u r t h e r s t u d i e s i n d i c a t e that the 35S RNA i s the main species present i n polyribosomes p r e c i p i t a b l e w i t h anti-p30 mono-specific serum (46). Studies i n a v i a n systems a l s o suggest that the f u l l - l e n g t h genomic RNA i s the mRNA f o r 'gag and ' p o l ' gene products (38, 47). These observations make i t u n l i k e l y that there i s a s m a l l s i z e mRNA (-16S) on which the m a j o r i t y o f the 'gag gene products are synthesized and d i s f a v o r the p o s s i b i l i t y that a l a r g e s i z e species (-35S) o f v i r a l mRNA, such as one able to code f o r P r 2 0 0 , c o n s t i t u t e a minor p o p u l a t i o n . I t i s d i f f i c u l t t o e l i m i n a t e the p o s s i b i l i t y that there are indeed two c l a s s e s o f 35S o r f u l l - l e n g t h genomic s i z e RNAs, a major one coding f o r 'gag' gene products and a minor one coding f o r 'gag' and ' p o l ' gen that s t o r e d 35S v i r a l RN gene a t the expense o f the 'gag' gene precursors than f r e s h batches 35S v i r a l RNA (Murphy and A r l i n g h a u s , unpublished r e s u l t s ) . This r e s u l t i s c o n s i s t e n t w i t h the idea that some s t r u c t u r a l a l t e r a t i o n ^ ) of 35S RNA genomic i s r e s p o n s i b l e f o r P r 2 0 0 g g ~ P s y n t h e s i s . This a l t e r a t i o n could be brought i n t o p l a y by i n t e r a c t i o n o f the 35S v i r a l RNA w i t h a second molecule. A candidate f o r t h i s second molecule would be a RNA s i n c e pronease-treated v i r a l 35S RNA does not a l t e r the frequency o f P r 2 0 0 ~ P synt h e s i s over non-treated RNA (Murphy and A r l i n g h a u s , unpublished results). Our r e s u l t s i n d i c a t e t h a t the envelope gene i s probably never t r a n s l a t e d from f u l l length genomic s i z e RNA but that a -22S i n t r a c e l l u l a r mRNA serves as a mRNA f o r the envelope p r o t e i n . The p o l a r i t y o f t h i s mRNA i s unknown but a s i m i l a r v i r a l envelope mRNA from a v i a n sarcoma v i r u s i n f e c t e d c e l l s has the same p o l a r i ty as v i r a l genomic RNA (47, 48, 49). Therefore, we would assume that -22S mRNA composed o f sequences from the 3' o n e - t h i r d o f R-MuLV genomic RNA i s the mRNA f o r the envelope p r o t e i n s . We would a l s o assume that t h i s RNA i s capped and contains some unt r a n s l a t e d sequences found a t the 5' end o f the 35S genomic RNA (48). 1
1
g a g p
o
a
g a g
o1
o 1
Model f o r the Synthesis and P r o c e s s i n g o f V i r a l Envelope P r o t e i n s As the model i n F i g . 10 shows, the i n i t i a l t r a n s l a t i o n product of the -22S 'env' mRNA i s a 68,000 mol. wt. non-glycosylated polypeptide that contains peptide sequences o f the major g l y c o p r o t e i n (gp69/71) and non-glycosylated p l 5 ( E ) . The next step i s shown to be the g l y c o s y l a t i o n o f the p o l y p r o t e i n producing g P r 9 0 . A l t e r n a t e l y , i t i s possible that inside infected c e l l s g l y c o s y l a t i o n occurs p r i o r t o formation o f the complete 'env' gene product which would r e s u l t i n the immediate formation o f e n v
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
e n v
Membrane
Proteins
195
e n v
g P r 9 0 . I f t h i s were the case, P r 6 8 would only be seen under c o n d i t i o n s o f c e l l - f r e e t r a n s l a t i o n and not i n whole c e l l s . However, i t has not been e s t a b l i s h e d whether or not some P r 6 8 i s formed i n i n f e c t e d c e l l s . Cleavage and f u r t h e r g l y c o s y l a t i o n (fucose residues are added) of g P r 9 0 v y i e l d s gp69/71 and pl5(E) which probably e x i s t s as a complex held together by d i s u l f i d e bonds and p o s s i b l y a d d i t i o n a l non-covalent bonds. The b a s i c u n i t of the p r o t r u d i n g s p i k e - g l y c o p r o t e i n knob i s b e l i e v e d to be t h i s gp69/71-pl5(E) complex. The a s s o c i a t i o n of such b a s i c u n i t s would then r e s u l t i n formation of the spike-knob s t r u c t u r e that t r a v e r s e s and p r o j e c t s through the l i p i d envelope of the v i r u s ( F i g . 11). The r o l e of v i r a l p r o t e i n pl2(E) i n the s t r u c t u r e of the v i r i o n i s unknown. Since, i t does not appear to form a complex w i t h gp69/71 (50), we assume that i t may be an a r t i f a c t of aged v i r u s r e s u l t i n g from the p r o t e o l y s i s o f the spike-knob s t r u c t u r e which i n t u r n leads to a s m a l l p o r t i o n of the e n v
en
V i r u s Assembly Cleavage of v i r a l p o l y p r o t e i n s c o n s t i t u t e s a predominant f e a t u r e i n the assembly of many animal and b a c t e r i a l v i r u s e s ( f o r reviews see 51, J52, J53, 54) . RNA tumor v i r u s e s are no exception. We have shown that actinomycin D reduced the r a t e of cleavage of Pr65gag (55) and have suggested that the r a t e o f cleavage o f Pr65S £ to the mature v i r a l - p r o t e i n s i s c a t a l y z e d by the v i r a l genomic RNA. Recent experiments have a l s o shown that the c l e a v age r a t e of Pr200g "~ i s a l s o a f f e c t e d by i n h i b i t i n g v i r a l RNA s y n t h e s i s (Kopchick and A r l i n g h a u s , unpublished r e s u l t s ) . We note that the cleavage of Pr80S S to P r 6 5 g i s not a f f e c t e d under these c o n d i t i o n s nor i s the cleavage of g P r 9 0 . However, cleavages of Pr65gag and P r 2 0 0 " " P do occur (but at a slower r a t e ) i n the absence of v i r a l RNA and, i n t e r e s t i n g l y , the reverse t r a n s c r i p t a s e can be incorporated i n t o v i r i o n s that are d e f i c i e n t of v i r a l RNA (56). Based on a number of s t u d i e s from my own labor a t o r y (55), we have proposed a model f o r the assembly of R-MuLV p a r t i c l e s ( F i g . 11). In t h i s model, P r 6 5 g g r a t h e r than the mature v i r a l p r o t e i n s c o n s t i t u t e the i n i t i a l assembly u n i t . U n i t s of P r 6 5 g g assemble, presumably at the c e l l membrane, i n t o a s t r u c t u r e that remains open on the i n s i d e , due to the e l e c t r o s t a t i c r e p u l s i o n of the b a s i c p o r t i o n (plO) of P r 6 5 g g , which i s then cleaved to form the v i r a l p r o t e i n s i n c l u d i n g plO. V i r a l plO i n t u r n i s found i n the v i r i o n i n a s s o c i a t i o n w i t h the v i r a l RNA (5, 57). This model a l s o proposes that P r 2 0 0 g ~ i s incorpor a t e d i n t o the v i r i o n by means of i t s s i m i l a r i t y i n s t r u c t u r e t o P r 6 5 g . In f a c t , P r 2 0 0 g g " * contains a l l the a n t i g e n i c d e t e r minants and peptide sequences that are found i n P r 6 5 g g plus those of the reverse t r a n s c r i p t a s e . Thus, Pr200g g~P°l would s u b s t i t u t e f o r s m a l l number (30-50) o f the P r 6 5 molecules r e s u l t i n g a
ag
p
a
g a
e n v
gag
o1
a
a
a
g a
a g
a
p o 1
po1
a
a
g a g
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
196
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES gp69/71
20-22S
mRNA
-p15(E) -...poly (A)
Cap.
3'
Translation
Pr68
,
env
p15(E)
K
prvrvn
1
s Translation and / Glycosylation
Glycosylation
gPr90
LTTl
erv
|
V
f
l a E I
COOH
Carbohydrate Chains Cleavage and Fucosylation
gp69/71 -p15(E) Complex
gp69/71
p15(E)
s-s
Viral Spike and Knob Assembly Figure 10.
Synthesis of Rauscher murine leukemia virus envelope proteins
9a9 f>0
^-Pr200 - ' vRNA
-Unc leaved Pr65W
| Cleavage of Pr65W and Pr200W-i>
01
Mature core proteins Budding virus
Cell membrane Figure 11. A model showing the assembly and budding of Rauscher murine leukemia virus particles
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
197
i n the i n c l u s i o n o f RT i n the v i r i o n . _ A s u b s t a n t i a l amount o f cleavage o f P r 6 5 fend P r 2 0 0 ° ) i s proposed i n the model i n order to a l l o w v i r u s r e l e a s e . V i r a l RNA, due t o charge n e u t r a l i z a t i o n , c a t a l y z e s the cleavage o f Pr65§ and P r 2 0 0 " . T h i s r e s u l t s i n a more r a p i d r a t e o f assembly of RNA-containing v i r a l c o r e s , and, i f p r e c u r s o r c l e a v age i s viewed to p l a y a p a r t i n the budding process, i n a f a s t e r r a t e o f r e l e a s e o f RNA-containing v i r u s p a r t i c l e s . We f u r t h e r propose that host c e l l RNA can s u b s t i t u t e t o a c e r t a i n degree f o r v i r a l RNA i n c a t a l y z i n g the cleavages of P r 6 5 and P r 2 0 0 " P , but host c e l l u l a r RNA i s packaged to a much l e s s e r extent than v i r a l RNA s i n c e v i r u s i s o l a t e d a f t e r actinomycin D treatment exh i b i t e d a lower r a t i o o f RNA to p r o t e i n than c o n t r o l p r e p a r a t i o n s of v i r u s (55). T h i s suggests the e x i s t a n c e of s p e c i f i c r e c o g n i t i o n by the assembled v i r a l s t r u c t u r e s o f v i r a l genomic RNA. Whether the b a s i s f o r t h i s r e c o g n i t i o n l i e s i n the s p e c i f i c i n t e r a c t i o n between some o f the v i r a v i r a l RNA (6) remains t In the above mentioned model of assembly, we have p o s t u l a t e d t h a t cleavages o f P r 6 5 and P r 2 0 0 8 ~ P i s probably r e q u i r e d f o r v i r u s budding and r e l e a s e . There i s a p o s s i b i l i t y , however, that Pr658 8 and P r 2 0 0 ~ may be cleaved not d u r i n g budding but a f t e r r e l e a s e o f the v i r u s from the c e l l . For many y e a r s , C-type p a r t i c l e s have been observed to undergo a p o s t - r e l e a s e maturation process i n which the core center of the v i r i o n c o l lapses i n t o a condensed s t r u c t u r e that i s c l e a r l y d i f f e r e n t from the organized c o n c e n t r i c c o i l s observed i n the core of budding p a r t i c l e s (58, 59, 60). The s i g n i f i c a n c e o f t h i s maturation process remains u n c e r t a i n . The recent r e s u l t s of Yoshinaka and L u f t e g (61) suggest that there i s a p o s i t i v e c o r r e l a t i o n between the amount of P r 6 5 8 8 i n v i r a l cores and the amount o f immature cores. However, i t remains to be shown whether o r not the c l e a v age o f Pr658 8 can occur i n r e l e a s e d v i r i o n s and i f so that the changes i n r e l e a s e d v i r i o n s (immature to mature) a r e r e l a t e d t o precursor cleavage. A number o f _ l i n e s evidence suggest that cleavage o f P r 6 5 8 8 and P r 2 0 0 ° occur mainly p r i o r t o v i r a l r e l e a s e (62) and p r i o r t o the immature t o mature v i r u s t r a n s i t i o n that takes p l a c e i n the c u l t u r e f l u i d . 1
g a g
ag
g a g
P
p o
g a g
g a g
a
g a g
g a g
a g
g a g
o 1
o 1
P
a
a
a
g a g
P
Literature Cited 1. Pinter, A. and Fleissner, E. Virology (1977) 83, 417. 2. Leamnson, R. N., Shander, M. H. M. and Halpern, M. S. Virology (1977) 76, 437. 3. Witte, O. N., Tsukamoto-Adey, A. and Weissman, I . L. Virology (1977) 76, 539. 4. Bolognesi, D. P . , Montelaro, R. C., Frank, H. and Schafer, W. Science (1978) 199, 183. 5. Davis, J., Scherrer, M., Tsai, W. P . , and Long, C. J . Virol. (1976) 18, 709.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
198
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
6. Sen, A . , Sherr, C. J., and Todaro, G. J . Cell (1976) 7, 21. 7. Panet, A . , Baltimore, D., and Hanafusa, T. J . Virol. (1975) 16, 146. 8. Stromberg, K . , Hurley, N. E . , Davis, N. L., Rueckert, R. R., and Fleissner J . Virol. (1974) 13, 513. 9. Bolognesis, D. P., Langlois, A. J., and Schaffer, W. Virology (1975) 58, 550. 10. Baltimore, D. Cold Spring Harbor Symp. Quant. Biol. (1974) 39, 1187. 11. Kennel, S. H . , Del Villano, B. C., Levy, R. L., and Lerner, R. A. Virology (1973) 55, 464. 12. Witte, O. N . , Weissman, I. L., Kaplan, H. S. Proc. Natl. Acad. Sci. (1973) 70, 36. 13. McLellan, W. and August, J . T. J . Virol. (1976) 20, 627. 14. Leamnson, R. N. and Halpern, M. S. J . Virol. (1976) 18, 956. 15. Hunsmann, G., Moennig V . Pister L . Seifert E . and Schaefer, W. Virolog 16. Ikeda, H . , Pincus, Boyse, E . , and Mellars, R. C. J . Virol. (1974) 14, 1274. 17. Famulare, N. G., Buchhagen, D. L., Klenk, H. D., and Fleissner, E. J . Virol. (1976) 20, 501. 18. Ihle, J . N . , Domotor, J r . , J . J., Bengali, K. M. J . Virol. (1976) 18, 124. 19. Ihle, J . N . , Hanna, J r . , M. G., Schafer, W., Hunsman, G., Bolognesi, D. P., Huper, G. Virology (1975) 63, 60. 20. Arcement, L. J., Karshin, W. L., Naso, R. B . , Jamjoom, G. A. and Arlinghaus, R. B. Virology (1976) 69, 763. 21. Naso, R. B . , Arcement, L. J., and Arlinghaus, R. B. Cell (1975) 4, 31. 22. Naso, R. B . , Arcement, L. J., Karshin, W. L., Jamjoom, G. A . , and Arlinghaus, R. B. Proc. Natl. Acad. Sci. USA (1976) 73, 2325. 23. Karshin, W. L., Arcement, L. J., Naso, R. B . , and Arlinghaus, R. B. J . Virol. (1977) 23, 787. 24. Kraemer, P. "Biomembranes" Vol. 1, p. 67, Ed. Mauson, L. A. (1971). 25. Bosmann, H. B . , Hagopien, A . , and Eylar, E. H. Arch. Biochem. Biophys. (1968) 128, 470. 26. Jamjoom, G. A . , Naso, R. B . , and Arlinghaus, R. B. Virology (1977) 73, 11. 27. Kopchick, J. J., Jamjoom, G. A . , Watson, K. F . , and Arlinghaus, R. B. Proc. Natl. Acad. Sci. USA (1978) In press. 28. Murphy, Jr., E. C., Kopchick, J. J., Watson, K. F., and Arlinghaus, R. B. Cell (1978) 13, 369. 29. Evans, L. H . , Dreslar, S., and Kabat, D. J . Virol. (1977) 24, 865. 30. Arcement, L. J., Karshin, W. L., Naso, R. B . , and Arlinghaus, R. B. Virology (1977) 81, 284. 31. Taber, R., Rekosh, D., and Baltimore, D. J . Virol. (1971) 8, 395.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
10.
ARLINGHAUS E T A L .
Murine
Leukemia
Virus
Membrane
Proteins
199
32. Butterworth, B. E . , and Rueckert, R. R. J . Virol. (1972) 9, 823. 33. Naso, R. B . , Arcement, L. J., Wood, T. G., Saunders, T. E . , and Arlinghaus, R. B. Biochim. Biophys. Acta (1975) 383, 195. 34. Murphy, J r . , E. C., and Arlinghaus, R. B. Virology (1978) In press. 35. Kerr, I. M., Olshevsky, U . , Lodish, H . , and Baltimore, D. J. Virol. (1975) 18, 627. 36. Pawson, T., Martin, G. S., and Smith, A. E. J . Virol. (1976) 19, 950. 37. Von Den Helm, K . , and Duesberg, P. H. Proc. Natl. Acad. Sci. (1975) 72, 614. 38. Paterson, B. M., Marciani, D. J., and Papas, T. S. Proc. Natl. Acad. Sci. USA (1977) 74, 4951. 39. Philipson, L., Andersson, P., Olshevsky, U . , Weinberg, R., and Baltimore, D. Cell (1978) 13 189 40. Fan, H. and Baltimore 41. Gielkens, A. L. J., Bloemendal, H. Proc. Natl. Acad. Sci. USA (1976) 73, 356. 42. Shanmugam, G., Bhadure, S. and Green, M. Biochim. Biophys. Res. Commun. (1974) 56, 697. 43. Shincarial, A. and Joklik, W. Virology (1973) 56, 532. 44. Duesberg, P. H . , Wang, L. H . , Mellan, P . , Mason, W. S., and Vogt, P. K. "ICN-UCLA Symposia on Molecular and Cellular Biology" Vol. IV, pp 107. Edited by D. Baltimore, A. S. Huang, and C. F. Fox. Academic Press, Inc., New York, New York. 1976. 45. Chen, J . H. and Hanafusa, H. J . Virol. (1974) 13, 340. 46. Mueller-Lantsch, N. and Fan, H. Cell (1976) 9, 579. 47. Haywood, W. S. J . Virol. (1977) 24, 47. 48. Mellon, P. and Duesberg, P. H. Nature (1977) 270, 63. 49. Stacey, D. W., Allfrey, V. G., and Hanafusa, H. Proc. Natl. Acad. Sci. USA (1977) 270, 631. 50. Montelaro, R. C., Sullivan, S. J., and Bolognesi, D. P. Virology (1978) In press. 51. Casjens, S. and King, J . Annu. Rev. Biochem. (1975) 44, 555. 52. Hershko, A. and Fry, M. Annu. Rev. Biochem. (1975) 44, 775. 53. Korant, B. D. "Cold Spring Harbor Symposium on Control of Cell Proliferation" Vol. 21, p. 621. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1975. 54. Showe, M. H. and Kellenberg, E. "Control Processes in Virus Multiplication" p. 407. Cambridge University Press, Cambridge, England. 1975. 55. Jamjoom, G. A . , Naso, R. B . , and Arlinghaus, R. B. J . Virol. (1976) 19, 1054. 56. Levin, J . G., Grimley, P. M., Ramseur, J . M., and Berezesky, I. K. J . Virol. (1974) 14, 152. 57. Fleissner, E. and Tress, E. J . Virol. (1973) 12, 1612. 58. Fleissner, E . , Ikeda, H . , Tung, J . S., Vitetta, E . , Tress, E., Hard, J r . , W., Stockert, E . , Boyse, E. A . , Pincus, T., and
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
200
O'Donnell, P. Cold Spring Harbor Symp. Quant. Biol. (1975) 39, 1057.
59. Dalton, A. J. and Haguenau, F. "Ultrastructure of Animal Viruses and Bacteriophages" pp. 255. Eds. A. J. Dalton and F. Haguenau. Academic Press, New York, New York. 1973. 60. deHarven, E. Adv. Virus Res. (1974) 19, 221. 61. Yoshinaka, Y. and Luftig, R. B. Proc. Natl. Acad. Sci. USA (1977) 74, 3445.
62. Jamjoom, G. A . , Ng, V. L., and Arlinghaus, R. B. J . Virol. (1978) 25, 408. RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11 Glycoprotein Inhibitor of Lipoprotein Lipase from Aortic Intima—Its Possible Role in Atherosclerosis PREMANAND V. WAGH Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, AR 72206
Early observations th f glycoprotein i th mammalian arterial wal -Spreer et a l , 1960; Berenso , 1962) Althoug siderable research interest is centered upon the relationship of glycoproteins in the aortic wall to atherosclerosis and ageing, little is known in regard to the structure and function of these macromolecules. Several soluble glycoproteins have been purified from bovine aorta after extraction of the tissue with neutral buffers (Radhakrishnamurthy et a l , 1964, Maier and Buddecke, 1971). The concentration of arterial glycoproteins in atherosclerosis and in ageing is a subject of question and is being actively investigated. The wet weight of the aorta has been found to be increased in atherosclerosis and among the aged (Manley and Mullinger, 1967). Our data (Wagh et a l , 1973), shown in Table I, provide evidence that the glycoprotein concentration increased significantly in atherosclerotic tissue as expressed by an increase in total neutral sugars, hexosamine and sialic acid. These results suggest that the increased glycoprotein concentration may have a functional role in the formation of atherosclerotic plaque. Several functions have been ascribed to arterial glycoproteins such as transplantation rejection and maintenance of structural integrity (Anderson, 1976). Furthermore, Ishii (1971) demonstrated that preparations of crude glycoprotein obtained from various organs including the aorta of dog inhibited dextran sulfate released lipoprotein lipase (LPL) activity of human plasma. LPL catalyzes the hydrolysis of the triglyceride moiety of chylomicrons and very low density lipoproteins (Eisenberg and Levi, 1975), and is normally located on the surface of endothelial cells where it is physiologically active in the normal clearance of lipoprotein-bound triglycerides (Robinson, 1970). Since the surface of the intimal layer of aortic wall is susceptive to continuous hemodynamic insult and thus may be i n volved in the genesis of atherosclerosis and other physiological events, we focussed our attention here for the purpose of isolation of glycoprotein. One of the shortcomings in the isolation 201 This chapter not subject to U . S . copyright Published 1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
202
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
of p u r i f i e d g l y c o p r o t e i n from the i n t i m a l region has been to separate i t from the r e s t of the a o r t i c w a l l . A new procedure (dermatome procedure) was developed i n our l a b o r a t o r y (Roberts et a l , 1974) that r a p i d l y and r e l i a b l y separates the intima i n s u f f i c i e n t q u a n t i t i e s from both porcine and human a o r t a . Studies on Porcine I n t i m a l G l y c o p r o t e i n ( L i p o l i p i n ) . We r e ported on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a novel g l y c o p r o t e i n from the i n t i m a l r e g i o n of porcine aorta (Wagh and Roberts 1972). Swine a o r t a was chosen because i n t h i s s p e c i e s , the aorta i s r e l a t i v e l y f r e e from a t h e r o s c l e r o t i c l e s i o n s and the m a t e r i a l i s r e a d i l y a v a i l a b l e . The p u r i f i c a t i o n procedure involved e x t r a c t i o n of i n t i m a l t i s s u e with n e u t r a l b u f f e r , (Nlfy^SO^ p r e c i p i t a t i o n between 40-90% s a t u r a t i o n and two DEAE-cellulose chromatographic steps. The p u r i f i e d g l y c o p r o t e i n was homogeneous upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n (4.86 S) and by polyacrylamide disc-gel electrophoresis p r o t e i n was obtained fro Due to the presence of equimolar glucose and galactose and the absence of hydroxylysine i n the molecule, i t was suggested that t h i s g l y c o p r o t e i n was unique i n i t s c h a r a c t e r i s t i c s and t h e r e f o r e of a new type. The i s o e l e c t r i c p o i n t of the p u r i f i e d glycoprot e i n was 4.3 as observed by a n a l y t i c a l i s o e l e c t r i c focusing (Baig and Ayoub, 1976). Subsequent s t u d i e s (Roberts and Wagh, 1976) revealed that sodium dodecyl s u l f a t e - polyacrylamide g e l e l e c t r o p h o r e s i s of the n a t i v e and i t s S-carboxyamidomethyl d e r i v a t i v e a t d i f f e r e n t polyacrylamide concentrations d i d not a f f e c t the molecular weight (72,000 daltons) i n d i c a t i n g the absence of subunits. The carboxyt e r m i n a l amino a c i d was found to be s e r i n e . Attempts to determine the i d e n t i t y of the amino a c i d i n d i c a t e d that the amino group was not f r e e . The g l y c o p r o t e i n d i d not contain an a l k a l i - l a b i l e (0g l y c o s i d i c ) carbohydrate-peptide linkage as tested by $-eliminat i o n r e a c t i o n . The r e l e a s e of monosaccharides from the i n t a c t g l y c o p r o t e i n as a f u n c t i o n of time was studied employing m i l d a c i d h y d r o l y s i s (0.5 M HC1, 80°C) and a l s o by the use of neuraminidase, a-D- and $-D-glucosidases and $-D-N-acetylglucosaminidase. From the observations on the r e l e a s e of monosaccharides and analogy with standard features determined by other i n v e s t i g a t o r s on s o l u b l e a o r t i c g l y c o p r o t e i n s (Radhakrishnamurthy e t a l , 1964; Radhakushnamurthy and Berenson, 1966; Klemer and Nager, 1967; Maier and Buddecke, 1971), a p r e d i c t i o n was made as to the general features of the carbohydrate moiety of the g l y c o p r o t e i n ( F i g . 1 ) . This p o s t u l a t e d s t r u c t u r e must s t i l l withstand r e s u l t s of f u t u r e i n v e s t i g a t i o n s i n c l u d i n g methylation s t u d i e s , o x i d a t i o n with p e r i o date and the use of other s p e c i f i c g l y c o s i d a s e s . The p u r i f i e d porcine i n t i m a l g l y c o p r o t e i n was tested f o r i t s LPL i n h i b i t o r y a c t i v i t y by employing post-heparin dog plasma as the source of enzyme and E d i o l ( s t a b i l i z e d coconut o i l emulsion) as the t r i g l y c e r i d e s u b s t r a t e . Because i t i n h i b i t e d post-heparin
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
203
Lipase
TABLE I C o n c e n t r a t i o n of Component Sugars of Normal and A t h e r o s c l e r o t i c Aortae Component
Sclerotic
Normal
2 Wet weight Xmg/cm ) DDAF (mg/cm ) T o t a l carbohydrate (mg/cm ) Hexose (ug/cm ) Hexosamine (ug/cm ) Uronic a c i d (ug/cm^) S i a l i c a c i d (ug/cm^) T o t a l carbohydrate (ug/m Hexose (ug/mg DDAF) Hexosamine (ug/mg DDAF) Uronic a c i d (ug/mg DDAF) S i a l i c a c i d (ug/mg DDAF)
250.4 (a) 36.9 (b) 2.30 (a) 978 (a) 698 (c) 308 (NS) 317 (b)
195.7 39.3 2.10 870 651 297 285
2
(a) 19.4 (a) 8.5 (NS) 8.8 (a)
17.0 7.9 7.5
The c o n c e n t r a t i o n o f components are expressed as weight per u n i t area and weight per u n i t dry, d e f a t t e d , a s h - f r e e r e s i d u e (DDAF). A l l v a l u e s represent averages from 15 samples of aortae obtained from i n d i v i d u a l s of 7 0 . 3 + 2 . 8 (mean + S.E.M.) years of age. P a i r e d t t e s t : (a) = P<0.01; (b) = P<0.05; (c) = P<0.02; (NS) = not s i g n i f i c a n t . From Wagh e t a l (1973), w i t h p e r m i s s i o n from E l s e v i e r / N o r t h - H o l l a n d Biomedical P r e s s .
NeuAc
Glu
Gal
Gal
Glu
GlcNAc
GlcNAc
GlcNAc
y
x ^
Maojf
(Fuc)?
I
i
^jyian
Glu
GlcNA^c
Gal Biochimica et Biophysica Acta AspNH (?) Figure 1. A predicted structure for the carbohydrate moiety of porcine intimal glycoprotein. NeuAc, N acetyl-neuraminic acid; GlcNAc, N-acetyl-D-glucosamine. (From Roberts and Wagh [1976]) 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
204
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
plasma LPL, the molecule was named " l i p o l i p i n " (from l i p o p r o t e i n l i p a s e i n h i b i t o r ) (Wagh, 1975). The i n h i b i t i o n of LPL a c t i v i t y at v a r i o u s l i p o l i p i n concent r a t i o n s and a t r i g l y c e r i d e c o n c e n t r a t i o n of 2.5 mM i s shown i n F i g . 2. I n h i b i t i o n of LPL increased e x p o n e n t i a l l y with l i p o l i p i n c o n c e n t r a t i o n . At the t r i g l y c e r i d e concentration used, the r e a c t i o n v e l o c i t y was reduced 50% when the concentration of l i p o l i p i n was 1.5 x 10~5 M. To determine the nature of i n h i b i t i o n , we measured the v e l o c i t y of l i p o l y s i s at v a r i o u s concentrations of t r i g l y c e r i d e and the i n h i b i t o r . The i n h i b i t i o n was observed to be non-competitive ( F i g . 3). Further s t u d i e s (unpublished data) i n our l a b o r a t o r y i n d i c a t e d that porcine l i p o l i p i n i n h i b i t s p u r i f i e d bovine m i l k LPL (Egelrud and O l i v e c r o n a , 1972) when r a d i o a c t i v e t r i g l y c e r i d e emulsion s t a b i l i z e d by gum a r a b i c ( H e r n e l l et a l , 1975) was used as the s u b s t r a t e . However, the mechanism by which l i p o l i p i stood due to complex natur The s t a b i l i t y of l i p o l i p i n under v a r i o u s c o n d i t i o n s was examined by e l e c t r o p h o r e s i s on polyacrylamide g e l . L i p o l i p i n i s unstable a f t e r approximately 2 weeks when stored i n n e u t r a l Tris« HC1 b u f f e r or as a f r e e z e - d r i e d powder. The i n s t a b i l i t y i s i n d i cated by a d d i t i o n a l bands i n the g e l and the l o s s of i t s i n h i b i t o r y property (Wagh, 1975). Studies on Human I n t i m a l G l y c o p r o t e i n . We extended our i n v e s t i g a t i o n of LPL i n h i b i t o r to human a t h e r o s c l e r o t i c intima (Wagh and O l i v e c r o n a , 1978). A t h e r o s c l e r o t i c human aortae (Grade III-IV) were obtained at necropsy from male veterans 60-80 years of age. I n t i m a l l a y e r was separated by the "dermatome procedure" and intima powder was prepared as described p r e v i o u s l y (Roberts et a l , 1974). The y i e l d of intima powder was 1.5 - 2.0 g per 100 g of wet weight of aortae. P u r i f i c a t i o n of human LPL i n h i b i t o r demanded that the o r i g i n a l procedure f o r porcine t i s s u e (Wagh and Roberts, 1972) be modi f i e d (Wagh and O l i v e c r o n a , 1978). B r i e f l y , intima powder (5 g) was e x t r a c t e d twice with 20 v o l of acetone at 4°C f o r 30 min. The acetone powder was d r i e d at 25°C f o r 30 min under vacuum. This step r e s u l t e d i n the removal of 0.75 g l i p i d s and was found to be necessary because the presence of these l i p i d s i n t e r f e r e d i n subsequent e x t r a c t i o n procedures. The acetone powder was ext r a c t e d two times each f o r a p e r i o d of 24 h with 20 v o l of e x t r a c t i o n b u f f e r (0.05 M Tris-HHCl - 1 mM EDTA - 0.3 M NaCl, pH 7.4). The ammonium s u l f a t e p r e c i p i t a t e of the e x t r a c t between 40-80% s a t u r a t i o n was c o l l e c t e d , d i s s o l v e d i n 5 mM T r i s * H C l cont a i n i n g 1 mM EDTA and 50 mM NaCl, pH 7.4 b u f f e r and d i a l y z e d exh a u s t i v e l y against the same b u f f e r . The d i a l y z e d s o l u t i o n was designated as the ammonium s u l f a t e f r a c t i o n . T h i s f r a c t i o n upon chromatography on a DEAE-cellulose column r e s u l t e d i n three peaks which contained m a t e r i a l that i n h i b i t e d LPL a c t i v i t y ( F i g . 4).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
Lipase
Advances in Experimental Medicine and Biology
Figure 2. Inhibition of LPL as a function of lipolipin concentration. Incubation mixture contained 0.2 mL of postheparin plasma, 0.5 mL of buffer (0.2M NH,,OH-NH Cl at a pH of 8.6) containing various amounts of lipolipin and 0.1 mL of Ediol (20 micromoles triglyceride per milliliter). Final concentration of triglyceride was 2.5mM. Percent inhibition was calculated as the decrease in LPL activity as related to assay mixtures without added lipolipin. (From Wagh [1975]) h
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
205
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
180-
Advances in Experimental Medicine and Biology Figure 3. Dixon plot of LPL activity. Incubation mixture contained 0.2 mL of post-heparin plasma, 0.5 mL of buffer (0.2M NH, OH-NH,,Cl at a pH of 8.6 containing various amounts of triglycerides). Velocity is expressed as micromoles of free fatty acids liberated per milliliter of post-heparin plasma per minute. Final concentrations of triglyceride were: (•—0.7mM; (A—A; 1.6mM; (X—X) 2.5mM; and (O—O) 4.3mM. (From Wagh [1975]) t
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of
Lipoprotein
Lipase
207
Peak 1 contained l a r g e amounts of hemoglobin which s e r i o u s l y i n t e r f e r r e d with the LPL assay. The p r o t e i n m a t e r i a l i n peak 2 e l u t e d at the same i o n i c strength of the gradient as that f o r porcine l i p o l i p i n . I t s homogeneity was assessed by polyacrylamide g e l e l e c t r o p h o r e s i s , a n a l y t i c a l i s o e l e c t r i c focusing and sediment a t i o n v e l o c i t y measurement. E l e c t r o p h o r e t i c a n a l y s i s as observed by scanning the g e l stained with Coomassie B r i l l i a n t Blue dye at 520 nm revealed one major and three minor components. The major component c o n s t i t u t e d approximately 80% of the t o t a l p r o t e i n . The e l e c t r o p h o r e t i c m o b i l i t y of the major component a f t e r s t a i n i n g f o r both the p r o t e i n and carbohydrate ( p e r i o d i c a c i d - S c h i f f s t a i n ) was s i m i l a r to that of h i g h l y p u r i f i e d porcine l i p o l i p i n . A n a l y t i c a l i s o e l e c t r i c f o c u s i n g i n a pH 4-6 ampholyte system r e s u l t e d i n a p a t t e r n of one major and two minor p r o t e i n bands as v i s u a l i z e d by Coomassie Blue s t a i n i n g . The major component e l e c t r o f o c u s e d at pH 4.3. Upon u l t r a c e n t r i f u g a t i o n major species which sedimente content of the peak 2 m a t e r i a l was 2.55%. The carbohydrate contained hexosamine, glucose, galactose, mannose, fucose and s i a l i c a c i d i n a molar r a t i o of 3:1:2:2:1:1, r e s p e c t i v e l y with s i a l i c a c i d taken as u n i t y . This m a t e r i a l e l u t e d from Bio-Gel P-150 i n the same p o s i t i o n as described f o r porcine l i p o l i p i n (Roberts and Wagh, 1976). A p a r t i a l l y p u r i f i e d p r e p a r a t i o n was employed f o r the k i n e t i c s t u d i e s . Instead of chromatography on DEAE-cellulose column, a batch-wise DEAE-cellulose f r a c t i o n a t i o n method was used (Wagh and O l i v e c r o n a , 1978). The p r e p a r a t i o n contained a l l the p r o t e i n i n peak 2 with some overlapping p r o t e i n s contributed from peak 3 (approximately 20%) but was f r e e from p r o t e i n s i n peak 1 ( F i g . 4). This m a t e r i a l i n h i b i t e d the a c t i v i t y of milk LPL against longchain t r i g l y c e r i d e s under a l l c o n d i t i o n s that we have studied (Wagh and O l i v e c r o n a , 1978). The r e l e a s e of f r e e f a t t y a c i d s was l i n e a r with time both with and without i n h i b i t o r , i n d i c a t i n g that the i n h i b i t o r d i d not cause a p r o g r e s s i v e i n a c t i v a t i o n of the enzyme with time. I n h i b i t i o n of both the b a s a l a c t i v i t y and the serum-stimulated a c t i v i t y was the same f o r upto a 7-fold increase of enzyme concentration. I t was observed that the i n h i b i t i o n was non-competitive with respect to serum ( F i g . 5). When the amount of t r i g l y c e r i d e substrate was increased at a constant l e v e l of serum, a c t i v i t y i n the absence of i n h i b i t o r f i r s t increased but then decreased at high concentrations of t r i g l y c e r i d e ; however, the i n h i b i t i o n was r e l i e v e d at high l e v e l s of t r i g l y c e r i d e subs t r a t e ( F i g . 6). Therefore, the i n h i b i t i o n may be competitive with respect to t r i g l y c e r i d e s u b s t r a t e . Concluding Remarks: I should l i k e to end by posing four questions to which we p r e s e n t l y do not have answers or our knowledge i s at best incomplete. F i r s t , are porcine and human i n t i m a l l i p o l i p i n s s i m i l a r ? There i s some evidence that both of these molecules share c e r t a i n c h a r a c t e r i s t i c s i n common, e.g., molecular
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Atherosclerosis Figure 4. Chromatography of ammonium sulfate fraction on DEAE-cellulose. The ammonium sulfate fraction was chromatographed on a DEAE-cellulose column. The elution was initiated with 5mM Tris • HCl - ImM EDTA-0.05M NaCl, pH = 7.4 (standard buffer). The elution was initiated with 450 mL of standard buffer and 0.25M NaCl in the same buffer. Fractions of 6 mL were collected. The flow rate was 53 mL/hr. (From Wagh and Olivecrona [1978])
0
10
20 30 40 50 60 SERUM (/il/ml assay mixture)
70
80
Atherosclerosis Figure 5. The effect of various concentrations of human serum on LPL inhibition. (O—O) control; (•—•) 356 fig inhibitor per mL assay. For details see Kef. Wagh and Olivecrona (1978).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
0
0-5
Inhibitor
1-0
of Lipoprotein
1-5
0 L I V E 01L
2-0
Lipase
2-5
209
3-0
6-0
(mg/ml assay mixture)
Atherosclerosis
Figure 6. inhibition.
The effect of various concentrations of olive oil on LPL (O—O) control; (•—•) 625 fig inhibitor per mL assay. For details see Kef. Wagh and Olivecrona (1978).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
210
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
weight, e l e c t r o p h o r e t i c m o b i l i t y and i s o e l e c t r i c p o i n t . Although the s t r u c t u r e of the carbohydrate moiety f o r the porcine molecule has been p r e d i c t e d (Roberts and Wagh, 1976) the p r e c i s e sequence of sugars remains to be confirmed and compared w i t h that of the human molecule. The second question i s - what i s the mechanism by which l i p o l i p i n i n h i b i t s LPL a c t i v i t y i n v i t r o ? We have p o s t u l a t e d from k i n e t i c s t u d i e s that the i n h i b i t i o n of LPL may be at l e a s t p a r t l y due to covering of the l i p i d - w a t e r interphase by the i n h i b i t o r thereby denying the enzyme access to the l i p i d s u b s t r a t e (Wagh and O l i v e c r o n a , 1978). Although t h i s has been suggested as a mechanism f o r the i n h i b i t i o n of p a n c r e a t i c l i p a s e by bovine serum albumin (Brockerhoff, 1971), f u r t h e r s t u d i e s are needed to confirm our hypothesis. T h i r d , which c e l l s i n the i n t i m a synthesize l i p o l i p i n ? I t has been demonstrated tha glycoprotein similar t 1976). Further immunological s t u d i e s on the c r o s s - r e a c t i o n between a n t i - l i p o l i p i n against i n t i m a l l i p o l i p i n and v a l v u l a r g l y c o p r o t e i n i n d i c a t e d that the p r e c i p i t i n bands formed due to c r o s s - r e a c t i o n fused w i t h each other (unpublished d a t a ) . I t seems t h e r e f o r e , that l i p o l i p i n i s present i n the mesenchymal t i s s u e s . Further h i s t o l o g i c a l and immunological s t u d i e s should provide i n f o r m a t i o n as to the l o c a l i z a t i o n of l i p o l i p i n i n the v a s c u l a r tissue. F i n a l l y , we come to the question of the f u n c t i o n of LPL i n h i b i t o r i n v i v o . Large blood v e s s e l s such as bovine t h o r a c i c a o r t a c o n t a i n LPL (Henson and Schotz, 1975; D i c o r l e t o and Z i l v e r smit, 1975). Although the c o n c e n t r a t i o n of LPL i n h i b i t o r i n the normal and diseased s t a t e s i s not known, i t i s p o s s i b l e that i n creased s y n t h e s i s of the i n h i b i t o r during atherogenesis may r e s u l t i n decreased LPL a c t i v i t y thereby a f f e c t i n g the normal l i p o l y t i c process. I f the i n h i b i t o r i s a major c o n s t i t u e n t of t o t a l g l y c o p r o t e i n content i n a t h e r o s c l e r o t i c a o r t a , the higher c o n c e n t r a t i o n of g l y c o p r o t e i n s i n a t h e r o s c l e r o t i c plaques as compared to that i n the uninvolved regions of the a o r t a (Wagh et a l , 1973) may be a r e f l e c t i o n of an increased c o n c e n t r a t i o n of the i n h i b i t o r i n diseased s t a t e . This s p e c u l a t i o n remains to be explored. Acknowledgements. This work was supported i n p a r t by the Veterans A d m i n i s t r a t i o n Research Funds ( P r o j e c t No. 9166) and a grant from the N a t i o n a l I n s t i t u t e s of Health HL 20549. The k i n e t i c s t u d i e s on LPL i n h i b i t i o n by human i n t i m a l g l y c o p r o t e i n were done i n c o l l a b o r a t i o n w i t h P r o f e s s o r Thomas O l i v e c r o n a at the U n i v e r s i t y of Umea, Sweden. I wish to thank Mrs. Betty Blaydes f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Ms. Diane B u t l e r for s e c r e t a r i a l help. A b s t r a c t . This paper briefly reviews the c u r r e n t understanding of a g l y c o p r o t e i n i s o l a t e d from porcine and human aortic i n t i m a . Because the g l y c o p r o t e i n from e i t h e r source inhibits
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
11.
WAGH
Glycoprotein
Inhibitor
of Lipoprotein
Lipase
211
l i p o p r o t e i n l i p a s e activity, in vitro of post-heparin plasma and of bovine m i l k , it is named " l i p o l i p i n " ( l i p o p r o t e i n l i p a s e in hibitor). The carbohydrate moiety of the porcine molecule (MW 72,000) contains 1 mole each of fucose and s i a l i c a c i d , 2 moles of mannose, 3 moles each of glucose and galactose and 4 moles of N -acetylglucosamine. The s t r u c t u r e of the carbohydrate i s p r e d i c t e d by s e q u e n t i a l a n a l y s i s . Although the molecule from the human a t h e r o s c l e r o t i c i n t i m a is not completely c h a r a c t e r i z e d , it appears that the porcine and human lipolipins share s e v e r a l f e a t u r e s in common: molecular weight, i s o e l e c t r i c p o i n t ( p I 4.3), e l e c t r o p h o r e t i c m o b i l i t y and ratio of carbohydrate c o n s t i t u e n t s . Theref o r e , porcine t i s s u e may serve as a model system t o study the human molecule. K i n e t i c s t u d i e s revealed that human lipolipin decreased both the b a s a l and serum-stimulated a c t i v i t y o f l i p o p r o t e i n l i p a s e . The inhibition was non-competitive w i t h respect to serum. However, hig ed t o relieve the i n h i b i t o r y lipin might be i n v o l v e d in an i n t e r a c t i o n w i t h e m u l s i f i e d lipid denying l i p o p r o t e i n l i p a s e access t o i t s s u b s t r a t e . I f lipolipin is a major c o n s t i t u e n t of t o t a l g l y c o p r o t e i n content in atheros c l e r o t i c a o r t a , the observed higher c o n c e n t r a t i o n of glycoprot e i n s i n a t h e r o s c l e r o t i c plaques as compared t o that i n the uni n v o l v e d regions of the a o r t a may be a reflection of an increased c o n c e n t r a t i o n of l i p o l i p i n i n a t h e r o s c l e r o s i s .
Literature Cited Anderson, J.C. (1976), International Rev. Conn. Tiss. Res., 7, 278. Baig, M.M., and Ayoub, E.M. (1976), Biochemistry 15, 2585. Berenson, G.S., and Fishkin, A.F. (1962), Arch. Biochem. Biophys. 97, 18. Berenson, G.S., Radhakrishnamurthy, B . , Dalferes, E.R., and Srinivasan, S.R. (1971), Human Path. 2, 57. Brockerhoff, H. (1971), J . Biol. Chem. 246, 5828. Buddecke, E. (1960), J . Physiol. Chem. 318, 33. Dicorleto, P.E., and Zilversmit, D.B. (1975), Proc. Soc. Exptl. Biol. Med. 148, 1101. Eisenberg, S., and Levi, R . I . (1975), Adv. Lipid Res. 13, 1. Egelrud, T., and Olivecrona, T. (1972), J . Biol. Chem. 247, 6212. Henson, L.C., and Schotz, M.C. (1975), Biochim. Biophys. Acta 409, 360. Hernell, O., Egelrud, T., and Olivecrona, T. (1975), Biochim. Biophys. Acta 381, 233. I s h i i , M. (1971), Jap. Heart J. 12, 22. Klemer, A . , and Nager, C. (1967), Z. Naturforsch 22, 456. Maier, V . , and Buddecke, E. (1971), Hoppe-Seyler Z. Physiol. Chem. 352, 1338. Manley, G., and Mullinger, R.N. (1967), Brit. J . Path. 58, 529. Muller-Spreer, H.C., Werber, U . , and Voight, K.D. (1960), Klin. Wschr. 38, 28.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
212
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Radhakrishnamurthy, B . , and Berenson, G.S. (1966), J . Biol. Chem. 241, 2106. Radhakrishnamurthy, B . , Fishkin, A . F . , Hubbell, G . J . , and Berenson, G.S. (1964), Arch. Biochem. Biophys. 104, 19. Roberts, B . I . , and Wagh, P.V. (1976), Biochim. Biophys. Acta 439, 26. Roberts, B . I . , White, H . J . , Read, R.C., and Wagh, P.V. (1974), Atherosclerosis 20, 533. Robinson, D.S. (1970), Compr. Biochem. 19, 51. Wagh, P.V. (1975), Adv. Exptl. Med. Biol. 52, 81. Wagh, P.V., and Olivecrona, T. (1978), Atherosclerosis 29, 195. Wagh, P.V., and Roberts, B.I. (1972), Biochemistry 11, 4222. Wagh, P.V., Roberts, B . I . , White, H . J . , and Read, R.C. (1973), Atherosclerosis 18, 83. RECEIVED
February 13, 1978
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12 Carbohydrate Moieties of the Collagens and Collagen-Like Proteins in Health and Disease WILLIAM T. BUTLER Institute of Dental Research, University of Alabama in Birmingham, Birmingham, AL 35294 The fact that interstita proteins contain covalentl for many years. In 1935 Grassmann and Schleich (1) found that hide collagen had equal quantities of firmly bound glucose and galactose, and later studies from this group (2) suggested that the hexoses were bound through O-glycosidic linkages. The stability of the binding of hexoses to collagen was demonstrated by Kühn, et al (3) who found that repeated reprecipitation of citrate-soluble collagen removed all the hexosamine, but only half the hexose. After three reprecipitations, the hexose level was 0.48%, and it remained constant throughout seven additional reprecipitations. This study revealed that hexose, but not hexosamine was an integral part of i n t e r s t i t i a l collagen. Gross, et al (4) examined the amino acid and sugar contents of collagens from a variety of tissues and species. They found that for vertebrate collagens, purification or gelatinization drastically reduced the hexosamine content, but was less effective i n removing hexose. The carbohydrate was identified as glucose and galactose with traces of other hexoses, pentoses and amino sugars. The hexose content was usually less than 1 % for vetebrate and from 3 to 11% i n invertebrate collagens. Similarly Blumenfeld, et al (5) found that ichthyocol (carp swim bladder collagen) contained glucose and galactose but no hexosamine or other sugars. In contrast to the low content of sugars generally found in i n t e r s t i t i a l collagens of vertebrates, the collagen-like proteins of basement membranes contain much higher levels of carbohydrate (6-8). As i n collagen, one type of carbohydrate consists of glucose and galactose units, but heteropolysaccarides with hexoses and hexosamines are also present.
0-8412-0452-7/78/47-080-213$05.00/0 © 1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
214
GLYCOPROTEINS
AND GLYCOLIPIDS IN DISEASE PROCESSES
THE NATURE OF THE LINKAGE P r i o r to 1965 s e v e r a l s t u d i e s on the nature o f the carbohydrate of c o l l a g e n were r e p o r t e d , but the r e s u l t s l e f t many u n c e r t a i n t i e s . The s t u d i e s of B u t l e r and Cunningham (9,10) c l a r i f i e d the nature o f the hexose attached to c o l l a g e n . S t a r t ing w i t h s o l u b l e guinea p i g s k i n c o l l a g e n and d i g e s t i n g to s h o r t e r u n i t s w i t h collagenase and t r y p s i n , glycopeptides were i s o l a t e d . The compositions of the peptides suggested that glucose and galactose might be attached to the 6-hydroxyl group of h y d r o x y l y s i n e . The r e s i s t a n c e of the h y d r o x y l y s i n e to p e r i o d a t e o x i d a t i o n supported t h i s c o n c l u s i o n (9). The nature of the l i n k a g e was c o n c l u s i v e l y shown by the i s o l a t i o n of a glycopeptide w i t h s t o i c h i o m e t r i c amounts of h y d r o x y l y s i n e , glucose, and g a l a c t o s e recovered a f t e r cleavage of the peptide bonds of the c o l l a g e n g l y c o p e p t i d w i t h 2N NaOH t 90° f o 10 hr. The r e s i s t a n c e t o glycosidic; this conclusio supporte y t i o n t h a t m i l d a c i d h y d r o l y s i s (2N HC1, 110°, 30 min.) r e l e a s e d 2 mol of reducing sugar. A l i n k a g e to the e-amino group of h y d r o x y l y s i n e was r u l e d out by the r e a c t i v i t y of t h i s group to f l u o r o d i n i t r o b e n z e n e and by the e l e c t r o p h o r e t i c m o b i l i t y of the glycopeptide. These experiments thus showed that c o l l a g e n cont a i n e d a d i s a c c h a r i d e c o n s i s t i n g of glucose and g a l a c t o s e attached O - g l y c o s i d i c a l l y to h y d r o x y l y s i n e ( G l c - G a l - H y l ) . STRUCTURE OF THE CARBOHYDRATE MOIETY The s t r u c t u r e of the carbohydrate was e l u c i d a t e d by S p i r o (11) u s i n g collagenous components from glomerular basement membranes. A f t e r i s o l a t i o n of a mixture of glycopeptides c o n t a i n ing h y d r o x y l y s i n e - l i n k e d glucose and g a l a c t o s e , he showed t h a t glucose, but not g a l a c t o s e , was r e a d i l y l i b e r a t e d w i t h 0.1 N ^SO^ a t 100° f o r 2-20 h r . Thus glucose was i n an e x t e r n a l p o s i t i o n , w i t h galactose l i n k e d to h y d r o x y l y s i n e . Next a glucose and g a l a c t o s e - c o n t a i n i n g d i s a c c h a r i d e was obtained a f t e r N - a c e t y l a t i o n and m i l d a c i d h y d r o l y s i s of the glycopept i d e s . Glucose was shown to be attached to C-2 of galactose by s t u d i e s employing p e r i o d a t e and g a l a c t o s e oxidase. This l i n k a g e was cleaved by a-glucosidase but not 3-glticosidase. Next G l c Gal-Hyl was i s o l a t e d a f t e r a l k a l i n e h y d r o l y s i s of g l y c o p e p t i d e s ; the anomeric c o n f i g u r a t i o n of the g a l a c t o s e l i n k a g e to hydroxyl y s i n e was shown to be 3 by i t s s u s c e p t i b i l i t y to 3 g a l a c t o s i d a s e . These experiments thus i n d i c a t e d that G l c - G a l - H y l had the s t r u c ture 2-0-a-D-glucopyranosy1-0-3-D-galac t o p y r a n o s y l h y d r o x y l y s i n e (Figure 1). A s i m i l a r s t r u c t u r e was proposed by K e f a l i d e s (12). The s t r u c t u r e of G l c - G a l - H y l i n c o l l a g e n s from v e r t e b r a t e and i n v e r t e b r a t e sources has been shown to be the same as that found i n basement membranes. Thus Cunningham and Ford (13) observed that glucose was s p l i t from the d i s a c c h a r i d e of guinea -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
Moieties
of the
Collagens
215
p i g s k i n c o l l a g e n by m i l d a c i d h y d r o l y s i s , w h i l e galactose r e mained attached. They a l s o reported f i n d i n g g a l a c t o s y l h y d r o x y l y s i n e (Gal-Hyl) a f t e r a l k a l i n e h y d r o l y s i s of s k i n c o l l a g e n . Using the amino a c i d a n a l y z e r , S p i r o (14) introduced a separat i o n technique f o r q u a n t i t a t i o n of G l c - G a l - H y l and Gal-Hyl. Chromatography o f a l k a l i n e h y d r o l y s a t e s of s e v e r a l c o l l a g e n samples showed that they contained the same mono- and d i s a c c h a r i d e u n i t s l i n k e d to h y d r o x y l y s i n e as basement membranes. Katzman, jet a l (15) u t i l i z e d G l c - G a l - H y l i s o l a t e d from the sponge and showed that the s t r u c t u r e was that proposed f o r basement membranes by S p i r o (11). The h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e i s o l a t e d from sea anenome, sea cucumber, and bovine cornea was shown to have an i d e n t i c a l s t r u c t u r e by a v a r i e t y of techniques (15). Since these e a r l y s t u d i e s , G l c - G a l H y l and Gal-Hyl have been shown to occur i n a v a r i e t y of c o l l a gen and c o l l a g e n - l i k e p r o t e i n s OCCURRENCE IN THE DIFFEREN The d i s c o v e r y that c a r t i l a g e contained a type o f c o l l a g e n d i s t i n c t from t h a t o f s k i n and bone (16) ushered i n a new e r a i n c o l l a g e n biochemistry. We now understand that a t l e a s t three c o l l a g e n s w i t h unique p r o p e r t i e s e x i s t i n the e x t r a c e l l u l a r m a t r i x of connective t i s s u e s (Table I ) . For more d e t a i l s conc e r n i n g these c o l l a g e n s , the reader i s r e f e r r e d t o the review by M i l l e r (17). S c i e n t i s t s have long recognized t h a t the basement membranes c o n t a i n collagenous components (6,18,19). A f i n a l r e s o l u t i o n of the s t r u c t u r e s o f the molecular species present i n basement membranes has not been accomplished. A t l e a s t one o f these appears to have a t r i p l e - h e l i c a l conformation r e f e r r e d t o as [ a l ( I V ) ] 3 (20). However i t i s apparent that s e v e r a l other s t r u c t u r e s are present (21-23). A l l three o f the i n t e r s t i t i a l c o l l a g e n s c o n t a i n hexose attached to h y d r o x y l y s i n e , but type I I c o l l a g e n has a s i g n i f i c a n t l y greater q u a n t i t y of h y d r o x y l y s i n e and o f h y d r o x y l y s i n e l i n k e d carbohydrate (Table I ) . I n general the collagenous p o r t i o n of basement membranes c o n t a i n much higher l e v e l s o f h y d r o x y l y s i n e and of the a s s o c i a t e d glucose and g a l a c t o s e moieties. I t should be noted that other h y d r o x y p r o l i n e - c o n t a i n i n g p r o t e i n s w i t h c o l l a g e n - l i k e ( i . e . Gly-X-Y repeating) sequences e x i s t . For example, the complement p r o t e i n C l q contains s e v e r a l g l y c o s y l a t e d hydroxylysines i n a c o l l a g e n - l i k e sequence (24). The exact l o c a t i o n s o f s e v e r a l o f the carbohydrate m o i e t i e s of the collagens has been documented during the s t u d i e s on the primary s t r u c t u r e s o f the v a r i o u s a chains. One s i t e of g l y c o s y l a t i o n common to the f o u r chains of the three i n t e r s t i t i a l c o l l a g e n s i s Hyl-87 (Figure 2). The amino a c i d sequences around t h i s s i t e are s i m i l a r i n these chains (25-29). The occurrence of the d i s a c c h a r i d e a t t h i s s i t e suggests that i t performs some
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
2
[al(II)]
[al(III)]
Several
II
III
IV Basement Membranes (Glomerulus, e t c . )
Ubiquitous ( E s p e c i a l l y Prominent i n F e t a l S k i n , Arteries, Cirrhotic Liver)
Cartilage, Intervetebral D i s c , Notocord
Ubiquitous ( S k i n , Bone, Tendon Dentin, etc.)
Tissue D i s t r i b u t i o n
40-50
5-8
20-25
5-15
b
55
2
b
15-30
2.5
a
Hexose Content (Residues/1000)
Data f o r the c o l l a g e n - l i k e p o r t i o n of glomerular basement membranes. Taken from r e f e r e n c e 19.
Glucose and galactose
3
[al(I)] o2
I
3
Molecular Composition
Type
Hydroxylysine Content (Residues/1000)
TABLE I : S t r u c t u r e s and C h a r a c t e r i s t i c s of I n t e r s t i t a l Collagens and C o l l a g e n - L i k e P r o t e i n s of Basement Membranes
BUTLER
Carbohydrate
Moieties of the
Collagens
CH OH 2
HC* V^~° OH
"^v
6H -NH © 2
3
®H N-CH-C-O© 3
CH OH
^
2
HO/KJ VOH
° \ Hy H
" /
CH OH 2
»yfr—°\H/°
g
HO
old)
2
Q
CH2-NH3®
Figure I. The structures of the glucose- and galactose-containing units isolated from the collagens d collagen-like proteins. (A) / ? - D galactopyranosylhydroxylsine (Gal an
hyl),
H/^ OH
(B)
2-O-a-D-glucopyranosyl
O - /? - D- galactopyranosylhydroxyly sine (Glc-Gal-Hyl),
- GLY -
Glc - Gal
a2
- GLY - L E U - HYP - GLY - P H E - HYL - GLY - I L E - ARG 1
Glc - Gal
a I (II)
- GLY - L E U - HYP - GLY - V A L - HYL - GLY - H I S - ARG -
Glc - Gal
a l(III)
- GLY - PHE - HYP - GLY - MET - H Y L - G L Y - H I S - ARG 1
Glc - Gal
Figure 2. Amino acid sequences around the disaccharide-bearing hydroxylysines common to types J, II, and III collagens
old)
174 - G L Y - A L A . A L A - G L Y - A L A - L Y S - GLY - G L U - A L A -
al(III)
- GLY - SER - HYP - GLY - A L A - L Y S - GLY - GLU - V A L -
ol(II)
- G L Y - A L A - HYP - G L Y - A L A - H Y L - G L Y - GLU - A L A •
Gal
o2
- G L Y - A L A - HYP - GLY - PRO - HYL - G L Y - GLU - L E U 1
Gal
Figure 3.
The amino acid sequences of a chains around the monosaccharide-bearing hydroxylysine of a2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
218
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
f u n c t i o n a l r o l e v i t a l to the three collagens (e.g. i n c r o s s l i n k i n g ) . The a2 chain of type I c o l l a g e n has a Gal-Hyl at p o s i t i o n 174 (26,27) not found i n a l ( I ) or a l ( I I I ) but present i n a l ( I I ) (Figure 3). Although these appear to be the major s i t e s of carbohydrate i n a l ( I ) , a2 and a l ( I I I ) , other p o s i t i o n s may be p a r t i a l l y g l y c o s y l a t e d . For i n s t a n c e s u b i n t e g r a l l e v e l s of Gal-Hyl are found near the COOH-terminus of a l ( I ) i n c a l f s k i n c o l l a g e n (26). As s t a t e d b e f o r e , the a l ( I I ) chain of c a r t i l a g e c o l l a g e n has many more s i t e s of carbohydrate attachment than the above. Studies on the covalent s t r u c t u r e of bovine and c h i c k a l ( I I ) chains (28,30,31) from t h i s l a b o r a t o r y have a l r e a d y l o c a t e d 13 g l y c o s y l a t e d hydroxylysines (Figure 4 ) , although only about 60% of the sequence i s known. These h y d r o x y l y s i n e s are not confined to any one area but occur throughout the chain. Comparison of the p o s i t i o n s of these g l y c o s y l a t e d h y d r o x y l y s i n e s w i t h homolo gous sequences i n a l ( I occupied by l y s i n e i n th chains have s i m i l a r amino a c i d sequences w i t h p o t e n t i a l s i t e s f o r carbohydrate attachment, but only f o r a l ( I I ) are the postt r a n s l a t i o n a l m o d i f i c a t i o n s ( i . e . h y d r o x y l a t i o n and g l y c o s y l a t i o n ) made. I w i l l r e t u r n to p o s s i b l e explanations f o r the h i g h l e v e l of Glc-Gal-Hyl and Gal-Hyl i n a l ( I I ) i n a l a t e r s e c t i o n . BIOSYNTHETIC ATTACHMENT OF HEXOSE TO PROCOLLAGEN a CHAINS Collagen i s synthesized w i t h i n connective t i s s u e c e l l s by the u s u a l p r o t e i n s y n t h e t i c mechanism 032) but has s e v e r a l f e a t u r e s which are unique (Figure 5). The i n i t i a l form of the three a chains are elongated, compared to that found i n f i b r i l l a r c o l l a g e n (33-36). These " e x t e n s i o n s " which occur on the NH2~and COOH-terminal ends of the c h a i n s , are cleaved from the molecules during or a f t e r e x i t from the c e l l . During and a f t e r the t r a n s l a t i o n of these p r o c o l l a g e n chains on ribosomal complexes, seve r a l p o s t - t r a n s l a t i o n m o d i f i c a t i o n s take p l a c e . L y s y l and p r o l y l residues are hydroxylated to form h y d r o x y l y s y l and h y d r o x y p r o l y l r e s i d u e s , and galactose and glucose m o i e t i e s are attached to c e r t a i n of the h y d r o x y l y s i n e s . Next the three chains of a p r o c o l l a g e n u n i t a s s o c i a t e , i n t e r c h a i n d i s u l f i d e bonds i n the COOH-terminal extensions are formed, and f o l d i n g i n t o a t r i p l e - c h a i n c o l l a g e n h e l i x takes p l a c e . I t i s obvious that the attachment of galactose and glucose to c o l l a g e n must be preceded by h y d r o x y l a t i o n of l y s y l r e s i d u e s . This r e a c t i o n i s c a t a l y z e d by l y s y l hydroxylase (32,29,40), an enxyme which i s s i m i l a r i n many respects to p r o l y l hydroxylase. Each of these mixed f u n c t i o n oxidases r e q u i r e s molecular oxygen, a - k e t o g l u t a r a t e , ascorbate and f e r r o u s i r o n (Figure 6) and each c a t a l y z e s h y d r o x y l a t i o n of r e s i d u e s i n the Y p o s i t i o n of the r e p e a t i n g Gly-X-Y c o l l a g e n sequence (37), though l y s y l hydroxyl a s e w i l l apparently a l s o a c t upon l y s i n e s i n the X p o s i t i o n (40).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
Moieties
of the
219
Collagens
al(l) Bovine al(ll) Bovine a 2 Bovine
87 GLY - LEU - HYP. - GLY - MET - HYL - GLY - HIS - ARG - GLY - PHE - SER GLY - LEU - HYP • GLY - VAL - HYL - GLY - HIS - ARG - GLY - THR - HYP GLY - LEU - HYP - GLY - PHE - HYL - GLY - ILE - ARG - GLY - HIS - ASN
al(l) Bovine al(ll) Bovine a 2 Bovine
99 GLY - LEU - ASP - GLY - ALA - LYS - GLY - ASP - ALA - GLY - PRO - A L A GLY - LEU - ASP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - ALA - HYP GLY - LEU - ASP - GLY - LEU - THR - GLY - GLN - HYP - GLY - ALA - HYP
al(l) Bovine al(ll) Bovine a 2 Bovine
108 GLY - PRO - ALA - GLY - PRO - LYS - GLY - GLU - HYP - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - SER - GLY - SER - HYP GLY - ALA - HYP - GLY - VAL - HYL - GLY - GLU - HYP - GLY - ALA - HYP
al(l) Rat a 1(11) Bovine a 2 Bovine
174 GLY - ALA - ALA - GLY - ALA - LYS - GLY - GLU '- ALA - GLY - PRO - GLN GLY - ALA - HYP - GLY - ALA - HYL - GLY - GLU - ALA - GLY - PRO GLY - ALA - HYP - GLY - PRO - HYL - GLY - GLU - LEU - GLY - PRO - VAL
al(l) Rot a 1(11) Bovine a 2 Bovine
GLY - GLN - HYP - GLY GLY - ILE - HYP - GLY GLY - LEU - HY
al(l)Rat al(l 1) Bovine a 2 Bovine
GLY - ALA GLY - PRO GLY - ALA
al(l) Bovine a 1(11) Bovine a 2 Chick
GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY GLY - PHE - HYP - GLY
a 1(1) Bovine al(ll)Bovine a 2 Chick
GLY - LYS GLY - LYS GLY - LYS
al(l) Bovine alODBovine a 2 Chick
531 GLY - ASN • ASP - GLY - ALA - LYS - GLY GLY - THR •• ASP - GLY - PRO - HYL - GLY GLY - PRO - ASP - GLY - ASN - LYS - GLY
al(l) Bovine a 1(11) Bovine a 2 Chick
564 GLY - LEU - HYP •• GLY - PRO - LYS - GLY - ASP - ARG - GLY - ASP - ALA GLY - ILE - ALA - GLY - PRO - HYL - GLY •• ASP - ARG - GLY - ASP - VAL GLY " VAL - HYP - GLY • GLY • LYS - GLY - GLU - LYS - GLU - A L A - HYP
a 1(1) Bovine alODBovine a 2 Chick
573 GLY - ASP - ALA •• GLY - PRO •• LYS •• GLY - A L A - ASP - GLY " A L A - PRO GLY - ASP - VAL •• GLY - GLU • LYS •• GLY - PRO - GLU - GLY - A L A - PRO GLY - ALA - HYP - GLY - LEU ••ARG - GLY - ASP - THR - GLY - ALA - THR
a 1(1) Bovine alODBovine a 2 Chick
603 GLY - ALA - HYP - GLY - ASP - LYS - GLY - GLU -• ALA - GLY - PRO - SER GLY - ASP • VAL - GLY - GLU - HYL - GLY • GLU - VAL - GLY - PRO - HYP GLY - GLY •• ALA - GLY - ASP - ARG - GLY - GLU -• GLY - GLY - PRO - ALA
al(l) Bovine alODBovine a 2 Chick
648 GLY •• GLN - HYP - GLY - ALA - LYS - GLY GLY - GLN - PRO - GLY - ALA - HYL - GLY GLY -• GLU • HYP - GLY - ALA - LYS - GLY
al(l) Bovine alODBovine a 2 Chick
657 GLY - ASP - ALA - GLY - ALA - LYS - GLY - ASP - A L A - GLY - PRO " GLY - GLU - A L A - GLY - GLN - HYL - GLY - ASP - ALA - GLY " ALA GLY - PRO - LYS - GLY - PRO - LYS - GLY - GLU - THR - GLY - PRO -
Figure 4.
-
- HYP - GLY - LEU - GLY - THR - GLY
219 - ALA - LYS - GLY - ALA - HYL - GLY
- ALA - ASN - GLY - A L A - HYP - SER - ALA - GLY - ALA - HYP HYP
- PRO - LYS - GLY - ASN • SER - PRO - HYL - GLY - ALA - ARG - GLY - LEU - VAL
408 - PRO - LYS - PRO - HYL - PRO - LYS
- GLY - GLY - GLY
• GLY • GLY
- ALA - ALA - GLY - GLU - HYP - ALA - ASN - GLY - GLU - HYP - PRO - THR - GLY - GLU - HYP
420 - ALA - GLY - GLU - ARG - GLY - VAL - HYP - GLY - PRO - HYP - ALA - GLY - GLU - HYL •- GLY - LEU - HYP " GLY - ALA - HYP - HYP •• GLY - GLU " LYS • GLY - ASN • VAL " GLY - L E U - ALA
- ASP - ALA - GLY - ALA - HYP - ALA - ALA - GLY - PRO - ALA • GLU - HYP - GLY - ASN - VAL
- GLU - HYP •• GLY - ASP - ALA - GLU - GLN •• GLY - GLU - A L A •• GLU - ARG •• GLY - PRO - LYS
The sequences around several hydroxy lysines and lysines of al(I), al(II), and a2 chains (taken from Ref. 31)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
220
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
LOCATION
BIOSYNTHETIC EVENTS
Polysomes Translation of Procollagen Chains Hydroxylation of Prolyl and Lysyl Residues
Cysternae of Endoplasmic Reticulum Chain Alignment
Helix Formation Disulfide Bond Formation
Figure 5. Intracellular steps in the biosynthesis of procollagen. Individual procollagen a chains are made by the usual protein biosynthetic mechanisms. The chains are then subjected to several post-translational modifications. After release from ribosomal complexes, three chains align, and triple-helices and interchain disulfide bonds at the COOH-terminal extremities are formed. Post-translational modifications cease upon formation of the triple-helix.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
o -NH-CH-CCH CH CH CH - N H ©
Moieties
of the Collagens
LYSYL HYDROXYLASE
221
0 -NH-CH-C CH CH HO-CH CHI -NH © 2
2
2
2
2
V
2
0
0 -NH-CH-CCH CH HO-CH CH -NH * 2
B.
-NH-CH-CGALACTOSYL TRANSFERASE
CH CH, i * O-CH
CH OH
2
2
2
2
3
.©
UDP-Gol MN"
H
OH
CH -NH *.© 2
3
Figure 6. Three enzymatic steps in the biosynthetic attachment of hexose to collagen. (A) Formation of hydroxylysyl residues from lysines on the nascent procollagen a chains; (B) attachment of galactose to certain hydroxylysyl residues; and (C) attachment of glucose to certain Gal-Hyl moieties.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
222
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
R e s u l t s of experiments i n v i t r o (37,38) and u s i n g c e l l s i n c u l ture (41), i n d i c a t e that the h y d r o x y l a t i o n of l y s y l r e s i d u e s does not occur a f t e r t r i p l e - h e l i c a l conformation of the proc o l l a g e n molecules i s formed ( F i g u r e 5). A f t e r h y d r o x y l a t i o n , g a l a c t o s y l t r a n s f e r a s e c a t a l y z e s the t r a n s f e r of g a l a c t o s e from UDP-Gal to c e r t a i n h y d r o x y l y s i n e s (Figure 6 ) ; then the t r a n s f e r of glucose from UDP-Glc to the C-2 of the g a l a c t o s e r e s i d u e s i s c a t a l y z e d by g l u c o s y l t r a n s f e r a s e . Both enzymes r e q u i r e d i v a l e n t c a t i o n s , p e r f e r a b l y manganese and are probably l o c a t e d i n the c i s t e r n a e of the endoplasmic r e t i culum. G l u c o s y l t r a n s f e r a s e has been p u r i f i e d to homogeneity (42) and g a l a c t o s y l t r a n s f e r a s e 1000-fold (43). S i m i l a r to the h y d r o x y l a t i o n r e a c t i o n s , the attachment of carbohydrate to p r o c o l l a g e n chains ceases when they f o l d i n t o a n a t i v e , t r i p l e - h e l i c a l s t r u c t u r e (44). Using c h i c k embryo tendon and c a r t i l a g e c e l l s i n c u l t u r e O i k a r i n e n et a l (45) have shown t h a t the g l y c o s y l a t i o a f f e c t e d by the r a t e o chains. For example, when the r a t e of h e l i x formation was i n h i b i t e d by i n c u b a t i o n i n the presence of 0.6 mM d i t h i o t h r e i t o l , the g l y c o s y l a t i o n was more than doubled. Conversely when c a r t i lage c e l l s r e c o v e r i n g from anoxia were used (a c o n d i t i o n known to a c c e l e r a t e t r i p l e - h e l i x f o r m a t i o n ) , there was a marked decrease i n the extent of h y d r o x y l y s i n e formation and an even greater decrease i n g l y c o s y l a t i o n of h y d r o x y l y s i n e r e s i d u e s . FACTORS CONTROLLING HYDROXYLYSINE FORMATION AND GLYCOSYLATION
SUBSEQUENT
The f o r e g o i n g d i s c u s s i o n may help e x p l a i n some observations on the v a r i a t i o n s i n the content and occurrence of h y d r o x y l y s i n e s and g l y c o s y l a t e d h y d r o x y l y s i n e s . The h y d r o x y l y s i n e content of a l ( I ) chains d i f f e r s c o n s i d e r a b l y i n the type I c o l l a g e n s of d i f f e r e n t t i s s u e s . For example a l ( I ) of r a t d e n t i n c o l l a g e n contains about three times as much h y d r o x y l y s i n e as does s k i n a l ( I ) , though these chains have the same primary s t r u c t u r e (4648). This r e l a t i v e i n c r e a s e could be due to a higher a c t i v i t y of l y s y l hydroxylase i n odontoblasts; a l t e r n a t i v e l y a slower r a t e of f o l d i n g of d e n t i n p r o c o l l a g e n a chains i n t o the t r i p l e - h e l i c a l s t r u c t u r e could occur, a l l o w i n g a more complete h y d r o x y l a t i o n of l y s y l r e s i d u e s . The problem w i t h the l a t t e r hypothesis i s that one might expect an increased g l y c o s y l a t i o n of the h y d r o x y l y s i n e s over t h a t i n s k i n a l ( I ) c h a i n s , i f the r a t e of h e l i x formation i s the major f a c t o r c o n t r o l l i n g h y d r o x y l a t i o n and g l y c o s y l a t i o n of nascent p r o c o l l a g e n a chains. However the a d d i t i o n a l hydroxylys i n e s do not appear to have increased l e v e l s of hexose (48). K i v i r i k k o and R i s t e l l i (32) speculate that the d i f f e r e n c e s i n h y d r o x y l y s i n e and g l y c o s y l a t e d h y d r o x y l y s i n e contents of c o l l a g e n s
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
Carbohydrate
Moieties
of the
Collagens
223
i n d i f f e r e n t t i s s u e s could be due i n p a r t to d i f f e r e n c e s i n the r a t e s of t r i p l e - h e l i x formation during s y n t h e s i s i n the v a r i o u s connective t i s s u e c e l l s . The extensive comparison of the amino a c i d sequences near g l y c o s y l a t e d hydroxylysines i n the a l ( I I ) chain w i t h r e l a t e d sequences i n a l ( I ) and a2 (Figure 4 ) , suggests that the r e l a t i v e l y h i g h l e v e l of carbohydrate i n type I I c o l l a g e n i s not due to d i f f e r e n c e s i n amino a c i d sequences which favor the enzymatic r e a c t i o n s ( i . e . h y d r o x y l a t i o n and g l y c o s y l a t i o n ) . I t appears that almost every l y s i n e i n p r o c o l l a g e n a l ( I I ) chains which i s i n the Y p o s i t i o n of the Gly-X-Y r e p e a t i n g sequences, i s hydroxylated and g l y c o s y l a t e d . On the. other hand, most of these l y s i n e s i n a l ( I ) are e i t h e r hydroxylated to a minimal extent or not at a l l and, are thus not g l y c o s y l a t e d . I t i s c u r i o u s that the extent of h y d r o x y l a t i o n of a2 chains i s greater than that of a l ( I ) , but f o r th t p a r t t h i phenomeno i confined to the N H - t e r m i n a l t h i r The more extensive h y d r o x y l a t i o o l y s i n e s and g l y c o s y l a t i o n of the r e s u l t a n t hydroxylysines i n type I I p r o c o l l a g e n could be due to a slower r a t e o f t r i p l e - h e l i x formation. I n s t u d i e s on the b i o s y n t h e s i s o f type I I p r o c o l l a g e n by c h i c k embryo c a r t i l a g e c e l l s , U i t t o and Prockop (49) found that c h a i n a s s o c i a t i o n and t r i p l e - h e l i x formation of p r o c o l l a g e n a l ( I I ) chains r e q u i r e d almost twice as long as that f o r type I c o l l a g e n synthesized by tendon c e l l s . These data t h e r e f o r e support the above hypothesis. A l t e r n a t i v e l y c a r t i l a g e c e l l s may simply have a higher a c t i v i t y f o r l y s y l hydroxylase and the g l y c o s y l t r a n s f e r a s e s . 2
DISEASES AFFECTING THE LEVELS OF COLLAGEN-ASSOCIATED CARBOHYDRATE One o f the symptoms of diabetes i s a t h i c k e n i n g of basement membranes. Beisswenger and S p i r o (50) have reported that d i a b e t i c glomerular basement membranes c o n t a i n increased l e v e l s of h y d r o x y l y s i n e and of h y d r o x y l y s i n e - l i n k e d d i s a c c h a r i d e u n i t s , compared to c o n t r o l specimens. S p i r o (51) a l s o found an i n creased a c t i v i t y of c o l l a g e n g l u c o s y l t r a n s f e r a s e a c t i v i t y i n a l l o x a n - d i a b e t i c r a t kidneys, which was p a r t i a l l y reversed by i n s u l i n a d m i n i s t r a t i o n . Cohen and K h a l i f a (52) s t u d i e d the e f f e c t of diabetes on p r o l y l and l y s y l hydroxylase a c t i v i t i e s i n the r a t glomerulus. Data from r a t s made d i a b e t i c by s t r e p t o z o t o c i n i n j e c t i o n s was compared t o that from age-matched c o n t r o l s and from i n s u l i n - t r e a t e d , s t r e p t o x o t o c i n - d i a b e t i c r a t s . They found that l y s y l hydroxylase a c t i v i t y was s i g n i f i c a n t l y higher i n d i a b e t i c r a t kidneys than i n c o n t r o l animals, but that p r o l y l hydroxylase was unaffected . A d m i n i s t r a t i o n of i n s u l i n reduced the l y s y l hydroxylase a c t i v i t y to normal. These observations suggest that a l t e r a t i o n s o f h y d r o x y l a t i o n and g l y c o s y l a t i o n of l y s i n e s i n basement membrane c o l l a g e n s might r e l a t e t o the nephropathology o f diabetes.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
224
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
I t should be pointed out that the d i f f e r e n c e s i n hydroxylys i n e and g l y c o s y l a t e d h y d r o x y l y s i n e i n d i a b e t i c kidneys seen by Beisswenger and S p i r o (50) have not been noted by other i n v e s t i g a t o r s (53,54). This p o i n t i s t h e r e f o r e one of controversy. Osteogenesis imperfecta congenita (01) i s a genetic disease r e s u l t i n g i n bones of severe f r a g i l i t y . Since c o l l a g e n i s the m a t r i x o r framework onto which a p a t i t e c r y s t a l s a r e l a i d during bone formation, i t has long been assumed that the b a s i c d e f e c t i n 01 i s some d e f e c t i n c o l l a g e n s t r u c t u r e (55). D i r e c t chemical a n a l y s i s of t i s s u e s from 01 p a t i e n t s have shown that bone and d e n t i n c o l l a g e n s have i n c r e a s e d l e v e l s of h y d r o x y l y s i n e (56). Recently T r e l s t a d , ^ t ail (57) reported a more complete biochemi c a l a n a l y s i s of c o l l a g e n from s e v e r a l t i s s u e s of an 01 i n f a n t , and compared the r e s u l t s to these of t i s s u e s from age-matched c o n t r o l s . Hydroxylysine was doubled i n 01 bone c o l l a g e n and increased by 55% i n c a r t i l a g e I n a d d i t i o n the galactose and glucose l e v e l s of 01 c o l l a g e creased. The data sugges a s s o c i a t e d w i t h i n c r e a s e s i n g l y c o s y l a t e d h y d r o x y l y s i n e s i n type I c o l l a g e n of bone and type I I c o l l a g e n of c a r t i l a g e . The a c t u a l e f f e c t which increased l e v e l s of h y d r o x y l y s i n e and the a s s o c i a t e d g l y c o s i d e s might have on the s t r u c t u r e and f u n c t i o n of c o l l a g e n s i s unknown a t t h i s time. But i t seems apparent from the foregoing d i s s c u s s i o n that the c o n t r o l of postt r a n s l a t i o n a l m o d i f i c a t i o n s i s c r i t i c a l i n order to i n s u r e that the e x t r a c e l l u l a r products ( i . e . c o l l a g e n monomers, f i b r i l s , and f i b e r s ) a r e able to perform the intended f u n c t i o n s . ACKNOWLEDGEMENT The research from t h i s l a b o r a t o r y r e f e r r e d to i n t h i s p u b l i c a t i o n was supported by United States P u b l i c Health S e r v i c e Grant DE-02670.
LITERATURE CITED 1. 2. 3. 4. 5. 6. 7.
Grassmann, W. and Schleich, J . (1935). Biochem. Z. 27, 230. Grassmann, W., Hörmann, H . , and Hafter, R. (1957). HoppeSeyler's Z. Physiol. Chem. 307, 87. Kühn, K. Grassmann, W., and Hoffmann, U. (1959). Z Naturforsch. Teil B. 14, 436. Gross, J., Dumsha, B., and Glazer, N. (1958) Biochim. Biophys. Acta 30, 293. Blumenfeld, O.O., Paz, M.A., Gallop. P.M., and Seifter, S. (1963). J . Biol. Chem. 238, 3835. Kefalides, N.A. and Winzler, R.J. (1966). Biochemistry 5, 702. Spiro, R.G. (1967). J . Biol. Chem. 242, 1915.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
12.
BUTLER
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Carbohydrate
Moieties
of the
Collagens
225
Dische, Z. (1964). In Involvement of Small Blood Vessels in Diabetes, Ed. by Siperstein, E . , Washington: Amer. Inst. Biol. S c i . , p. 201. Butler, W.T., and Cunningham, L.W. (1965). J . Biol. Chem. 240, 3449. Butler, W.T., and Cunningham, L.W. (1966). J . Biol. Chem. 241, 3882. Spiro, R.G. (1967). J . Biol. Chem. 242, 4813. Kefalides, N.A. (1967). in "Proceedings of the Sixth Congress of the International Diabetes Foundation", Ed. by J. Ostman, Excerpta Med. Found., Amsterdam, P. 307. Cunningham, L.W., and Ford (1968). J . Biol. Chem. 243, 2390. Spiro, R.G. (1969). J . Biol. Chem 244, 602. Katzman, R.L., Halford, M.H., Reinhold, V.N., and Jeanloz, R.W. (1972). Biochemistry 11 1161 Miller, E . J . , and Sci., USA 64, 1264 Miller, E.J. (1976). Mol. Cell. Biochem. 13, 165. Goodman, M., Greenspon, S.A., and Krakower, C.A. (1955). J. Immunol. 75, 96. Kefalides, N.A. (1973). Intl. Rev. Connect. Tiss. Res. 6, 63. Kefalides, N.A. (1971). Biochem. Biophys. Res. Commun. 45, 226. Daniels, J.R. and Chu, G.H. (1975). J . Biol. Chem. 250, 3531. Chung, E . , Rhodes, R.K., and Miller, E.J. (1976). Biochem. Biophys. Acta 71, 1167. Hudson, B.G., and Spiro, R.G. (1972). J . Biol. Chem. 247, 4229. Redi, K.B.M. (1974). Biochem. J . 141, 189. Butler, W.T. (1970). Biochemistry 9, 44. Fietzek, P.P. and Kühn, K. (1976). Intl. Rev. Connect. Tis. Res. 7, 1. Aguilar, H . , Jacobs, H.G., Butler, W.T., and Cunningham, L.W. (1973). J . Biol. Chem. 248, 5106. Butler, W.T., Miller, E.J. and Finch, J . E . , J r . , (1976). Biochemistry 15, 3000. Seyer, J.M., and Kang, A.H. (1977). Biochemistry 16, 1158. Butler, W.T., Finch, J . E . , J r . , and Miller E.J. (1977). Biochemistry 16, 4981. Francis, G., Butler, W.T., and Finch, J . E . , J r . , (1978). Biochem. J., in press. Kivirikko, K . I . and R i s t e l i , L. (1976). Med. Biol. 54, 154. Bornstein, P. (1974). Annu. Rev. Biochem. 43, 567. Fessler, L.I., Morris, N.P., and Fessler, J.H. (1975). Proc. Natl. Acad. Sci. USA 72, 4905.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
226
35. Martin, G.R., Byers, P.H., and Piez, K.A. (1975). Adv. Enzymol. 42, 167. 36. Grant, M.E., Schofield, D . J . , Kefalides, N.A., and Prockop, D.J. (1973). J . Biol. Chem. 248, 7432. 37. Kivirriko, K.I., Ryhänen, L., Anttinen, H . , Bornstein, P., and Prockop, D.J. (1973). Biochemistry 12, 4966. 38. Ryhänen, L. and Kivirikko, K . I . (1974). Biochim. Biophys. Acta 343, 129. 39. Ryhänen, L. (1975). Biochim. Biophys. Acta 397, 50. 40. Ryhänen, L. (1976). Biochim. Biophys. Acta 438, 71. 41. Oikarinen, A . , Anttinen, H., and Kivirikko, K . I . (1976). Biochem. J. 156, 545. 42. Myllylä, R., Anttinen, H . , Ristelli, L. and Kivirikko, K.I. (1977). Biochim. Biophys. Acta 480, 113. 43. R i s t e l i , L . , Myllä, R., Kivirikko, K . I . (1976). Europ. J . Biochem. 67, 197 44. Myllylä, R., R i s t e l l i Europ. J . Biochem 45. Oikarinen, A . , Anttinen, H . , and Kivirikko, K . I . (1977). Biochem. J. 166, 357. 46. Butler, W.T. (1973). Ala. J. Med. Sci. 10, 103. 47. Butler, W.T., Finch, J . E . , Jr. and Desteno. C.V. (1972). Biochim. Biophys. Acta 257, 157. 48. Butler, W.T. (1972). Biochem. Biophys. Res. Commun. 48, 1540. 49. Uitto, J . and Prockop, D.J. (1974). Biochemistry 13, 4586. 50. Beisswenger, P., and Spiro, R.G. (1970). Science 168, 596. 51. Spiro, R.G. (1973). New Engl. J . Med. 288, 1337. 52. Cohen. M.P. and Khalifa, A. (1977). Biochim. Biophys. Acta. 496, 88. 53. Kefalides, N.A. (1973). Adv. Metab. Disorders Suppl. 2, 167. 54. Westberg. N.A. , and Michael, A.F. (1973). Acta Med. Scand. 194, 39. 55. McKusick, V.A. (1972). Hereditable Disorders of Connective Tissue, St. Louis: Mosby, p. 230. 56. Eastoe, J . E . , Martens, P . , and Thomas, N.R. (1973). Calc. Tiss. Res. 12, 91. 57. Trelstad, R . L . , Rubin, D., and Gross, J . (1977). Lab. Invest. 36, 501. RECEIVED
March 13, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13 Molecular Pathology of Vascular Elastic Fiber—The Importance of the Glycoprotein Coat M. M. LONG and D. W. URRY Laboratory of Molecular Biophysics and the Cardiovascular Research and Training Center, University of Alabama Medical Center, Birmingham, AL 35294 J. BAKER and B. CATERSON Department of Rheumatology, University of Alabama Medical Center, Birmingham, AL 35294 The tissues of ligaments maintain their resilency Concentrating on the arterial wall, one sees that in the vascular tree, three major regions comprise the arterial wall; they are from lumen to vessel wall: the intima, the media and the adventia. Each are separated from one another by two dense bands of connective tissue; the internal elastic lamina and the external elastic lamina. Collagen fibers and elastic fibers are predominantly, but not exclusively, in these lamina. Further subdivision finds that the elastic fiber consists of two distinct molecular species, a single protein, elastin, which is surrounded by a glycoprotein sheath (1). Elastin, as a 70,000 m.w. protein, is insoluble because of covalent cross-links achieved primarily by combination and condensation of four lysine side chains to form desmosines and isodesmosines (2). These cross-links result in large bundles with a diameter of about 5-6µm(3). The molecular details which ultimately render elastin elastomeric also render it susceptible to disease, for arterial wall elastic fiber is a site of calcium phosphate deposition and l i p i d binding (4,5). This calcification is a progressive process; in the first decade of l i f e calcium salts comprise 0.6% of aorta dry weight, while in the eighth decade the percentage is from 6% to 12% (6). It i s also rather specific to the elastic fiber. Martin and colleagues (7) showed that in the internal elastic lamina of rat aorta, zones of heavy mineralization occurred on the elastic fiber. Only when calcification was extensive did it s p i l l over onto collagen fibers. How this elastin calcifies has been a long standing problem of not inconsiderable interest, especially in view of the fact that the molecule is largely hydrophobic with few negatively charged groups. 90% of the amino acids of elastin have non-functional side chains; 33% are glycine; and 58% are hydrophobic residues. Of the remaining 10%, glutamyl and aspartyl residues constitute 2-3%, but 90% of these are blocked by being in their amide form, i . e . asparagine and glutamine (8). In 1971 (9), the 0-8412-0452-7/78/47-080-227$07.25/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
228
idea of n e u t r a l s i t e binding/charge n e u t r a l i z a t i o n was developed to e x p l a i n e l a s t i n s c a l c i f i c a t i o n , an idea consonant w i t h e l a s t i n s amino a c i d composition and primary s t r u c t u r e (Figure 1). The thought was that e l a s t i n bound calcium a t the f o r m a l l y n e u t r a l yet p o l a r i z a b l e carbonyl oxygens of the peptide m o i e t i e s o r i e n t e d i n a c o n f i g u r a t i o n that o f f e r e d s e l e c t i v e b i n d i n g . Calcium c a t i o n s would be c o n t i n u o u s l y bound u n t i l p o s i t i v e charge-charge r e p u l s i o n between calcium ions balanced out the b i n d i n g s i t e s calcium a f f i n i t y . This i n i t i a l calcium c h e l a t i o n then s e t the stage f o r the next step i n c a l c i f i c a t i o n , phosphate d e p o s i t i o n , because once calcium i n t e r a c t e d w i t h the e l a s t i n m a t r i x , i t became p o s i t i v e l y charged and sequestered p o l y v a l e n t anions such as phosphate. Phosphate n e u t r a l i z e d the p o s i t i v e charge of the c a l c i u m , allowed more calcium b i n d i n g which i n t u r n brought down more phosphate anions, forming the foundation f o r hydroxyapatitegrowth. Experimental data charge n e u t r a l i z a t i o n theory b i n d i n g to n e u t r a l s i t e s - the peptide c a r b o n y l s , and (2) subsequent c a l c i f i c a t i o n of n e u t r a l e l a s t i n molecules. Figure 2 presents the c a t i o n t i t r a t i o n s of blocked a - e l a s t i n w i t h i n c r e a s ing c o n c e n t r a t i o n of CaCl2 and NaCl. Binding was f o l l o w e d by monitoring the c i r c u l a r d i c h r o i s m s i g n a l a t 220 nm a t each s p e c i f i c i o n increment and p l o t t i n g the d i f f e r e n c e between the i n i t i a l e l l i p t i c i t y and the e l l i p i c i t y a t each i o n a d d i t i o n . I n TFE, c a l c i u m bound blocked a - e l a s t i n r e a d i l y , sodium d i d not. These data are supportive of the n e u t r a l s i t e carbonyl b i n d i n g concept because the sample of a - e l a s t i n used was t o t a l l y blocked. A l l the f r e e aminos were blocked w i t h formyl and a l l the f r e e c a r boxyls w i t h methyl, making the molecule uncharged. By e l i m i n a t i o n , one would p r e d i c t then that calcium was b i n d i n g a t n e u t r a l s i t e s , the c a r b o n y l . I n f r a r e d spectroscopy provided data which d i r e c t l y i m p l i c a t e d the peptide backbone as the calcium b i n d i n g s i t e . IR was used because i t can f o l l o w d i f f e r e n t v i b r a t i o n a l modes of the peptide moiety i n molecules too l a r g e to be s t u d i e d w i t h nuclear magnetic resonance. The amide I C-0 s t r e t c h of the carbonyl of the peptide moiety of blocked a - e l a s t i n coacervate was monitored as a f u n c t i o n of c a l c i u m a d d i t i o n . F i g u r e 3 shows t h a t c a l c i u m caused the carbonyl frequency of 1662 c m to s h i f t to lower wave numbers and to become b i p h a s i c w i t h minima a t 1655 cm" and 1630 cm" (10). This i s the same p a t t e r n e x h i b i t e d by a s y n t h e t i c e l a s t i n peptide which bound calcium c a t i o n s v i a i t s peptide carbonyls as shown by PMR and CMR (11). These data l e d to the c o n c l u s i o n that the calcium i o n s p e c i f i c s i t e i s the peptide carbonyl oxygen i n blocked a - e l a s t i n coacervate. Since the coacervate approximates an i n v i v o conformation of e l a s t i n ( 1 2 ) , these s t u d i e s support the idea of n e u t r a l s i t e b i n d i n g t o e l a s t i n when i t i s i n a s t a t e having d i r e c t bearing on i t s b i o l o g i c a l state. Not only does blocked a - e l a s t i n bind c a l c i u m and a t n e u t r a l f
1
1
-1
1
1
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
LONG E T A L .
Vascular
Elastic
Fiber
229
Technical Publishing Company
Figure 1. Schematics of neutral-site calcium ion binding to the elastin matrix, showing calcium sequestration followed by deposition of polyvalent anions, in this case, phosphate anions. Adapted from Ref. 46.
Blocked
a-Elastin
T i t r a t i o n in T F E
Co**
Figure 2. Ion titration of N-formyl-Omethyl-ester a-elastin from aorta in trifluordethanol (TFE) with aqueous increments of 0.1M CaCl and 0.1 M NaCl followed by circular dichroism. A[6] is the difference between the ellipticity of the free state and each state with incrementing amounts of cation. 2
2 [Cation]
3 mM
4
5
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Coacervate of a-elastin . Blocked + NaCl (2M) + N a H P 0 (2M) + CaCI (2M) 2
4
2
1700
1600
1500
cm"
1400
1
Biochemical and Biophysical Research Communications Figure 3. IR spectra with percent transmission plotted vs. wave number of blocked a-elastin coacervate in the presence and absence of calcium, ( ; 2 M CaCl ( ) 2M NaCl, ( ) 2M NaCl + KH PO (10). 2y
2
/f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
Fiber
231
s i t e s , but i t a l s o i n i t i a t e s c a l c i f i c a t i o n . E l a s t i n was incubated at 37°C f o r 20 hours i n a serum c a l c i f y i n g medium w i t h (Figure 5) and without (Figure 4) calcium and phosphate (12). The secondary e l e c t r o n image o f the c o n t r o l i s seen i n F i g u r e 4b. The e l a s t i n m a t r i x had cracked along the top and l e f t hand corner, r e v e a l i n g the p l e x i g l a s support beneath. The s u r f a c e appears smooth, the X-ray spectrum (Figure 4a) contains a peak f o r thealuminum coat and a s m a l l one f o r s u l f u r , and the X-ray map (Figure 4c) i n d i cates no c o n c e n t r a t i o n o f c a l c i u m ions on the sample when the EDAX was programmed t o detect only c a l c i u m X-rays. The other sample, having been exposed t o calcium and phosphate, now has a rough textured topography (Figure 5b), an X-ray spectrum i n d i c a t i n g calcium (Figure 5a) and phosphate and a c a l c i u m map (Figure 5c) which r e v e a l s a uniform d i s t r i b u t i o n o f calcium, l o c a l i z e d only on a - e l a s t i n . When Epon embedded c a l c i f i e d m a t e r i a l i s cross sectioned perpendicular t o i t s s u r f a c e (Figure 6), i t s secondary image s t r a t e that c a l c i f i c a t i o m a t e r i a l (14). I n summary, the CD s t u d i e s and the SEM-EPM data demonstrate the v a l i d i t y of the n e u t r a l s i t e / c h a r g e n e u t r a l i z a t i o n theory w i t h respect t o e l a s t i n calcium b i n d i n g and c a l c i f i cation. a - E l a s t i n , being of 70,000 m.w., i s much too l a r g e t o study i n molecular s t r u c t u r a l d e t a i l w i t h nuclear magnetic resonance, both PMR and CMR which would be techniques of choice t o d e l i n e a t e the calcium b i n d i n g s i t e s . Gray and Sandberg have sequenced about one h a l f o f t r o p o e l a s t i n , the precursor p r o t e i n of e l a s t i n (15,16) and found r e p e a t i n g sequences, a tetramer, a pentamer, and a hexamer o c c u r r i n g approximately, f o u r , s i x and f i v e times i n s i n g l e sequences, r e s p e c t i v e l y . They are as f o l l o w s : Val-Pro-Gly-Gly Val-Pro-Gly-Val-Gly Ala-Pro-Gly-Val-Gly-Val Our l a b o r a t o r y synthesized these repeat peptides as monomers, dimers, t r i m e r s and high polymers. Because o f t h e i r s m a l l molec u l a r weight, r e l a t i v e t o e l a s t i n and t h e i r defined number of d i f f e r e n t amino a c i d r e s i d u e s , these peptides are amenable t o study w i t h s p e c t r o s c o p i c techniques, e s p e c i a l l y NMR to e l u c i d a t e f u r t h e r the idea of n e u t r a l s i t e binding/charge n e u t r a l i z a t i o n . F i g u r e 7a i s the i o n t i t r a t i o n of the pentamer N-formyl-Val-ProGly-Val-Gly-O-Methyl followed w i t h c i r c u l a r d i c h r o i s m as i n F i g u r e 2. Of the three d i v a l e n t c a t i o n s , s t r o n t i u m c h e l a t e d the best f o l l o w e d by calcium and then magnesium. I n t e r a c t i o n w i t h sodium and potassium was n e g l i g i b l e — w i t h i n i n s t r u m e n t a l e r r o r (17). Calcium and magnesium had the same i n i t i a l s l o p e , but a f t e r an [ i o n ] / [ p e p t i d e ] r a t i o of about 0.25, the magnesium curve became b i p h a s i c , as i f the pentamer peptide were l o s i n g the i o n . This i s undoubtedly the case because, w h i l e the peptide i s
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
232
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Calcified Tissue Research Figure 4. Scanning electron microscopy and electron probe microanalysis data for blocked a-elastin incubated in a calcifying medium without calcium and phosphate (a) x-ray spectrum, (b) secondary electron image, and (c) calcium elemental map. The identification of the peaks in the x-ray spectrum (a) is as follows: 1.48 KeV peak is a scatter peak originating from the aluminum sample holder; 2.3 KeV peak is the K sulfur peak. Calcium and phosphorus x-rays were not detected. The vertical scale is from 0 to 250 cps. The secondary electron image (b) is at 850X magnification and demonstrates that the noncalcified elastin coacervate is a smooth film below which is visible the Plexiglas support in the cracked areas. Figure (c) is the calcium map of the area shown in Figure (b) with the ED AX set at 3,690 KeV energy (13). al
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
233
Fiber
Calcified Tissue Research
Figure 5. This figure is arranged identically to Figure 4 and presents data obtained from blocked a-elastin coacervate which has been incubated in a calcifying medium with CaCl and KH PO^. The x-ray spectrum (a) contains a peak at 2.013 KeV corresponding to phosphorus K x-rays, a peak at 3.690 KeV corresponding to calcium K x-rays, and a small peak at 2.307 for sulfur. The vertical scale is from 0 to 10,000 cps. The secondary image is at 850X magnification showing that the calcified coacervate is very rough in appearance. Figure (c) is the calcium x-ray map which shows a dense population over areas of the field covered by coacervate. At 850X the calcium distribution appears uniform over the elastin matrix (IS). 2
2
al
a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
234
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Calcified Tissue Research Figure 6. Again the same arrangement as in Figures 4 and 5. In the x-ray spectrum (a), the peaks at 2.013 KeV and 3.690 KeV are from the phosphorus K x-rays and calcium Koc x-rays respectively. The peak at 1.48 KeV is from the aluminum coating of the material. The secondary electron image is of cross-sectioned calcified elastin coacervate at 0.5/x thickness taken at 780X magnification. The depth of the coacervate calculates to he 10p. The calcium x-ray map (c) shows that calcification extends the whole depth of the coacervate, although concentrated on the serum side (14). al
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
Fiber
235
d i s s o l v e d i n t r i f l u o r o e t h a n o l , the i o n i s added i n 1 u £ aqueous a l i q u o t s . With water a d d i t i o n , a c o m p e t i t i o n f o r the c a t i o n i s set up between the peptide carbonyl oxygen and the water oxygen, each t r y i n g t o a t t r a c t the p o s i t i v e l y charged s p e c i e s . This comp e t i t i o n can be used t o an advantage t o compare b i n d i n g a f f i n i t i e s among ions and peptides which have d i f f e r e n t s t o i c h i o m e t r i e s and d i f f e r e n t water s e n s i t i v i t i e s . F i g u r e 7b i s a b a c k - t i t r a t i o n of both the c a l c i u m and magnesium peptide complexes w i t h water t o assess the s t r e n g t h w i t h which the peptide carbonyl can secure the two c a t i o n s . The midpoint o f each complex's t i t r a t i o n i s i n d i c a t e d by an arrow. This p o i n t i s i n t e r p r e t e d t o be the conc e n t r a t i o n of water where one h a l f of the c a t i o n i s bound t o water and one h a l f t o the peptide c a r b o n y l . Once the r a t i o o f water c o n c e n t r a t i o n t o peptide carbonyl c o n c e n t r a t i o n a t t h i s p o i n t i s c a l c u l a t e d i t can be used t o compare the b i n d i n g a f f i n i t i e s o f the pentamer monomer r e l a t i v e t o i t s h i g h polymer o r r e l a t i v e t o the hexame summarizes these v a l u e s TABLE I * Peptide
Calcium [H2O] Complex [TJRj]
Magnesium [H2O] Complex [TJSTJ]
215
53
350
97
270
66
535
167
HCO-Val-Pro-Gly-ValGly-OMe HCO-(Val-Pro-Gly-ValGly) -Val-OMe n
HCO-Val-Ala-Pro-GlyVal-Gly-OMe HCO-(Val-Ala-Pro-Gly-Val-Gly) *Expanded from Reference 17 Val-OMe
F i g u r e s 8a and 8b a r e comparable data f o r the hexamer repeat e l a s t i n peptide (18). I n both i t s monomeric and polymeric form i t c h e l a t e d c a l c i u m and magnesium b e t t e r than the pentamer repeat e l a s t i n p e p t i d e . P o l y m e r i z a t i o n of both repeats a l s o enhanced t h e i r b i n d i n g p o t e n t i a l . I n numerical terms the c a l c i u m a f f i n i t y constant f o r the hexamer monomer was about 0.5x10^ a t l i m i t i n g peptide c o n c e n t r a t i o n (18). Since a l l o f these s y n t h e t i c peptides were n e u t r a l , the f a c t that they bound calcium t i g h t l y and s e l e c t i v e l y provides f u r t h e r support f o r n e u t r a l s i t e b i n d i n g . NMR t i t r a t i o n s o f the s y n t h e t i c e l a s t i n repeat peptide both prove t h a t the calcium b i n d i n g s i t e i s composed o f peptide carbon y l s and d e l i n e a t e which carbonyls a c t u a l l y a r e i n v o l v e d (11,18).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
236
HCO-Val,-Pro2-Gly -Val -Gly5-OMe 3
4
Ion Titration in T F E
0.5
1.0
1.5
2.0
[lon]/[Peptide]
Calcium Binding Proteins and Calcium Function Figure 7a.
Ion titration of the repeat pentapeptide of elastin; cf. Figure 2 and text for discussion (17).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
LONG E T A L .
Vascular
Elastic
237
Fiber
HCO-Val|-Pro2-Gly -Val4-Gly -OMe 3
Water Titration of C a * *
Complex
5
and M g * * Complex
Calcium Binding Proteins and Calcium Function
Figure
7b.
Water titration of the calcium and magnesium complexes of the pentapeptide. See text for discussion (17).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
238
N F (VAPGVG) OMe .6-
0
T F E + Varying [ H O ] 2
0.5
1.0
1.5
2.0
2.5
[PEPTIDE]
Archives of Biochemistry and Biophysics Figure 8a. Ion titration of the repeat hexapeptide of elastin in trifluoroetnanol (TFE) with ion additions as aqueous aliquots of 0.1M chloride salts (18).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
LONG E T A L .
Vascular
Elastic
Fiber
239
NF (VAPGVG) OMe - C o " , Mg**, Sr* + H0 2
x I0~
Titration
4
.10
6
8
10
12
14
H 0 % (v/v) 2
Archives of Biochemistry and Biophysics
Figure 8b.
Water titration of the strontium, calcium, and magnesium ion complexes of the hexapeptide repeat (IS).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND
240
GLYCOLIPIDS IN DISEASE PROCESSES
PMR f o l l o w e d the calcium t i t r a t i o n of the pentamer i n t r i f l u o r o e t h a n o l w i t h 3% water (Figure 9 ) . The PMR s i g n a l , the chemical s h i f t , i s p l o t t e d as a f u n c t i o n of i n c r e a s i n g c a l c i u m concentrat i o n , w i t h the peptide c o n c e n t r a t i o n h e l d constant. P l o t t e d are the protons of the formyl p r o t e c t i n g group, V a l i NH, Gly3 NH, Val4 NH and Gly5 NH (17). Because of experimental c o n d i t i o n s the v a l i n e and g l y c i n e NH were not d e l i n e a t e d . The carbonyl of each r e s p e c t i v e peptide moiety i n t e r a c t e d because a l l the resonances s h i f t e d downfield. These are the C=0 of the Pro2, the Gly3 and the Val4 r e s i d u e s . Since the V a l ^ C=0 precedes the p r o l i n e and s i n c e the Gly5 C=0 i s p a r t of the methyl e s t e r there are no pept i d e NH data f o r them. Stepwise t i t r a t i o n of the pentamer s carbonyls f o l l o w e d by C-13 nuclear magnetic resonance f i l l e d i n the gaps i n and confirmed the PMR data. I n F i g u r e 10 i s p l o t t e d the peptide moiety carbonyl carbon resonances as a f u n c t i o n of c a l c i u m concentration There are four carbonyl Gly3 C=0, the Val4 C=0, p r e d i c t e d by the previous PMR data, w h i l e no PMR data were a v a i l able f o r the l a s t . The Pro2 C=0 curve i s anomalous; w h i l e PMR i n d i c a t e d t h a t the Pro2 C=0 d i d b i n d , here there i s l i t t l e i n d i c a t i o n of a downfield s h i f t . The e x p l a n a t i o n may l i e i n the f a c t that the Pro2 C=0 i s i n the end peptide moiety of a beta t u r n which, w i t h c a l c i u m b i n d i n g , would be d i s r u p t e d . The downfield s h i f t upon c a l c i u m c h e l a t i o n could be obscurred by a p a r a l l e l u p f i e l d s h i f t as the carbonyl came out of the beta t u r n . Table I I summarizes these NMR data. f
TABLE I I Pentamer C=0 Candidates PMR
CMR
Yes
Yes
C=0
-
No
HCO Val
±
f o r Ca B i n d i n g
Pro
C=0
Yes
?
2
Gly
3
C=0
Yes
Yes
Val
4
C=0
Yes
Yes
Gly
5
C=0
-
Yes
A l l the c a r b o n y l s , but the V a l ^ , were i n v o l v e d i n the calcium b i n d i n g s i t e of the pentamer. PMR and CMR t i t r a t i o n s of the hexamer (11) repeat peptide i n d i c a t e that again a l l the carbonyls of the HCO-Val -Ala2-Pro3-Gly4-Val5-Gly6-OMe, but the V a l ^ bound 1
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 0.5
2
3
titration 2
4
2
2
[caCI ]/[Peptide]
1.0
1.5
2.0
Calcium Binding Proteins and Calcium Function
5
2
Figure 9. PMR titration data of CaCl and the pentapeptide. The PMR signal, the chemical shift of each peptide NH, is plotted as a function of [CaCl ] which was added as a powder to the peptide in TFE (17).
0.0
2
in T F E with 3% H 0
CaCI
H C O - V a l i - Pro - G I y - V a l - G l y - O M e
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
242
0
0.5
1.0
1.5
2.0
2.5
[caCI ]/[peptide] 2
Calcium Binding Proteins and Calcium Function Figure
10.
CMR titration data as in Figure 9. Here the carbonyl chemical shift is plotted as a function of [CaCl ] (17). 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
Fiber
243
c a l c i u m . The major p o i n t from these s t u d i e s i s that the n e u t r a l e l a s t i n repeat peptides bind c a l c i u m v i a t h e i r c a r b o n y l s . As w i t h n a t i v e a - e l a s t i n , the repeat e l a s t i n s y n t h e t i c pept i d e s a l s o i n i t i a t e d c a l c i f i c a t i o n f o r when c r o s s - l i n k e d , they formed i n s o l u b l e matrices which served as s u b s t r a t e s f o r hydroxya p a t i t e growth (19). F i g u r e 11 provides the scanning e l e c t r o n micrographs, Ca X-ray maps, and X-ray s p e c t r a of both the p o l y pentapeptide (a,b,c) and polyhexapeptide (d,e,f) (20). Each was incubated w i t h serum augmented w i t h 3.0 mM exogenous CaCl2 and KH2PO4 a t 37°C f o r 72 hours. F i g u r e l i b , which i s the c a l c i u m X-ray map corresponding t o the secondary e l e c t r o n image o f F i g u r e 11a, shows that the polypentapeptide c a l c i f i e d over i t s e n t i r e s u r f a c e . I n a d d i t i o n once c r o s s - s e c t i o n e d t h i s c a l c i f i e d m a t e r i a l has c a l c i u m and phosphate evenly d i s t r i b u t e d throughout i t s b u l k (21). I n c o n t r a s t , the hexamer c a l c i f i e d o n l y i n d i s c r e t e patches. The i n f o r m a t i o n presente neutral site/charge n e u t r a l i z a t i o y e l a s t i n c a l c i f i c a t i o n . The evidence i s so c o n v i n c i n g t h a t one could ask, s i n c e e l a s t i n and the repeat e l a s t i n peptides bind c a l c i u m and c a l c i f y r e a d i l y i n v i t r o , why does not e l a s t i n c a l c i f y j u s t as spontaneously and r a p i d l y i n v i v o ? The f a c t that i t does not p o i n t s t o the n e c e s s i t y o f c o n t r o l mechanisms and/or p r o t e c t i v e b a r r i e r s . Other i n v i v o observations suggest the same. The extent o f e l a s t i c f i b e r m i n e r a l i z a t i o n v a r i e s from t i s s u e t o t i s s u e ; the a o r t a and l a r g e a r t e r i e s c a l c i f y w e l l , w h i l e normal s k i n ( w i t h the notable exception o f the s k i n o f p a t i e n t s w i t h Pseudoxanthoma elasticum) and lung c a l c i f y p o o r l y ( 4 ) . As mentioned before, e l a s t i c f i b e r c a l c i f i c a t i o n i s a progressive d i s e a s e , as i f w i t h time some i n t r i n s i c defense i s breached. M i c r o s c o p i c examination of the e l a s t i c f i b e r shows that i t i s composed of two s t r u c t u r a l l y d i f f e r e n t components, a c e n t r a l area which i s e l a s t i n p r o t e i n and a p e r i p h e r a l sheath o f microf i b r i l s which i n c r o s s s e c t i o n appear t u b u l a r and o f 10-12 nm i n diameter (22-29). During the embryological development o f e l a s t i c t i s s u e , the m i c r o f i b r i l s appear f i r s t i n the e x t r a c e l l u r m a t r i x (22). I n t i s s u e c u l t u r e , f o r example, a f t e r seven days the m i c r o f i l a m e n t s are observable f o l l o w e d by the e l a s t i n by 14 and 21 days (30). A t t h i s stage the e l a s t i c f i b e r has a t t a i n e d roughly i t s mature EM appearance of a c i r c u l a r s t r u c t u r e w i t h a c l e a r center and e l e c t r o n dense border. Once mature, the e l a s t i c f i b e r , e s p e c i a l l y the human f i b e r , i s 10% m i c r o f i b r i l s and 90% e l a s t i n (26,31). The p r o p o r t i o n o f m i c r o f i b r i l t o e l a s t i n v a r i e s w i t h age though, from p r e n a t a l t o mature, t o senescent f i b e r s (26,32,33). Aged e l a s t i c f i b e r s appear t o be d e f i c i e n t i n p e r i p h e r a l m i c r o f i b r i l s (26). Ross and B o r n s t e i n (22), f o l l o w e d by Robert e t a l . (25) prov i d e d the chemical d i f f e r e n t i a t i o n and c h a r a c t e r i z a t i o n o f the m i c r o f i b r i l s v i s a v i s the e l a s t i n p r o t e i n core. Using chemical i s o l a t i o n techniques and monitoring the r e a c t i o n s w i t h e l e c t r o n
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
244
Fifth American Peptide Symposium Figure 11. Figure 11a is an SEM photomicrograph of the cross-linked polypentapeptiae which has been calcified in serum augmented with exogenous 3.0mM CaCl and KH PO . Figure 11c is the x-ray elemental spectrum indicating the presence of calcium at 3.7 KeV, phosphorus at 2.0 KeV, and aluminum (from the coating) at 1.4 KeV in the sample. Figure lib is the calcium x-ray map demonstrating that calcium phosphate clearly deposited on the synthetic elastin framework. Figures lid, e, and f fright) are the same as a,b and c except for the cross-linked polyhexapeptide (20). 2
2
h
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
LONG E T A L .
Vascular
Elastic
Fiber
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
245
246
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
microscopy, Ross and B o r n s t e i n demonstrated that the e l a s t i c f i b e r m i c r o f i l a m e n t s were d i g e s t e d by p r o t e o l y t i c enzymes i n c l u d i n g t r y p s i n , chymotrypsin and pepsin which d i d not touch e l a s t i n . In c o n t r a s t , hyaluronidase and 3-glucuronidase were without e f f e c t . A n a l y s i s of the amino a c i d composition of the m i c r o f i b r i l s s o l u b i l i z e d by p r o t e o l y t i c enzymes, revealed a s t r i k i n g l y d i f f e r e n t d i s t r i b u t i o n from t h a t seen i n e l a s t i n . They contained more p o l a r and a c i d i c amino a c i d s , more s u l f u r presumably as c y s t i n e , and l e s s n e u t r a l residues such as g l y c i n e , v a l i n e , a l a n i n e and p r o l i n e . They lacked h y d r o x y p r o l i n e , h y d r o x y l y s i n e and desmosines, c l e a r l y d i s t i n g u i s h i n g them from both e l a s t i n and c o l l a g e n . The high c y s t i n e content, 70-80 residues/1000 r e s i d u e s , was thought to represent numerous d i s u l f i d e l i n k a g e s which rendered the m i c r o f i b r i l s r e l a t i v e l y i n s o l u b l e (26), and a l s o provided a non enzymatic way to s o l u b i l i z e the sheath. The i n v e s t i g a t o r s found that d i t h i o t h r e i t o l , a d i s u l f i d e bond reducin r e l e a s e d the m i c r o f i b r i l another sample whose amino a c i d composition was that of the e n z y m a t i c a l l y cleaved m a t e r i a l . Numerous sugar m o i e t i e s were a l s o found i n d i c a t i n g the m a t e r i a l was made up of one or more g l y c o p r o t e i n s . The f i b r i l s were 4.7% by weight hexose as measured w i t h the anthrone reagent and 0.7% hexosamine as determined by i o n exchange chromatography (22). The 1976 paper by Muir, B o r n s t e i n and Ross (34) d e s c r i b e s the i s o l a t i o n of one of the presumptive p r o t e i n s of the g l y c o p r o t e i n sheath and shows that i t i s of l a r g e molecular weight, i . e . 270,000. With t h i s s i z e and the amino a c i d and carbohydrate composition i n mind, one could suggest that the g l y c o p r o t e i n sheath i n h i b i t s premature c a l c i f i c a t i o n by forming a s h i e l d around the p r o t e i n core of the e l a s t i c f i b e r , screening i t from calcium i o n s . To t e s t t h i s i d e a , i d e a l l y one would wish to i n v e s t i g a t e the i n v i t r o i n t e r a c t i o n of e l a s t i n and i t s g l y c o p r o t e i n ( s ) and i n a c a l c i f y i n g medium, to see i f c a l c i f i c a t i o n i s i n h i b i t e d . To date, these experiments have not been c a r r i e d out, due i n great p a r t , to the d i f f i c u l t y i n o b t a i n i n g the g l y c o p r o t e i n . S e v e r a l s t u d i e s have been done, though, f o l l o w i n g the i n t e r a c t i o n of e l a s t i n (35) and t r o p o e l a s t i n (36) w i t h more r e a d i l y o b t a i n a b l e bovine n a s a l c a r t i l a g e proteoglycan. S i g n i f i c a n t i n t e r a c t i o n was found by Radhakrishnamurthy, Ruiz and Berenson (37) w i t h heparan s u l f a t e and dermatan s u l f a t e . In c o n t r a s t , Wusteman and G i l l a r d (38) demonstrated t h a t mature f i b e r s of e l a s t i c t i s s u e from e l a s t i c c a r t i l a g e had l i t t l e s p e c i f i c a s s o c i a t i o n w i t h h y a l u r o n i c acid. Our l a b o r a t o r y s t u d i e d these i n t e r a c t i o n s w i t h a - e l a s t i n and then extended t h i s work to begin to d e f i n e the s i t e s on the e l a s t i n macromolecule which could be s p e c i f i c f o r i n t e r a c t i o n . This was done by i n v e s t i g a t i n g the i n t e r a c t i v e p r o p e r t i e s of each of the repeat e l a s t i n peptide high polymers synthesized here. Bovine n a s a l c a r t i l a g e , i s o l a t e d by Baker w i t h a s l i g h t l y
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
Fiber
247
EQUILIBRIUM SOLUTION
SOLUTION OF ELASTIN PEPTIDES (tropoelastin, a-elastin, polypentapeptide, polyhexapeptide)
COACERVATE 1. stable state 1n aqueous solution at body T. 2. 60% H 0 by volume as 1n fibrous elastin 3. filamentous with periodicities similar to native elastin 2
FIBROUS
ELASTIN
TROPOELASTIN (precursor protein which contains repeat peptides J*
a-ELASTIN (oxalic add fragmentation product)
International Journal of Quantum Chemistry
Figure 12. Diagram of the process of coacervation. Blocked a-elastin, tropoelastin, and polymers of the repeat peptides of elastin are soluble in aqueous solutions at low temperature. As the temperature increases, the solution becomes cloudy and with time a phase separation occurs with a yellow, viscous glue-like precipitate forming below an equilibrium solution. The temperature profile of coacervation follows this process by measuring turbidity as a function of temperature as depicted in Figures 15 and 16 (47).
American Chemical Society Library 1155 16th St. N. w. In Glycoproteins and Glycolipids Disease Processes; Walborg, E.; Washington, D.inC. 20038 ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
1.0
-
0. a-elastin in solution b. a-elastin coacervate film C. a-elastin coacervate film corrected spectrum
c A
A.I
- 1.0
-
240
200
X (nm) International Journal of Quantum Chemistry Figure 13. CD spectra of a-elastin in solution (a) in 0.01M sodium acetate, pH-5, and in its coacervated state (b). Since the coacervate formed a light scattering film, curve b was corrected, giving curve c (41).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
(b) Electron micrograph of polypentapeptide coacervate negatively stained to bring out the fibrillar nature of the material. Twisted rope-like structures are observable (cf. left-handed structure at the arrow) (3).
Perspective in Biology and Medicine
Figure 14. (a) High resolution electron micrographs of tropoelastin coacervate which has been negatively stained, showing parallel aligned 5-nm filaments. As temperature increases, tropoelastin molecules go from a random distribution to the parallel array seen here, showing that elevated temperature enhances fiber formation (42).
Biochimica et Biophysica Acta
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
N-formyl-O-methyl Concentration
10
20
30 Temperature
a-elastin
Dependence
40
50
60
°C
Perspective in Biology and Medicine Figure 15. Blocked a-elastin temperature profiles of coacervation (cf. Figure 12) with varying concentrations of protein. That intermolecular interactions are responsible for coacervation is seen by its concentration dependency. That the process is cooperative is indicated by the increased steepness of the curve with increasing concentration, that is when two a-elastin molecules associate, the resultant conformation facilitates further association (3).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
LONG E T A L .
Vascular
Elastic
251
Fiber
HC0-(Val,-Pr02-6ly3-Val4-Gly ) -Val-0ry1e 5
Concentration
20
30
40
n
Dependence
50 Temperature
60
70
80
°C
Perspective in Biology and Medicine
Figure 16. Polypentapeptide temperature profiles of coacervation with varying concentration of peptide. As with blocked a-elastin in Figure 15, the increased steepness with increased concentration indicates cooperativity (3).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
252
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
m o d i f i e d technique of H a s c a l l and Sajdera (39), and blocked a e l a s t i n , the p o l y t e t r a p e p t i d e , the polypentapeptide, and the polyhexapeptide were mixed together and t h e i r temperature p r o f i l e of c o a c e r v a t i o n recorded. At t h i s p o i n t , a b r i e f d e s c r i p t i o n of e l a s t i n c o a c e r v a t i o n and a method t o f o l l o w i t i s warranted. F i g u r e 12 o u t l i n e s the process of c o a c e r v a t i o n (40-41). Both t r o p o e l a s t i n , which i s the precursor p r o t e i n of f i b r o u s e l a s t i n , and a - e l a s t i n which i s an o x a l i c a c i d fragmentation product d e r i v e d from the f i b e r , coacervate as do the s y n t h e t i c repeat e l a s t i n peptide polymers. When aqueous s o l u t i o n s of these p r o t e i n s and p o l y p e p t i d e s go from room temperature to e l e v a t e d temperature, f o r example from 25°C to body temperature a t 37°C, they undergo a phase t r a n s i t i o n . The s o l u t i o n s become cloudy and w i t h time, a y e l l o w g l u e l i k e p r e c i p i t a t e s e t t l e s t o the bottom of the r e a c t i o n v i a l f o r each species except the polyhexapeptides I t s coacervate w i t h time s e t t l e s to a w h i t e , f l o c u l a r p r e c i p i t a t e increased i n t r a m o l e c u l a the c i r c u l a r d i c h r o i s m s p e c t r a of blocked a - e l a s t i n i n s o l u t i o n at 25°C (curve a ) , and i n i t s coacervate form (curve b ) . Since the coacervate forms a l i g h t s c a t t e r i n g f i l m on the s p e c t r o s c o p i c c e l l , i t s spectrum was c o r r e c t e d (curve c) (42). The i n c r e a s e i n the 222 nm minimum and the appearance of the 192 nm maximum c o r r e l a t e w i t h an i n c r e a s e i n i n t r a m o l e c u l a r order t o between 20% and 25% a - h e l i x (43). The n e g a t i v e l y s t a i n e d micrograph of the t r o p o e l a s t i n coacervate (Figure 14a) proves i t s filamentous nature, one of i n c r e a s e d i n t e r m o l e c u l a r order (44). The p o l y pentapeptide coacervate has the same ordered filamentous ropel i k e s t r u c t u r e (Figure 14b) ( 3 ) . A r a p i d , s e n s i t i v e technique can be used to f o l l o w the coac e r v a t i o n process (45). Since a s o l u t i o n of e l a s t i n or i t s repeat peptides t u r n s cloudy w i t h e l e v a t i o n of temperature, the t u r b i d i t y due t o l i g h t s c a t t e r i n g a t 300 nm can be monitored as a f u n c t i o n of temperature. F i g u r e 15 i s an example of the nature of the data one o b t a i n s . I t i s note-worthy that c o a c e r v a t i o n of blocked a - e l a s t i n i s a d i s t i n c t f u n c t i o n of c o n c e n t r a t i o n . This i s t r u e , t o o , f o r the polypentapeptide ( F i g u r e 16). We found that a d d i t i o n of bovine n a s a l c a r t i l a g e t o coacervatable s o l u t i o n s o f blocked a - e l a s t i n , the p o l y t e t r a p e p t i d e , the polypentap e p t i d e , or two d i f f e r e n t p r e p a r a t i o n s of the polyhexapeptide r e s u l t e d i n changes i n t h e i r temperature p r o f i l e of coacervate m i r r o r i n g changes i n c o n c e n t r a t i o n observed w i t h the f r e e s o l u t i o n s . These o b s e r v a t i o n s a r e summarized i n Table I I I .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
LONG E T A L .
Vascular
Elastic
Fiber
253
TABLE I I I Proteoglycan I n t e r a c t i o n w i t h E l a s t i n and E l a s t i n Repeat P e p t i d e s * Blocked α-Elastin
+
H(VPGG) VOMe n
NF(VPGVG) VOMe n
H(APGVGV) VOMe
+
n
HiAPGifrGV^vOMe
1
+
*Reaction c o n d i t i o n s were as f o l l o w s : [proteoglycan] = 5 mg/ml, [Blocked α-elastin] = 5 mg/ml, [H(VPGG) VOMe] =1.0 mg/ml, [NF(VPGVG) VOMe] =0.5 mg/ml d 0.25 mg/ml [H(APGVGV) VOMe] 1 mg/ml and 0.5 mg/ml an i n aqueous s o l u t i o n s . Th proteoglyca weighe amounts of d r i e d m a t e r i a l d i r e c t l y to the e l a s t i n s o l u t i o n i n the spectrophotometer c e l l which was continuously v i b r a t e d to i n s u r e proper mixing as the complex c o a c e r v a t i o n proceeded. 1: φ i s V a l or L y s . n
n
ACKNOWLEDGMENT : This work was supported i n p a r t by N a t i o n a l I n s t i t u t e s of H e a l t h , Grant No. HL-11310. The authors thank Dr. W. D. Thompson f o r a s s i s t a n c e w i t h the proteoglycan-peptide temperature p r o f i l e s of c o a c e r v a t i o n .
LITERATURE CITED: 1. Ross, R., Bornstein, P., J . Cell. Biol. 40, 366, 1969. 2. Partridge, S. Μ., Gerontologia, 15, 85, 1969. 3. Urry, D. W., Persp. Biol. Med., 21, 265, 1978. 4. Yu, S. Y . , Blumenthal, H. T., in The Connective Tissue, ed. by Β. M. Wagner and D. E. Smith, Williams and Williams Co., Baltimore, 1967, pp. 17-49. 5. Kramsch, D. M., Franzblau, C., Hollander, W., Adv. Exp. Med. Biol., 43, 193, 1974. 6. Yu, S. V . , Blumenthal, H. T., J . Gerontol., 18, 119, 1963. 7. Martin, G. R., Schiffman, E . , Bladen, Η. Α., Nylen, Μ., J. Cell. B i o l . , 16, 243, 1963. 8. Kagan, Η. M., Lerch, R. Μ., Biochim. Biophys. Acta, 436, 223, 1976. 9. Urry, D. W., Proc. Natl. Acad. S c i . , U.S.A., 68, 810, 1971. 10. Long, M. M., Urry, D. W., Starcher, B. C., Biochem. Biophys. Res. Commun., 56, 14, 1974. 11. Urry, D. W., Ohnishi, T., Bioinorg. Chem., 3, 305, 1974. 12. Cox, Β. Α., Starcher, B. C., Urry, D. W., Biochim. Biophys. Acta, 317, 209, 1973. 13. Cox, Β. Α., Starcher, B. C., Urry, D. W., Calcif. Tissue Res., 17, 219, 1975.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
254
14. 15. 16. 17.
18. 19. 20.
21. 22. 23.
24. 25.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
U r r y , D. W., Hendrix, C. F., Long, M. M., Calcif. Tissue Res., 21, 57, 1976. F o s t e r , L. A., Bruenger, E., Gray, W. R., Sandberg, L. B., J . Biol. Chem., 248, 2876, 1973. Gray, W. R., Sandberg, L. B., F o s t e r , J . A., Nature (London), 246, 461, 1973. Long, M. M., Urry, D. W., Thompson, W. D., in Calcium B i n d i n g P r o t e i n s and Calcium F u n c t i o n , ed. Wasserman, R. H. et al., E l s e v i e r North-Holland, N.Y., 1977, p. 419. Long, M. M., O h n i s h i , T., U r r y , D. W., Arch. Biochem. Biophys., 166, 187, 1975. U r r y , D. W., Long, M. M., Okamoto, K., V o l p i n , D., R o v e r i , N., Ripamonte, A., A l a . J. Med. Sci., 14, 255, 1977. Rapaka, R. S., Okamoto, K., Long, M. M., U r r y , D. W., Fifth American P e p t i d e Symposium, U n i v e r s i t y of California, San Diego, La Jolla, California, 1977 U r r y , D. W., Long B i o c h e m i s t r y , 15, Ross, R., B o r n s t e i n , P., J . Cell. Biol., 40, 366, 1969. Ross, R., B o r n s t e i n , P., in Chemistry and M o l e c u l a r B i o l o g y of the Intercellular M a t r i x , ed. B a l a z s , E. A., Academic P r e s s , London and New York, 1970, p. 641. Ross, R., J . Cell. Biol., 50, 172, 1971. Robert, B., Szigetti, M., Derouette, J. C., Robert, L., Bouissou, H., Fabre, M. T., Eur. J . Biochem., 21, 507, 1971. Ross, R., J . Histochem. and Cytochem., 21, 199, 1973. Robert, L., Kadar, A., Robert, B., Adv. Exp. Med. Biol., 43, 85, 1974. Robert, L., Biol. Med. ( P a r i s ) , 4, 1, 1975. Ross, R., P h i l o s . Trans. R. Soc. Lond. (Biol), 271, 247, 1975. Daoud, A. S., Fritz, K. E., Jarmolych, J., Augustyn, J., Mawhinney, T. P., Adv. Exp. Med. Biol., 82, 928, 1977. McCullagh, K. G., Derouette, S., Robert, L., Exp. Mol. P a t h o l . , 18, 202, 1974. DeBacker, M., P i z i e u x , O., Moschetto, Y., P a r o i Arterielle, 3, 71, 1975. G i r o , M. G., C a s t e l l a n i , I . , V o l p i n , D., Connect. Tissue Res., 2, 231, 1974. M u i r , L. W., B o r n s t e i n , P., Ross, R., Eur. J . Biochem., 64, 105, 1976. Podrazky, V., Adam, M., E x p e r i e n t i a , 31, 523, 1975. Adam, M., Podrazky, V., E x p e r i e n t i a , 32, 430, 1976. Radhakrishnamurthy, B., Ruizitt, and Berenson, G. S., Adv. Exp. Med. Biol., 82, 160, 1977. Wusteman, F. S., Gillard, G. C., E x p e r i e n t i a , 33, 721, 1977. H a s c a l l , V. C., Sajdera, S. W., J . Biol. Chem., 244, 2384, 1969.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
13.
40. 41. 42. 43. 44. 45. 46. 47.
LONG E T A L .
Vascular
Elastic
Fiber
255
Urry, D. W., Faraday Disc. Chem. Soc., No. 61, 205, 1976. Urry, D. W., Long, M. M. and Mitchell, L. W., Int. J . Quantum Chem.: Quantum Biology Symp. No. 3, 107, 1976. Urry, D. W., Biochim. Biophys. Acta, 265, 115, 1972. Starcher, B. C., Saccomani, G., Urry, D. W., Biochim. Biophys. Acta, 310, 481, 1973. Cox, B. A . , Starcher, B. C., Urry, D. W., J . Biol. Chem., 249, 997, 1974. Urry, D. W., Long, M. M., Cox, B. A . , Ohnishi, T., Mitchell, L. W., Jacobs, M., Biochim. Biophys. Acta, 371, 597, 1974. Urry, D. W., Res./Develop., 24, 30, 1973. Urry, D. W., Long, M. M., Mitchell, L. W., Int. J . Quantum Chem.: Quantum Biol. Symp., No. 3, p. 107, 1976.
RECEIVED
December 29, 1977.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14 Membrane Glycoproteins—Dynamics and Affinity Isolation REUBEN LOTAN and GARTH L. NICOLSON Department of Developmental and Cell Biology, University of California, Irvine, CA 92717 Although the exac membranes has not been determine information has been assembled indicating that cellular membranes generally conform to several basic principles. The first of these is that the bulk membrane lipids such as the phospholipids are arranged in a planar bilayer configuration which i s in a predominantly fluid state under physiological conditions (1-4). The lipid bilayer i s interrupted at certain sites by tightly bound integral (or intrinsic) membrane proteins and glycoproteins which are i n serted to varying degrees into the bilayer, and these proteins and glycoproteins are capable of rapid lateral movement within the fluid planar lipid matrix. Cellular membranes are asymmetric with respect to the distribution of specific lipids in each half of the bilayer and proteins and glycoproteins at either surface, and plasma membranes often have non-uniform distributions of proteins, glycoproteins, lipids and glycolipids in the membrane plane (5). In addition, most plasma membranes are not autonomous structures; they are linked to other cellular organelles by a cytoskeletal system composed of microfilaments, microtubules and perhaps intermediate filaments (Fig. 1). However, in its simplest form a biological membrane can be viewed as a two-dimensional solution of a mosaic of integral membrane proteins and glycoproteins embedded in a fluid l i p i d bilayer with peripheral (loosely bound) proteins and glycoproteins attached at the inner and outer membrane surfaces, respectively. The striking asymmetry of this structure allows receptors for hormones, antibodies, viruses, lectins and other agents to be present exclusively on the outer surface where they are exposed to the extracellular environment. Asymmetric arrangement of these components is also well suited to allow the vectorial flow of information across the membrane. The other most important feature of this type of molecular arrangement is that components can diffuse laterally within the membrane plane permitting rapid and reversible changes in membrane topography. Measurements of the lateral mobility of membrane glycoproteins indicate that some diffuse freely in the membrane while others are 0-8412-0452-7/78/47-080-256$05.00/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
L O T A N AND NICOLSON
Membrane
Glycoproteins
257
Cell Surface Reviews
Figure 1. Hypothetical structure of a plasma membrane (PM) including possible interactions between membrane-associated microtubule (MT) and microfilament (MF) systems involved in trans-membrane control over cell surface receptor mobility and distribution (5)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
258
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
r e s t r a i n e d and are unable t o undergo r a p i d l a t e r a l movements (4,5_) . This graded h i e r a r c h y o f m o b i l i t i e s f o r d i f f e r e n t components i n the plasma membrane and even w i t h i n c e r t a i n s p e c i f i c regions o f the plasma membrane suggests t h a t a c e l l can c o n t r o l the m o b i l i t i e s o f c e r t a i n membrane components i n order t o maintain c e r t a i n s p e c i f i c topographic arrays o r "patterns " o f c e l l surface recept o r s t h a t are probably important i n i d e n t i f i c a t i o n and a v a r i e t y of other c e l l u l a r phenomena r e q u i r i n g trans-membrane communication. Since these topographic arrangements are dynamic and not s t a t i c , r a p i d and r e v e r s i b l e changes i n membrane topography could occur i n response t o both i n t r a - and e x t r a c e l l u l a r s t i m u l i . MEMBRANE GLYCOPROTEINS The most important c l a s s o f c e l l surface recpetors i n v o l v e d i n trans-membrane receptor processes are the plasma membrane g l y c o p r o t e i n s . Most o f these appear t o be i n t e g r a l components which are i n t e r c a l a t e d i n t o the hydrophobic plasma membrane core t o depths dependent on th by three-dimensional f o l d i n s t r u c t u r e s . Some o f these i n t e g r a l membrane g l y c o p r o t e i n s span the plasma membrane and have h y d r o p h i l i c regions o f t h e i r s t r u c t u r e s p r o t r u d i n g a t both the e x t r a c e l l u l a r and i n t r a c e l l u l a r membrane surfaces (6-10). A l s o , i t has been found t h a t c e r t a i n membrane g l y c o p r o t e i n s can e x i s t i n o l i g o m e r i c complexes (11-13) o r i n complexes w i t h p e r i p h e r a l membrane p r o t e i n s (14-16). The m a j o r i t y o f c e l l membrane g l y c o p r o t e i n s are i n t e g r a l components s t a b i l i z e d by hydrophobic f o r c e s , and t h e i r s o l u b i l i z a t i o n r e q u i r e s the use o f c h a o t r o p i c agents o r detergents (JL) . Once s o l u b i l i z e d and s t a b i l i z e d i n b u f f e r e d detergent s o l u t i o n s , membrane g l y c o p r o t e i n s can be p u r i f i e d by conventional methods o f g e l f i l t r a t i o n and i o n exchange chromatography ( i n non-ionic detergents) o r by a f f i n i t y chromatography on i n s o l u b i l i z e d l e c t i n s . LECTIN AFFINITY CHROMATOGRAPHY L e c t i n s are p r o t e i n s o r g l y c o p r o t e i n s t h a t b i n d t o mono- and o l i g o s a c c h a r i d e s w i t h remarkable s p e c i f i c i t y (17,18). They usua l l y possess more than one saccharide b i n d i n g s i t e p e r molecule, and t h e i r i n t e r a c t i o n s w i t h carbohydrate-containing polymers resemble antibody-antigen r e a c t i o n s . For example, l e c t i n s can p r e c i p i t a t e p o l y s a c c h a r i d e s and g l y c o p r o t e i n s and a g g l u t i n a t e c e l l s by c r o s s l i n k i n g s a c c h a r i d e - c o n t a i n i n g biopolymers on the surfaces o f adjacent c e l l s . A l a r g e number o f l e c t i n s have been i s o l a t e d and p u r i f i e d , and i n Figure 2 we have l i s t e d some common l e c t i n s a v a i l a b l e t h a t b i n d c e r t a i n o l i g o s a c c h a r i d e s found i n s o l u b l e g l y c o p r o t e i n s (19-23J and c e l l membrane g l y c o p r o t e i n s (2428). Binding o f l e c t i n s t o g l y c o p r o t e i n s o r even c e l l s can be i n h i b i t e d and i n many cases reversed by use o f s p e c i f i c simple sugars o r g l y c o s i d e s , and t h i s i n f o r m a t i o n can be used t o e l u c i date p o s s i b l e carbohydrate determinants on c e l l surface components (Figure 2). However, l e c t i n b i n d i n g t o macromolecules c a r r y i n g h e t e r o - o l i g o s a c c h a r i d e s i d e chains can be i n f l u e n c e d by a v a r i e t y of f a c t o r s which cannot be adequately reproduced i n experiments
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
L O T A N AND NICOLSON
Membrane
Glycoproteins
259
where simple monosaccharide-lectin i n t e r a c t i o n s are measured. Nevertheless, the b i n d i n g o f a l e c t i n i n a s p e c i f i c and r e v e r s i b l e manner t o a macromolecule i s g e n e r a l l y accepted as an i n d i c a t i o n t h a t the molecule i n question contains c e r t a i n carbohydrate sequences, but the unequivocal c h a r a c t e r i z a t i o n o f carbohydrate s e quence i n a l e c t i n receptor r e q u i r e s i t s i s o l a t i o n i n a pure form from the c e l l membrane f o l l o w e d by chemical e l u c i d a t i o n o f i t s structure. Since most membrane g l y c o p r o t e i n s are almost i n s o l u b l e i n n e u t r a l aqueous s o l u t i o n s , p r o t e o l y t i c enzymes have been used t o cleave and remove s o l u b l e surface glycopeptides from c e l l s . Such glycopeptides were then t e s t e d f o r t h e i r a b i l i t y t o i n h i b i t c e l l a g g l u t i n a t i o n , lymphocyte s t i m u l a t i o n o r b i n d i n g o f l e c t i n s t o c e l l s (29-32). The chemical s t r u c t u r e o f a few l e c t i n - r e a c t i v e glycopeptides has been determined (31-32). However, s i n c e the behavior o f membrane g l y c o p r o t e i n depend t onl thei o l i g o s a c c h a r i d e s i d e chain moiety w i t h other membran components, r a t h e r than i n v e s t i g a t i o n s o f fragmented p o r t i o n s o f the membrane components are r e q u i r e d . For t h i s purpose techniques have been developed t o i s o l a t e and p u r i f y membrane g l y c o p r o t e i n s i n b u f f e r e d detergent s o l u t i o n s by l e c t i n a f f i n i t y chromatography. Using a wide v a r i e t y o f i n s o l u b i l i z e d l e c t i n s , a f f i n i t y chromatography has proven t o be very u s e f u l i n p u r i f y i n g an i n c r e a s i n g number o f membrane g l y c o p r o t e i n s (Table 1 ) . L e c t i n A f f i n i t y P u r i f i c a t i o n o f RBC G l y c o p r o t e i n s The e r y t h r o c y t e membrane has been i n t e n s i v e l y i n v e s t i g a t e d i n an attempt t o understand the s t r u c t u r e and f u n c t i o n o f i t s components (33). As a r e s u l t o f these e f f o r t s the human r e d blood c e l l (HuRBC) membrane has proven t o be a u s e f u l model f o r the development o f methods f o r i s o l a t i o n o f l e c t i n r e c e p t o r s . Indeed, s e v e r a l l a b o r a t o r i e s have succeeded i n p u r i f y i n g HuRBC membrane components to homogeneity using l e c t i n a f f i n i t y chromatography techniques (Table 1 ) . F i n d l a y (34) a p p l i e d s o l u b i l i z e d RBC ghost membrane g l y c o p r o t e i n s onto Con A-Sepharose o r LCA-Sepharose, and a f t e r the unbound m a t e r i a l was removed by washing, the a d d i t i o n o f methyl a-mannoside t o Con A-Sepharose detached a g l y c o p r o t e i n which was i d e n t i f i e d as the major i n t e g r a l g l y c o p r o t e i n o f HuRBC — the component m i g r a t i n g on g e l e l e c t r o p h o r e s i s as Band I I I , a g l y c o p r o t e i n having a mol. wt. o f ^100,000 t h a t contains 5-8% carbohydrate. Use o f the LCA-Sepharose column y i e l d e d the major s i a l o g l y c o p r o t e i n , g l y c o p h o r i n , which contains n e a r l y 60% carbohydrate (24). Band I I I has a l s o been p u r i f i e d on Con A-Sepharose by Ross and McConnell (35) who combined i t w i t h egg l e c i t h i n , e r y t h r o c y t e l i p i d s , c h o l e s t e r o l and g l y c o p h o r i n t o o b t a i n v e s i c l e s which were capable o f s u l f a t e t r a n s p o r t . Two d i s t i n c t c l a s s e s o f HuRBC membrane g l y c o p r o t e i n s w i t h d i f f e r i n g carbohydrate compositions and l e c t i n r e a c t i v i t i e s have been separated by A d a i r and K o r n f e l d (36) using a f f i n i t y chromatography on WGA- and RCAj-Sepharose. The WGA-Sepharose column bound and s p e c i f i c a l l y r e l e a s e d a s i n g l e
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
f
MW >100 000 IgM, IgD H-2K, H-2D, I a Synaptic g l y c o p r o t e i n s Synaptic g l y c o p r o t e i n s Nicotinic acetylcholine receptor
f
Band I I I Band I I I + g l y c o p h o r i n Band I I I Band I I I + 2 other glycoprote ins Glycophorin Glycophorin A A s i a l o g l y c o p h o r i n (T-Ag) Major g l y c o p r o t e i n Glycoproteins Glycoprote i n s HLA antigens Component 5.1 T-25, T-200 Major antigen H-2K, H-2D, I a IgM IgD Glycoproteins: MW 20,000-80,000
Material Purified
Table 1:
Mu lymphocytes Mu B lymphocytes Mu lymphocytes Rat b r a i n Rat b r a i n Rat b r a i n o r e l e c t r i c e e l
TX-100 TX-100 TX-100 NaDOC SDS TX-100
PA-^CA^CAj48 Seph WGA, BPA-Seph 48 PA-Seph 48 PA-Seph 48 LCA-, WGA-Seph 49 Con A-, UEA-Seph 50,51 Con A, WGA-Seph, 52 RCAj-glass beads
TX-100
Mu lymphocytes
36 37 38 40 41 42 43 44 45 46 47 47
36
RCAj-Seph WGA-Seph WGA-Seph PNA-Seph RCAj-Seph Con A-Seph LCA-Seph LCA-Seph Con A-Seph Con A, PA-Seph LCA-Seph Con A-Seph Con A-Seph
TX-100 TX-100 SDS DDG (Empigen BB) Emulphogene NaDOC NaDOC NaDOC TX-100 NP-40, NaDOC NaDOC NP-40 NP-40
34 34 35
Ref.
Con A-Seph LCA-Seph Con A-Seph
Affinity Adsorbent
HuRBC
TX-100 TX-100 DTAB
Membrane S o l u b i l i z i n g Agent
HuRBC HuRBC HuRBC + NANase Bovine RBC P i g lymphocytes P i g lymphocytes Hu lymphocytes Ra thymocytes Mu thymocytes Rat thymocytes Mu thymocytes Mu B lymphocytes
HuRBC HuRBC HuRBC
Source o f Membrane
ISOLATION AND CHARACTERIZATION OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Rat spermatozoa Enveloped v i r u s e s ( i n f l u e n z a , Sendai) Mu mammary tumor v i r u s D i c t y o s t e l i u m discoidum
Con A-receptors V i r a l glycoprotein
TX-100 NaDOC
Membrane S o l u b i l i z i n g Agent NaDOC CTAB LIS LIS LIS SDS DDG NaDOC
Affinity Adsorbent Con A-Seph Con A-Seph Con A-Seph WGA-Seph Con A-Seph WGA-Seph RCAj-Seph RCAj-, Con A-, WGA, SBA-Seph Con A-Seph LCA-Seph 61 62
Ref. 53 54 55 56 57 58 59 60
Serdox NNP10 Con A-Seph Major g l y c o p e p t i d e s 63 Membrane-bound enzymes NaDOC Con A-Seph 64 (cf, 5'-nucleotidase, a l k a l i n e phosphatase) A b b r e v i a t i o n s : BPA, Bauhinia purpurea a g g l u t i n i n ; CTAB, cetyltrimethylammonium bromide; DDG, d i m e t h y l dodecylglycine (Empigen BB); DTAB, dodecyltrimethy1ammonium bromide; Emulphogene, a l k o x y p o l y (ethyleneoxy)ethanol; Ham, hamster; Hu, human; HuRBC, human r e d blood c e l l s ; Mu, murine; NaDOC, sodium deoxycholate; NP-40, Nonidet P-40, p o l y o x y e t h y l e n e g l y c o l ( 6 - 7 ) - p - t - o c t y l p h e n o l ; Ra, r a b b i t ; SDS, sodium dodecyl s u l f a t e ; Seph, Sepharose; Serdox NNP10, a n o n i o n i c detergent, Servo, Delden, The Netherlands; TX-100, T r i t o n X-100, p o l y o x y e t h y l e n e g l y c o l ( 9 - 1 0 ) - p - t - o c t y l p h e n o l .
Source o f Membrane Bovine b r a i n Bovine r e t i n a Hu p l a t e l e t s Mu LI210 lymphoma Mu L-929 c e l l s Hu HeLa c e l l s Ham embryo f i b r o b l a s t s (NIL) Mu E h r l i c h a s c i t e s carcinoma
OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS
Material Purified Tissue f a c t o r Rhodopsin Major g l y c o p r o t e i n WGA-receptors Con A-receptor WGA-receptors G a l a c t o p r o t e i n s a and b Glycoproteins
Table 1 ( c o n ' t . ) : ISOLATION AND CHARACTERIZATION
GLYCOPROTEINS AND
262
GLYCOLIPIDS IN
DISEASE PROCESSES
g l y c o p r o t e i n (glycophorin) i n h i b i t o r of WGA, Agaricus bisporus a g g l u t i n i n and PHA. The RCAj-Sepharose column adsorbed s e v e r a l g l y c p r o t e i n s c o n t a i n i n g b i n d i n g s i t e s f o r RCAj and Abrus p r e c a t o r i u s a g g l u t i n i n , while most of the g l y c o p r o t e i n receptors f o r A. bisporus a g g l u t i n i n , PHA, LCA and WGA were not r e t a i n e d by the column. P u r i f i c a t i o n of g l y c o p h o r i n i n a one-step p r e p a r a t i v e procedure was obtained by Kahane et a l . (37) a f t e r SDS s o l u b i l i z a t i o n of HuRBC ghosts i n the presence of r e l a t i v e l y high s a l t and a d s o r p t i o n to WGA-Sepharose. The r e c e p t o r f o r peanut a g g l u t i n i n was i s o l a t e d from neuraminidase-treated, dimethyl d o d e c y l g l y c i n e s o l u b i l i z e d HuRBC by chromatography on PNA-polyacrylhydrazidoSepharose (38). In t h i s l a s t procedure c e l l surface galactose residues on i n t a c t neuraminidase-treated HuRBC were r a d i o l a b e l e d by o x i d a t i o n w i t h galactose oxidase f o l l o w e d by r e d u c t i o n w i t h N a B H t p r i o r to t h e i r p u r i f i c a t i o n . The use of p o l y a c r y l h y d r a zido-Sepharose a f f o r d e d a high c a p a c i t y charge-free and nonl e a c h i n g supporting m a t r i a d s o r p t i o n and high r e c o v e r i e PNA-polyacrylhydrazido-Sepharose w i t h l a c t o s e c o n s i s t e d of two g l y c o p r o t e i n s (MW ^82,000 and ^46,000, r e s p e c t i v e l y ) which resemb l e d a s i a l o g l y c o p h o r i n i n e l e c t r o p h o r e t i c m i g r a t i o n and amino a c i d composition. L i k e a s i a l o g l y c o p h o r i n , the PNA receptor formed p r e c i p i t i n bands w i t h SBA and RCA-j- but f a i l e d to p r e c i p i t a t e w i t h Con A, Dolichos b i f l o r u s a g g l u t i n i n and WGA (38). In an e f f o r t to determine the degree to which the carbohydrate s i d e chains of HuRBC membrane g l y c o p r o t e i n s vary between s p e c i e s , Emerson and K o r n f e l d (40) used non-ionic detergent s o l u b i l i z e d bovine e r y t h r o c y t e membranes as a source of g l y c o p r o t e i n s . RCAjSepharose adsorbed the 230,000 mol. wt. major g l y c o p r o t e i n of bovine e r y t h r o c y t e membrane which contains ^80% carbohydrate, mostly l i n k e d o - g l y c o s i d i c a l l y . V i r t u a l l y a l l the carbohydrate of the membrane (excluding g l y c o l i p i d s ) was borne by t h i s g l y c o p r o t e i n . L e c t i n A f f i n i t y P u r i f i c a t i o n of Lymphoid C e l l Membrane Components Lymphoid c e l l membranes c o n t a i n a wide v a r i e t y of g l y c o p r o t e i n molecules compared to RBC; t h e r e f o r e , l e c t i n a f f i n i t y columns are g e n e r a l l y u s e f u l f o r p a r t i a l p u r i f i c a t i o n or enrichment of g l y c o p r o t e i n s . For example, almost a l l the g l y c o p r o t e i n s of p i g lymphocyte plasma membrane s o l u b i l i z e d i n sodium deoxycholate (NaDOC) were adsorbed on columns of Con A-Sepharose (41) or LCA-Sepharose (42). The l a t t e r immobilized l e c t i n was much more e f f i c i e n t i n p u r i f y i n g the s o l u b i l i z e d membrane g l y c o p r o t e i n s . Con A-Sepharose bound 20% of the a p p l i e d m a t e r i a l i n a n o n s p e c i f i c manner, and the y i e l d of g l y c o p r o t e i n s o f f the Con A-Sepharose column was low ( 5 % ) , whereas LCA-Sepharose y i e l d e d more than twice the amount obtained w i t h Con A-Sepharose, and the recovery was 95%. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) of the s a c c h a r i d e - e l u t e d LCA-Sepharose f r a c t i o n s revealed a t l e a s t ten p r o t e i n bands t h a t s t a i n e d f o r carbohydrate w i t h p e r i o d a t e - S c h i f f reagent. P u r i f i e d LCAreceptors i n h i b i t e d LCA-induced lymphocyte t r a n s f o r m a t i o n 10 times 3
+
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
LOTAN AND NICOLSON
Membrane
Glycoproteins
263
more e f f e c t i v e l y compared t o the u n f r a c t i o n a t e d membrane g l y c o p r o t e i n s , and a t l e a s t one o f the LCA-receptor g l y c o p r o t e i n s was a l s o capable o f i n h i b i t i n g PHA-induced lymphocyte s t i m u l a t i o n . I n the above s t u d i e s f u r t h e r c h a r a c t e r i z a t i o n was not performed t o e l u c i d a t e the nature and f u n c t i o n o f the l e c t i n r e a c t i v e m a t e r i a l s ; however, i n other r e p o r t s defined components have been i s o l a t e d by l e c t i n a f f i n i t y chromatography. For example, the h i s t o c o m p a t i b i l i t y antigens o f human lymphocytes (HLA) were s o l u b i l i z e d i n sodium deoxycholate and then p u r i f i e d by chromatography on LCA-Sepharose (43) . Improvements have been made i n l e c t i n a f f i n i t y chromatography techniques t o increase s p e c i f i c i t y and y i e l d as w e l l as reduce nons p e c i f i c a d s o r p t i o n . In an attempt t o increase the s e l e c t i v i t y o f lectin-Sepharose chromatography, S c h m i d t - U l l r i c h e t a l . (4£) adsorbed T r i t o n X - 1 0 0 - s o l u b i l i z e d r a b b i t thymocyte membrane g l y c o p r o t e i n s onto Con A-Sepharos i othe procedures th e l u t i o n was performed i c e n t r a t i o n s o f the hapte methy a-glucoside procedur lowed p r e f e r e n t i a l e l u t i o n o f receptors according t o t h e i r r e l a t i v e b i n d i n g a f f i n i t i e s t o Con A. Using these procedures a s i a l o g l y c o p r o t e i n t h a t has been designated component 5.1 because o f i t s e l e c t r o p h o r e t i c m o b i l i t y i n SDS-PAGE was e l u t e d i n a homogeneous form. This Con A receptor has an apparent MW o f 55,000 d a l t o n s but tends t o aggregate i n s o l u t i o n . When t h i s p r e p a r a t i o n was examined f u r t h e r , two molecules were found t h a t d i f f e r i n t h e i r carbohydrate side chains, and these could be recognized by cross immune e l e c t r o p h o r e s i s . Trowbridge e t a l . (45) reported t h a t specific I - l a b e l e d surface antigens (T25 and T200) on mouse thymocytes could be p u r i f i e d from NaDOC-solubilized membranes by a d s o r p t i o n t o e i t h e r Con A- o r PA-Sepharose. Upon a n a l y s i s by SDS-PAGE these components separated i n t o s e v e r a l s p e c i e s , p o s s i b l y due to heterogeneity o f t h e i r carbohydrate s i d e chains. Fabre and W i l l i a m s (46) i s o l a t e d a major antigen from the surfaces o f r a t thymocyte membranes by s p e c i f i c a d s o r p t i o n on LCA-Sepharose, and t h i s behavior demonstrated t h a t the antigen i s a g l y c o p r o t e i n . N i l s s o n and Waxdal (47) a l s o l a b e l e d p r o t e i n s on the surface o f murine lymphocytes by l a c t o p e r o x i d a s e - c a t a l y z e d I - i o d i n a t i o n or by c u l t u r i n g the c e l l s i n media c o n t a i n i n g H - l e u c i n e o r H fucose. C e l l membranes were s o l u b i l i z e d i n NP-40 before p a s s i n g the s o l u b i l i z e d g l y c o p r o t e i n s through Con A-Sepharose. The m a t e r i a l s p e c i f i c a l l y e l u t e d from Con A-Sepharose was c h a r a c t e r i z e d by imm u n o p r e c i p i t a t i o n w i t h s p e c i f i c a n t i s e r a , and i t was found t h a t Con A-receptors i n c l u d e d antigens coded by the h i s t o c o m p a t i b i l i t y - 2 complex such as H-2K, H-2D and l a . I n a d d i t i o n , the immobilized Con A a l s o adsorbed immunoglobulins M and D from s o l u b i l i z e d B lymphocyte membranes. E s s e n t i a l l y the same r e s u l t s were obtained r e c e n t l y by Iwata e t a l . (48) who e x t e n s i v e l y f r a c t i o n a t e d murine lymphoid c e l l surface components on a b a t t e r y o f lectin-Sepharose columns (see Table 1). This l a s t study demonstrated t h a t the use of s e v e r a l lectin-Sepharose columns f o r f r a c t i o n a t i o n o f c e l l 1 2 5
1 2 5
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3
GLYCOPROTEINS AND
264
GLYCOLIPIDS IN
DISEASE PROCESSES
membrane components narrows the range of i s o l a t e d components. For example, PLA-, LCA- and RCAj-Sepharose s p e c i f i c a l l y bound g l y c o p r o t e i n s of MW i n the range 20,000-80,000 d a l t o n s , whereas g l y c o p r o t e i n s i n the higher MW (>100,000) range t h a t were not adsorbed on the l a t t e r columns were p r e f e r e n t i a l l y bound by WGA- and BPASepharose (48). L e c t i n A f f i n i t y Chromatography of Nerve C e l l Components L e c t i n a f f i n i t y chromatography has been e s p e c i a l l y u s e f u l i n the p u r i f i c a t i o n of carbohydrate-containing macromolecules from nervous t i s s u e . G l y c o p r o t e i n s present i n NaDOC-solubilized syna p t i c plasma membranes prepared from r a t b r a i n s t h a t were i n j e c t e d w i t h H-fucose 16 hr e a r l i e r have been f r a c t i o n a t e d by chromatography on LCA- or WGA-Sepharose (49). In these s t u d i e s LCA-Sepharose adsorbed 40-45% of the a p p l i e d r a d i o a c t i v i t y and bound most of the 7-8 g l y c o p r o t e i n s known to be present i n the s y n a p t i c membranes; whereas WGA-Sepharose bound o n l y 25-30% of the r a d i o a c t i v i t y suggesting t h a LCA-Sepharose were not boun and Mahler (49) decided t o use both of these a f f i n i t y columns i n tandem such t h a t the sample a p p l i e d onto LCA-Sepharose was allowed to pass i n t o the WGA-Sepharose column. A f t e r washing both columns and s e q u e n t i a l l y e l u t i n g w i t h the appropriate monosaccharide i n h i b i t o r s f o r each l e c t i n , four f r a c t i o n s were obtained: LCA-negative, WGA-negative; LCA-negative, WGA-positive; L C A - p o s i t i v e , WGA-negat i v e ; and L C A - p o s i t i v e , WGA-positive. When analyzed by SDS-PAGE, each of the f r a c t i o n s had d i s t i n c t p a t t e r n s w i t h some o v e r l a p of molecular weight c l a s s e s . Zanetta e t a l . (50,51) have f r a c t i o n a t e d r a t b r a i n s y n a p t i c membranes d i s s o l v e d i n SDS on Con A- or UEASepharose. E l u t i o n from the former a f f i n i t y column was w i t h methyl a-glucoside; however, L-fucose was unable to r e l e a s e bound m a t e r i a l but c o u l d i n h i b i t b i n d i n g t o the UEA-Sepharose. I n c r e a s i n g the SDS c o n c e n t r a t i o n i n the e l u t i o n b u f f e r (without saccharides) r e leased the adsorbed m a t e r i a l , and subsequent PAGE a n a l y s i s revealed a l a r g e number of carbohydrate c o n t a i n i n g components. P a r t of the heterogeneity i n g l y c o p r o t e i n s e l u t e d by these procedures was a t t r i b u t e d t o the f a c t t h a t the s y n a p t i c membranes were d e r i v e d from a heterogeneous p o p u l a t i o n of neurons. S p e c i f i c , f u n c t i o n a l g l y c o p r o t e i n s have been i s o l a t e d from s o l u b i l i z e d b r a i n t i s s u e by lectin-Sepharose chromatography. Nicot i n i c a c e t y l c h o l i n e r e c e p t o r , T r i t o n X-100 s o l u b i l i z e d from a crude, p o s t - n u c l e a r membrane f r a c t i o n of r a t c e r e b r a l c o r t e x or from membranes of Torpedo c a l i f o r n i c a e l e c t r o p l a x , was r e t a i n e d on immobilized Con A, WGA or RCAj (52) . In another study p a r t i a l p u r i f i c a t i o n of coagulant arylamidase and a l k a l i n e phosphatase enzymes s o l u b i l i z e d i n NaDOC from bovine b r a i n was achieved by a d s o r p t i o n on Con A-Sepharose (53). L e c t i n A f f i n i t y Chromatography of Components from Other C e l l Types In c o n t r a s t to the m u l t i t u d e of l e c t i n receptors found i n s y n a p t i c membranes, s e v e r a l systems have been described i n which 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
L O T A N AND NICOLSON
14.
Membrane
Glycoproteins
265
a s i n g l e receptor was obtained. For example, s o l u b i l i z a t i o n o f bovine r e t i n a l r o d outer segments w i t h cetyltrimethylammonium bromide f o l l o w e d by chromatography on Con A-Sepharose y i e l d e d a homogeneous rhodopsin (54). The major human p l a t e l e t membrane g l y c o p r o t e i n (MW ^100,000) was s o l u b i l i z e d by l i t h i u m d i i o d o s a l i c y l a t e -phenol e x t r a c t i o n o f I - l a b e l e d membranes and p u r i f i e d on Con ASepharose (55). Using s i m i l a r e x t r a c t i o n methods, four WGA-recept o r s o f MW 40,000-60,000 were i s o l a t e d from L1210 c e l l s (560, and a s i n g l e Con A receptor having an apparent MW o f 100,000 and a t l e a s t f i v e carbohydrate s i d e chains was i s o l a t e d from mouse L929 c e l l s (57J. M u l t i p l e WGA receptors o f MW i n the range 40,000300,000 were i s o l a t e d from S D S - s o l u b i l i z e d membranes o f il a b e l e d HeLa c e l l s (58). E x t r a c t i o n o f hamster embryo f i b r o b l a s t s (NIL) l a b e l e d w i t h galactose-oxidase and NaB^H^ w i t h 8M urea o r s o l u b i l i z a t i o n w i t h d i e m t h y l d o d e c y l g l y c i n e f o l l o w e d by chromatography on RCASepharose allowed p u r i f i c a t i o f galactoprotei (LETS) and g a l a c t o p r o t e i by an a c t i n - l i k e p r o t e i n galactoprotei appeare composi t i o n a l l y heterogeneous. While most o f the above i n v e s t i g a t o r s d i d not r e p o r t problems i n u s i n g lectin-Sepharose columns, an e x t e n s i v e study by Nachbar e t a l . (60) demonstrated t h a t i n c e l l membranes o f E h r l i c h a s c i t e s carcinoma c e l l s s o l u b i l i z e d i n NaDOC there are s e v e r a l carbohydrate c o n t a i n i n g components t h a t adsorb t o Con A-, WGA-, SBA- and RCA-Sepharose columns. Using C-glucosaminel a b e l e d membrane components the r e c o v e r i e s from the v a r i o u s columns were determined, and i t was found t h a t s p e c i f i c e l u t i o n o f the WGAand the Con A-Sepharose columns r e s u l t e d i n r e l e a s e o f only 55-70% of the bound m a t e r i a l . The remaining t i g h t l y bound m a t e r i a l may have been i r r e v e r s i b l y h e l d t o the column by hydrophobic i n t e r a c t i o n s . The r e c e p t o r s f o r the d i f f e r e n t l e c t i n s appeared s i m i l a r on SDS-PAGE, and some immobilized l e c t i n s p u r i f i e d the same g l y c o p r o t e i n r e c e p t o r s . P u r i f i c a t i o n o f g l y c o p r o t e i n s from s o l u b i l i z e d enveloped v i r u s e s (62,63) and o f membrane-associated enzymes on l e c t i n a f f i n i t y columns has a l s o been d e s c r i b e d (Table 1 ) . Although one-step p u r i f i c a t i o n t o homogeneity o f a membrane g l y c o p r o t e i n has only been reported f o r HuRBC g l y c o p h o r i n (37), most s t u d i e s i n d i c a t e t h a t a f f i n i t y chromatography on immobilized l e c t i n columns i s the method o f c h o i c e , a t l e a s t i n p r e l i m i n a r y stages o f membrane g l y c o p r o t e i n p u r i f i c a t i o n . In e a r l i e r s t u d i e s (34,36,41,42) no attempt was made t o r a d i o a c t i v e l y l a b e l c e l l membrane components p r i o r t o s o l u b i l i z a t i o n and chromatography, b u t more recent i n v e s t i g a t i o n s have demonstrated t h a t p r e l a b e l i n g membrane components e i t h e r by l a c t o p e r o x i d a s e - c a t a l y z e d I-iodinat i o n (47,55) o r neuraminidase f o l l o w e d by galactose oxidase and NaB Hi (_38/57) f a c i l i t a t e s the f r a c t i o n a t i o n procedure and i n s u r e s t h a t the i s o l a t e d components o r i g i n a t e from the plasma membrane s u r f a c e . I n a d d i t i o n , the l a b e l e d components may be detected by autoradiography o r fluorography a f t e r s e p a r a t i o n by SDS-PAGE, even i f they are obtained i n s m a l l q u a n t i t i e s . Many o f the saccharides found i n membrane g l y c o p r o t e i n s are 1 2 5
1 2 5
ll+
1 2 5
3
+
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
266
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
u b i q u i t o u s ; t h e r e f o r e , o n l y p a r t i a l p u r i f i c a t i o n should be expected when a s i n g l e lectin-Sepharose column i s used. The use o f a hapten g r a d i e n t i n the e l u t i o n step (44) o r combinations o f immobilized l e c t i n s w i t h d i f f e r e n t sugar s p e c i f i c i t i e s (48,49) has proven t o be very powerful i n the s e p a r a t i o n o f membrane g l y c o p r o t e i n s . The choice o f detergent f o r the s o l u b i l i z a t i o n o f membranes and s t a b i l i z a t i o n o f membrane g l y c o p r o t e i n s p r i o r t o a p p l i c a t i o n onto lectin-Sepharose columns seems i n many r e p o r t s t o have been random, or i n the best examples, e m p i r i c a l . This problem has been e l i m i nated i n p a r t by a systematic a n a l y s i s o f the e f f e c t s o f s e v e r a l z w i t t e r i o n i c , c a t i o n i c , a n i o n i c and non-ionic detergents on g l y c o p r o t e i n b i n d i n g t o and s p e c i f i c e l u t i o n from f i v e lectin-Sepharose columns (39). Our r e s u l t s (Figure 3) i n d i c a t e t h a t f o r most l e c t i n s , NaDOC caused marked decreases i n b i n d i n g a f f i n i t y . The z w i t t e r i o n i c detergent dimethyl d o d e c y l g l y c i n e decreased the e f f i c i e n c y o f the Con A- and SBA-Sepharose w h i l e the non-ionic detergents NP-40 and T r i t o b i l i z e d l e c t i n s t e s t e d (39_ n i t y chromatography techniques w i l l be g r e a t l y f a c i l i t a t e d by opt i m i z i n g v a r i o u s l e c t i n s , detergents, s o l u t i o n c o n d i t i o n s , columns and techniques f o r s p e c i f i c e l u t i o n o f bound g l y c o p r o t e i n s . DYNAMICS OF CELL SURFACE LECTIN RECEPTORS One o f the most u s e f u l approaches f o r studying c e l l s u r f a c e g l y c o p r o t e i n dynamics has been the u t i l i z a t i o n o f l e c t i n s as probes f o r f l u o r e s c e n t o r e l e c t r o n microscopy (18,65). Observing the movement and r e d i s t r i b u t i o n o f c e l l surface-bound l e c t i n s has allowed estimates t o be made on the r a t e s o f l a t e r a l d i f f u s i o n and, i n c e r t a i n cases, i n t e r n a l i z a t i o n o f the l e c t i n - r e c e p t o r comp l e x e s (_5) . These experiments have been g e n e r a l l y conducted w i t h f l u o r e s c e i n - l a b e l e d l e c t i n s and UV-fluorescent microscopy o r f e r r i t i n - , hemocyanin- o r p e r o x i d a s e - l a b e l e d l e c t i n s and e l e c t r o n microscopy (65). By l a b e l i n g p r e f i x e d o r u n f i x e d c e l l s u r f a c e s w i t h these probes and then observing the a l t e r a t i o n s i n s u r f a c e d i s t r i b u t i o n s o f the probes under a v a r i e t y o f experimental c o n d i t i o n s , a dynamic p i c t u r e o f l e c t i n r e c e p t o r movements has been obtained i n s e v e r a l b i o l o g i c a l systems. These observations have l e d i n v e s t i g a t o r s t o conclude the f o l l o w i n g : F i r s t , the i n h e r e n t topographic d i s t r i b u t i o n s o f most l e c t i n r e c e p t o r s (as found on p r e f i x e d c e l l s o r c e l l s l a b e l e d a t 0°C) are uniform and random across the e n t i r e c e l l s u r f a c e . Some exceptions have been noted, however, i n s p e c i a l i z e d c e l l s o f h i g h asymmetry such as sperm where WGA-receptors were found t o be l o c a l i z e d o n l y i n c e r t a i n regions o f the sperm head (66) and nerve c e l l s where Con A recept o r s were found t o be r e l a t i v e l y immobile and predominately i n the s y n a p t i c c l e f t (67). Most l e c t i n r e c e p t o r r e d i s t r i b u t i o n s t u d i e s have r e v e a l e d t h a t l i g a n d - r e c e p t o r complexes undergo r a p i d change from random, uniform d i s t r i b u t i o n s t o form c l u s t e r s and patches (68-72). I n some c e l l types the c l u s t e r e d l i g a n d - r e c e p t o r complexes coalesce t o form a s i n g l e p o l a r "cap" o r aggregate o f c r o s s l i n k e d l i g a n d - r e c e p t o r complexes. A f t e r r e d i s t r i b u t i o n the c l u s -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
L O T A N AND NICOLSON
Membrane
Glycoproteins
I. Serum Glycoprotein-Type
LECTIN
267
Oligosaccharide
NANA-Gal-GlcNAc-Man. Man-GlcNAc-GlcNAc-Asn
LPA Gal-GlcNAc-Man*
t Fuc
RCA,SBA,PNA PHA WGA Con A,LCA,PA LTA,UEA I I . Mucin-Type
Oligosaccharide NANA-Gal-GalNAc-Ser(Thr)
LPA Gal-GalNAc-Ser(Thr) PNA
I
1
SBA,RCA Figure 2. Possible binding region for lectins on oligosaccharide side chains of a hypothetical cell membrane glycoprotein. LPA, Limulus polyphemus agglutinin; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; PNA, peanut agglutinin; PHA, phytohemagglutinin from Phaseolus vulgaris; WGA, wheat germ agglutinin; Con A, concanavalin A; LCA, Lens culinaris agglutinin; PA, pea agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europeus agglutinin.
• RC
60 20
1 t 1 I it 1 J I 11 TXIOOPj,
100 60 20
3
100 60 20 100
a SBA • Con Am WGA
PNA
NP40
100
-D
DDG
1 m 1 ii y DOC
1
60 20 100 60 20
•E
c
i! i 2
0.1
T A B
1.0
DETERGENT CONC. (%)
2.5
Biochemistry
Figure 3. Effect of increasing concentrations of detergents on the glycoprotein binding efficiencies of immobilized lectins (for experimental details see Ref. 39)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
268
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
t e r e d o r capped l i g a n d - r e c e p t o r complexes may be i n t e r n a l i z e d by endocytosis o r shed from the c e l l . The extents and r a t e s o f r e d i s t r i b u t i o n , i n t e r n a l i z a t i o n and shedding appear t o be c o n t r o l l e d by the c e l l type and the biochemical nature o f the l i g a n d and i t s r e c e p t o r s (reviewed i n 4,18,73). I t has been noted t h a t w h i l e a l e c t i n a t one c o n c e n t r a t i o n can induce r e d i s t r i b u t i o n o f i t s r e ceptors i n t o p o l a r caps, a t other c o n c e n t r a t i o n s ( u s u a l l y much h i g h e r ) , i t can i n h i b i t the movement o f d i f f e r e n t c e l l surface r e c e p t o r s (74,751 Q u a n t i t a t i o n o f the r o t a t i o n a l and l a t e r a l m o b i l i t i e s o f c e l l surface g l y c o p r o t e i n s on c e l l surfaces has been achieved by use o f f l u o r e s c e i n i s o t h i o c y a n a t e - l a b e l e d Con A. S h i n i t z k y e t a l . (76) and Inbar e t a l . (77) have s t u d i e d the r o t a t i o n a l m o b i l i t i e s o f Con A receptor complexes on c e l l surfaces by fluorescence p o l a r i z a t i o n . The recent i n t r o d u c t i o n o f the fluorescence photobleaching recovelry technique allowed Jacobso a l (78) d Schles s i n g e r e t a l (79) t o measur the Con A-receptor complexes w i t h i s u r f a c e membranes o s e v e r a c u l t u r e d c e l l s . Such s t u d i e s and others i n d i c a t e d t h a t the m o b i l i t i e s o f c e l l membrane g l y c o p r o t e i n s are determined t o a l a r g e ext e n t by t h e i r i n t e r a c t i o n s w i t h other membrane components and membrane-associated s t r u c t u r e s such as m i c r o f i l a m e n t s and microtubules (4,5^) . I t i s hoped t h a t i s o l a t i o n and c h a r a c t e r i z a t i o n of s p e c i f i c membrane g l y c o p r o t e i n s w i l l increase the understanding of the exact molecular s t r u c t u r e and o r g a n i z a t i o n o f c e l l membranes. Acknowledgments; Supported by PHS NCI Contract CB-74153, PHS NCI Grant CA-15122 and American Cancer S o c i e t y Grant BC-211A t o G.L. Nicolson.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
L O T A N AND NICOLSON
Membrane
Glycoproteins
269
LITERATURE CITED
1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Singer, S.J. & Nicolson, G.L. (1972) Science 175, 720. Edidin, M. (1974) Ann. Rev. Biophys. Bioeng. 2, 179. Singer, S.J. (1974) Ann. Rev. Biochem. 43, 805. Nicolson, G.L. (1976) Biochim. Biophys. Acta 457, 57. Nicolson, G.L., Poste, G. & Ji, T.H. (1977) In "Dynamic Aspects of Cell Surface Organization", Vol. 3 of "Cell Surface Reviews" (G. Poste & G.L. Nicolson, eds.), p. 1, NorthHolland, Amsterdam. Bretscher, M.S. (1971) J . Mol. Biol. 59, 351. Segrest, J.P., Kahne, I . , Jackson, R.L. et a l . (1973) Arch. Biochem. Biophys. 155, 167. Morrison, M., Mueller, T.J. & Huber, C.T. (1974) J . Biol. Chem. 249, 2658. Hunt, R.C. & Brown, J.C. (1975) J . Mol. Biol. 97, 413. Gahmberg, C.G. (1977) In"DynamicAspects of Cell Surface Organization", Vol G.L. Nicolson, eds.) Pinto da Silva, P. & Nicolson, G.L. (1974) Biochim. Biophys. Acta 363, 311. Wang, K. & Richards, F.M. (1974) J . Biol. Chem. 249, 8005. Hynes, R.O. & Pearlstein, E.S. (1976) J . Supramol. Struct. 4, 1. Nicolson, G.L. & Painter, R.G. (1973) J . Cell Biol. 59, 395. Ji, T.H. & Nicolson, G.L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2212. Elgsaeter, A . , Shotton, D.M. & Branton, D. (1976) Biochim. Biophys. Acta 426, 101. L i s , H. & Sharon, N. (1973) Ann. Rev. Biochem. 43, 541. Nicolson, G.L. (1974) Int. Rev. Cytol. 39, 89. Spiro, R.G. (1973) Adv. Protein Chem. 27, 349. Spiro, R.G. & Bohyroo, V.D. (1974) J . Biol. Chem. 249, 5704. Toyoshima, S., Fukada, M. & Osawa, T. (1974) Biochemistry 11, 4000. Thomas, D.B. & Winzler, R.J. (1969) J . Biol. Chem. 244, 5943. Kornfeld, S. & Kornfeld, R. (1971) In "Glycoproteins in Blood Cells and Plasma" (G.A. Jamieson & T.J. Greenwalt, eds.), p. 50, J.B. Lippincott, Philadelphia. Tomita, M. & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964. Newman, R.A., Glöckner, W.M. & Uhlenbruck, G.G. (1976) Eur. J . Biochem. 64, 373. Finne, J . (1975) Biochim. Biophys. Acta 412, 317. Funakoshi, I . , Nakada, H. & Yamashina, I. (1974) J . Biochem. (Tokyo) 76, 319. Nakada, H., Funakoshi, I . & Yamashina, I. (1975) J . Biochem. (Tokyo) 78, 863. Kornfeld, S. & Kornfeld, R. (1969) Proc. Natl. Acad. Sci. U.S.A. 62, 1439. Kornfeld, R. & Kornfeld, S. (1970) J . Biol. Chem. 245, 2536.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
270
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
31.
Kornfeld, S., Rogers, J . & Gregory, W. (1971) J . Biol. Chem. 246, 6581. Akiyama, Y. & Osawa, T. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 323. Marchesi, V.T., Furthmayr, H. & Tomita, M. (1976) Ann. Rev. Biochem. 46, 667. Findlay, J.B.C. (1974) J . Biol. Chem. 249 4398. Ross, A.H. & McConnell, H.M. (1977) Biochem. Biophys. Res. Commun. 74, 1318. Adair, W.L. & Kornfeld, S. (1974) J . Biol. Chem. 249 4696. Kahane, I . , Furthmayr, H. & Marchesi, V.T. (1976) Biochim. Biophys. Acta 426, 464. Carter, W.G. & Sharon, N. (1977) Arch. Biochem. Biophys. 180, 570. Lotan, R., Beattie, G., Hubbell, W.L. et a l . (1977) Biochemistry 16, 1787 Emerson, W.A. & Kornfeld Allan, D., Auger, J Crumpton, (1972) 236, 23. Hayman, M.J. & Crumpton, M.J. (1972) Biochem. Biophys. Res. Commun. 47, 923. Snary, D., Goodfellow, P., Hayman, M.J. et a l . (1974) Nature New Biol. 247, 457. Schmidt-Ullrich, R., Wallach, D.F.H. & Hendricks, J . (1975) Biochim. Biophys. Acta 382, 295. Trowbridge, I.S., Nilsen-Hamilton, M., Hamilton, R.T. et a l . (1977) Biochem. J . 163, 211. Fabre, J.W. & Williams, A.F. (1977) Transplantation 23, 349. Nilsson, S.F. & Waxdal, M.J. (1976) Biochemistry 15, 2698. Iwata, M., Ide, H., Terao, T. et a l . (1977) J . Biochem. (Tokyo) 82, 661. Gurd, J.W. & Mahler, H.R. (1974) Biochemistry 13, 5193. Zanetta, J.P., Morgan, I.G. & Gombos, G. (1975) Brain Res. 83, 337. Zanetta, J.P., Reeber, A . , Vincendon, G. et a l . (1977) Brain Res. 138, 317. Salvaterra, P.M., Gurd, J.M. & Mahler, H.R. (1977) J . Neurochem. 29, 345. P i t l i c k , F.A. (1976) Biochim. Biophys. Acta 428, 27. Steinemann, A. & Stryer, L. (1973) Biochemistry 12, 1499. Nachman, R.L., Hubbard, A. & Ferris, B. (1973) J . Biol. Chem. 248, 2928. Jansons, V.K. & Burger, M.M. (1973) Biochim. Biophys. Acta 291, 127. Hunt, R.C., Bullis, C.M. & Brown, J.C. (1975) Biochemistry 14, 109. Kramer, R.H. & Canellakis, E.S. (1977) Proc. Am. Assoc. Cancer Res. 18, 56. Carter, W.G. & Hakomori, S.-I. (1977) Biochem. Biophys. Res. Commun. 76, 299.
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14.
60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79.
L O T A N AND NICOLSON
Membrane
Glycoproteins
271
Nachbar, S.M., Oppenheim, J.D. & Aull, F. (1976) Biochim. Biophys. Acta 419, 512. Fournier-Delpech, S., Danzo, B . J . & Orgebincrist, M.C. (1977) Ann. Biol. Anim. Biochem. Biophys. 17, 207. Hayman, M.J., Skehel, J . J . & Crumpton, M.J. (1973) FEBS Lett. 29, 185. Westenbrink, F . , Koonstra, W. & Bentvelzen, P. (1977) Eur. J . Biochem. 76, 85. Crean, E.V. & Rossomando, E.F. (1977) Biochem. Biophys. Res. Commun. 75, 488. Nicolson, G.L. (1978) In "Advanced Techniques in Biological Electron Microscopy" (J.K. Koehler, ed.), Vol. 2, SpringerVerlag, New York (in press). Nicolson, G.L., Usui, N . , Yanagimachi, R. et a l . (1977) J . Cell Biol. 74, 950. Kelly, P., Cotman C.W. Gentry C a l (1976) J Cell Biol. 71, 487. de Petris, S. & Rabb, M.C. (1974) Eur. J . Immunol. 4, 130. Rosenblith, J . Z . , Ukena, T.E., Yin, H.H. et al. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1625. Nicolson, G.L. (1973) Nature New Biol. 243, 218. de Petris, S., Raff, M.C. & Mallucci, L. (1973) Nature New Biol. 244, 275. Goldman, R., Sharon, N. & Lotan, R. (1976) Exp. Cell Res. 99, 408. de Petris, S. (1977) In "Dynamic Aspects of Cell Surface Organization", Vol. 3 of "Cell Surface Reviews" (G. Poste & G.L. Nicolson, eds.), p. 643, North-Holland, Amsterdam. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J . Immunol. 2, 203. Yahara, I. & Edelman, G.M. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 608. Shinitzky, M., Inbar, M. & Sachs, L. (1973) FEBS Lett. 34, 247. Inbar, M., Shinitzky, M. & Sachs, L. (1973) J . Mol. Biol. 81, 245. Jacobson, K., Wu, E.-S. & Poste, G. (1976) Biochim. Biophys. Acta 433, 215. Schlessinger, J., Koppel, D.E., Axelrod, D. et a l . (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2409.
RECEIVED
April 4, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15 Masking of Cell-Surface Antigens by Ectoglycoproteins JOHN F. CODINGTON Laboratory for Carbohydrate Research, Massachusetts General Hospital, Boston, MA 02114
The surfaces ofmammalian antigenic activities, tha to individual (1, 2), and tissue (3) of origin. Specific antigens at the cell surface may reflect the stage of embryonic development (4), and antigens may result from environmental factors, such as exposure to viruses (5), chemicals (6), or radiation (7). The impact of such factors can result i n neoplasia, and the surfaces of neoplastic cells then possess antigens which have resulted from these factors (7, 8, 9) or they may possess derepressed embryonic or fetal antigens (10). These antigens, known as tumor associated or tumor specific antigens, cannot usually be detected at the surfaces of normal cells similar to those from which the neoplasm was derived. The effectiveness of an immune surveillance system i n the defense against cancer i n humans has been debated widely (11, 12). An immune response to tumor specific antigens has often proven sufficient to cause rejection of some experimental tumors, although i n established neoplasms the manifestation of a similarly successful defense mechanism is rare (12). A number of explanations have been proposed to explain the capacity of tumor cells to survive and grow progressively i n immunologically hostile environments. Of particular concern i s the survival mechanism(s) employed by metastatic or suspended tumor cells i n the blood, lymph, or other body fluids. Masking of cell-surface antigens to protect them from immune defense factors of the host has been proposed by a number of investigators (13-22). Evidence to support the existence of at least four different types of masking has been presented. These include masking of cryptic antigenic sites, usually galactose terminated, by sialic acid (14, 15); nonspecific coulombic or charge repulsion due to cell-surface s i a l i c acid residues (16); masking by adsorbed exogenous glycoprotein (17, 18) or proteoglycan (19) material, which may be specific Sor particular cell-surface receptors; and masking of nearby antigenic sites by large endogenous glycoprotein molecules (13, .20-22). The present discussion will be limited to evidence 0-8412-0452-7/78/47-080-277$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE
278
PROCESSES
i n support of masking by endogenous glycoproteins and the possible relevance of this phenomenon to human cancer. Occurrence Evidence i n support of antigen masking by large endogenous ectoglycoproteins has been presented, to my knowledge, for only two ascites tumor systems, the mammary adenocarcinoma, TA3, of the s t r a i n A mouse and the IgA-synthesizing plasmacytoma c e l l , 58-8, of the BALB/c mouse. In each case, evidence to support this mechanism has involved comparison with c e l l s derived from the same tumor i n which no evidence f o r masking was found. In the TA3 tumor system several nonstrain-specific sublines were investigated, as w e l l as s i x nonstrain-specific TA3-Ka/A.CA hybrid cloned l i n e s , which had resulted from fusion of c e l l s from the nonstrain-specific TA3-H sublin d l embryoni f i b r o blasts of the A.CA mous the major histocompatibility antige (II-* ) investigated Each nonstrain-specific c e l l l i n e was found to possess at i t s c e l l surface an abundance of rod-like glycoprotein (epiglycanin) molecules of high molecular weight. These molecules are believed to be involved i n masking. Fortuitously, a s t r a i n - s p e c i f i c ascites subline, TA3-St, which had been derived from the same spontaneous s o l i d tumor as the TA3-Ha c e l l , was available f o r comparison. The TA3-St ascites c e l l was not found to possess any epiglycanin at i t s surface. As described below, immunological, chemical, immunochemical, and physical studies support the hypothesis of masking by epiglycanin molecules. In the plasma c e l l tumor, 56-8, which has been less extensively studied than the TA3 tumor, the capacities to absorb anti-H-2 antisera or rabbit anti-58-8 antisera were observed to decrease over f i v e to s i x passages i n the ascites form i n the mouse (passage 8 to passage 13) (23). After passage 13 no absorption of either antibody could be observed. Removal of protein material by b r i e f treatment with pronase, however, restored the capacity to absorb each antibody. Moreover, after incubation of the pronase-treated c e l l s for several hours i n culture at 37°, the c e l l s again resisted absorption of antisera, suggesting resynthesis of a masking glycoprotein. These characteristics, which are closely related to those of nonstrains p e c i f i c TA3-Ha c e l l s , suggest masking by endogenous molecules. To my knowledge, no chemical or imrnunochemical evidence for a masking glycoprotein has been reported. Glycoproteins related i n composition to epiglycanin have been isolated from hepatoma ascites c e l l s by Funakoshi and coworkers (24) and from B-16 melanoma ascites c e l l s by Bhavanandan and Davidson (2_5). To my knowledge, no firm evidence i n support of a masking function for the glycoproteins has been published. a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
Antigens
279
Early Chemical and. Imrr.uiiochemical Studies of the TA3 Ascites Sublines" In 1962 Gasic and Gasic reported, on the basis of histochemical studies, that the TA3-Ha c e l l possessed at i t s surface a high concentration of s i a l i c acid-containing glycoprotein material (26). This finding was confirmed by our laboratory, which reported i n 1970 that large amounts of carbohydrate and protein material could be removed by proteolysis from viable TA3Ha ascites c e l l s (27). Scon thereafter we described the i s o l a t i o n of a glycoprotein fraction of high molecular weight, which had been removed from viable ascites c e l l s by proteolysis (28). This material possessed, a high concentration c f serine, threonine, and N-acetylgalactosamine, components associated with glycoproteins with a high proportion of O-glycosyl-linked carbohydrate chains. The cell-surface glycoprotein from which these fragments were derived was l a t e r terme this finding was not recognize s p e c i f i c subline of the same tumor, TA3-St, revealed that the TA3-St ascites c e l l did not possess any detectable amount of this fraction (20, 21). At about t h i s time several laboratories described some s t r i k i n g b i o l o g i c a l differences between the same two ascites sublines, the nonstrain-specific TA3-Ha c e l l and the s t r a i n s p e c i f i c TA3-St subline (21, 30-35). Friberg (30) reported marked differences i n karyotypes of the two c e l l l i n e s , and Hauschka et a l . (32) confirmed a near d i p l o i d chromosome number for the TE"3-Ha c e l l . The work of several groups (30-34) showed c l e a r l y that the TA3-Ka c e l l was s i g n i f i c a n t l y less immunogenic than the TA3-St c e l l and far more immunoresistant. Of particular significance was the finding of Friberg (31) that the TA3-St c e l l possessed the capa.city i n v i t r o to absorb many f o l d more anti-H-2 antibody to the major histocompatibility antigen (H-2 ) than the TA5-Ha c e i l . Sanford et a l . (21) l a t e r confirmed this observation and showed, as did FriEerg and L i l l i h o o k (35), that disruption of the c e l l structures by l y o p h i l i z a t i o n resulted i n a marked increase i n the number of H-^! antigenic sites exposed on the surface of the TA3-Ha c e l l , but t h i s treatment resulted i n no increase i n antigenic s i t e s exposed on the TA3-St c e l l surface. These observations, coupled with the finding that the TA3-Ha, but not the TA3-St, c e l l possessed at i t s surface an abundance of epiglycanin molecules, led us to suggest at that time that a l l o t r a n s p l a n t a b i l i t y and the reduced capacity to absorb anti-H2 antibody was due to masking of surface antigens by epiglycanin molecules (20, 21). Springer et_ a l . (36) demonstrated that epiglycanin was a potent i n h i b i t o r oF hemagglutination by the l e c t i n from V i c i a graminea. seeds, a l e c t i n of high s p e c i f i c i t y for structures at the surfaces of human erythrocytes of N blood group s p e c i f i c i t y . G
a
a
a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
280
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
Later Cooper et a l . (57) developed a method for the quantitation of epiglycanin~T)y the use o f this l e c t i n . Although receptors f o r the V i c i a graminea l e c t i n , which are present i n epiglycanin, are abundant at the TA3-Ha c e l l surface, no receptors for this l e c t i n were detected at the surface of the TA3-St ascites c e l l (20, 38). The Origin of Nonstrain S p e c i f i c i t y i n the TA3 Ascites Cells The TA3-Ha ascites subline was i n i t i a l l y s t r a i n s p e c i f i c (32). Loss o f s t r a i n s p e c i f i c i t y i n the TA3-Ha subline occurred during routine passage as an ascites c e l l i n the syngeneic s t r a i n A mouse (32). Although t h i s event occurred i n ascites c e l l s following passage 200, neither the exact passage (s) nor the circumstances of the occurrence were reported. Although epiglycanin may have appeared at the TA3-Ha c e l l surface at this time, i t s presence was not observed u n t i l after passage 520 by means of chemical method Another opportunit occurred recently i n our laboratory (39, 40). Strain specific TA3-St c e l l s were inoculated intraperitoneally into s t r a i n A mice suffering from incipient acute i n t e r s t i t i a l pneumonia. Later two micoorganisms, Pasteurella pneumotropica and Pasteurella multocida, were isolated from the sick mice. Cells were harvested routinely on day 7 and tested for the presence o f receptors for the l e c t i n from V i c i a graminea seeds at t h e i r surfaces. The determination of l e c t i n receptors was part o f a project i n progress at that time i n collaboration with a graduate student, D. K. M i l l e r , and with the laboratory of Dr. A. G. Cooper. Whereas, TA3-St ascites c e l l s normally demonstrate essentially no l e c t i n receptors, the c e l l s were now able to adsorb a high level of V i c i a graminea l e c t i n (39). I t was shown that the new c e l l l i n e , which was c a l l e d TA3-MM, possessed at i t s surface a high concentration of epiglycanin molecules (40). Similar to the nonstrain s p e c i f i c TA3-Ka ascites c e l l , the TA3-MM c e l l l i n e was able to grow progressively i n mice of foreign strains. The o r i g i n and characteristics of the TA3-MM subline are described i n two papers prepared for publication (39, 40). The new allotransplantable c e l l t i n e , TA3-MM, was approximately tetraploid with an average of about 80 chromosomes, which was 12 more than was possessed by the TA3-St c e l l (approximately 68) and twice the number of the TA3-Ha c e l l (41-42) (39). Although about 20% greater i n diameter than the TA3-Ha ascites c e l l , i t possessed about the same amount (2.3 mg/10 c e l l s ) of epiglycanin per c e l l (40). Comparison of the compositions of p u r i f i e d fractions o f epiglycanin from TA3-Ha and TA3-NM c e l l s are presented i n Table I . Values are almost i d e n t i c a l for the two c e l l l i n e s . One may speculate that nonstrain s p e c i f i c i t y i n the TA3-Ha ascites c e l l l i n e also occurred under the influence of pneumonia associated microorganisms, but no evidence i s available to support 9
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
281
Antigens
TABLE I Carbohydrate and Amino Acid Compositions of Epiglycanin Isolated from the Ascites F l u i d of Syngeneic Mice Bearing TA3 Ascites Cells TA3-Ha Carbohydrate Components
TA3-M^l
(moles r e l a t i v e to N-acetylgalactosamine)
N-Acetylgalactosamine
1.0
1.0
Galactose
1.4
1.4
N-Acetylglucosamine
0.
N-Acetylneuraminic Acid
0.4
Amino Acid Components
0.6
(residues per 1000 residues) 24
31
Threonine
307
306
Serine
252
223
Glutamic Acid
32
36
Proline
93
107
Glycine
81
92
Alanine
131
131
2o
12
1
11
Leucine
33
49
Lysine
12
n.d.
Aspartic Acid
Valine Isoleucine
a
a
Values were obtained for the TA3-MM/1 subline (40).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE
282
PROCESSES
this contention. Moreover, the two c e l l lines demonstrate many different characteristics at this time. I t i s , of course, conceivable that after many passages the TA3-MM c e l l w i l l become similar to the TA3-Ha c e l l , but i t i s also plausible that the two o r i g i n a l s t r a i n s p e c i f i c ascites sublines, TA3-Ha and TA3-St, were dissimilar i n important respects and some of t h e i r d i s s i m i l a r i t i e s have persisted after loss of s t r a i n s p e c i f i c i t y . I t i s clear, however, that i n each subline a l l o t r a n s p l a n t a b i l i t y i s associated with the presence at the c e l l surface of a large number (approximately 4 x 10 ) of molecules of epiglycanin. 6
Topographical Features o f the TA3-Ha and TA3-St Cells Electron microscopy revealed major differences i n the topography of the TA3-Ha and TA3-St ascites c e l l s (41). Scanning electron microscopy showed the surface of the TA3-Ha c e l l to consist of long m i c r o v i l l surface, as shown i n Figur , surface consisted of irregularly-spaced ridges or folds (Figure 2) (41). By dark f i e l d light microscopy, m i c r o v i l l i of viable TA3-Ha c e l l s i n suspension appeared to be of greater length than the diameter of the c e l l s and the m i c r o v i l l i appeared to have a c i l i a l i k e motion (42). By transmission electron microscopy the two c e l l types were hardly distinguishable at a magnification of about 10,000, with the exception of the morphological differences i n the microextensions, those of the TA3-Ha c e l l being more erect, more regularly spaced, and longer than those of the TA3-St c e l l (41). Each c e l l type appeared to contain two d i s t i n c t types of virus particles (41). This observation was consistent with immunologic findings which demonstrated that the TA3-Ha, TA3-St, and TA3-MM ascites c e l l s contained the same three types of virus p a r t i c l e s , the murine mammary tumor v i r u s , the Rauscher leukemia v i r u s , and. the pneumonia virus of mice (39). After glutaraldehyde and osmium tetroxide f i x i n g , high resolution electron microscopy (> 50,000 times magnification) revealed a network of fine filaments extending from the surface of the TA3-Ha ascites c e l l . On occasion these filaments appeared to aggregate into thicker, more rod-like structures, as shown i n Figure 3. These extended 200-400 nm, sometimes 500 nm, from the plasma membrane. By contrast, the TA3-St c e l l , when fixed s i m i l a r l y , possessed no v i s i b l e extracellular coat (41). Evidence that these extracellular structures at the TA3-Ha c e l l surface represented s i a l i c acid-containing glycoproteins, probably epiglycanin, was obtained by the use of pclycationic f e r r i t i n labeling. Anionic groups, labeled with the electron-dense polycationic f e r r i t i n , appeared to exist several hundred nm from the plasma membrane of the TA3-Ma ascites c e l l . By contrast, this reagent appeared to be attached only i n close proximity to the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
Antigens
283
plasma membrane of TA3-St c e l l s treated i n a similar way. After p r i o r treatment with neuraminidase to remove the c e l l surface s i a l i c acid r e l a t i v e l y few f e r r i t i n p a r t i c l e s were visualized at the surface of either c e l l (41). I t has been shown that i n the TA3-Ha c e l l epiglycanin contains approximately 60% of the surface s i a l i c acid (43). Isolation of Epiglycanin Epiglycanin was o r i g i n a l l y i d e n t i f i e d as glycopeptide frapients cleaved from viable c e l l s by proteolysis and fractionated by gel f i l t r a t i o n (28). The molecular weight of t h i s material (Fraction I ) , as calculated from sedimentation equilibrium and electron microscopy data, ranged from 80,000 to 460,000 (44). The yields of epiglycanin obtained by t h i s method averaged about 2.3 mg/10 c e l l s for either the TA3-Ha or TA5-NIM ascites sublines (Table I ) . Further fractionatio i n the proteolysis product (44). This v a r i a b i l i t y i n the composition of carbohydrate i s due to variations i n the r e l a t i v e proportion of each of f i v e different types of 0-glycosyl-linked carbohydrate chains (29, 43, 45). We have observed that epiglycanin i s shed from the c e l l surfaces of each nonstrain-specific TA3 c e l l l i n e which we have investigated. I t may be detected i n the growth medium at levels below 5 ng/ml by a hemagglutination i n h i b i t i o n method (37). By this method epiglycanin has been detected i n the serum 6T s t r a i n A mice bearing TA5-Ha ascites c e l l s on day 2 or 3 following i n t r a peritoneal inoculation of the c e l l s . Isolation of epiglycanin from the ascites f l u i d followed precipitation of the bulk of other protein and glycoprotein material by b r i e f exposure to 0.3 M perchloric acid at 0°. Fractionation by passage of the perchloric acid-free solution through a long column of Sspharose 4B gave nearly pure epiglycanin of composition similar to that of proteolysis derived material (43). Yields of epiglycanin by t h i s method averaged about 0.2 to 0.3 mg/10 c e l l s , and the molecular weight of the materials isolated from either TA3-Ha or TA3-MM ascites c e l l s was approximately 500,000, as determined by sedimentation equilibrium or electron microscopy (40). By shadow-casting electron microscopy (H. S. Slayter, personal communication), approximately 60-70% of the material obtained i n t h i s way ranged from 450 to 500 nm i n length. Whether epiglycanin sned into the growtli medium represents the entire native molecule or only the major portion cleaved from the c e l l surface by membrane-bound proteases has not been determined. This material demonstrated l i t t l e tendency to aggregate, i n contrast to epiglycanin isolated after s o l u b i l i z a t i o n with detergents or, a l t e r n a t i v e l y , with lithium diiodosalicylate, followed by phenol extraction (43). By gel f i l t r a t i o n chromatography small yields of non-aggregated 9
9
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
284
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 1. Scanning electron micrograph of the TA3-Ha ascites cell. Long, regularly-spaced microvilli cover the cell surface. X4300. (Courtesy of S. C. Miller and E. D. Hay.)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
CODINGTON
Masking
of Cell-Surface
Antigens
Figure 2. Scanning electron micrograph of the TA3-St ascite cell. The cell surface is characterized by numerous irregularly spaced folds or ridges. X4300. ( Courtesy of S. C. Miller and E. D. Hay.)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
286
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Figure 3. Transmission electron micrograph of the TA3-Ha ascites cell surface. Cells were fixed with glutaraldehyde and osmium tetroxide. Extramembranous structures 200-500 nm in length are observed on the cell surface. X 43,000. (Courtesy of S. C. Miller and E. D. Hay.)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
Antigens
287
epiglycanin were obtained by either of these two methods. From gel f i l t r a t i o n effluent volumes and by measurement of the molecular lengths on electron micrographs (II. S. Slayter, personal communication) the molecular size of epiglycanin obtained i n these ways appeared to be s i m i l a r to that o f ascites fluid-derived epiglycanin. I t seems probable, however, that i n the case of a molecule as large as epiglycanin (approximately 1300 amino acids i n an extended chain), the addition of a peptide segment of 20 to 30 amino acids could not be readily detected. Physicocnemical
Characterization of Epiglycanin
The carbonydrate and amino acid compositions of epiglycanin from the TA3-MM and TA3-IIa sub-lines are s i m i l a r (Table I ) . The percentage of carbohydrate, however, appears to be consistently higher (30-90%) i n TA3-MM material tha i TA3-H epiglycani (7^-80%). Five types o from epiglycanin as t h e i N-acetylgalactosaminito , and t h e i r sequences proposed (13, 43, 4o). A pentasaccharide, containing two galactose, one N-acetylglucosamine, one N-acetylgalactosamine, and one s i a l i c acid residue was suggested on the basis of compositional data obtained by gas-liquid chromatography (43). Recent analyses, however, (45) suggest that a hexasaccharide with two s i a l i c acid residues, rather than one, as suggested for the pentasaccharide, may be present. In this chain i t would be expected that the second s i a l i c acid residue i s attached by a 2-H3 linkage to N-acetylgalactosamine. A s i a l i c acid (2-*3) galactose linkage has been previously proposed (29). The number of carbohydrate chains present i n a molecule of 500,000 molecular weight, consisting of approximately 1300 amino acid residues, has been found to be 500-600 (29, 43). A l l samples of epiglycanin whicli we have investigated have contained a small amount of marjiose (approximately 0.3%). I t was not known whether the presence o f this residue indicated that a glycoprotein impurity were present or i f the mannose were a component of an asparagine-linked carbohydrate chain i n epiglycanin. By the use of a f f i n i t y chromatography with a Concanavalin A - Sepharose 4B column i t has recently been possible to obtain from proteolytically-cleaved fragments (Fraction I) a fraction of epiglycanin free of mannose. I t was found, however, that epiglycanin isolated from the ascites f l u i d (molecular weight, 500,000) was completely adsorbed on a s i m i l a r a f f i n i t y column and could be eluted from the column with methyl-a-mannopyranoside. These results suggest that epiglycanin does contain N-glycosyl-linked carbohydrate material. On the basis of 0.3% mannose there could be no more than nine residues of mannose per molecule of epiglycanin, enough for one, possibly two, asparaginelinked carbohydrate chains. I t has not been possible to obtain amino acid sequence data for epiglycanin isolated from the ascites f l u i d , since the amino
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
288
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
group at the N-terminal end of tiie molecule i s blocked. This observation suggests that the N-terminal amino group i s extrac e l l u l a r , and that the carboxy terminal i s rooted i n the c e l l , as i t i s i n the erythrocyte glycoprotein which contains M and N blood group a c t i v i t y (46). By shadow casting electron microscopy a l l samples of epiglycanin have appeared as s i m i l a r rod-like p a r t i c l e s . Widths were consistently 2.J nm, and lengths varied from about 70 nm for the smallest proteolytically-cleaved fragment (44) to 450-500 nm for material isolated without proteolytic digestion (40). Further Evidence for Masking i n the TA3 Tumor The hypothesis of masking was tested i n a series of s i x hybrid c e l l lines which had resulted from fusion i n v i t r o of TA3-Ha c e l l s and normal embryoni fibroblast f tli A.CA (47). These lines had bee i n s t r a i n A mice and possesse y majo histocompatibility complex of the TA3-Ha parent, H-2 (48, 49). Each of the hybrid lines exhibited the capacity to grow progressively i n mice of foreign s t r a i n s ; each showed a reduced capacity, compared to the TA5-St s t r a i n s p e c i f i c c e l l l i n e , to absorb anti-H-2 antibody; and each of the hybrid lines possessed epiglycanin at i t s c e l l surface (22). The r e l a t i v e values for the three parameters, allotransp l a n t a b i l i t y {% of foreign mice k i l l e d by the tumor), resistance to antibody absorption (absorptive capacity plotted i n reverse order of magnitude), and amount (mg) of epiglycanin per 10 c e l l s are plotted i n Figure 4. The results strongly suggest that with increasing amounts of epiglycanin the c e l l s increase t h e i r capacity to grow i n mice of foreign strains and possess a reduced capacity to absorb antibody to H-2 antigen. These results support the hypothesis that i n the TA3 murine tumor system a function of epiglycanin i s the masking of cell-surface antigens (22). a
a
9
a
Relevance to Human Cancer of Masking by Endogenous Glycoproteins Tne discovery of epiglycanin and the correlation of i t s presence at the c e l l surface with a l l o t r a n s p l a n t a b i l i t y was unprecedented. Furthermore, since the i n i t i a l report of i t s i s o l a t i o n (28) i n 1972 and b i o l o g i c a l function (2£, 23) i n 1973 very l i t t l e evidence for the occurrence of a s i m i l a r phenomenon i n other tumor systems has been reported. Three plausible explanations for this may be suggested: (a) I t may be that masking of this type does not often occur i n neoplasia, but i s associated only with certain stages i n the cancer process and occurs only under c e r t a i n conditions, (b) Suitable experimental models for the study of t h i s phenomenon have not been available, (c) Investigators have not always u t i l i z e d experimental procedures
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
Antigens
289
TA3-H* l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l f l
i iiiiiiiiiiiMiiiiiiiiiiiiiiiiiiiiiiiiHiiiiiiiiiiiiiiiiiiiiiiiiiiuriiiiii
TA3-Ha//LCA/lO
TA3-Ha/*.CA/ll
IIIIIIIIIIIIIIIIIIIIMIIMMIIIMIMIIIHIIIIII
MIIIIIIIIIIIIIIIIMIIIIII
Figure 4. Comparison of three characteristics of six TA3-Ha/A.Ca hybrid, the TA3-Ha, and the TA3-St ascites cell lines. The relative amounts of epiglycanin on the cell surfaces ( ) are plotted with the capacities of the cells to resist absorption of anti-H-^ antibody ( millmi) and their capacities to grow progressively in mice of foreign strains (i J ). The units and scales used in plotting this graph are similar to those described in Ref. 22.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
290
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
suitable for a study o f t h i s type. I t i s my contention that a l l of these explanations may apply to t h i s situation. The appearance of large extended molecules, such as epiglycanin, on tumor c e l l surfaces probably occurs only under c e r t a i n circumstances i n established tumors - with probably disastrous consequences for the host. Furthermore, i t may be d i f f i c u l t to establish "spontaneously-developed" masking-glycoprotein-synthesizing c e l l s as experimental models for study, or i n the past when c e l l s of t h i s type did develop i n experimental systems they may have been overlooked. Loss of s t r a i n s p e c i f i c i t y i n the TA3 tumor system i s known to have occurred only twice, and only on the second occurrence were the circumstances recorded (39, As described i n t h i s report the capacity o f s t r a i n - s p e c i f i c c e l l s to synthesize epiglycanin occurred i n the TA3-St subline i n vivo i n hosts infected by microorganisms Th mechanis B which t h i chang took place i n the TA3-H biochemical event which synthesi epiglycani could occur under the influence o f other, seemingly unrelated factors. During the period o f tumor development i n humans, which i s often of many years duration, the patient may be subjected to many types of environmental influences, among them invasions by viruses and bacteria. Under such circumstances changes i n the character of tumors may possibly occur. Indeed, the occurrence of changes i n the morphology and growth characteristics of human, tumors i s w e l l documented (50). Of p a r t i c u l a r relevance to t h i s discussion may be the appearance o f invasiveness, metastasis, and a more rapid rate o f c e l l p r o l i f e r a t i o n . Indeed, evidence from the TA3 murine mammary carcinoma system suggest that the presence of epiglycanin-like glycoprotein molecules at the surfaces of human tumor c e l l s could possibly explain an increase i n virulence i n some human cancers. Comparison o f the growth rates o f TA3 ascites c e l l s i n the peritoneal cavity o f syngeneic mice revealed that c e l l s bearing epiglycanin at t h e i r surfaces, namely those o f the TA3-Ha and TA3-NM l i n e s , grew more r a p i d l y than those (Ta3-St) without epiglycanin. Friberg (30) attributed differences i n growth rates o f TA3-Ha and TA3-St l i n e s to an i n i t i a l lag period i n TA3-St growth. We have found that during a 7 day growth period the average doubling time f o r the TA3-MM and TA3-Ha c e l l s i s approximately 15 hours, compared to a doubling time of about 20 hours f o r the TA3-St c e l l . A plausible explanation for the increased growth rate i n the epiglycanin-bearing c e l l s may involve masking of tumor associated antigens, although to my knowledge no comparison of the properties o f the tumor-associated antigens i n the TA3 sublines has been reported. Recent reports o f M i l l e r et a l . (51) and Cooper and coworkers (52) describe properties i n TA3"suBlines which are consistent with a role i n metastasis by epiglycanin. I t was shown that the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
Masking
of Cell-Surface
Antigens
291
appearance of epiglycanin depends upon environmental factors and that, under certain circumstances epiglycanin may become l o s t , or become nondetectable, at the TA3-Ha c e l l surface. After several weeks of growth i n culture epiglycanin could not be detected at the surface of the TA3-Ha c e l l (51). However, after passage i n the mouse i n ascites form, epiglycanin was again present i n abundance at the c e l l surface. It was recently reported by Hagmar and Ryd (53) that the TA3-Ha c e l l was not transplantable when passaged from the ascites form i n the s t r a i n A mouse to a subcutaneous s i t e i n a mouse of a foreign s t r a i n . However, Cooper et a l . (52) have demonstrated that with continuous passage of TA3-Ha ascites c e l l s subcutaneously i n syngeneic mice the concentration of epiglycanin increased, as evidenced by the appearance of an increased concentration of receptors for the V i c i a graminea l e c t i n (38), with the appearance of subcutaneous a l l o t r a n s p i a n t a b i l i t y These circumstances f o r tumor growth may more nearl growing s o l i d tumors i correlaticn between the presence of epiglycanin and allotransp i a n t a b i l i t y i n s o l i d tumors and suggest that masking by endogenous glycoprotein molecules may occur i n s o l i d tumors. A possible relationship between epiglycanin concentration and metastatic properties was also demonstrated by this group (52). Variants derived from TA3-Ha c e l l s by prolonged suEcutaneous growth i n syngeneic mice w ere selected on the basis of t h e i r capacity to grow i n s i t e s distant from the t a i l vein injection s i t e , i . e . , beyond the lung, i n the l i v e r , spleen, of kidney. I t was found that those variants which possessed the greatest concentration of epiglycanin demonstrated the greatest metastatic property. The most "metastatic" of the c e l l s investigated was a TA3-Ha variant which possessed 2-3 times as much epiglycanin at i t s surface as the established TA3-Ha ascites c e l l l i n e . Thus, a c o r r e l a t i o n has been demonstrated between the presence of epiglycanin. and the capacity of c e l l s i n the TA3 murine carcinoma to pass f r e e l y i n the circulatory system to distant sites before establishing f o c i of tumor growth. Any conclusions regarding the relationship between masking by large endogenous ectoglycoproteins and cancer i n humans must be considered as purely speculative at t h i s time. Nevertheless, the investigations of the TA3 mamirary carcinoma i n the mouse described here suggest, as was stated i n 1975 (13), that "similar-type transformations, resulting i n the appearance of epiglycanin-Iike macromolecules, could occur i n other spontaneously derived tumors. Such phenomena may have relevance to human cancers, p a r t i c u l a r l y to the occurrence of metastases or to the recurrence of cancer i n seemingly cured patients." r
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
292
Acknowledgements This investigation was supported by grants from the National Institutes of Health, U.S. Public Health Service (CA-18600 to J. F. Codington and CA-08418 to R. W. Jeanloz).
Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Kahan, B. D., and Reisfeld, R. A . , Eds., "Transplantation Antigens", Academic Press, New York, 1972. Nathenson, S. G., and Cullen, S. E . , Biochim. Biophys. Acta (1974) 344, 1-25. Moscona, A.A., Proc. Soc. Exptl. Biol. Med. (1956) 92, 410416. Moscona, A., Ed., "The Cell Surface in Development", J . Wiley and Sons, Inc., New York 1974 Old, L. J., and Boyse Prehn, R. T., Canad Conf. (l963), 387. Klein, G., in Davis, B. D., and Warren, L., Eds., "The Specificity of Cell Surfaces", pp. 165-180, Prentice-Hall, Inc., New Jersey, 1967. McKhann, C. F . , and Jagarlamoody, S. M . , in Day, S. B . , and Good, R. A . , Eds.,"Membranesand Viruses in Immunopathology", pp. 577-599, Academic Press, New York, 1972. Prehn, R. T., in Nowotny, A . , Ed., "Cellular Antigens", pp. 308-313, Springer-Verlag, New York, 1972. Granatek, C. H . , Hanna, M. G., Jr., Hersh, E. M., Gutterman, J . U . , Mavligit, G. M . , and Candler, E. L., Cancer Res. (1976) 36, 3464-3470. Smith, R. T., and Landy, M., Eds., "Immune Surveillance", Academic Press, New York, 1970. Klein, G., and Klein, E . , Proc. Natl. Acad. S c i . U.S.A. (1977) 74, 2121-2125. Codington, J. F . , in "Cellular Membranes and Tumor Cell Behavior", pp. 399-419, Williams and Wilkins Co., Baltimore, 1975. Jeanloz, R. W., and Codington, J . F . , in Rosenberg, A . , and Schengrund, C. -L., Eds., "Biological Roles of Sialic Acid", pp. 201-238, Plenum Press, New York, 1976. Bekesi, J. G., Roboz, J. P., and Holland, J. F . , Ann. N.Y. Acad. S c i . (1976) 277, 313-331. Apffel, C. A . , and Peters, J . H . , J. Theor. Biol. (1970) 26, 47-59. Wnitehead, R. H . , Roberts, G. P., Thatcher, J., Teasdale, C., and Hughes, L. E., J. Natl. Cancer Inst. (1977) 58, 1573-1576. Dorval, G., Witz, I. P . , Klein, E., and Wigzell, H . , J. Natl. Cancer Inst. (1976) 56, 523-527. Lippman, M., Nature (1968) 219, 33-36. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1973) 51, 585-591.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
15.
CODINGTON
21.
Sanford, B. H . , Codington, J. F . , Jeanloz, R. W., and Palmer, P. D., J . Immunol. (1973) 110, 1233-1237. Codington, J. F., Klein, G., Cooper, A. G., Lee, N . , Brown, M. C., and Jeanloz, R. W., J. Natl. Cancer Inst. (1978) in press. Ohno, S., Natsu-ume, S., and Migita, S., J. Natl. Cancer Inst. (1975) 55, 569-578. Funakoshi, I., Nakada, H . , and Yamashina, I., J . Biochem. (1974) 76, 319-533. Bhavanandan, V. P., and Davidson, E. A . , Biochem. Biophys. Res. Commun. (1976) 70, 139-145. Gasic, G., and Gasic, T . , Nature (1962) 196, 170. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1970) 45, 637-647. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., Biochemistry (1972) 11 2559-2564 Codington, J. F . , Linsley and Osawa, T., Carbchydr (1975) , Friberg, S., Jr., J . Natl. Cancer Inst. (1972) 48, 1463-1476. Friberg, S., J r . , J . Natl. Cancer Inst. (1972) 48, 1477-1489. Hauschka, T. S., Weiss, L., Holdridge, B. A . , Cudhey, T. L., Zunpft, M., and Planinsek, J. A . , J. Natl. Cancer Inst. (1971) 47, 343-359. Grohsman, J., and Nowotny, A . , J. Immunol. (1972) 109, 10901095. Lippman, M. M., Venditti, J. M . , Kline, I . , and Elam, D. L., Cancer Res. (1973) 33, 679-684. Friberg, S., J r . , ana Lilliehook, B . , Nature New Biol. (1973) 241, 112-113. Springer, G. F . , Codington, J. F . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1972) 49, 1469-1470. Cooper, A. G., Codington, J. F . , and Brown, M. G., Proc. Natl. Acad. S c i . U.S.A. (1974) 71, 1224-1228. Codington, J. F . , Cooper, A. G., Brown, M. C., and Jeanloz, R. W., Biochemistry (1975) 14, 855-859. Cooper, A. G., Codington, J. F., Miller, D. K . , and Brown, M. C., prepared for publication. Codington, J. F . , Cooper, A. G., Miller, D. K . , Slayter, H. S., and Jeanloz, R.W., prepared for publication. Miller, S. C., Hay, E. D., and Codington, J. F . , J. Cell Biol. (1977) 72, 511-529. Codington, J. F . , and Bruns, R. R., unpublished results. Codington, J. F . , Van den Eijnden, D. H . , and Jeanloz, R. W., in Harmon, R. E., Ed., "Cell Surface Carbohydrate Chemistry", pp. 49-66, Academic Press, New York, 1978. Slayter, H. S., and Codinton, J. F . , J. B i o l . Chem. (1973) 248, 3405-3410. Van den Eijnden, D. H . , Evans, N., Codington, J. F . , and Jeanloz, R. W., prepared for publication.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
Masking
of Cell-Surface
Antigens
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
293
294
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
46. Marchesi, V. T., and Furthmayr, H . , Ann. Rev. Biochem. (1976) 45, 667-698. 47. Wiener, G., Klein, G., and Harris, H . , J. Cell S c i . (1971) 8, 681-692. 48. Klein, G., Friberg, S., J r . Wiener, F . , J. Natl. Cancer Inst. (1973) 50, 1259-1268. 49. Friberg, S., Jr., Klein, G., Wiener, F . , J. Natl. Cancer Inst. (1973) 50, 1269-1286. 50. Berenblum, I., i n Florey, H. W., "General Pathology", pp. 645-667, W. B. Saunders Co., Philadelphia, 1970. 51. Miller, D. K . , Cooper, A. G., Brown, M. C., and Jeanloz, R. W., J. Natl. Cancer Inst. (1975) 55, 1249-1252. 52. Cooper, A . , Morgello, S., Miller, and Brown, M., in "Cancer Invasion and Metastasis: Biologic Mechanisms and Therapy", pp. 49-64, Raven Press, New York, 1977. 53. Hagnar, B . , and Ryd W. Transplantation (1977) 23 93-97 RECEIVED
February 27, 1978
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16 Glycoconjugate Alterations in Malignant and Inflammatory Disease of the Colon YOUNG S. KIM, JAMES S. WHITEHEAD, BADER SIDDIQUI, and DEAN TSAO Gastrointestinal Research Laboratory, Veterans Administration Hospital and University of California, San Francisco, CA 94121 In the United State dence among all malignan easily detectable and curable skin cancer. As is the case with most tumors, the survival rate and prognosis of patients with colonic cancer depends upon the grade and the extent of malignancy. Low grade, more differentiated colonic tumors as well as disease confined to the mucosa and submucosa are associated with longer patient survival. The prognosis is dismal for those who have a high rate of tumor growth or nonlocalized disease (1). The final outcome rests with a balance between the biological properties of the tumor and the type and extent of the host response. This appears to be reflected in the wide variation in the clinical development of the disease and in the growth rate and changes in biochemical and immunochemical properties of the tumor. In considering development of methods for early diagnosis of colonic cancer in those patients of high risk, such as individuals with inflammatory bowel diseases or intestinal polypoid lesions, and development of rational methods to control colonic neoplasia, it i s important to identify and characterize organ specific tumor markers and to clarify the molecular mechanisms involved in the altered biochemical and biological properties that accompany malignant transformation in the colon. The involvement of c e l l surface components in c e l l - c e l l interaction, in growth regulation of cells and in the process of malignant transformation has been reported by many investigators (2-5). Much of our current knowledge has been obtained from studies on virally transformed murine or avian fibroblasts. Although most human carcinomas, including colonic tumors, are epithelial in type and origin, relatively little i s known about cell surface properties of normal and cancerous epithelial cells. Since glycoproteins and glycolipids are major components of surface membranes of mammalian cells, especially those of the intestinal mucosa, and glycoconjugates have been implicated in diverse cellular phenomena, our laboratory has been examining these 0-8412-0452-7/78/47-080-295$05.00/0 ©
1978 A m e r i c a n C h e m i c a l Society
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
296
GLYCOPROTEINS AND
GLYCOLIPIDS IN DISEASE PROCESSES
components i n normal and cancerous c o l o n i c mucosal c e l l s . Three experimental models are being used f o r our i n v e s t i g a t i o n : 1) human cancerous and normal c o l o n i c t i s s u e s obtained a t surgery, 2) human c o l o n i c cancer c e l l s maintained i n monolayer c u l t u r e , and 3) experimental c o l o n i c cancer i n rodents induced by a chemi c a l carcinogen, 1,2-dimethylhydrazine. Some o f the approaches t h a t we are u s i n g i n the study of membrane glycoconjugates i n normal and cancerous c o l o n i c c e l l s i n c l u d e : 1) the i s o l a t i o n and chemical and immunochemical chara c t e r i z a t i o n of membrane glycoconjugates, 2) the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of both membrane and cytoplasmic glycoconjugates u s i n g f l u o r e s c e i n - or r a d i o - l a b e l e d l e c t i n s , 3) e x t e r n a l c e l l s u r f a c e r a d i o " l a b e l i n g w i t h g a l a c t o s e oxidase and iodium b o r o t r i t i d e or w i t h l a c t o p e r o x i d a s e - c a t a l y z e d i o d i n a t i o n , and 4) a study of the metabolism and turnover of membrane g l y c o c o n j u gates u s i n g i n v i v o and i n v i t r o i n c o r p o r a t i o n of r a d i o - l a b e l e d p r e c u r s o r s . In the presen p r i m a r i l y of our data o t i e s of human c o l o n i c tumors and the i s o l a t i o n and c h a r a c t e r i z a t i o n of s u r f a c e membrane g l y c o p r o t e i n s . Studies i n Human C o l o n i c Adenocarcinomatous T i s s u e s . Many a n t i g e n i c a c t i v i t i e s have been a s c r i b e d t o g l y c o p r o t e i n s and g l y c o l i p i d s . For example, g a s t r o i n t e s t i n a l mucins and i n t e s t i n a l mucosal c e l l surface membrane glycoconjugates have been reported to possess blood group a c t i v i t i e s . Immunofluorescence s t u d i e s on t i s s u e s i n d i c a t e d t h a t ABH blood group a c t i v i t y was low or absent i n primary g a s t r o i n t e s t i n a l tumors as w e l l as i n other e p i t h e l i a l tumors (60 . The l o s s of these a n t i g e n i c a c t i v i t i e s may be due t o e i t h e r decreased synt h e s i s or i n c r e a s e d degradation of these antigens i n tumor t i s sues. To determine i f d e l e t i o n of blood group a c t i v i t y occurs i n c o l o n i c cancer t i s s u e s and t o c l a r i f y the metabolic b a s i s f o r such a l t e r a t i o n s , the l e v e l s of blood group antigens were measured i n cancerous and adjacent normal c o l o n i c mucosa u s i n g hemagglutination i n h i b i t i o n methods (7^) . The r e s u l t s are shown i n Table I . The data r e p o r t e d here are f o r t i s s u e s taken from blood group A p a t i e n t s . The l e v e l s of these antigens were determined by measuring the a b i l i t y of membrane g l y c o p e p t i d e s t o i n h i b i t the a g g l u t i n a t i o n of red blood c e l l s by v a r i o u s agglut i n i n s . These a g g l u t i n i n s b i n d the s p e c i f i c sugar s t r u c t u r e s i n d i c a t e d i n the parentheses. A negative s i g n i n d i c a t e s no det e c t a b l e a n t i g e n . The g r e a t e r the number of p l u s e s , the higher the l e v e l of a n t i g e n . In a l l p a t i e n t s examined, blood group A a c t i v i t y could not be detected i n the cancer t i s s u e but normal l e v e l s were present i n adjacent t i s s u e s . Two p a t i e n t s who had h i g h l e v e l s of L e a c t i v i t y i n normal mucosa had no d e t e c t a b l e Lewis A a c t i v i t y i n the cancer t i s s u e . The use o f R i c i n u s communis l e c t i n (RCA), which recognizes s p e c i f i c sugar s t r u c t u r e s a
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and Inflammatory
Colon
Disease
297
i n c l u d i n g the immediate p r e c u r s o r o f the H s t r u c t u r e , detected h i g h e r l e v e l s o f t e r m i n a l g a l a c t o s e r e s i d u e s i n the cancerous t i s sues. No c o n s i s t e n t change i n Con A-binding g l y c o p e p t i d e s was observed. Since a l t e r a t i o n s i n blood group a n t i g e n i c i t y and l e c t i n r e a c t i v i t y suggested changes i n carbohydrate composition, the membrane f r a c t i o n o f cancerous and adjacent t i s s u e s was subjected to chemical a n a l y s i s . We measured the amounts o f 3 n e u t r a l sugars, 2 aminosugars and s i a l i c a c i d i n normal and cancerous t i s s u e s . The r e s u l t s i n d i c a t e d t h a t i n general the g l y c o p r o t e i n f r a c t i o n o f the cancer t i s s u e s contained l e s s o f each o f the sugars than d i d the normal t i s s u e s (Figure 1 ) . Fucose, glucosamine, galactosamine and s i a l i c a c i d were reduced s i g n i f i c a n t l y i n the cancerous t i s s u e s , w h i l e galactose was reduced t o a l e s s e r e x t e n t . The mannose content was o n l y s l i g h t l y reduced Not o n l y was the t o t a was reduced t o a d i f f e r e n t e x t e n t . s s e v i d e n t whe the mola r a t i o s o f each sugar normalized t o mannose are compared (Figure 2). For example, N-acetylgalactosamine which had a molar r a t i o to mannose o f 1.5 i n normal t i s s u e was reduced t o 0 . 4 i n cancer t i s s u e s . This r e d u c t i o n may be d i r e c t l y r e l a t e d t o the l o s s i n blood group A a c t i v i t y s i n c e N-acetylgalactosamine i s a p a r t o f t h i s blood group determinant. To e s t a b l i s h whether the observed a l t e r a t i o n s i n antigen l e v e l s and i n carbohydrate content were due t o decreased s y n t h e s i s o r t o i n c r e a s e d degradation o f the carbohydrate moiety, we examined two groups o f enzymes: g l y c o s y l t r a n s f e r a s e s , i n v o l v e d i n t h e i r b i o s y n t h e s i s , and g l y c o s i d a s e s , which degrade them. The A, B o r 0 blood group o f an i n d i v i d u a l i s determined by the presence o r absence o f two s p e c i f i c g l y c o s y l t r a n s f e r a s e s which are products o f the A o r B gene. The r e l a t i o n s h i p between blood groups o f an i n d i v i d u a l and the presence o f these two g l y c o s y l t r a n s f erases i s shown i n Table I I . Only i n d i v i d u a l s o f blood type A or AB c o n t a i n a s p e c i f i c N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e i n t h e i r i n t e s t i n a l mucosa, w h i l e o n l y those o f blood types B and AB have a s p e c i f i c g a l a c t o s y l t r a n s f e r a s e . Subjects who are blood type 0 l a c k both o f these enzymes. The immediate p r e c u r s o r f o r the A and B determinants i s the 0(H) s t r u c t u r e , so t h a t i t can be assumed t h a t a l l i n d i v i d u a l s who s e c r e t e A. B or 0 blood group substances have the g l y c o s y l t r a n s f e r a s e s r e q u i r e d t o s y n t h e s i z e the p r e c u r s o r o l i g o s a c c h a r i d e . Four o t h e r g l y c o s y l t r a n s f e r a s e s shown i n Table I I are not dependent on the i n d i v i d u a l ' s blood type. F i g u r e 3 shows some o f the g l y c o s y l t r a n s f e r a s e a c t i v i t i e s assayed i n tumors and i n t i s s u e s taken from the h i s t o l o g i c a l l y normal adjacent mucosa. A l s o shown are the l e v e l s o f these glycos y l t r a n s f erases i n normal c o l o n i c t i s s u e s taken from p a t i e n t s w i t h diseases other than cancer ( d i v e r t i c u l i t i s o r v o l v u l u s o f the c o l o n ) . There were no s i g n i f i c a n t d i f f e r e n c e s between the two
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
298
Table I Hemagglutination I n h i b i t i o n by the Membrane Glycopeptides of the Normal and Cancerous Mucosa (rz) Dolichos ,_ A n t i - L e . (A) b i f lorus serum
a
_ Ricinus _ Con A (Le ) . (Gal) communis
x
Patients
NL
1 2 3 4 5 6
++* ++++ ++++ ++++ ++++ +++
CA -
/ T
a
x
x
(Man .Glc N)
NL
CA
NL
CA
NL
CA
++++ ++++
-
-
+ ++ ++
+++ ++ ++++
+++ ++++ ++++
++++
++++
+
++++
++
*-, no i n h i b i t i o n up t o 300 ug membrane p r o t e i n ; +, i n h i b i t e d by 200-300 ug; ++, i n h i b i t e d by 100-200 ug; +++ i n h i b i t e d by 50-100 ug; and ++++, i n h i b i t e d by 50 ug. A, blood group A; Lea, blood group Lewis A; G a l , galactose; Man, mannose; GlcN, N-acetylglucosamine. f
Z
i
60-
•
NORMAL
•
CANCER
o
3
40-
i
o z
20-
MAN
Figure 1.
GAL
FUC
i
GLC GAL NEUAC -NAC -NAC
Carbohydrate composition of the membrane fraction of normal and cancerous colonic mucosa
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and Inflammatory
Colon
Disease
299
<
cr. <
Figure 2. Molar ratio of carbohydrate relative to mannose in membrane fraction of normal and cancerous colonic mucosa
Table I I G l y c o s y l t r a n s f e r a s e A c t i v i t i e s i n Human Colonic Mucosa Blood Type Glycosyltransferase
A (6)
AB (4)
B (4)
0 (6)
cpm/mg p r o t e i n / h r (X 10-3) N-acetylgalactosaminylt r a n s f e r a s e (A) Galactosylt r a n s f e r a s e (B) Galactosyltransferase I Galactosylt r a n s f e r a s e II Sialyltransferase Fucosyltransferase
210 + 97
176 + 30
0
0
59 + 12
98 + 27
0
13 + 3
12 + 4
15 + 4
9 + 4
77 + 17
90 + 24
110 + 32
79 + 29
54 + 8
45 + 6
32 + 6
44 + 8
117 + 32
110 + 30
90 + 23
119 + 27
0
Mean + S.D.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
300
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
normal groups. In view of the absence o f d e t e c t a b l e A antigen i n cancerous t i s s u e s , i t might be expected that the l e v e l of the t r a n s f e r a s e r e s p o n s i b l e f o r i t s synthesis might be low. Indeed the tumors had only 20% o f the a c t i v i t y found i n the normal t i s s u e s . A l though not a l l o f the enzymes are shown, two f u c o s y l t r a n s f e r a s e s and two g a l a c t o s y l t r a n s f e r a s e s were found to be lower i n the cancer t i s s u e s , but the d i f f e r e n c e was s t a t i s t i c a l l y s i g n i f i c a n t only f o r one g a l a c t o s y l t r a n s f e r a s e . The l e v e l of a s i a l y l t r a n s ferase was not s i g n i f i c a n t l y d i f f e r e n t i n normal and cancerous tissues. In two p a t i e n t s with blood group B, the g a l a c t o s y l t r a n s f e r a s e a c t i v i t y r e s p o n s i b l e f o r producing the B determinant was markedly reduced i n cancer t i s s u e s . These data suggest t h a t the absence o f blood group a c t i v i t y i n tumor t i s s u e s i s a consequence of a r e d u c t i o n or l o s s of s p e c i f i c g l y c o s y l t r a n s f e r a s e s . The s i a l y l t r a n s f e r a s e a c t i v i t y was unchanged i n the c o l o n i c cancer t i s s u e s d e s p i t neuraminic a c i d i n th Several s i a l y l t r a n s f e r a s e s i n mammalian t i s s u e s have been described; the one shown i n Figure 3 u t i l i z e s d e s i a l y z e d O C - a c i d g l y c o p r o t e i n as an acceptor. This enzyme c a t a l y z e s the a d d i t i o n of s i a l i c a c i d (N-acetylneuraminic acid) to t e r m i n a l galactose residues of o l i g o s a c c h a r i d e s . With d e s i a l y z e d f e t u i n as an acceptor, i t i s l i k e l y t h a t the same enzyme i s being measured. However, with d e s i a l y z e d ovine submaxillary mucin as an acceptor, an eznyme which c a t a l y z e s the t r a n s f e r of s i a l i c a c i d to Nacetylgalactosamine i s measured. Warren and h i s coworkers observed t h a t v i r a l l y transformed c e l l s have a higher l e v e l o f a s i a l y l t r a n s f e r a s e than the normal c e l l s when the membrane g l y c o peptides, obtained from transformed f i b r o b l a s t s and from Novikoff a s c i t e s tumor c e l l s , were used as acceptors. Table I I I shows the a c t i v i t i e s of four s i a l y l t r a n s f e r a s e s measured i n tumors, adjacent normal t i s s u e and c o l o n i c mucosa from p a t i e n t s with inflammatory bowel disease {8) . S i a l y l t r a n s f e r a s e a c t i v i t y was e s s e n t i a l l y the same u s i n g three o f the acceptors i n c l u d i n g glycopeptides from Novikoff a s c i t e s tumor c e l l s . However, when d e s i a l y z e d OSM was the acceptor, the s i a l y l t r a n s f e r a s e a c t i v i t y was s u b s t a n t i a l l y lower i n the cancer t i s s u e s . From these data, only the a c t i v i t y of a s i a l y l t r a n s f e r a s e involved i n the a d d i t i o n of N-acetylneuramine a c i d to a mucin acceptor was reduced i n tumors. Experimentally determined a c t i v i t i e s of the g l y c o s y l t r a n s f e r ases i n mixing experiments agreed with the t h e o r e t i c a l p r e d i c t i o n s , demonstrating t h a t no i n h i b i t o r s or a c t i v a t o r s of these enzymes are present i n tumor or normal t i s s u e s . Thus, the observed r e d u c t i o n i n g l y c o s y l t r a n s f e r a s e a c t i v i t y appears to be due to an a c t u a l r e d u c t i o n of the enzymes. A l t e r a t i o n s i n a n t i g e n i c determinants and carbohydrate comp o s i t i o n s of cancerous t i s s u e could a l s o be due to changes i n glycosidase a c t i v i t y . F i v e g l y c o s i d a s e a c t i v i t i e s were measured
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Figure
Malignant
3.
and Inflammatory
Colon
Disease
301
Glycosyltransf erase activities in normal and cancerous colonic mucosa
Table I I I S i a l y l t r a n s f e r a s e s o f Normal and Cancerous Colonic Mucosa Acceptors
C^-acid glycoprotein Fetuin Tumor glycopeptides Ovine s u b m a x i l l a r y mucin *A11 the acceptors
Normal (6)
Cancer (6)
Inflammatory Bowel Disease (3)
cpm/mg p r o t e i n / h r (X 10~3) 36.4 + 12.4 34.2 + 16.1 33.4 + 11.2 40.7 + 14.6 41.4 + 16.9 44.1 + 18.8 5.1 + 1.5 4.9 + 1.8 5.0 + 1.6 14.2 + 9.6
3.7 + 1.7**
are d e s i a l y z e d .
**P < 0.025.
17.6 + 4.9 Cancer Research
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
302
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
u s i n g 4-methylumbelliferone g l y c o s i d e s as s u b s t r a t e s . To d e t e r the l e v e l of blood group A degrading g l y c o s i d a s e s , a r a d i o a c t i v e l y l a b e l e d blood group A o l i g o s a c c h r i d e was prepared by i n c u b a t i n g f u c o s y l l a c t o s e w i t h 14c-labeled UDP-N-acetylgalactosaminy1transferase. Of 6 g l y c o s i d e s shown i n F i g u r e 4, no a p p r e c i a b l e d i f f e r e n c e was seen between normal and cancerous t i s s u e . In f a c t , the mean l e v e l of the enzyme capable of degrading the A antigen was lower i n the cancer t i s s u e s . From t h i s i n f o r m a t i o n , the lower l e v e l o f A antigen i n cancerous t i s sue probably i s due to i n c r e a s e d g l y c o s i d a s e a c t i v i t y . Similar r e s u l t s were a l s o obtained w i t h t i s s u e s from p a t i e n t s w i t h O and B blood types. One should be c a u t i o u s i n c o r r e l a t i n g the carbohydrate content of the t i s s u e s or c e l l s w i t h the l e v e l s of a c t i v i t i e s o f these enzymes s i n c e the g l y c o p r o t e i n or o l i g o s a c c h a r i d e acceptors or s y n t h e t i c s u b s t r a t e s used may not r e f l e c t a c c u r a t e l y the s t r u c t u r e of n a t u r a l acceptors or s u b s t r a t e s i n the t i s s u e s or c e l l s Figure 5 d e p i c t s s c h e m a t i c a l l tumor c e l l t r a n s f o r m a t i o n . The t e r m i n a l t r i s a c c h a r i d e sequence a t the non-reducing end of the carbohydrate s i d e chain i s the blood group A determinant. When g a l a c t o s e i s s u b s t i t u t e d f o r N-acetylgalactosamine, the B determinant i s formed. Our data show t h a t A o r B blood group a c t i v i t i e s which were present i n the normal adjacent mucosa disappeared or were markedly reduced i n c o l o n i c cancer c e l l s and t h a t there was an i n c r e a s e d exposure o f t e r m i n a l g a l a c t o s e r e s i d u e s . This suggests t h a t the carbohydrate s i d e chains of blood group a c t i v e membrane g l y c o p r o t e i n s may be incomplete i n the c o l o n i c cancer tissue. A s i m i l a r d e l e t i o n or r e d u c t i o n i n the a n t i g e n i c i t y of ABH blood group a c t i v e i n g a s t r o i n t e s t i n a l tumors has been r e p o r t e d p r e v i o u s l y by Dr, Hakomori s group (9} and r e c e n t l y by Dr, S i d d i q u i i n our l a b o r a t o r y (10), Table IV g i v e s the g l y c o l i p i d a s s o c i a t e d blood group ABH a c t i v i t i e s obtained from normal and cancerous c o l o n i c t i s s u e s (10). In the e i g h t specimens exami n e d , there was a decrease or absence o f g l y c o l i p i d a s s o c i a t e d blood group a c t i v i t y i n a l l cancer t i s s u e s compared to adjacent normal t i s s u e s . An incompletion of the carbohydrate s i d e chains of blood group a c t i v e g l y c o l i p i d s and g l y c o p r o t e i n s r e s u l t i n g i n the appearance o f new, p r e c u r s o r , blood group antigens such as T and i has been r e p o r t e d to occur i n g a s t r o i n t e s t i n a l and b r e a s t tumors by s e v e r a l i n v e s t i g a t o r s i n c l u d i n g Drs. Springer (11) and F e i z i (12). These data and e a r l i e r work by Drs. Hakomori (13), Fishman 05) and Robbins (14) u s i n g transformed f i b r o b l a s t s have been the b a s i s f o r the hypothesis t h a t a d e l e t i o n or r e d u c t i o n i n carbohydrate antigens i s accompanied by an i n c r e a s e i n i t s l e s s complex p r e c u r s o r s . Our recent s t u d i e s on g l y c o l i p i d compositions of normal and cancerous c o l o n i c t i s s u e s i n d i c a t e t h a t a s i m p l i f i c a t i o n of f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and
Inflammatory
Colon
Disease
303
^ N O R M A L (NONCANCER) •NORMAL
3
. |
300-
CANCER
o
Z>
-10
x
o
x 200-
o ->
100Q_
u B GAL
B GAL (9GLC «<MAN o
°
r
Figure 4.
Glycosidase
activities in normal and cancerous colonic mucosa
Fuc
GAL
I ^ (1-2) G A L N A C ^ ^ G A L
I ,41-3
OR
4)
GLCNAC
GLCNAC
GAL ^ ^ . G L C N A C
^ 3 ) ,
^
4(1^6) / J ^ ( M ) GLCNAC
GAL^i^UGLcNAcM
|
L
/•CM)
GAL PRECURSOR
ABO & LEWIS
t***i*t*+t*'*>f****tt ************* MALIGNAN
BENIGN
Figure 5.
Glycoprotein-associated ABO(H) glycoproteins in colonic cancer
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
304
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
carbohydrate s i d e chains does not occur i n a l l c l a s s e s o f g l y c o l i p i d s (1(3) G a n g l i o s i d e s , both simple and complex s i a l i c a c i d - c o n t a i n i n g g l y c o l i p i d s such as GM^, GD^, GM^ and b D i , were increased over t w o - f o l d i n 3 out o f 8 cancer t i s s u e s (10). In 5 cases no d i f f e r ence was observed. Of the l a t t e r group, none o f the tumors showed lymph node involvement w h i l e two o f the former had substant i a l lymph node m e t a s t a s i s . P a t h o l o g i c a l examination showed the t h i r d tumor o f t h i s group t o be an extensive mucoid carcinoma but examination o f lymph node involvement was incomplete. Thus, more complex g l y c o l i p i d s seem t o be abundant i n some human c o l o n i c cancer t i s s u e s , u n l i k e v i r a l l y - t r a n s f o r m e d malignant f i b r o b l a s t s which become enriched i n l e s s complex p r e c u r s o r s . Another example o f the appearance o f more complex carbohydrate s i d e chains i n tumor t i s s u e s i s the appearance o f Forssman a n t i genic a c t i v i t y i n some human c o l o n i c cancer t i s s u e s from i n d i v i d u a l s who are Forssman negativ by Dr. Hakomori. To understan to compare s t r u c t u r e s and processes between the tumor and normal t i s s u e s . However, some problems or s t u d i e s a r e best approached by examining tumor c e l l s grown i n c u l t u r e . To study some aspects o f tumor c e l l b i o c h e m i s t r y , we have begun t o examine human c o l o n i c adenocarcinoma c e l l l i n e s maint a i n e d i n monolayer c u l t u r e . a
C e l l Surface G l y c o p r o t e i n s o f Human Colonic Adenocarcinoma C e l l s We have used a method f o r s p e c i f i c a l l y l a b e l i n g galactose and galactosamine residues which a r e present a t non-reducing t e r m i n i of o l i g o s a c c h a r i d e s w i t h galactose oxidase and t r i t i a t e d sodium borohydride. This method was used t o i n v e s t i g a t e the o r g a n i z a t i o n and chemical nature o f surface membrane carbohydrates, such as those a s s o c i a t e d w i t h g l y c o p r o t e i n s and g l y c o l i p i d s o f c o l o n i c cancer c e l l s . The r a d i o a c t i v e p r o f i l e s o f t r i t i u m - l a b e l e d c e l l surface g l y c o p r o t e i n s , f o l l o w i n g c e l l surface l a b e l i n g and SDS-gel e l e c t r o p h o r e s i s from 5 human f e t a l i n t e s t i n a l c e l l s and 6 c o l o n i c adenocarcinoma c e l l s were compared. F e t a l c e l l s produced a s i m i l a r l a b e l i n g p a t t e r n o f l a r g e molecular weight g l y c o p r o t e i n s , whether the c e l l l i n e s were d e r i v e d from the small i n t e s t i n e o r from the c o l o n . Colonic cancer c e l l s showed l a b e l i n g p a t t e r n s d i s t i n c t from f e t a l c e l l s w i t h some v a r i a b i l i t y among them. When c e l l s o f a human c o l o n i c adenocarcinoma l i n e , SKCO-1 were l a b e l e d w i t h galactose oxidase and t r i t i a t e d NaBH^ a f t e r treatment w i t h neuraminidase, the p r e - e x i s t i n g bands were l a b e l e d more i n t e n s e l y compared t o the c o n t r o l as shown i n F i g u r e 6 (16). In a d d i t i o n , a few more bands became l a b e l e d a f t e r neuraminidase treatment. T r y p s i n treatment o f l a b e l e d c e l l s produced a d i m i n u t i o n o f only few bands. Some o f the t r i t i a t e d g a l a c t o p r o t e i n s were a l s o found t o be r a d i o i o d i n a t e d by l a c t o p e r o x i d a s e .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and Inflammatory
Colon
Disease
305
Table IV G l y c o l i p i d Blood Group A c t i v i t i e s i n C o l o n i c Mucosa (to) Patient
Blood Type
1 2 3 4 5 6 7 8
A AB 0 0 0 0 0 0
ABH blood group a c t i v i t y Normal Cancer _
++* ++++ ++++ ++++ ++ ++++ ++++ ++++
+
-
++ +
-
*++++/ h i g h e s t d i l u t i o n o f g l y c o l i p i d which i n h i b i t s hemagglutination; no i n h i b i t i o n
Figure 6. Autoradiographic pattern of H (A to F) and I-laheled (G to I) SKCO-1 cell membrane proteins separated in SDS-polyacrylamide slab gel. A , no treatment; B, galactose oxidase treatment; C, galactose oxidase followed by trypsin treatment; D, treatment with neuraminidase; E, treatment with neuraminidase followed by galactose oxidase; F, treatment with neuraminidase followed by galactose oxidase with subsequent trypsin treatment; G, lactoperoxidase alone; H, lactoper-oxidase and H 0 ; I, lactoperoxidase and H 0 with subsequent trypsin treatment (16). 3
125
2
2
2
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
306 1
2
5
Additional I l a b e l e d membrane p r o t e i n s not l a b e l e d by t r i t i u m could a l s o be detected. F o l l o w i n g e x t e r n a l c e l l surface l a b e l i n g w i t h galactose o x i dase and t r i a t e d borohydride, the membrane f r a c t i o n o f SKC0-1 c e l l s was s o l u b i l i z e d w i t h detergent and f r a c t i o n a t e d by a s e r i e s of chromatographic methods c o n s i s t i n g o f a f f i n i t y chromatography on R i c i n u s communis and Concanavalin A-Sepharose columns, and g e l f i l t r a t i o n u s i n g Sephadex G-200. Using t h i s procedure, we succeeded i n i s o l a t i n g a l a b e l e d g l y c o p r o t e i n which we designated as G a l a c t o p r o t e i n I . This prot e i n corresponded t o the slow m i g r a t i n g major band which was present o n l y i n SKCO-1 c e l l s . The i s o l a t e d G a l a c t o p r o t e i n I was found t o be homogenous on SDS-gel e l e c t r o p h o r e s i s when s t a i n e d f o r p r o t e i n o r carbohydrate. When compared t o carcinoembryonic antigen on SDS-gel e l e c t r o p h o r e s i s , t h i s g a l a c t o p r o t e i n was found t o have an e l e c t r o p h o r e t i c mobility i d e n t i c a l to tha ent molecular weight o When immunodiffusion o f G a l a c t o p r o t e i n I a g a i n s t CEA antiserum was c a r r i e d out, a s i n g l e p r e c i p i t i n l i n e o f f u s i o n formed i n d i c a t i n g t h a t G a l a c t o p r o t e i n I shares immunological i d e n t i t y w i t h CEA. No p r e c i p i t i n l i n e was formed between anti-CEA antiserum and other g a l a c t o p r o t e i n s . The r e s u l t s o f q u a n t i t a t i v e p r e c i p i t a t i o n i n d i c a t e t h a t G a l a c t o p r o t e i n I could be completely p r e c i p i t a t e d by CEA antiserum.. G a l a c t o p r o t e i n I could n o t be r e l e a s e d from the plasma membranes o f SKCO-1 c e l l s w i t h e i t h e r EDTA, b u f f e r e d s a l i n e o r s o n i c a t i o n b u t could be s o l u b i l i z e d e f f e c t i v e l y w i t h detergents. These p r o p e r t i e s i n d i c a t e t h a t G a l a c t o p r o t e i n I i s an " i n t e g r a l " p r o t e i n o f t h e membrane. The amino a c i d and carbohydrate composition o f G a l a c t o p r o t e i n I was determined and compared, w i t h those obtained by v a r i o u s i n v e s t i g a t o r s , f o r carcinoembryonic a n t i g e n . Although not shown, the amino a c i d composition was s i m i l a r between g a l a c t o p r o t e i n I and CEA. Carbohydrate a n a l y s i s , shown i n Table V r e v e a l e d t h a t Galactop r o t e i n I contained the same n e u t r a l and amino sugars as CEA reported by others (_1) . However, the r e l a t i v e p r o p o r t i o n o f each sugar i n G a l a c t o p r o t e i n I and CEA d i f f e r e d c o n s i d e r a b l y . For i n s t a n c e , much l e s s fucose and more s i a l i c a c i d was present i n G a l a c t o p r o t e i n I than the p u b l i s h e d values f o r CEA. This d i f f e r ence may, i n p a r t , be due t o the d i f f e r e n c e i n t i s s u e s from which these m a t e r i a l s a r e prepared. CEA m a t e r i a l s are l i k e l y t o have been prepared from c o l o n i c cancer metastasized t o the l i v e r , while SKC0-1 c e l l s were d e r i v e d from a primary c o l o n i c tumor. I t i s p o s s i b l e t h a t the decreased s i a l i c a c i d and i n c r e a s e d fucose content o f t h e surface membrane g l y c o p r o t e i n o f these cancer c e l l s may be r e l a t e d t o the c a p a c i t y o f c o l o n i c tumor c e l l s t o invade and metastasize t o other organs. A l t e r n a t i v e l y , the d i f f e r e n c e i n the p r o p o r t i o n o f v a r i o u s carbohydrates may be due t o the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and Inflammatory
Colon
Disease
307
Table V Carbohydrate Content o f G a l a c t o p r o t e i n I (\ b) (Weight o f monosaccharide/weight t o t a l carbohydrate) X 100
Fucose Mannose Galactose S i a l i c acid N-acetylgalactosamine N-acetylglucosamine
6..1 20..5 24..8 18..8 6..3 23..6
11..3 12..8 19..9 8..1 2..6 43..3
12..0 21..7 15..6 7..9 N.S.* 43..3
15.,2 14..3 17..9 10..7 1..8 40..2
21,.0 11..3 26..6 3..9 1..1 35..7
*N.S. = not s i g n i f i c a n t ^Carbohydrate analyses o f CEA were r e p o r t e d by: A, Banjo e t a l . , B, Kupchik et_ a l . , C, C o l i g a n ejt a l . , and D, Westwood e t a l .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
308
GLYCOPROTEINS AND
GLYCOLIPIDS IN
DISEASE PROCESSES
d i f f e r e n c e between t i s s u e s and c u l t u r e d c e l l s . We a l s o examined the disappearance from c e l l membrane o f r a d i o a c t i v i t y a s s o c i a t e d w i t h G a l a c t o p r o t e i n I as a f u n c t i o n of time a f t e r galactose oxidase and t r i t i a t e d sodium borohydride l a b e l i n g . The turnover r a t e of l a b e l e d G a l a c t o p r o t e i n I i n d i c a t e d t h a t i t was r e l e a s e d from the membrane s l o w l y i n t o the medium w i t h a h a l f l i f e of about 5 days. When l a b e l e d G a l a c t o p r o t e i n I i s o l a t e d from c e l l s immediately a f t e r and 5 days a f t e r galactose oxidase l a b e l ing was compared, no d i f f e r e n c e i n the m o b i l i t y on SDS g e l e l e c t r o p h o r e s i s was observed (16). When the s u b c e l l u l a r d i s t r i b u t i o n of the carcinoembryonic antigen (CEA) was examined i n 5 human c o l o n i c cancer c e l l l i n e s , 85-90% of the a c t i v i t y was found t o be a s s o c i a t e d w i t h the membrane f r a c t i o n of each c e l l . Considerable v a r i a t i o n i n the amount of CEA content was observed among the 5 c e l l l i n e s . Only one c e l l l i n e produced an a p p r e c i a b l e amount of CEA, namely SKCO-1. This l i n e contained 14 ug o I t i s a l s o of i n t e r e s t o n l y 6% o f the c e l l u l a r CEA w h i l e another l i n e secreted over 300% o f the c e l l u l a r CEA i n 3 days. Although the numbers of c e l l l i n e s examined are s m a l l , these r e s u l t s suggest t h a t membrane-bound CEA from each c o l o n i c cancer c e l l l i n e has a d i f f e r e n t turnover or shedding r a t e . I t may be s i g n i f i c a n t t h a t the SKCO-1 l i n e was the l e a s t tumorigenic when i n j e c t e d to nude mice and produced the l a r g e s t amount of CEA. Inflammatory bowel disease such as u l c e r a t i v e and granulomatous c o l i t i s are precancerous c o n d i t i o n s , and p a t i e n t s w i t h such diseases are a t higher r i s k f o r developing colon cancer than are h e a l t h y i n d i v i d u a l s . The more extensive the involvement of the bowel and the longer the d u r a t i o n of the inflammatory d i s e a s e s , the higher the i n c i d e n c e of c o l o n i c cancer. Although c o n s i d e r a b l e h i s t o c h e m i c a l changes have been observed i n the mucosal c e l l s i n d i c a t i n g a l t e r a t i o n i n glycoconjugates, o n l y l i m i t e d data are a v a i l a b l e on many of the biochemical parameters i n these disease s t a t e s . In the few p a t i e n t s w i t h i n f l a m matory bowel diseases t h a t we have s t u d i e d , we were unable to observe s i g n i f i c a n t a l t e r a t i o n s i n the a c t i v i t i e s o f g l y c o s y l t r a n s f e r a s e s or g l y c o s i d a s e s seen i n p a t i e n t s w i t h c o l o n i c tumors (8) . C l e a r l y , there i s a need to c a r e f u l l y examine glycocon j u gates and t h e i r a s s o c i a t e d enzymes i n the i n t e s t i n a l mucosa of these p a t i e n t s . By understanding how and which membrane g l y c o p r o t e i n s and g l y c o l i p i d s are synthesized and degraded i n normal, premalignant and cancerous mucosa, we should be i n a much b e t t e r p o s i t i o n to understand c a r c i n o g e n e s i s . Summary As i n f o r m a t i o n i s accumulated, i t i s becoming i n c r e a s i n g l y apparent t h a t a l t e r a t i o n s i n carbohydrate content and metabolism are a s s o c i a t e d w i t h the malignant process. Our approach has been
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
16.
KIM E T AL.
Malignant
and Inflammatory
Colon
Disease
309
to examine g l y c o p r o t e i n s and g l y c o l i p i d s , e s p e c i a l l y those on the c e l l surface, i n t i s s u e s and c u l t u r e d c e l l s and the enzymes i n volved i n the s y n t h e s i s and degradation o f t h e i r o l i g o s a c c h a r i d e s i d e chains* With regard t o the s t u d i e s comparing tumors w i t h adjacent normal t i s s u e s , we found a d e l e t i o n o r marked r e d u c t i o n i n A and B blood group a c t i v i t i e s i n tumor t i s s u e s w i t h a concomitant increase i n R i c i n u s communis b i n d i n g components. The carbohydrate contents o f the g l y c o p r o t e i n f r a c t i o n from membranes and cytoplasm were g e n e r a l l y reduced i n cancerous t i s s u e s . Sugars a s s o c i a t e d with mucin type g l y c o p r o t e i n s were those most affected. The a c t i v i t i e s o f some g l y c o s y l t r a n s f e r a s e s r e s p o n s i b l e f o r the formation o f the A and B blood group determinants were markedly lower i n the tumors w h i l e g l y c o s i d a s e a c t i v i t i e s were r e l a t i v e l y unchanged. Regarding s t u d i e s o f tumor c e l l s grown i n c u l t u r e , we have i s o l a t e d a membrane g l y c o p r o t e i n antigen from the surface o f a c u l t u r e d human adenocarcinom i d e n t i c a l t o CEA. Thi weight i n t e g r a l membrane p r o t e i n w i t h a carbohydrate composition q u a l i t a t i v e l y s i m i l a r , but q u a n t i t a t i v e l y d i s t i n c t from CEA. Studies o f the s u b c e l l u l a r d i s t r i b u t i o n o f CEA have i n d i c a t e d t h a t CEA i n s e v e r a l c u l t u r e d human c o l o n i c tumor c e l l s i s a membrane p r o t e i n and that the r a t e o f appearance o f CEA i n the c u l t u r e medium was d i f f e r e n t among these c e l l l i n e s . Acknowledgement This work was supported i n p a r t by United States P u b l i c Health S e r v i c e Grant CA-14905 from the N a t i o n a l Cancer I n s t i t u t e through the N a t i o n a l Large Bowel Cancer P r o j e c t and by the Veterans A d m i n i s t r a t i o n Medical Research S e r v i c e . Reprint requests should be addressed t o Young S* Kim, M,D %
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
310
GLYCOPROTEINS A N D GLYCOLIPIDS IN DISEASE PROCESSES
Literature Cited 1.
Kim, Y. S., Tsao, D., and Whitehead J . Cancer (1977) 40:24732478. 2. Meezan, E . , Wu, H. C., Black, P. H. and Robbins, P. W. Biochemistry (1969) 8:2518-2524. 3. Warren, L . , Fuhrer, J . P . , and Buck, C. A. Fed. Proc. (1973) 32:80-85. 4. Gahmberg, C. G., and Hakomori, S. Proc. Natl. Acad. Sci. U.S. (1973) 70:3329-3333. 5. Fishman, P. H., Brady, R. O., Bradley, R. M., Aronson, S. A . , and Todaro, G. J. Proc. Natl. Acad. Sci. U.S. (1974) 71: 298-301. 6. Davidsohn, I., Kovarik, S., and Lee, C. L. Arch. Pathol. (1966) 81:381-390. 7. Kim, Y. S., Issacs R., and Perdomo J M Proc Natl Acad Sci. U.S. (1974) 71:4869-4873. 8. Kim, Y. S., and Issacs, (1975) 9. Stellner, K. and Hakomori, S. Biochem. Biophys. Res. Commun. 55:439-445. 10. Siddiqui, B . , Whitehead, J., and Kim, Y. S. J . Biol. Chem. (1978) 253:2168-2175. 11. Springer, G. F., and Desai, P. R., Yang, H. J., and Murthy, M. S. Clin. Immun. and Immunopath. (1977) 7:426-441. 12. Feizi, T., Turberville, C., and Westwood, J . H. Lancet (1975) 2:391-393. 13. Nakomori, S., and Murakami, W. T. Proc. Natl. Acad. S c i . U.S. (1968) 59:254-261. 14. Robbins, P. W., and MacPherson, I. Nature (1971) 229:569570. 15. Hakomori, S., Wang, S. W., and Young, J r . , W. W. Proc. Natl. Acad. Sci. U.S. (1977) 74:3023-3027. 16. Tsao, D. and Kim, Y. S. J . Biol. Chem. (1978) 253:2271-2278. RECEIVED
March 14, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
17 Human Carcinoma-Associated Precursors of the Blood Group MN Antigens G.F.SPRINGER,P.R. DESAI, and M. S. MURTHY Department of Immunochemistry Research, Evanston Hospital, 2650 Ridge Ave., Evanston, IL 60201 E.F.SCANLON Department of Surgery and Microbiology—Immunology, Evanston Hospital, 2650 RidgeAve.,Evanston, IL 60201 M and N are the majo group system (1). Glycoprotein the human red blood cell MN specificities which are determined by carbohydrates (cf. 2,3). A look at the structural basis of the interrelation of blood group MN specificities, which also occur on human epithelial tissues (4,5,6), and their carbohydrate precursor specificities T and Tn is important for an understanding of the human carcinoma-associated antigenic specificities T and Tn, as is also the appreciation that all humans possess anti-T and anti-Tn but not anti-M and anti-N antibodies. The biosynthesis of T-, N- and M- specific structures and the surprising significance of the carcinoma-associated precursor antigens T and Tn for the human immune response, humoral as well as cell-mediated, against adenocarcinoma are the main topics of this paper. Materials and Methods Bacteria and Vaccines. Bacteria were obtained from the American Type Culture Collection (Washington, DC); bacterial antigens were prepared in this laboratory or given by Drs. D.A.L. Davies, O. Lüderitz and E. Rietschel. Gram-negative bacteria were grown on fully defined media using procedures described previously (7). Vaccines were from the individuals and Drug Houses listed in Table IV. Carcinomatous and Control Tissue. Mammary gland tissues originated from ductal adenocarcinomata Stage I (International Nomenclature) which had not invaded regional lymph nodes and from Stage II and III ductal carcinomata as well as from nonmalignant breast lesions (fibroadenomatosis and fibrocystic disease) of U.S. women 18 to 86 years old. Glandular tissue of the primary tumor as well as distant metastases were obtained under aseptic conditions during surgery. Specimens left in the Pathology Department for more than a few minutes and autopsy tissues were not used because of autolytic changes. No specimen showed obvious 0-8412-0452-7/78/47-080-311$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
312
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
b a c t e r i a l contamination and none t e s t e d was pyrogenic. The glandular s t r u c t u r e s were s l i c e d and f r e e d of other t i s s u e s . H i s t o l o g i c s e c t i o n s were made to assure that only predominantly malignant regions were used i n case of carcinoma. C e l l membranes were prepared by hypotonic l y s i s as described p r e v i o u s l y f o l l o w i n g procedures used by others ( J 3 , 9 ) . The two human colon carcinoma-derived t i s s u e c u l t u r e c e l l l i n e s were given by Dr. B. Tom (10); c e l l s derived from normal colon e p i t h e l i u m were not a v a i l a b l e . P r e p a r a t i o n o f T antigen from Human Blood Group 0,MN Red Cells. The mode o f p r e p a r a t i o n has been described e a r l i e r (11,12, 13). From r e d blood c e l l stroma the MN antigens were e x t r a c t e d with aqueous 45% phenol p l u s e l e c t r o l y t e a t 23-25°C. A c t i v e m a t e r i a l was i s o l a t e d by f r a c t i o n a l u l t r a c e n t r i f u g a t i o n and ethanol f r a c t i o n a t i o n . The g l y c o p r o t e i n which p r e c i p i t a t e d between 45 and 70% ethano l y z e d , f r e e z e - d r i e d an gen was uncovered by the removal o f NAN from the i s o l a t e d 0,MN antigens with V i b r i o cholerae neuraminidase (RDE) a t pH 6.8; RDE was then i n a c t i v a t e d by h e a t i n g at 100°C f o r 5 min. T antigen was prepared a s e p t i c a l l y . Absorption o f A n t i s e r a . Because o f t h e i r s t r i c t i n t r a species s p e c i f i c i t y human a n t i s e r a only were used against blood group antigens M, N, T, and Tn, unless s t a t e d otherwise; a l l s e r a used have been described before (14,37). The a n t i s e r a were absorbed once with the membrane-cytoplasm preparations o r homogenized glandular t i s s u e or colon carcinoma-derived t i s s u e c u l t u r e c e l l s or b a c t e r i a under standard c o n d i t i o n s (7,14,15). C o n t r o l absorptions o f human a n t i - b l o o d group B s e r a with blood group H ( 0 ) - s p e c i f i c human breast glandular t i s s u e , benign o r malignant, r e s u l t e d i n no t i t e r decrease, nor d i d absorption o f human a n t i Rh (D) s e r a with benign o r malignant t i s s u e s from blood groups A l and H(0) persons (see a l s o Table I ) . Q
Blood C o l l e c t i o n and S e r o l o g i c Procedures. Bloods f o r determination of a n t i - T t i t e r s were c o l l e c t e d 24 to 48 h r before o r immediately a f t e r surgery, unless s t a t e d otherwise. The c l o t t e d bloods were coded and recorded by i n d i v i d u a l s who were not i n v o l v e d i n the assays. Sera were prepared and used a t once o r s t o r e d i n a l i q u o t s a t -20°C. For s e r o l o g i c a l assays 0,MM and 0,NN erythrocytes were obtained, s t o r e d , washed, and employed as such o r a f t e r T - a c t i v a t i o n with RDE (2). Tn r e d blood c e l l s were donated by Drs. G. Leonard (Cla.Ric.) and W.D. Bowman (Car.Lip.) ( c f . 16,17). Hemagglutination and hemagglutination i n h i b i t i o n t e s t s i n c l u d i n g c o n t r o l s and standards were performed and i n t e r p r e t e d by r o u t i n e procedures (7,13). Tests were scored ( c f . 18) independently by three i n d i v i d u a l s to whom nature of t i s s u e s used f o r absorption
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Anti-T
58 -
0 0
98
10 - 25
100
83
50 -
70 (30 - > 90) 64 (50 - > 80)
53 (33 - 75)
42 (25 - 50)
< 1 ( o -
58
6)
59
< 10 ( o - 25)
49 -
3
OMM erythrocytes
0
0
2
"Buffy coat"
a b c — Number of cases. — No normal e p i t h e l i a l ^ c e l l s a v a i l a b l e . — A r i t h m e t i c averages i f > 2 persons t e s t e d , f i g u r e s i n parentheses = range. — M a c t i v i t i e s were found, with one exception, i f M antigen was expressed on the subject's r e d c e l l s (see t e x t ) . — Membranes from MM i n d i v i d u a l .
Anti-Tn
> 90)
/ m
v
Colon carcinoma . ^ \ (Tissue c u l t u r e ) ' 2-
53 - 100
66 ( 0 * .
15
Stages I I & I I I carcinoma
77 (50 - > 80)
72 (60 - 80)
3
Stage I carcinoma
53 (20 - 70)-
(50 - 66)
Anti-N
(A
5
ign
52^ (30 - 75)
B e n
Anti-M
sera
Human
TT
Breast glandular t i s s u e
Table I . Percent Absorption o f A n t i - B l o o d Group M, N, and Precursor A g g l u t i n i n s with Human Breast Gland Membrane-Cytoplasm P r e p a r a t i o n s , Colon Carcinomata and Controls
314
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
and source o f the sera were unknown; three serum standards were included as c o n t r o l s i n a l l determinations. Specimens were decoded only a f t e r completion o f the t e s t s . B i o s y n t h e t i c Procedures. For enzymatic transformation o f Tn to T group B r e d c e l l s o f C l a . R i c , which are completely Tntransformed, were used (19 ,20). Sera o f blood group B and A]B persons f r e e d o f a n t i - T and -Tn a g g l u t i n i n s served as p-galactos y l t r a n s f e r a s e source, UDP-Gal as Gal donor, and ATP, M n C l and MgCl2 as a c t i v a t o r s . A n a l y t i c a l grade chemicals were used; UDPGal and ATP were from Sigma Chemicals. Tn r e d c e l l s incubated i n absence o f one or more o f the other reactants served as c o n t r o l s ; they d i d not a c q u i r e T s p e c i f i c i t y , nor d i d normal MN r e d blood c e l l s when incubated under experimental c o n d i t i o n s with transf e r a s e - c o n t a i n i n g serum. For b i o s y n t h e s i s o f N and M s p e c i f i c determinants T antigens prepared by d e s i a l a t i o each from i s o l a t e d huma were used as s u b s t r a t e s , and f o r M - a c t i v a t i o n o f N, three d i f f e r ent human r e d c e l l NN g l y c o p r o t e i n preparations i s o l a t e d under gentle c o n d i t i o n s (13) were employed. Sera from three to s i x d i f f e r e n t donors o f NN as w e l l as MM blood types served as s i a l y l t r a n s f e r a s e source and CMP-NAN as NAN donor. CMP-NAN was prepared i n Dr. H. Schachter's l a b o r a t o r y as d e s c r i b e d by others (21+ 22) and C-CMP-NAN was from New England Nuclear Corp. In " c o l d experiments, the r e a c t i o n mixtures c o n s i s t e d o f 5 mg (0.5% f i n a l cone.) o f antigen with 2.0 (jmoles CMP-NAN and 500 serum as t r a n s f e r a s e source. In "hot" experiments, a l l r e a c t a n t s were reduced to 12.5% o f those i n the " c o l d " experiment. Incubation and work up were as described p r e v i o u s l y (JL9,_20). C o n t r o l samples were run throughout e x a c t l y as the experiment proper; they c o n s i s t e d o f r e a c t i o n mixtures l a c k i n g i n e i t h e r substrate or CMP-NAN. Substrates incubated with t r a n s f e r a s e i n absence o f CMP-NAN showed no change i n s p e c i f i c i t y . In a l l instances r e covery o f substrate was < 507o a t the end o f the experiment. Controls without added s u b s t r a t e o c c a s i o n a l l y acquired t r a c e s o f activity. 2
14
11
11
In v i v o Measurement o f Delayed-Type " R e c a l l H y p e r s e n s i t i v i ty. T antigen was d i s s o l v e d a t 1% f i n a l c o n c e n t r a t i o n i n buff e r e d s a l i n e c o n t a i n i n g 0.25% phenol; c o n t r o l s were solvent alone and solvent c o n t a i n i n g 1% MN a n t i g e n . A l l reagents were s t e r i l e and free o f pyrogens and Au-antigen. These s o l u t i o n s (0.1 ml each) were i n j e c t e d i . d . i n t o the upper arm. P a t i e n t s with mastectomy were i n j e c t e d i n the c o n t r a l a t e r a l arm. Induration and erythema were measured c a . 24 and 48 h r l a t e r and scored as by H o l l i n s h e a d ejt a l . (23). Reactions, i f any, given by the cont r o l s were s u b t r a c t e d from those due to T antigen. In v i t r o Measurement o f Cell-Mediated Immune Response.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Cell-
17.
SPRINGER E T A L .
Cancer-Associated
Blood Group
MN Precursors
315
u l a r immunity towards T antigen was assessed by the leukocyte migr a t i o n i n h i b i t i o n (LMI) assay i n agarose p l a t e s e s s e n t i a l l y as by Clausen (15,24). As c o n t r o l , leukocytes o f an apparently healthy person were t e s t e d i n p a r a l l e l on the same p l a t e s with those o f the p a t i e n t f o r MI by T antigen (5 o r 10 yg T antigen per 10 u l ) i n each agarose p l a t e . Each t e s t was run i n t r i p l i c a t e , and a r i t h m e t i c averages o f the r e s u l t s were used. C o n t r o l t e s t s with MN i n s t e a d o f T antigen were performed under l i k e c o n d i t i o n s . For healthy i n d i v i d u a l s , the average migration index a t 5 and 10 ug o f T antigen was 1.00; a migration index o f <_ 0.90 i s considered a p o s i t i v e r e a c t i o n (25). Results T - s p e c i f i c haptenic s t r u c t u r e s on erythrocytes were r e a d i l y b i o s y n t h e s i z e d by the procedure o u t l i n e d i n M a t e r i a l s and Methods S p e c i f i c r e a c t i v i t y of absorption t e s t s amounte mally T - a c t i v a t e d normal human r e d c e l l s . Hemagglutination o f Ta c t i v a t e d Tn c e l l s by four doses of a n t i - T was completely i n h i b i t ed by 0.005-0.02 mg/ml T antigen, depending on the serum used, but not by 100-times as much o f MN antigens. We have a l s o transformed T antigens prepared from i s o l a t e d NN or MM antigens i n t o those c a r r y i n g N- and M - s p e c i f i c i t i e s . The s p e c i f i c i t y o f t h i s transformation depended s o l e l y on the MN-type of the t r a n s f e r a s e donor. S i a l y l t r a n s f e r a s e s from MN and MM donors produced N- and M- a c t i v a t i o n , those from NN donors only N specificity. I s o l a t e d NN antigen was transformed to that possessi n g M s p e c i f i c i t y with s e r a from MN and MM but not NN donors. In hemagglutination i n h i b i t i o n assays, a c t i v i t i e s o f the p a r t i a l l y p u r i f i e d products, when compared to the corresponding i s o l a t e d e r y t h r o c y t e antigen, ranged from two to > 75% f o r N - a c t i v a t i o n o f T, from four to > 34% f o r M - a c t i v a t i o n o f T and from two to > 75% f o r M - a c t i v a t i o n o f N antigen depending on the human o r animal antiserum i n h i b i t e d . Incorporation of ^C-NAN i n t o T- and Nantigens during incubation with t r a n s f e r a s e - c o n t a i n i n g s e r a was i n keeping with the s e r o l o g i c a l a c t i v i t i e s of the products obtained. S e r o l o g i c a l r e s u l t s were s i m i l a r f o r M - a c t i v a t i o n o f the a-1 glycopeptide i s o l a t e d from NN e r y t h r o c y t e s . The b i o s y n t h e t i c experiments are summarized i n Figure 1. We were s t r u c k by the s t r u c t u r a l s i m i l a r i t y o f the N- and Ms p e c i f i c groups o f the human red c e l l g l y c o p r o t e i n s and glycoprot e i n I ( e p i g l y c a n i n ) (26) of the TA3 mouse mammary adenocarcinoma; we found that t h i s g l y c o p r o t e i n indeed possessed blood group Nl i k e s p e c i f i c i t y (27). TA3 g l y c o p r o t e i n I o f s t r a i n A mice i s carcinoma-associated and does not occur i n normal s t r a i n A mouse t i s sue (28). Thus, a l i n k was e s t a b l i s h e d f o r the f i r s t time between an animal carcinoma-associated antigen and a human blood group substance (N and precursor T ) . The human a n t i - T a g g l u t i n i n of Thomsen and F r i e d e n r e i c h r e a d i l y k i l l e d TA3-St carcinoma c e l l s
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
316
GLYCOPROTEINS
A N D GLYCOLIPIDS
I N DISEASE
cx-GalNAc-OSer/Thr —
Tn
Figure 1. Biosynthetic pathway of the M- and Nspecific immunodeterminant structures of human blood group M and N glycoproteins
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
17.
SPRINGER E T A L .
Cancer-Associated
Blood
Group
MN Precursors
317
i n the presence o f guinea p i g complement (28). We therefore turned our a t t e n t i o n from murine mammary glands and the TA3 carcinoma to human breast t i s s u e and i t s malignancies. Human breast gland c e l l membrane preparations were used to absorb human a n t i s e r a . The r e s u l t s of some o f these s t u d i e s are l i s t e d i n Table I . M- and N- s p e c i f i c s t r u c t u r e s are present i n both benign and malignant human breast glands, T- and Tn- s p e c i f i c i t i e s occurred i n a l l of the human b r e a s t (primary) and colon carcinomata t e s t e d but not i n corresponding preparations from benign o r normal b r e a s t t i s s u e s . T a c t i v i t y was low i n one i n v a s i v e Stage I I I b r e a s t carcinoma. A l l but one gland membrane p r e p a r a t i o n s p e c i f i c a l l y absorbed anti-M and anti-N a n t i b o d i e s i n agreement with the r e p r e s e n t a t i o n o f these antigens on the p a t i e n t ' s r e d cells. There was one exception where N but n o t M was expressed on the p a t i e n t ' s carcinoma c e l l membranes, while both antigens were present on her red c e l l s . We have t e s t e d metastase gery f o r primary d u c t a t i e n t s . A l l contained T- and the two t e s t e d f o r Tn contained a l s o T n - s p e c i f i c s t r u c t u r e s . This promising observation i n d i c a t e s that T- and Tn- s p e c i f i c i t i e s are not e l i m i n a t e d by e i t h e r somatic mutation or modulation and thus remain a c c e s s i b l e to p o t e n t i a l immunodiagnosis and immunotherapy. The T - s p e c i f i c i t y on adenocarcinomata prompted our determinat i o n of a n t i - T t i t e r scores o f human s e r a , which o r d i n a r i l y range from 22 to 24. They were s e v e r e l y depressed i n a h i g h l y s i g n i f i cant number o f a l a r g e p o p u l a t i o n o f breast carcinoma p a t i e n t s as compared to 270 p a t i e n t s with benign breast d i s o r d e r s and 470 cont r o l s a l l together (Table I I ) . S i m i l a r r e s u l t s were obtained i n p a t i e n t s with lung and g a s t r o i n t e s t i n a l (G.I.) carcinomata (15; to be p u b l i s h e d ) . Of the j u s t described p a t i e n t s with breast disease, 32 with mastectomy f o r carcinoma and 32 with breast biopsy who s u f f e r e d from benign disease were r e b l e d once w i t h i n 14 months of surgery. Of the 32 breast carcinoma p a t i e n t s r e b l e d , 21 (65.6%) showed an i n c r e a s e of > 25 to 90% i n a n t i - T t i t e r , while only one o f 32 (3.1%) benign breast disease p a t i e n t s and none o f 11 p a t i e n t s w i t h major operations f o r non-malignant d i s o r d e r s showed a s i g n i f i c a n t i n c r e a s e i n a n t i - T . This d i f f e r e n c e between the carcinoma pat i e n t s and the c o n t r o l s was s i g n i f i c a n t (JD < 0.001). In some p a t i e n t s , b l e d a t h i r d time, c l i n i c a l recurrence o f the carcinoma was a s s o c i a t e d with renewed lowering o f serum a n t i - T l e v e l s (to be p u b l i s h e d ) . Table I I I summarizes the f i n d i n g s on delayed-type s k i n hypers e n s i t i v i t y (DTH) observed on i . d . i n j e c t i o n of T antigen, and those o f the leukocyte migration i n h i b i t i o n assay (LMI). I t can be c l e a r l y seen that the immune response towards T antigen of breast carcinoma p a t i e n t s extends from humoral to c e l l - m e d i a t e d r e a c t i v i t y and i s demonstrable i n v i v o as w e l l as i n v i t r o . F o r t y four breast carcinoma p a t i e n t s were s k i n t e s t e d (Table I I I ) , 36 o f
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3.62
A l l noncarcinomatous persons studied (B) C
17 /470
8.30
8.15
14^/270
£
39 /470
22^/270
61/189
32.28-
40/189
No. o f persons
No. of persons
12 or <
%
score
— B l i n d s t u d i e s ; standards: three pools of 36-1000 d i f f e r e n t ^ s e r a each; a r i t h m e t i c average t i t e r scores: 22-24 (U.S. p o p u l a t i o n ) . — j>: that d i f f e r e n c e between a n t i - T scores of breast carcinoma p a t i e n t s and c o n t r o l group (A) as w e l l as (B) i s due to chance i s < 0.001 throughout (X t e s t ) . £ Two of the 14 p a t i e n t s have s i n c e developed carcinoma.
5.19
Benign breast disease (A)
21.16^
%
10 or <
I n d i v i d u a l s with
A n t i - T A g g l u t i n i n T i t e r Score— i n P a t i e n t s with Breast Disease and C o n t r o l H o s p i t a l Population (cf.12)
Test group
II.
Breast carcinoma
Table
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 6
1^/95
0/23
tested—
— Only p a t i e n t s > 3 weeks a f t e r surgery or chemotherapy and > 12 months a f t e r r a d i a t i o n therapy were s t u d i e d , because of the known immunosuppression due to these i n t e r v e n t i o n s (46). Individuals of Chinese and Japanese descent not i n c l u d e d . — DTH p o s i t i v e = > 4.5 mm i n d u r a t i o n a f t e r 24-48 hr or erythema at 48 hr > 25 mm (average of 2 diameters measured p e r p e n d i c u l a r l y ) (23). — Leukocyte migration i n h i b i t i o n on agarose p l a t e ; 10 (jbg T antigen/1.5 x 10 leukocytes; not t o x i c up to > 45 jig. — Negative was the only i n f i l t r a t i n g l o b u l a r carcinoma. — The two negative persons had i n s i t u l o b u l a r carcinoma. — 2/38 p o s i t i v e . I n t r a d u c t a l papilloma was the most ominous s t r u c t u r e i n both. £ Vietnam war veteran with typhoid, c h o l e r a and tetanus t o x o i d v a c c i n a t i o n s .
"Healthy"
11/84
8/27
14-/16
I 2-/38
17/31
13/13
II
Benign
10/21
8^/9
7/14
Persons p o s i t i v e / P e r s o n s
III
tested
6/6
Persons positive—/Persons
LMI
IV
Disease stage
DTH
Table I I I . Delayed-Type R e c a l l Skin R e a c t i v i t y upon i . d . I n j e c t i o n of T and i n v i t r o Cell-Mediated Immune Response to T Antigen i n P a t i e n t s with Breast Carcinoma, Benign Breast Disease and i n Apparently Healthy Controls-^
GLYCOPROTEINS AND
320
GLYCOLIPIDS IN
DISEASE
PROCESSES
these s u f f e r e d from carcinoma of d u c t a l o r i g i n , 2 were noni n v a s i v e . The remaining 8 p a t i e n t s had l o b u l a r carcinoma. A l l p a t i e n t s with d u c t a l carcinoma gave a p o s i t i v e DTH r e a c t i o n regardless of Stage and invasiveness (15,29). F i v e with l o b u l a r carcinomata a l s o gave a p o s i t i v e r e a c t i o n but one of the p a t i e n t s with i n v a s i v e and two with non-invasive l o b u l a r tumors d i d not r e a c t (29). In c o n t r a s t , none of the 23 healthy i n d i v i d u a l s t e s t ed had any delayed h y p e r s e n s i t i v i t y r e a c t i o n toward T antigen, and 36 of the 38 p a t i e n t s diagnosed h i s t o l o g i c a l l y to have f i b r o adenoma-fibrocystic disease had a negative r e a c t i o n . The r i g h t hand s i d e of Table I I I shows that i n LMI 34 (51.5%) of the 66 p a t i e n t s with breast carcinoma Stages I I , I I I and IV had a p o s i t i v e r e a c t i o n , while of 27 p a t i e n t s with Stage I disease, eight (29.6%) were p o s i t i v e . Seven of the e i g h t p a t i e n t s with l o b u l a r carcinoma r e f e r r e d to above were t e s t e d i n LMI. The leukocytes of only two of these p a t i e n t s , both with i n f i l t r a t i n g tumor showed migration i n h i b i t i o n Of 84 p a t i e n t s wit p o s i t i v e i n LMI with T antigen. No e f f e c t of T antigen was found on the p e r i p h e r a l leukocytes of a l a r g e number of presumably healthy people. LMI was p o s i t i v e i n only one of 95 healthy i n d i v i d u a l s , a Vietnam war veteran v a c c i n a t e d repeatedly with S.typhi, V. choJLerae and Tetanus t o x o i d a l l of which possess T - s p e c i f i c i t y (see Table IV). T antigen up to 50 yg d i d not a f f e c t v i a b i l i t y (trypan blue) or migration of the white c e l l s of healthy persons, MN antigen at the same high concentration had no e f f e c t on migrat i o n of leukocytes of any of the p a t i e n t s and c o n t r o l s t e s t e d . Discussion In t h i s communication we report that we have reversed the degradative steps l e a d i n g from blood group M - s p e c i f i c s t r u c t u r e s to i t s precursors and synthesized T - s p e c i f i c groups (19,20) on Tn erythrocytes as w e l l as N- and M- s p e c i f i c groups on i s o l a t e d T antigens. The peptide core of T antigen i n M- d i f f e r s i n 2 amino acids from that i n N- i n d i v i d u a l s and M a n t i g e n i c determinants possess more carbohydrate (30,31,32,33,34). Since g l y c o s y l t r a n s ferases g e n e r a l l y recognize only l i m i t e d areas (one to two monosaccharides) u n d e r l y i n g the acceptor molecule, s i a l y l t r a n s f e r a s e s from MM and MN donors produced N and M a c t i v a t i o n , whereas those from NN donors produced only N regardless of the MN type o r i g i n of T antigen. I s o l a t e d N antigen could be transformed to M only with s e r a from MM and MN donors. The g e n e t i c a l sequence of these immunological s p e c i f i c i t i e s i s t h e r e f o r e : Tn-*- T* N->- M (M could conceivably a r i s e d i r e c t l y from T). These s t r u c t u r e s have a l s o been found on l i p i d i c c a r r i e r s , i n c l u d i n g those from a carcinoma (35). The discovery that human adenocarcinoma but not healthy human t i s s u e s and benign s t r u c t u r e s possess T- and Tn- s p e c i f i c i t i e s i n r e a c t i v e , unmasked form e s t a b l i s h e s that these precursors are
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
P a s t e u r e l l a pseudotuberculosis— Pneumococcus XIV p o l y s a c c h a r i d e — BCG, T i c e s t r a i n (Univ. of Illinois).^ T u b e r c u l i n , PPD ( T u b e r s o l ) - * Influenza v i r u s : A / V i c t o r i a + B/Hong Kong (Parke-Davis )— Pseudomonas—*^
VACCINES:
Aerobacter aerogenes K l e b s i e l l a pneumoniae 3/3 Proteus s t r a i n s
LPS:
BACTERIA:
Absent
(LPS) and
— Non-dialyzable p a r t . SL A c t i v e with some but not a l l human a n t i - T . — Donated by Dr. M. H e i d e l berger. ^ Donated by Dr. R. G. Crispen. — A p o s s i b l e c o n t r i b u t i o n to a c t i v i t i e s by growth^medium has not been excluded. -= From Connaught L a b o r a t o r i e s . Donated by Dr. J . U. Gutterman. — Donated by Wellcome L a b o r a t o r i e s , - i From L e d e r l e , Orimune t r i v a l e n t .
Salmonella t y p h i (Wyeth, E. L i l l y ) Corynebacterium parvum (Coparvax)—>|l C l o s t r i d i u m t e t a n i t o x o i d (Wyeth)—*— V i b r i o cholerae (Wyeth)—>— Poliomyelitis v i r u s ^ ' i
4/8 E. c o l i 6/9 Salmonella s t r a i n s S. typhimurium S. abortus equi S. t y p h i S. minnesota 2/3 S h i g e l l a s t r a i n s S e r r a t i a marcescens Bacterium t u l a r e n s i s 2/3 Chromobacterium violaceum
LPS:
VACCINES:
9/9 E s c h e r i c h i a c o l i 3/3 E. f r e u n d i i Salmonella poona 1/3 Pseudomonas s t r a i n s
T Specificity
T - S p e c i f i c Substances i n Microbes, t h e i r Lipopolysaccharides M i c r o b i a l Vaccines^
Present
IV.
BACTERIA:
Table
322
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE
PROCESSES
c h a r a c t e r i s t i c o f adenocarcinomata o f human b r e a s t , G.I. t r a c t and probably lung. The occurrence o f f u l l y r e a c t i v e T- and Tna n t i g e n i c s p e c i f i c i t i e s i n these cancers i s probably due to i n complete b i o s y n t h e s i s o f normal c e l l - s u r f a c e components, as i n d i cated by the presence of a l l these s p e c i f i c i t i e s except M, i n the breast carcinoma o f one p a t i e n t whose r e d c e l l s c a r r i e d M and N antigens as w e l l as by the extensive absorption (70%) of anti-N by b r e a s t carcinoma membranes from one p a t i e n t o f e r y t h r o c y t e group MM. T- and Tn- s p e c i f i c a n t i g e n i c determinants have r e c e n t l y been demonstrated d i r e c t l y with immunofluorescent l e c t i n s i n a l l carcinomatous b r e a s t glands t e s t e d but not i n n o n - c a r c i nomatous glandular areas o f the same s e c t i o n s (36; Dr. Cr. McNeil, p e r s o n a l communication). O r d i n a r i l y humans have no anti-N o r anti-M a n t i b o d i e s s i n c e a l l possess b l o o d group N, M, o r NM, and i n d i v i d u a l s homozygous f o r M uniformly have some N as w e l l (1,37). Healthy mammals do not form a n t i b o d i e s agains with t h e i r immune machinery are not ubiquitous as are A and B s p e c i f i c i t i e s (3,7,38). On the other hand, T- and Tn- s p e c i f i c s t r u c t u r e s are always masked i n healthy humans, but are widespread among i n t e s t i n a l and a i r b o r n e b a c t e r i a (see below); consequently a n t i b o d i e s against these s t r u c t u r e s are found i n a l l humans (16,39). Levels o f these a n t i bodies are q u i t e constant under o r d i n a r y circumstances (29,39). Anti-T was s e v e r e l y depressed i n p a t i e n t s with carcinoma o f the b r e a s t , lung and G.I. t r a c t much more f r e q u e n t l y when compared to populations o f comparable age and socio-economic s t a t u s who had e i t h e r benign disease o r were apparently h e a l t h y . Of a l l i n d i v i duals with s e v e r e l y depressed a n t i - T , > 80% s u f f e r e d from c a r c i noma. This depression was not due to IgG, IgM, o r IgA decrease. S i m i l a r observations on a n t i - T of p a t i e n t s with b r e a s t cancer have r e c e n t l y been reported by others (40). Anti-T antibody may be depressed because of i t s i n t e r a c t i o n with T - s p e c i f i c substances e i t h e r attached to cancer c e l l - s u r f a c e and/or r e l e a s e d from t h e c e l l - s u r f a c e i n t o the c i r c u l a t i o n . This hypothesis i s supported by the o b s e r v a t i o n of r e l e a s e o f T - s p e c i f i c antigens from human mammary carcinoma-derived t i s s u e c u l t u r e c e l l s i n t o the c u l t u r e f l u i d (41). We d i d not f i n d depressed a n t i - T i n p a t i e n t s with sarcoma, melanoma o r with b r a i n tumors t e s t e d so f a r , n o r Ts p e c i f i c s t r u c t u r e s on these tumors. As mentioned above, there i s evidence that humoral a n t i - T as w e l l as a n t i - T n r e s u l t l a r g e l y from immunization by the host's own i n t e s t i n a l f l o r a (7,14,38,42,43,44,45). We s t u d i e d numerous, predominantly gram-negative b a c t e r i a and/or t h e i r l i p o p o l y s a c c h a r i d e s , and some v a c c i n e s . Table IV shows that many microbes indeed possess T - s p e c i f i c s t r u c t u r e s as determined by hemagglut i n a t i o n i n h i b i t i o n and by absorption assays. Three of the 12 a c t i v e E s c h e r i c h i a c o l i and one o f the two a c t i v e Chromobacterium violaceum s t r a i n s , Salmonella milwaukee and JS. abortus equi i n h i b i t e d human a n t i - T only when t e s t e d a f t e r b o i l i n g . It is
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
17.
SPRINGER E T A L .
Cancer-Associated
Blood
Group
MN Precursors
323
noteworthy that Corynebacterium parvum v a c c i n e , i n wide use to s t i m u l a t e " u n s p e c i f i c " r e s i s t a n c e against cancer, possessed s i g n i f i c a n t T s p e c i f i c i t y , while the one commercial BCG t e s t e d and the Pseudomonas v a c c i n e (also used as " u n s p e c i f i c " s t i m u l a n t against cancer) and PPD t u b e r c u l i n had no T a c t i v i t y . Tn a c t i v i t y o f b a c t e r i a l p r e p a r a t i o n s was a l s o found i n 25 of 30 Enterobacteariaceae ( i n c l u d i n g S.typhi). Bacterium t u l a r e n s i s , and i n JC. parvum, typhoid, tetanus and p o l i o m y e l i t i s v a c c i n e s . Tn a c t i v i t y was not demonstrable i n BCG, PPD t u b e r c u l i n , three Pseudomonas s t r a i n s and some Enterobacteriaceae. The discovery o f T- and Tn- s p e c i f i c immunologically r e a c t i v e determinants i n the c e l l membranes of breast and colon adenocarcinomata but not i n healthy t i s s u e s e s t a b l i s h e s f o r the f i r s t time a p h y s i c a l l y and chemically w e l l - d e f i n e d immunospecific s t r u c t u r e c h a r a c t e r i s t i c a l l y a s s o c i a t e d with human carcinoma. These precursor s p e c i f i c i t i e d i r e a c t i v for be due to incomplete b i o s y n t h e s i normal c e l l - s u r f a c e components gen a l s o occurs i n other malignancies and o c c a s i o n a l l y i n some permanently benign human tumors. The T antigen q u i t e l i k e l y can be considered a paradigm f o r other p r e c u r s o r antigens, e.g. Tn, and a l s o f o r the Forssman antigens and r e l a t e d blood group antigens, with s i m i l a r f u n c t i o n s i n malignant processes. T antigen i s a v a i l a b l e i n u n l i m i t e d q u a n t i t y , i s not contaminated with HL-A- and Au- antigens, and i s o b t a i n a b l e pyrogen-free. I t can be r e a d i l y prepared by removal of NAN from i s o l a t e d human r e d c e l l blood group NM antigens. T antigen and the a n t i - T a n t i b o d i e s which are present i n a l l humans (conceivably coupled with r a d i o a c t i v e i s o t o p e s ) may be u s e f u l i n d i a g n o s i s , prognosis, and p o s s i b l y therapy and immunoprophylaxis of some adenocarcinomata. Acknowledgments G. F. S. i s J u l i a S. Michels I n v e s t i g a t o r i n S u r g i c a l Oncology. This work was supported by the N a t i o n a l I n s t i t u t e s o f Health grants CA 19083 and CA 22540. We thank surgeons and patho l o g i s t s of Evanston and St. F r a n c i s H o s p i t a l s , Evanston, IL and the B e r k s h i r e Medical Center, P i t t s f i e l d , MA f o r f r e s h t i s s u e s . We are g r a t e f u l t o Mrs. H. Tegtmeyer, Mrs. T. Mo, Mr. M. Coleman, Mrs. V. Scanlon, Mrs. E. M i t c h e l l and Mrs. J . K e l l o g g f o r e x c e l lent assistance.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
324 Literature Cited
1. Landsteiner, K.; Levine, P. J. Exp. Med. (1928), 47, 757-775. 2. Springer, G. F.; Ansell, N. J. Proc. Natl Acad. Sci. USA (1958), 44, 182-189. 3. Springer, G. F. Naturwissenschaften (1970), 57, 162-171. 4. Kosjakov, P. N.; Tribulev, G. P. J. Immunol. (1939), 37, 283-295. 5. Boorman, K. E.; Dodd, B. E. J. Pathol. Bacteriol. (1943), 55, 329-339. 6. Stalder, K.; Springer, G. F. Fed. Proc. (1960), 19, 70. 7. Springer, G. F.; Horton, R. E. J. Clin. Invest. (1969), 48, 1280-1291. 8. Davies, D. A. L. Immunology (1966), 11, 115-125. 9. Mann, D. L.; Rogentine, G. N., Jr.; Fahey, J. L.; Nathenson, S. G. Nature (1968), 217, 1180-1181. 10. Tom, B. H.; Rutzky C. I.; Kahan, B. D 11. Springer, G. F.; Desai, P. R.Carbohydr.Res. (1975), 40, 183-192. 12. Springer, G. F.; Desai, P. R.; Scanlon, E. F. Cancer (1976), 37, 169-176. 13. Springer, G. F.; Nagai, Y.; Tegtmeyer, H. Biochemistry (1966), 5, 3254-3272. 14. Springer, G. F.; Desai, P. R.; Banatwala, I. J. Natl. Cancer Inst. (1975), 54, 335-339. 15. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. J. Surg. Oncol. (1978), in press. 16. Moreau, R.; Dausset, J.; Bernard, J.; Moullec, J . Bull. Soc. Méd. Hôp. (1957), 73, 569-587. 17. Sturgeon, P.; McQuiston, D. T.; Taswell, H. F.; Allan, C. J. Vox Sang. (1973), 25, 481-497. 18. Stratton, F.; Renton, P. H. "Practical Blood Grouping", p. 34, Blackwell, Oxford, 1958. 19. Springer, G. F.; Desai, P. R.; Schachter, H.; Narasimhan, S. Naturwissenschaften (1976), 63, 488-489. 20. Desai, P. R.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. 21. Comb, D. G.; Roseman, S. J. Biol. Chem. (1960), 235, 25292537. 22. Kean, E. L. J. Biol. Chem. (1970), 245, 2301-2308. 23. Hollinshead, A. C.; Stewart, T. H. M.; Herberman, R. B. J. Natl. Cancer Inst. (1974), 52, 327-338. 24. Clausen, J. E. Acta Allergol. (Kbh.) (1971), 26, 56-80. 25. Springer, G. F.; Desai, P. R. Transplant. Proc. (1977), 9, 1105-1111. 26. Codington, J. F.; Sanford, B. H.; Jeanloz, R. W. Biochemistry (1972), 11, 2559-2564. 27. Springer, G. F.; Codington, J. F.; Jeanloz, R. W. J . Natl. Cancer Inst. (1972), 49, 1469-1470.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
17.
28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.
SPRINGER ET AL.
Cancer-Associated Blood Group MN Precursors
Springer, G. F.; Desai, P. R. Ann. Clin. Lab. Sci. (1974), 4, 294-298. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. Naturwissenschaften (1978), in press. Springer, G. F.; Yang, H. J.; Huang, I.-Y. Naturwissenschaften (1977), 64, 393. Springer, G. F.; Yang, H. J. Immunochemistry (1977), 14, 497-502. Yang, H. J.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Waśniowska, K.; Drzeniek, Z.; Lisowska, E. Biochem. Biophys. Res. Commun. (1977), 76, 385-390. Friedensen, B.; Yang, H. J.; Springer, G. F., in preparation. Cantrell, J. L.; Springer, G. F.; Desai, P. R., in preparation. Fischer, K.; Poschmann Wochenschr. (1977), , Springer, G. F.; Tegtmeyer, H.; Huprikar, S. V. Vox Sang. (1972), 22, 325-343. Hoskins, L. C.; Boulding, E. T. J. Clin. Invest. (1976), 57, 74-82. Friedenreich, V. "The Thomsen Hemagglutination Phenomenon" Levin and Munksgaard, Copenhagen, 1930. Luner, S. J.; Wile, A. G.; Sparks, F. C. Proc. 68th Annu. Meet. Am. Assoc. Cancer Res. (1977), Abstract 375. Anglin, J. H.; Lerner, M. P.; Nordquist, R. E. Nature (1977), 269, 254-255. Springer, G. F.; Desai, P. R. Annu. Meet. Am. Soc. Microbiol. (1977), Abstracts, E42, p. 88. Springer, G. F.; Desai, P. R.; Murthy, S. M.; Scanlon, E. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Boccardi, V.; Attinà, D.; Girelli, G. Vox Sang. (1974), 27, 268-272. Springer, G. F.; Horton, R. E.; Forbes, M. J . Exp. Med. (1959), 110, 221-244. Daban, A.; Schneider, M.; Calle, R. Bull, du Cancer (1975), 62, 21-28.
RECEIVED
March 30, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18 Alpha-fetoprotein as an Indicator of Early Events in Chemical Carcinogenesis STEWART SELL Department of Pathology, M-012, University of California, San Diego, La Jolla, CA 92093 Alphafetoprotein (AFP) concentration in fetal sera, y sera, has attracted increasing attention among oncologists and developmental biologists. AFP was discovered by the great Soviet scientist, Garri Israelevich Abelev, in 1963, when he noted the presence of a specific serum protein in neonatal mice and in mice bearing transplantable hepatomas (1). Several other laboratories soon were able to confirm this observation in other species, including man (2, 3, 4, 5, 6). However, further advances in understanding the biologic and diagnostic significance of AFP were hindered by lack of a sensitive accurate technique for measuring AFP. Accurate radioimmunoassays for AFP were established in the early 1970's (7, 8, 9, 10). This was followed by development of specific immunoassays for AFP synthesis by labeled amino acid incorporation and immunofluorescent labeling techniques. Using these assays, AFP production and cellular localization both in vivo and in vitro under a variety of experimental systems has been examined (11, 12). These experimental studies led to understandings which have been expanded clinically in regard to the use of AFP in the diagnosis of congenital anomalies, the differentiation of benign from malignant liver disease, and the determination of the effects of therapy on AFP-producing tumors, as well as to insights into the significance of AFP in normal development and of the sequence of events culminating in development of hepato-cellular carcinomas after exposure of rats to chemical hepato-carcinogens. Properties of AFP AFP is a single protein chain with physical chemical properties similar to albumin (Table I). In native configuration AFP is immunochemically distinct from albumin, but cross inhibition of antibody binding between AFP and albumin is observed after unfolding of the molecules by disulfide reduction and carboxyaminylmethylation (13). In addition, preliminary amino acid 0-8412-0452-7/78/47-080-326$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18.
SELL
Alpha-fetoprotein
as an Indicator
in
Carcinogenesis
327
sequencing i n d i c a t e s a 45% homology (14). AFP of murine species has a high estrogen b i n d i n g a f f i n i t y (10~^M) , whereas the AFP of other species does not (15, 16). TABLE I PROPERTIES OF RAT AFP AND ALBUMIN AFP Molecular Weight 70,000 I s o e l e c t r i c Point 4.9 CHO Content 4% Serum Concentration (ug/ml) Adult .03 Fetal 8,000 Estrogen Binding -9 Ka 10 pm/mg P r o t e i n 50 Sequence Homology Cross R e a c t i v i t y 0 Native + Unfolded*
ALBUMIN 60,000 5.6 None 60,000 100 -5 10 0.2
* D i s u l f i d e r e d u c t i o n plus carboxamidomethylation. A s e r i e s of systematic experimental s t u d i e s on the nature and s i g n i f i c a n c e of a l p h a f e t o p r o t e i n (AFP) i n normal development, during r e s t i t u t i v e h e p a t o c e l l u l a r p r o l i f e r a t i o n , during tumor growth and therapy, and a f t e r exposure to hepatocarcinogens has been c a r r i e d out i n our l a b o r a t o r y . A summary of the serum conc e n t r a t i o n s of AFP under v a r i o u s c o n d i t i o n s i s given i n Chart 1. Normal Development A l p h a f e t o p r o t e i n i s synthesized by the f e t a l l i v e r and y o l k sac (18, 19), crosses the p l a c e n t a i n t o the maternal c i r c u l a t i o n and i s r a p i d l y c a t a b o l i z e d by the mother (20). There i s a r e c i p r o c a l r e l a t i o n s h i p between the serum c o n c e n t r a t i o n of AFP and albumin. E a r l y i n development AFP concentrations are high and albumin concentrations low (4, 5, 21). When the serum concent r a t i o n s of AFP f a l l during neonatal development, the concentrat i o n of albumin r i s e s . The d e l i n e a t i o n of the feto-maternal synthesis and d i s t r i b u t i o n i n the r a t has e s t a b l i s h e d a base f o r understanding e l e v a t i o n s of AFP concentrations i n aminotic f l u i d or maternal serum a s s o c i a t e d with c o n g e n i t a l anomalies i n the human (Table II) (22, 25).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
328
GLYCOPROTEINS A N D
GLYCOLIPIDS IN
DISEASE
PROCESSES
TABLE I I INCREASED AMNIOTIC FLUID AFP IN ABNORMAL PREGNANCY ABNORMALITY MECHANISM F e t a l Death Release of F e t a l AFP i n t o AMF Neural Tube Defects Transfer of AFP from F e t a l CSF Esophageal A t r e s i a Reduced Turnover, Lack of Swallowing Congenital Nephrosis Loss of F e t a l AFP from Renal Defect M u l t i p l e Pregnancy Increased Production Rh I n c o m p a t i b i l i t y ?Increased Production A l p h a f e t o p r o t e i n i s synthesized by the l i v e r of the newborn r a t u n t i l four weeks of age where there i s an abrupt c e s s a t i o n of s y n t h e s i s (11). This s t r i k i n g c e s s a t i o n of AFP s y n t h e s i s i s a s s o c i a t e d with the termination of neonatal l i v e r c e l l p r o l i f e r a t i o n and change i n the ploid. There i s a c l o s e r e l a t i o n s h i p between the f e t a l t i s s u e s that synthesize AFP and tumors i n a d u l t s that produce AFP. Tissue s i t e s of s y n t h e s i s of AFP, measured by i n c o r p o r a t i o n of r a d i o l a b e l e d aminoacids, were determined using d i f f e r e n t f e t a l t i s s u e s (19). The f e t a l l i v e r and the y o l k sac elements are the f e t a l t i s s u e s which produce the highest amounts of AFP; i n a d u l t s l i v e r and teratocarcinoma (yolk sac) f e t a l tumors produce AFP with the highest frequency (26, 27, 28). The f e t a l GI t r a c t (stomach, small i n t e s t i n e and pancreas) a l s o produces AFP although at much lower amounts than l i v e r or y o l k sac; human tumors of the GI t r a c t may produce AFP but the frequence of AFP production by tumors of these t i s s u e s i s much l e s s than the production by hepatomas or teratocarcinomas (29). Small amounts of AFP are produced by f e t a l lung and a small number of bronchiogenic carcinomas produce (21, 29). F i n a l l y , although not produced d e t e c t a b l y by f e t a l mesenchymal t i s s u e s , i t i s p o s s i b l e that mesenchymal tumors may a l s o produce AFP (30). These r e s u l t s support the concept of d i f f e r e n t l e v e l s of expression of developmental products by tumors (31, 32). Such exposures a l s o a p p l i e s to e c t o p i c hormone product i o n and carcinoembryonic antigen by tumors (Table I I I ) .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18.
SELL
Alpha-fetoprotein
as an Indicator
in Carcinogenesis
329
TABLE I I I LEVELS OF EXPRESSION OF SOME ONCQDEVELQPMENTAL GENE PRODUCTS BY TUMORS AFP HORMONE CEA FIRST LEVEL (e.g. ACTH) F e t a l or Adult Pituitary Colonic Hepatoma, Tissue Which Adenoma Teratocarcinoma Carcinoma Normally With Yolk Sac Produce I t Elements SECOND LEVEL C l o s e l y Related Embryologically (Same C e l l Line) THIRD LEVEL More D i s t a n t l y Related (Same C e l l Line) FOURTH LEVEL Different Cell Line
Pancreas, GI CA
Pancreas, Gastric Liver CA
Medullary CA of Thyroid
Breast CA
of Lung
Sarcoma, Lymphoma
Pulmonary CA
Pulmonar
?Sarcoma Lymphoma
The c o n t r o l of AFP s y n t h e s i s by f e t a l hepatocytes has been determined by q u a n t i t a t i v e assay of AFP and albumin synthesis i n a f e t a l hepatocytes c u l t u r e system developed by Hyam L e f f e r t of the Salk I n s t i t u t e (18). The data i n d i c a t e that albumin i s synthesized throughout the c e l l c y c l e , whereas AFP i s l a r g e l y synthesized p r i o r to S and r e l e a s e d p r i o r to M (19). Factors such as hormones, which may a f f e c t AFP s y n t h e s i s , appear to do so through a l t e r a t i o n s i n the growth s t a t e of f e t a l hepatocytes. When f e t a l hepatocytes are not i n an a c t i v e growth s t a t e , product i o n o f AFP i s not d e t e c t a b l e . Hepatotoxic
L i v e r Injury
The l i v e r of adult r a t s subjected to 70% p a r t i a l hepatectomy (16) or f o l l o w i n g exposure to chemical hepatotoxic agents such as C C l ^ or galactosamine (35, 36) w i l l synthesize AFP during the r e s t i t u t i v e phase of l i v e r regeneration. The serum concentrat i o n s of AFP become elevated s h o r t l y a f t e r p r o l i f e r a t i o n i s observed and d e c l i n e to normal s h o r t l y a f t e r p r o l i f e r a t i o n stops (16). This e f f e c t i s r e p r o d u c i b l e i n v i t r o as adult hepatocytes c u l t u r e s w i l l re-express f e t a l phenotype during the a c t i v e growth phase but r e - e s t a b l i s h adult phenotype when becoming conf l u e n t (37). The absence of an e l e v a t i o n i n serum AFP f o l l o w i n g l i v e r i n j u r y i s a poor prognostic s i g n , as a l l r a t s not demons t r a t i n g an AFP e l e v a t i o n d i e d . In humans, AFP concentrations may be used i n a s i m i l a r manner to determine the extent of l i v e r
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
330
GLYCOPROTEINS
A N D GLYCOLIPIDS IN
DISEASE
PROCESSES
damage f o l l o w i n g l i v e r n e c r o s i s or h e p a t i t i s and to determine the occurrence of r e s t i t u t i v e p r o l i f e r a t i o n (38,39). By immunof l u o r e s c e n c e , AFP may be i d e n t i f i e d i n hepatocytes undergoing p r o l i f e r a t i o n (40). A s p e c i f i c f u n c t i o n f o r AFP has not been c o n v i n c i n g l y demonstrated. Some of the p o s t u l a t e d f u n c t i o n s of AFP are l i s t e d i n Table IV. Several of these deserve f u r t h e r comment. Those f u n c t i o n s r e l a t e d to the high b i n d i n g c a p a c i t y of AFP f o r estrogen seem not to be g e n e r a l l y a p p l i c a b l e s i n c e only the AFP of r a t and mouse have t h i s property. The p o s s i b i l i t y of AFP p r o t e c t i n g the fetus from maternal immune r e j e c t i o n i s an a t t r a c t i v e one, but c o n f l i c t i n g experimental data have been obtained (41, 42, 43, 44). I t i s c l e a r that AFP does c a r r y out some of the osmotic and c a r r i e r f u n c t i o n s of albumin I n the f e t u s s i n c e AFP i s the major serum p r o t e i n during a c o n s i d e r a b l e time i n the f e t u s (Table I ) .
SOME PROPOSED FUNCTION OBSERVATION S t r u c t u r e Homology with Albumin Inverse Serum Concentration to ALB Immunosuppressive In V i t r o Binds Estrogen Produced During Hepatocyte Proliferation Produced During L i v e r Lobule Formation
POSSIBLE FUNCTION F e t a l Albumin - C a r r i e r and
Osmotic
Blocks Maternal R e j e c t i o n of Fetus P r o t e c t s Fetus From Maternal Estrogen Growth C o n t r o l Developmental Signal
T i s s u e Organizing
The c o r r e l a t i o n of AFP production with normal neonatal l i v e r development and with r e s t i t u t i v e l i v e r c e l l p r o l i f e r a t i o n has suggested that the f u n c t i o n of AFP may be to serve as a t i s s u e o r g a n i z a t i o n s i g n a l f o r proper alignment of d i f f e r e n t c e l l types i n the l i v e r (31). In both neonatal development and r e s o t r a t i o n of the a d u l t l i v e r a f t e r p a r t i a l hepatectomy, not only must the number of hepatocytes be r a p i d l y i n c r e a s e d , but a l s o these c e l l s must assume the proper alignment with s i n u s o i d a l c e l l s , b i l e c a n n i c u l i and v e s s e l s . The r o l e of AFP as an o r g a n i z a t i o n s i g n a l i s supported by analogy to other "oncodevelopmental" markers such as the T locus markers of the mouse and the T c e l l antigens of lymphocytes, one being r e s p o n s i b l e f o r c e l l - c e l l r e c o g n i t i o n during normal development of the e a r l y embryo, the other b e l i e v e d to be r e q u i r e d f o r proper c e l l - c e l l i n t e r a c t i o n s i n immune responses. I t i s a t t r a c t i v e to assume other "oncodevelopmental products such as AFP might f u n c t i o n i n a s i m i l a r manner (31). 11
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18.
SELL
Alpha-fetoprotein
as an Indicator
in
Carcinogenesis
331
Tumor Growth The e l e v a t i o n s of serum concentrations of AFP f o l l o w i n g t r a n s p l a n t a t i o n of a v a r i e t y of hepatomas i n syngeneic r a t s has been analysed (45, 46, 47). Not a l l t r a n s p l a n t a b l e hepatomas produce elevated serum c o n c e n t r a t i o n s of AFP. The ]evel of product i o n of AFP i s r e l a t e d to m u l t i p l e f a c t o r s i n c l u d i n g degree of aneuploidy, growth r a t e and degree of h i s t o l o g i c d i f f e r e n t i a t i o n . Upon t r a n s p l a n t a t i o n of tumors that produce AFP an exponential continuous e l e v a t i o n of serum c o n c e n t r a t i o n s of AFP occurs (48) (see Chart 1). This i s i n c o n t r a s t to the t r a n s i e n t , u s u a l l y much l o w e r , e l e v a t i o n s found f o l l o w i n g l i v e r c e l l n e c r o s i s and proliferation. Thus, t h i s model suggests that s e r i a l determinat i o n s of AFP i n p a t i e n t s with undiagnosed hepatomas may be u s e f u l i n d i f f e r e n t i a t i n g c i r r h o s i s or h e p a t i t i s (with l i v e r c e l l p r o l i f e r a t i o n ) from hepatom (49 31) The serum c o n c e n t r a t i o may be used to f o l l o w the e f f e c t s of therapy (48, 50, 51). In a model system, s u r g i c a l removal of an AFP producing tumor r e s u l t s i n an immediate d e c l i n e of serum AFP concentrations. A prolonged r e t u r n to normal i s found i n long-term s u r v i v o r s ; r e - e l e v a t i o n a s s o c i a t e d wtih development of pulmonary or lymph node metastases. R a d i a t i o n to the lung s h o r t l y f o l l o w i n g tumor removal r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n the number of long-term s u r v i v o r s (51). Again, t h i s animal model p o i n t s out the p o t e n t i a l u s e f u l n e s s of AFP i n c l i n i c a l d i a g n o s i s and prognosis (31). Tumor Development For over twenty years the i n d u c t i o n of r a t h e p a t o c e l l u l a r carcinomas has been used as a model f o r chemical c a r c i n o g e n e s i s (52, 53, 54). In t h i s system a sequence of morphologic a l t e r a t i o n s i s observed during exposure of r a t s culminating i n the appearance of h e p a t o c e l l u l a r carcinomas a f t e r 16-20 weeks. The sequence of morphologic changes proceeds from the appearance of small f o c i of a few hepatocytes with a l t e r e d s t a i n i n g c h a r a c t e r i s t i c s through l a r g e r microscopic f o c i of c e l l s which impinge upon the surrounding parenchyrra and g r o s s l y v i s i b l e n e o p l a s t i c nodules to frank h e p a t o c e l l u l a r carcinomas (55, 56, 57, 58). The hypot h e s i s that t h i s sequence i s r e l a t e d and that the nodules r e p r e sent pre-malignant but r e v e r s i b l e l e s i o n s i s supported by the presence of a l t e r e d h e p a t i c enzyme expression i n the n e o p l a s t i c nodules that i s a l s o found i n h e p a t o c e l l u l a r carcinomas (59, 60). Data obtained from the a p p l i c a t i o n of AFP measurements and c e l l u l a r l o c a l i z a t i o n as an e a r l y i n d i c a t o r of events i n chemical carcinogenesis may r e s u l t i n s i g n i f i c a n t changes i n the current concept of the sequence of events l e a d i n g to development of hepatoc e l l u l a r carcinomas. This use of AFP has been made i n c o l l a b o r a t i o n with F.F. Becker, E. Smuckler, B. Lombardi and H. Shinozuka. Exposure of r a t s to hepatocarcinogens such as e t h i o n i n e (61),
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
332
GLYCOPROTEINS A N D
GLYCOLIPIDS IN
DISEASE
PROCESSES
acetylaminofluorene (62) and 3-methyl-4-dimethylaminoazobenzene (63) r e s u l t s i n e l e v a t i o n of the serum concentrations of AFP w i t h i n a few hours or days even though (?premalignant) morphologic changes may not be observed u n t i l a f t e r 6-8 weeks of exposure and frank hepatomas do not develop u n t i l 16-20 weeks a f t e r the i n i t i a l exposure (64). D i s c o n t i n u a t i o n of exposure to the carcinogen a f t e r AFP e l e v a t i o n s have occurred does not cause a c e s s a t i o n of AFP production. For instance, serum concentrations of AFP remain elevated f o r 12-18 weeks a f t e r one 3-week pulse of a non-carcinogenic dose of 2-AAF,and four such pulses are r e q u i r e d to produce tumors (62). Therefore, the k i n e t i c s of AFP f o l l o w i n g administ r a t i o n of non-hepatotoxic doses of hepatocarcinogens i s q u i t e d i f f e r e n t than the k i n e t i c s a f t e r a d m i n i s t r a t i o n of agents which induce l i v e r c e l l n e c r o s i s . On the other hand, continued feeding of 2-AAF does not maintain the serum AFP e l e v a t i o n as the serum AFP c o n c e n t r a t i o n of r a t s fed a continuous d i e t of 2-AAF w i l l f a l l back to j u s t about norma observed when exposure i i s that the serum c o n c e n t r a t i o n of AFP i s a c t u a l l y f a l l i n g at the time of maximal development of n e o p l a s t i c nodules (65) (Chart 2). The serum AFP r i s e s sharply with the appearance of an AFP producing h e p a t o c e l l u l a r carcinoma at 16-20 weeks. About two-thirds of the h e p a t o c e l l u l a r carcinomas that are produced by 2-AAF exposure a c t u a l l y produce AFP (65). The i d e n t i f i c a t i o n of the c e l l s producing AFP a f t e r exposure to chemical hepatocarcinogens has been the subject of much unrewarding study, but immunofluorescent s t u d i e s r e c e n t l y c a r r i e d out have c l a r i f i e d the s i t u a t i o n . Several groups reported that some of the " o v a l " or t r a n s i t i o n a l c e l l s which appear s e v e r a l weeks a f t e r exposure to diaminoazobenzene (DAB) contained AFP (66-71). However, DAB i s a potent n e c r o t i z i n g agent f o r l i v e r c e l l s so that the r e l a t i o n s h i p between o v a l c e l l s and carcinogenesis i s d i f f i c u l t to i n t e r p r e t . On the other hand, O k i t a et a l . reported that not o v a l c e l l s but some of the n o e p l a s t i c nodules that appear a f t e r exposure to e t h i o n i n e c o n t a i n AFP (72). They use t h i s evidence to support Farber's concept of a sequence of metab o l i c and morphologic changes l i n k i n g the n e o p l a s t i c nodule to the hepatoma as a premalignant l e s i o n . Other data does not support t h i s concept. As mentioned above, there i s a reverse c o r r e l a t i o n between serum AFP concentrations and the appearance of n e o p l a s t i c nodules i n r a t s fed FAA (65). In a d d i t i o n , the l i v e r s of r a t s t r e a t e d with e t h i o n i n e or DEN and fed a c h o l i n e d e f i c i e n t d i e t d i s p l a y a marked o v a l c e l l p r o l i f e r a t i o n that occurs w i t h i n 2-3 weeks i n the absence of d e t e c t a b l e l i v e r c e l l n e c r o s i s . This "oval c e l l " p r o l i f e r a t i o n i s a s s o c i a t e d with serum AFP concentrat i o n s two logs higher than that found with e t h i o n i n e or DEN alone (60 ug/ml vs. 0.6-1.0 ug/ml). Immunofluorescent l a b e l i n g r e v e a l s that many of these o v a l c e l l s c o n t a i n albumin and approximately 3% a l s o c o n t a i n AFP (27^, and unpublished o b s e r v a t i o n s ) . Those elements r e c o g n i z a b l e as b i l e ducts i n the p r o l i f e r a t i v e l e s i o n s are negative f o r AFP and albumin. Double immunofluorescent
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
SELL
Mpha-fetoprotein
as an Indicator
SERUM ALPHAFETOPROTEIN
in
Carcinogenesis
CONCENTRATIONS
IN
RATS TRANSPLANTED
PREGNANCY
2IOI2345
333
HEPATOMA 7777
012
2 3 4 5 6 7 8 9 10II 12 131415 16 17 1819 20 WEEKS
Chart 1. Serum alphafetoprotein trations of rats during normal gestation, after partial hepatectomy and chemically induced liver injury, after exposure to chemical carcinogens, and during growth of an AFP producing transplantable hepatoma are presented (from Ref. 17).
Chart 2. Lack of relationship between serum AFP concentrations and development of neoplastic nodule after FAA feeding of AC I rats. Following feeding of 0.06% FAA the serum concentrations of AFP become elevated with the first week of feeding, attain a maximum after six weeks, and then fall during the third and fourth feeding cycles when neoplastic nodule formation is most evident (see Ref. 65).
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Fk [ure 1. Edge of neoplastic nodule (left margin) and adjacent liver with marked increase in "oval cells" The oval cells are the smaller cells which alternate with larger hepatocytes. H & E X245. Figure 2. Same section as Figure 1 labeled for AFP by immunofluorescence. Note AFP containing oval cells. X245. Figures 3 and 4. Oval cells containing AFP. X245.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
Figure 5. Area of "atypical" hyperplasia of liver. Note gland-like collection of atypical hepatocytes. H & E X230. The rectangle outlines the area shown in Figures 7 and 8. Figure 6. AFP immunofluorescence staining of area of "atypical" hyperplasia. X90. Figure 7 (H & E) and 8 (AFP immunofluorescence). Area shown within Figure 5. AFP containing cells are in gland-like area of "atypical" hyperplasia. X-230.
336
GLYCOPROTEINS A N D
GLYCOLIPIDS IN
DISEASE
PROCESSES
s t a i n i n g f o r albumin and AFP r e v e a l s that the AFP c e l l s are a l s o p o s i t i v e f o r albumin. E a r l y f o c i of c e l l u l a r a l t e r a t i o n and small n e o p l a s t i c nodules are AFP negative although many of the c e l l s i n these h e p a t o c e l l u l a r l e s i o n s contain albumin. Extensive examinat i o n of the l i v e r s of c h o l i n e supplemented r a t s r e c e i v i n g 1 i n j e c t i o n of DEN (75 mg/kg) r e v e a l s that s t r o n g l y AFP p o s i t i v e " o v a l " c e l s l (approximately 1 per 5,000 l i v e r c e l l s ) may be i d e n t i f i e d as s i n g l e c e l l s i n the p o r t a l zones only one week a f t e r i n j e c t i o n , at a time when l i t t l e or no morphologic change i s recognizable. This f i n d i n g has stimulated a more extensive a n a l y s i s of the r e l a t i o n s h i p of AFP production to the development of h e p a t o c e l l u l a r carcinomas a f t e r exposure of r a t s to chemical carcinogens (74). P r e l i m i n a r y observations show that the n e o p l a s t i c nodules that develQp a f t e r feeding 2-FAA to F i s h e r male r a t s f o r f o u r , 2-week on 1-week o f f , c y c l e s do not contain c e l l s with AFP although many of the c e l l s of these nodules are p o s i t i v e f o r a l b u min. At the time of t h i v a r y i n g i n s i z e from eigh been examined, and none of these contained c e l l s with AFP. Neop l a s t i c nodules are recognizable as d i s c r e t e well-demarcated c o l l e c t i o n s of l i v e r c e l l s s i m i l a r to normal hepatocytes without normal s i n u s o i d (74). In zones of these l i v e r s where there are not i d e n t i f i a b l e n e o p l a s t i c nodules the s i n u s o i d s contain a d i f f u s e increase i n " o v a l " c e l l s . Approximately 3-5% of these o v a l c e l l s are p o s i t i v e f o r AFP. In a d d i t i o n , some zones of " a t y p i c a l h y p e r p l a s i a " of hepatocytes a l s o contain c e l l s with AFP. Representative immunofluorescent s t a i n i n g patterns are shown i n Figures 1-8. Since none of the " n e o p l a s t i c nodules" c o n t a i n AFP but many of the tumors that a r i s e from carcinogen exposure produce AFP (65), the r e l a t i o n s h i p between n e o p l a s t i c nodules and h e p a t o c e l l u l a r carcinoma must be re-evaluated (75). The dogma among workers i n chemical carcinogenesis has been that h e p a t o c e l l u l a r carcinomas a r i s e from n e o p l a s t i c nodules. On the b a s i s of the above observations as w e l l as the p r e l i m i n a r y observ a t i o n s made i n c o l l a b o r a t i o n with Dr. L e f f e r t , that c e l l s from n e o p l a s t i c nodules do not p r o l i f e r a t e as w e l l i n v i t r o as normal hepatocytes, i t i s proposed that h e p a t o c e l l u l a r carcinomas do not a r i s e from n e o p l a s t i c nodules, but from oval c e l l s which are not part of the nodules. The production of AFP by these c e l l s a f t e r exposure to chemical carcinogens most l i k e l y represents inapprop r i a t e synthesis. Chemical carcinogens may s e l e c t a very l i m i t e d c e l l population (?stem c e l l s ) to produce AFP and t h i s s i g n a l may be misread by the normally c o n t r o l l i n g environment or by the developing stem c e l l s so that the stem c e l l s p r o l i f e r a t e and d i f f e r e n t i a t e r a t h e r than form normal l i v e r l o b u l e s , and eventua l l y progress to h e p a t o c e l l u l a r carcinomas (Figure 9). In c o n c l u s i o n , by c o r r e l a t i o n and analogy, i t i s hypothesized that the f u n c t i o n of AFP i s to serve as a s i g n a l f o r normal t i s s u e ( l i v e r ) o r g a n i z a t i o n (31). The c o r r e l a t i o n i s that AFP i s produced by f e t a l l i v e r and by the adult l i v e r f o l l o w i n g p a r t i a l
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18.
SELL
Alpha-fetoprotein
as an Indicator
STIMULATION
OF AFP
RESTITUTIVE PROLIFERATION
in
Carcinogenesis
ADULT LIVER
CHEMICAL CARCINOGENESIS
PARTIAL HEPATECTOMY GALACTOSAMINE CCI 4
OVAL CELL PROLIFERATION
PORTAL
ATYPICAL DIFFERENTIATION HEPATOCELLULAR CARCINOMA
Figure 9. Representation of hypothesized targets for chemical toxins and chemical carcinogens in relationship to target cell and resulting AFP synthesis (for explanation, see text)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
337
338
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE
PROCESSES
hepatectomy or c h e m i c a l l y induced n e c r o s i s . In these s i t u a t i o n s , the developing or growing l i v e r must a l i g n a number of c e l l types i n c l u d i n g hepatocytes, s i n u s o i d s , b i l e c a n n i c u l i as w e l l as blood and lymphatic v e s s e l s . The analogy i s with other markers shared by f e t a l t i s s u e s and tumors i n a d u l t s (oncodevelopmental gene products) that have been demonstrated to play a r o l e i n c e l l - c e l l i n t e r a c t i o n s r e q u i r e d f o r normal development and t i s s u e o r g a n i zation. I t i s p o s t u l a t e d that chemical carcinogens a c t on a small number of u n d i f f e r e n t i a t e d h e p a t o c e l l u l a r stem c e l l s (?oval c e l l s ) causing the i n a p p r o p r i a t e s y n t h e s i s of AFP. Under the c o n t i n u i n g i n f l u e n c e of the carcinogen, these c e l l s develop i n t o h e p a t o c e l l u l a r carcinomas. T h i s occurs because e i t h e r the progeny of the stem c e l l s a r e unable to respond to environmental s i g n a l s or the appropriate environmental c o n t r o l s are not a v a i l a b l e .
Abstract Systematic quantitative sutdies and immunofluorescent c e l l u lar localization of alphafetoprotein (AFP) in experimental systems has provided a basis for understanding and applying AFP quantitation to the diagnosis of congenital anomalies, to the differentiation of benign from malignant l i v e r disease and to the determination of the effects of therapy upon AFP producing tumors. In addition, these studies have contributed to new insights into the significance of AFP in normal development and of the sequence of events in induction of hepatocellular carcinoma by chemical carcinogens. An hypothesis has been formulated that AFP functions during fetal development and in the adult during l i v e r undergoing regeneration as a marker for organization of growing l i v e r tissue into normal lobules. Production of AFP during exposrue to chemical hepatocarcinogens represents an inappropriate signal. Carcinogens induce a small number of immature hepatocytes (?stem cells) to proliferate. Under inappropriate signals for organization, some of these stem c e l l s , perhaps only one or two per l i v e r , eventually produce hepatocellular carcinomas. The previously accepted sequential evolution of hepatocellular carcinomas from foci of altered hepatocytes through neoplastic nodules to malignant tumors is not supported by the results of ongoing experiments. Neoplastic nodules may represent a non-cancerous response of differentiated hepatocytes to carcinogen exposure.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18. SELL
339 Alpha-fetoprotein as an Indicator in Carcinogenesis
Literature Cited 1. Abelev, G.I., Perova, S.D., Khramkova, N.I. et al., Transplantation (1956) 1:174. 2. Tatarinov, Y. Vopr. Med. Khim. (1964) 10:90. 3. Stanislawski-Birencwajg, M., Uriel, J., Grabar, P. Cane. Res. (1967) 27:1990. 4. Abelev, G.I. Cancer Res. (1968) 28:1334. 5. Abelev, G.I. Adv. Cancer Res. (1971) 14:295. 6. Sell, S., Wepsic, H.T. "The Liver. The Molecular Biology of Its Diseases" (Becker, F.F. Ed.), pp. 773, Marcel Dekker, New York, 1975. 7. Ruoslahti, E., Seppala, M. Int. J. Cancer (1971) 8:374. 8. Oakes, D.D., Shuster, J., Gold, P. Cancer Res. (1972) 32: 2753. 9. Sell, S., Gord, D Immunochem (1973) 10:439 10. Sizaret, P., Breslaw (1975) 3:201. 11. Sell, S., Nichols, M., Becker, F.F. et al. Cancer Res. (1974) 34:865. 12. Sell, S., Becker, F.F., Leffert, H.L. et al. Cancer Res. (1976) 36:4239. 13. Ruoslahti, E., Engvall, E. Proc. Natl. Acad. Sci. USA (1976) 73:4641. 14. Ruoslahti, E., Terry, W.D. Nature (1976) 260:804. 15. Liang-Tang, L.K., Soloff, M.S. Biochim. Biophys. Acta (1972) 263:753. 16. Nunez, E., Vallette, G., Benassayag, C., Jayle, M-F. Biochem. Biophys. Res. Commun. (1974) 57:126. 17. Sell, S., Becker, F.F. J. Natl. Cancer Inst. (1978) 60:19. 18. Gitlin, D., Boesman, M. J. Clin. Invest. (1967) 46:1010. 19. Sell, S., Skelly, H. J. Natl. Cancer Inst. (1976) 56:645. 20. Sell, S., Alexander, D. J. Natl. Cancer Inst. (1974) 52:1489. 21. Gitlin, D. Ann. N.Y. Acad. Sci. (1975) 259:7. 22. Seppala, M. Ann. N.Y. Acad. Sci. (1975) 259:59. 23. Adinolfi, A., Adinolfi, M., Lessof, M.H. J. Med. Genet. (1975) 12:138. 24. Norgaard-Pedersen, B. Scand. J. Immunol. (1976) 5:1. 25. Brock, D.J., Scrimgeour, J.B., Nelson, M.M. Clin. Genet. (1975) 7:163. 26. Masseyeff, R. Pathol. Biol. (Paris) (1972) 20:703. 27. Waldmann, T.A., McIntire, K.R. Cancer Res. (1974) 34:1510. 28. JcIntire, K.R., Bogel, C.L., Princler, G.L., et al. Cancer Res. (1972) 32:1941. 29. McIntire,K.R.,Waldmann, T.A., Moertel, C.G., et al. Cancer Res. (1975) 35:991. 30. Mihalev, A., Tzingilev, D., Sirakov, L.M. Neoplasma (1976) 23:103. 31. Sell, S. "Handbook of Cancer and Immunology" (Waters, H. Ed.). Garland Publishing Co., in press.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
340 32. G., 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Sell, S. "Basic Immunologic Mechanisms in Cancer" (Hanna, M. Jr., Ed.). Academic Press, New York, in press. Leffert, H.L., Sell, S. J. Cell Biol. (1974) 61:823. Sell, S., Leffert, H.L., Skelly, H., et al. Ann. N.Y. Acad. Sci. (1975) 259:45. Sell, S., Stillman, D., Gochman, N. Am. J. Clin. Pathol. (1976) 66:847. Watanabe, A., Miyazaki, M., Taket, K. Gann (1975)67:279. Leffert, H.L. et al. Proc. Natl. Acad. Sci., in press. Endo, Y., Kanai, K., Oda, T., et al. Ann. N.Y. Acad. Sci. (1975) 259:234. Murray-Lyon, A.M., Orr, A.H., Gazzard, B., Kohn, J., Williams, R. Gut (1976) 17:576. Engelhardt, N.V., Lazareva, M.N., Abelev, G.I., et al. Nature (1976) 263:146. Murgita, R.A., Tomasi T.B. Jr J Exp Med (1975) 141: 269. Murgita, R.A., Tomasi 440. Sheppard, H.W., Jr., Sell, S., Trefts, P., et al. J. Immunol. (1977) 119:91. Sell, S., Sheppard, H.W., Jr., Poler, M. J. Immunol. (1977) 119:98. Sell, S., Morris, H.P. Cancer Res. (1974) 34:1413. Becker, F.F., Klein, K.M., Wolman, S.R., et al. Cancer Res. (1973) 33:3330. Becker, F.F., Wolman, S.R., Asofsky, R. et al. Cancer Res. (1975) 35:3021. Sell, S., Wepsic, H.T., Nickel, R. et al. J. Natl. Cancer Inst. (1974) 52:133. Sell, S. "Proceedings of the Seventh Annual Conference on Environmental Toxicology" (Fries, S.L., Ed.), pp. 278, National Technical Information Service, Springfield, 1977. Sell, S., Sheppard, H.W., Jr., Nickel, R. et al. Cancer Res. (1976) 36:476. Sell, S., Stillman, D., Michaelsen, M., et al. Radiat. Res. (1977) 69:54. Farber, E. Cancer Res. (1956) 16:142. Orr, T.W. Path. Bact. (1940) 50:393. Price, J.M., Harmon, J.W., Miller, E.C., Miller, J.A. Cancer Res. (1952) 12:192. Epstein, S., Ito, N., Merkow, L . , Farber, E. Cancer Res. (1967) 27:1702. Farber, E. Adv. Cancer Res. (1963) 7:383 Farber. E. Cancer Res. (1973) 33:2537. Farber, E . , Parker, S., Gruenstein, M. Cancer Res. (1976) 36:3879. Knox, W.E. "Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues." S. Karger, Basel, 1976.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
18.
SELL
341 Alpha-fetoprotein as an Indicator in Carcinogenesis
60.
Pitot, H.C., Barsness, L., Goldsworthy, T., Kitagawa, T. Nature (1978) 271:456. 61. Smuckler, E.A., Koplitz, M., Sell, S. Cancer Res. (1976) 36:4558. 62. Becker, F.F., Sell, S. Cancer Res. (1974) 34:2489. 63. Watabe, H. Cancer Res. (1971) 31:1192. 64. Teebor, G.W., Becker, G.W. Cancer Res. (1971) 31:1. 65. Becker, F.F., Sell, S. Submitted. 66. Bannikov, G.A., Gel'Shtein, V.I., Chipysheva, T.A. Vestn. Akad. Med. Nank. SSSR (1977) 3:39. 67. Dempo, K.N., Chisaka, N., Yoshida, Y., Kaneko, A., Onoe, T. Cancer Res. (1975) 35:1282. 68. Fujita, S., Ishizuka, H., Kamimura, N., Kaneda, H., Ariga, K. Ann. N.Y. Acad. Sci. (1975) 259:217. 69. Onda, H. Gann (1976) 67:253. 70. Onoe, T., Kaneko, A. Dempo K. Ogawa K., Minase T Ann. N.Y. Acad. Sci 71. Tchipysheva, T.A. J. Cancer (1977) 20:388. 72. Okita, K., Gruenstein, M., Klaiber, M., Farber, E. Cancer Res. (1974) 34:2758. 73. Shinozuka, H., Lombardi, B., Sell, S., Iammarino, R.M. Cancer Res. in press. 74. Squire, R.A., Levitt, M.H. Cancer Res. (1975) 35:3214. 75. Sell, S. submitted. RECEIVED
December 29, 1977.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
19 Carcinoembryonic Antigen—A Marker of Human Colonic Cancer CHARLES W. TODD and JOHN E. SHIVELY Division of Immunology, City of Hope National Medical Center, Duarte, CA 91010
The current literatur and quantitative change nancy. Despite the potential that these antigens offer for the control of cancer through detection, therapy, or prevention by immunization, in most cases the antigens are poorly defined and practical applications are lacking. One antigen which has achieved considerable application in the diagnosis and monitoring of cancer therapy is the carcinoembryonic antigen (CEA). Our study of the chemical nature of this complex molecule, or perhaps more accurately stated molecular complex, was undertaken with the goal of improving its clinical utility in the management of cancer. The initial reports by Gold and Freedman (1,2) suggested that CEA was synthesized by the rudimentary digestive system in the human fetus, that synthesis ceased prior to birth, but that it resumed with the onset of colonic adenocarcinoma in the adult. This sequence lead them to name the substance the carcinoembryonic antigen. Their development of a radioimmunoassay for CEA (3) and the potential importance of such a tumor marker stimulated studies of the diagnostic value of CEA assays in a variety of pathological conditions, both benign and malignant. The l i t erature on these clinical studies has been recently reviewed (4). These studies demonstrated that CEA-reactivity was not universally elevated in adenocarcinoma of the digestive system, that it was often elevated in other malignancies notably of breast, bladder, and lung, and that moderate elevations are often present in nonmalignant inflammatory diseases. Moreover, normal sera showed variable low levels of CEA-reactivity. Clearly the situation was more complex than originally visualized. Since radioimmunoassays are not completely specific (5), more precise chemical knowledge of the substances being detected was necessary. Most of the studies done on CEA have employed material isolated from hepatic metastases of colonic adenocarcinoma using essentially the method originally described by Krupey et al. (6), although the final block electrophoresis step is usually omitted. The CEA employed in most of our studies was obtained by homogeni0-8412-0452-7/78/47-080-342$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
19.
TODD A N D SHIVELY
Carcinoembryonic
343
Antigen
z a t i o n of l i v e r metastases o f colon adenocarcinoma i n water, a d d i t i o n o f an equal volume o f 2M p e r c h l o r i c a c i d , c e n t r i f u g a t i o n , d i a l y s i s o f the supernatant, c o n c e n t r a t i o n , chromatography on Sepharose 4B, and rechromatography on Sephadex G200 ( 7 ) . The CEA a c t i v i t y during these i s o l a t i o n s was followed by a t r i p l e isotope double antibody radioimmunoassay (8.), which has been r e c e n t l y improved by the s u b s t i t u t i o n o f C o f o r N a as a v o l ume marker f o r the supernatant (9). CEA prepared i n t h i s way when examined by e l e c t r o n microscopy c o n s i s t e d o f twisted rod or c r u l l e r shaped p a r t i c l e s with dimensions o f 9 x 40 nm (10). Further p u r i f i c a t i o n o f CEA by a f f i n i t y chromatography on concanavalin A Sepharose removed a small amount (15%) of contaminants, which were not r e t a i n e d by the a f f i n i t y column. This m a t e r i a l was found to c o n t a i n mucopolysaccharides as i d e n t i f i e d by c e l l u l o s e acetate s t r i p e l e c t r o phoresis, depolymerization with bovine t e s t i c u l a r hyaluronidase and i d e n t i f i c a t i o n o f g l u c u r o n i d e r i v a t i v e o f i t s methy spectrometry (12). E a r l y s t u d i e s by Krupey et al. (13) revealed CEA to be a g l y c o p r o t e i n with a molecular weight about 180,000, comprising about 60% carbohydrate and H0% p r o t e i n . This high percentage o f carbohydrate may e x p l a i n the f a c t that the molecular dimensions as v i s u a l i z e d by e l e c t r o n microscopy i n d i c a t e a molecular volume about 16 times g r e a t e r than that c a l c u l a t e d f o r a c l o s e l y packed molecule o f 180,000 molecular weight. Presumably the frequency and s i z e o f the carbohydrate groupings i n t e r f e r e with compact f o l d i n g o f the chain. There appears to be only one polypeptide chain present i n CEA. Consistent with t h i s view i s the f i n d i n g that r e d u c t i o n and a l k y l a t i o n o f the d i s u l f i d e bonds p r i o r to e l e c t r o n microscopic examination o f CEA leads to the appearance of t h r e a d - l i k e s t r u c t u r e s . The maximum extended length observed, 220 nm, approaches that expected f o r a p r o t e i n o f the r e q u i s i t e s i z e a l l o w i n g 0.364 nm extension per amino a c i d r e s i d u e (14). C h a r a c t e r i z a t i o n o f CEA by i s o e l e c t r i c f o c u s i n g (15), i o n exchange chromatography (15,16) and a f f i n i t y chromatography on concanavalin A Sepharose ^11>17>_18) demonstrated considerable heterogeneity, as might be expected f o r a h i g h l y g l y c o s y l a t e d p r o t e i n . Indeed, v a r i a t i o n i s seen i n the r e l a t i v e amounts o f the carbohydrate components of CEA i s o l a t e s from d i f f e r e n t tumors (19). T y p i c a l r e s u l t s from a CEA p r e p a r a t i o n p u r i f i e d by a f f i n i t y chromatography on concanavalin A Sepharose (11_) are presented i n Table I . The carbohydrate compositions were determined by the method of Clamp (20) f o r n e u t r a l sugars, by the method o f Warren (21) f o r s i a l i c a c i d , and by the method o f L i u and Chang (22) with the amino a c i d a n a l y z e r f o r the amino sugars. The s i a l i c a c i d has been i d e n t i f i e d as N-acetylneuraminic a c i d (23)• These analyses r e q u i r e d samples o f approximately 250 yg. More r e c e n t l y we have developed a method approximately 2 orders o f magnitude more s e n s i t i v e employing methanolysis and t r i f l u o r o a c e t y l a t i o n to generate methyl N , 0 - t r i f l u o r o a c e t y l g l y c o s i d e s , which a r e separated by gas chromatography on 7m x 0.5 mm packed 5 7
2 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
344
GLYCOPROTEINS A N D GLYCOLIPIDS IN
DISEASE
PROCESSES
c a p i l l a r y columns and q u a n t i t a t e d by e l e c t r o n capture d e t e c t i o n (24). In c o n t r a s t to the v a r i a b i l i t y seen f o r the carbohydrate composition, the amino a c i d composition o f v a r i o u s CEA i s o l a t e s remains q u i t e constant (25). A t y p i c a l a n a l y s i s i s presented i n Table 1. Using the r e s u l t s i n Table 1 some deductions regarding the s t r u c t u r e o f CEA can be made. The t o t a l number o f amino a c i d residues i s 829. A s i n g l e polypeptide chain with 829 amino a c i d residues with a length of 0.364 nm per residue (14) would have a length of 302 nm, i f f u l l y extended. This compares f a v o r a b l y with the 220 maximum observed by e l e c t r o n microscopy c o n s i d e r i n g the approximations i n v o l v e d . The low content of N - a c e t y l galactosamine (1.5? of the sugar r e s i d u e s ) suggests that the o l i gosaccharide u n i t s are not bound to the p r o t e i n backbone through N-acetylgalactosamine l i n k e d to s e r i n e or threonine The f a i l ure to accomplish 3 - e l i m i n a t i o n a t i v e l y , i f the attachmen to asparagine, the t o t a l a s p a r t i c acid/asparagine content l i m i t s the number o f o l i g o s a c c h a r i d e u n i t s to 128 with an average s i z e of 4 r e s i d u e s . Subsequently we s h a l l see evidence that there are about h a l f as many u n i t s with an average of twice t h i s s i z e . The amount of N-acetyglucosamine i n d i c a t e s some o f these residues must be d i s t a l from the attachment s i t e s i n c e the r a t i o o f Nacetylglucosamine to a s p a r t i c acid/asparagine i s 1.5. The sum of s e r i n e (91 r e s i d u e s ) and threonine (74 r e s i d u e s ) i s 165. Thus there are ample residues to e s t a b l i s h the asp,x,ser/thr sequences i n the p r o t e i n chain, b e l i e v e d to code f o r N-acetylglucosamineasparagine attachment (26). More p r e c i s e information on the s t r u c t u r e o f the o l i g o saccharide u n i t s was obtained by methylation a n a l y s i s (27) employing gas chromatography and mass spectrometry as depicted i n Figure 1. Samples were methylated by the method of Hakomori (28_) i n which the m e t h y l s u l f i n y l anion (29) was used to generate the polysaccharide alkoxide before the a d d i t i o n o f methyl i o d i d e . A c e t o l y s i s , h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n o f the permethylated p o l y s a c c h a r i d e s were performed using the procedures described by S t e l l n e r et al. (30). I d e n t i f i c a t i o n o f the p a r t i a l l y methylated a l d i t o l acetates was performed by the method of Bjorndal et al. (31) f o r the n e u t r a l sugar d e r i v a t i v e s and by the method of S t e l l n e r et al. (30) f o r the amino sugar d e r i v a tives. The products are i d e n t i f i e d by t h e i r r e t e n t i o n times i n the gas chromatograph and t h e i r fragments as produced i n the mass spectrometer. There are c e r t a i n p r e f e r r e d cleavage p o i n t s that occur on e l e c t r o n impact i n the mass spectrometer. The order of the preference o f these cleavages i s i n d i c a t e d by the numbers 1, 2, and 3 (Figure 1). A d e t a i l e d procedure has been published (32). The r e s u l t s of these s t u d i e s (27) on 4 CEA samples are presented i n Table 2 together with the r e s u l t s on a sample analyzed by Hammarstrom et al. (33). I t i s apparent that considerable
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
TODD A N D SHIVELY
Carcinoembryonic
Antigen
TABLE 1 COMPOSITION OF CEA
Moles per 180,000 MW
Components
Sugars N-Acetylgalactosamine
188.2
N-Ace tylglucosamine N-Acetylneuraminic
7.5
acid
12.1
Fucose
102.4
Galactose
110.9
Mannose Amino Acids Alanine
49.3
Arginine
20.1
A s p a r t i c acid/Asparagine
127.8
Cysteine
15.8
Glutamic acid/glutamine
84.6
Glycine
47.7
Histidine
20.1
Isoleucine
31.3
Leucine
67.8
Lysine
20.3
Methionine
Trace
Phenylalanine
18.9
Proline
58.6
Serine
91.2
Threonine
74.0
Tryptophan
11.9
Tyrosine
36.1
Valine
53.5
Carbohydrate
49.9?
Protein
50.1?
These v a l v e s were c a l c u l a t e d from the data f o r F r a c t i o n V, Table 2, Reference 11.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
346
GLYCOPROTEINS
A N D GLYCOLIPIDS IN
DISEASE
PROCESSES
v a r i a t i o n e x i s t s among these samples, but that a b a s i c p a t t e r n i s being followed. As expected from patterns p r e v i o u s l y observed i n mammalian g l y c o p r o t e i n s (34_), a l l the fucose i s t e r m i n a l . S i m i l a r l y a l l the N-acetylneuraminic a c i d i s t e r m i n a l . This f o l l o w s not from the methylation a n a l y s i s , but from the observ a t i o n that removal of the N-acetylneuraminic a c i d with neuraminidase exposed to a t t a c k by periodate a d d i t i o n a l moles of galactose e q u i v a l e n t to the moles o f N-acetylneuraminic a c i d removed (23). This observation together with the n e g l i g i b l e amounts of branching galactose observed by methylation a n a l y s i s (Table 2) a l s o f i x e s the p o i n t of attachment of the N - a c e t y l neuraminic a c i d at the 3 - p o s i t i o n of galactose (23). Threefourths of the mannose r e s i d u e s i n a l l the CEA samples were l i n k e d to 3 other sugar residues (branching mannose). Except f o r the small amounts of 3,4- and 2,3-linked g a l a c t o s e , the only other branching sugar i s N-acetylglucosamine of which 32-41? i s present a t branching p o i n t s The data i n Table 2 ber of o l i g o s a c c h a r i d e u n i t s l i n k e d to the p r o t e i n chain and t h e i r average s i z e (35). The number of o l i g o s a c c h a r i d e u n i t s equals the sum of the nonreducing terminal residues minus the sum o f the branching r e s i d u e s . From t h i s the average s i z e can be obtained by d i v i d i n g the t o t a l number of residues by the number of o l i g o s a c c h a r i d e u n i t s . Thus the numbers of o l i g o s a c c h a r i d e u n i t s f o r the CEA samples i n Table 2 are 51, 77, 124, 64, and 69 r e s p e c t i v e l y . On t h i s b a s i s the comparable o l i g o s a c c h a r i d e u n i t s contain 9, 7, 4, 8, and 6 monosaccharide residues each. Considering the cumulative e r r o r s and the expected i n h e r ent v a r i a t i o n , the data are i n reasonably good accord. The p i c ture that emerges i s many small o l i g o s a c c h a r i d e u n i t s attached to about 1 out o f 10 of the amino a c i d residues u t i l i z i n g about 2/3 of the a s p a r t i c acid/asparagine r e s i d u e s . The high carbohydrate content of CEA suggested the p o s s i b i l i t y that the a n t i g e n i c s i t e as detected i n the radioimmunoassay might r e s i d e there. Although the p r e c i s e nature of t h i s s i t e i s s t i l l undefined and may vary depending on the a n t i s e r a used to define i t , the cumulative weight of evidence i n d i c a t e s that the p r o t e i n p o r t i o n of the molecule plays the major, i f not e x c l u s i v e , r o l e i n determining the s i t e . This does not exclude the p o s s i b i l i t y that many CEA a n t i s e r a contain a n t i b o d i e s to carbohydrate. Under the c o n d i t i o n s of the usual radioimmunoassay only a r a t h e r narrow p o p u l a t i o n of high a f f i n i t y a n t i b o d i e s p l a y s a s i g n i f i cant r o l e (4). A consequence of t h i s s e l e c t i o n i s that the antiserum used may contain l a r g e populations of a n t i b o d i e s p l a y i n g an i n s i g n i f i c a n t r o l e i n the immunoassay t i t r a t i o n , but quite capable of r e a c t i n g with the antigen under the more concentrated c o n d i t i o n s p r e v a i l i n g i n immunodiffusion t e s t s . For t h i s reason i t i s dangerous to assume that phenomena observed by various immunodiffusion techniques are n e c e s s a r i l y involved i n immunoassay t i t r a t i o n s ( 4 _ ) .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
residues
Terminal 4 3,4
Terminal 2 6 2,4 2,6 3,6
103 43 61 5 11 5 125 9 9 Trace 20 16 23 77 Trace 92 63 155
Terminal Terminal 3 2 6 3,4 and 2,3
a
x
CEA
23
linkage
Terminal
Glyoosidio
103 72 22 18 2 2 116 14 7 Trace 18 18 25 83 16 94 76 186
11
2
CEA
of h
95 63 18 9 4 4 98 13 7 Trace 13 18 22 73 20 110 83 213
13
CEA
CEA
96 4 14 Trace 18 23 18 77 5 83 41 129
110 36 11 36 13
18
h
CEA-14
A l l tumors were l i v e r
104 58 45 18 11 2 134 9 9 Trace 22 20 11 71 32 88 58 178
34
Z
CEA
5
8-10 g
T h e s u b s c r i p t s 1 to 4 i n d i c a t e CEA p u r i f i e d from d i f f e r e n t tumors. metastases that had o r i g i n a t e d i n the colon. ^Data from Hammarstrom et al. (33).
a
Total
Total N-Acetylglucosamine
Total Mannose
N-Acetylneuraminic acid Fucose Galactose
Carbohydrate
Moles/1.
STRUCTURAL UNITS OF THE POLYSACCHARIDE PORTION OF CEA
TABLE 2
348
GLYCOPROTEINS
A N D GLYCOLIPIDS
IN
DISEASE
PROCESSES
The evidence supporting the r o l e of the p r o t e i n p o r t i o n i n determining the a n t i g e n i c s i t e may be summarized as f o l l o w s . S e r i a l p e r i o d a t e o x i d a t i o n , Smith degradation (36), of CEA demonstrated p e r s i s t e n c e of a n t i g e n i c a c t i v i t y even a f t e r removal o f 90? of the carbohydrate ( 2 3 , 3 3 , 3 7 ) . A n t i s e r a to CEA can detect as l i t t l e as l O ' * M o f CEA. Such high b i n d i n g a f f i n i t i e s are c h a r a c t e r i s t i c a l l y achieved a g a i n s t p r o t e i n a n t i gens but not a g a i n s t carbohydrates (38). These high b i n d i n g a f f i n i t i e s can not be explained by b i v a l e n t antibody b i n d i n g to adjacent s i t e s on the CEA molecule, s i n c e e q u i v a l e n t concentrat i o n s of b i n d i n g s i t e s of monovalent Fab' fragments of CEA a n t i bodies were e q u a l l y e f f e c t i v e as the i n t a c t antibody i n the CEA radioimmunoassay (39_). D i l u t e a l k a l i treatment of CEA destroys i t s a n t i g e n i c a c t i v i t y , but does not a f f e c t i t s l e c t i n b i n d i n g ability (33). I t i s w e l l known that p r o t e i n s t r u c t u r e i s r a p i d l y degraded by a l k a l i , whereas the carbohydrate l i n k a g e s o f asparagine l i n k e d g l y c o p r o t e i n Westwood et al. (40) hav o f CEA destroys i t s a n t i g e n i c a c t i v i t y , producing N-terminal p r o l i n e peptides with i n t a c t carbohydrate chains except f o r the l o s s of s i a l i c a c i d and fucose. I t had p r e v i o u s l y been shown that d e s t r u c t i o n of s i a l i c a c i d and fucose by periodate o x i d a t i o n does not a l t e r the a n t i g e n i c a c t i v i t y (23). Reduction and a l k y l a t i o n o f the c y s t i n e d i s u l f i d e bonds i n CEA destroys i t s a n t i g e n i c a c t i v i t y (22,33,41,42). C l e a r l y the secondary and t e r t i a r y s t r u c t u r e s o f CEA are c r i t i c a l i n maintaining i t s a n t i genic nature. 1
The sequence o f the f i r s t 24 amino a c i d s at the N-terminus of CEA was r e a d i l y determined by standard automated Edman techniques. T h i s sequence i s presented i n Figure 2 (43). I t has been confirmed s e v e r a l times (44) and extended an a d d i t i o n a l 6 r e s i d u e s (45), although the choice between glutamic a c i d and glutamine at c e r t a i n r e s i d u e s has not been uniform (46). A d d i t i o n a l sequencing encountered t e c h n i c a l problems a r i s i n g from the high degree of g l y c o s y l a t i o n . These d i f f i c u l t i e s began with the r e s i s t a n c e to s p e c i f i c cleavage, presumably due to s t e r i c hinderance by the carbohydrate groups. Since the carbohydrate s u b s t i t u e n t s are h i g h l y heterogeneous i n degree of s u b s t i t u t i o n among a p o p u l a t i o n o f CEA molecules, s p e c i f i c cleavage methods d i d not g i v e uniform or s t o i c h i o m e t r i c y i e l d s of expected p e p t i d e s . Once peptides were obtained, t h e i r p u r i f i c a t i o n was rendered d i f f i c u l t by these heterogeneous carbohydrate substituents. Glycopeptides have broad, i l l d e f i n e d peaks i n separations based on c o n v e n t i o n a l s i z i n g techniques. Edman degradations o f g l y c o p e p t i d e s f r e q u e n t l y come to an abrupt h a l t when an amino a c i d to which carbohydrate i s attached i s encountered. The f i r s t of these d i f f i c u l t i e s was solved by the use of the detergent T r i t o n X100 (0.25?), which rendered CEA suscept i b l e to t r y p s i n cleavage (47). Although CEA c o n t a i n s s u f f i c i e n t a r g i n i n e and l y s i n e r e s i d u e s to produce about 40 t r y p t i c p e p t i d e s ,
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
TODD AND SHIVELY
Carcinoembryonic
Antigen
,0H
v
0-R
R-0
R-0
OCH,
HO
0-R
OCH,
OCH. 0
CH OH
CH OCCH
2
I
HCOCH3
HCOCH,
© (D (D
CH OCH
CH 0CH
3
3
I 0 HCOCCH,
HCOH
HCOCCH,
HCOH
I
I CH OCH 2
CH OCH 2
3
3
Cancer Research Figure 1. Methylation analysis. Numbers indicate preferred order of bond rupture under electron bombardment in the mass spectrometer.
CARCINOEMBRYONIC ANTIGEN A m i n o - T e r m i n a l Residues 5 10 Leu Thr He Glu Ser Thr Pro Phe A s n 15 Vol Ala Glu Gly Lys Glu Vol L e u Leu Leu vol His Asn L e u Lys
f at the N-terminus of CEA 2
S
e
q
u
e
n
c
e
0
a
m
i
n
0
a c i d s
—[AAAAAAAA)— ORIGINAL GENE
I —[AAAAAAAA]
|AAAAAAAA|—
DUPLICATED GENES ^ —fvvwvw] [wwwv^j— DIVERGENT GENES
t
Figure 3. Proposed genetic evolui f th protein chains of CEA and related antigens on
or
e
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
350
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
only 10 were i s o l a t e d . These were f r a c t i o n a t e d by a combinat i o n o f anion and c a t i o n exchange r e s i n s using high pressure l i q u i d chromatography. In some cases these peptides could be sequenced through 20 Edman degradation c y c l e s . D e t a i l s are presented elsewhere (47). C u r r e n t l y methods of d e g l y c o s y l a t i n g CEA are being evaluated with the o b j e c t i v e o f s i m p l i f y i n g the sequencing problem. I t has long been recognized that radioimmunoassay o f serum from normal i n d i v i d u a l s f o r CEA always showed some v a r i a b l e low l e v e l o f CEA r e a c t i v i t y . The demonstration i n normal t i s s u e s o f m a t e r i a l c r o s s r e a c t i v e with but d i s t i n g u i s h a b l e from CEA appeared to e x p l a i n the observed normal l e v e l s . These molecules included NCA (48), NGP (49), CCEA-2 (50), CEX (51), and 6 p r o t e i n (52). These molecules a l l have lower molecular weights than does CEA. Subsequently other groups (53-55) reported the presence i n normal serum and normal t i s s u e e x t r a c t s o f m a t e r i a l with CEA-like a c t i v i t y and s i m i l a r t umns. Unfortunately th mal t i s s u e was so low that s u f f i c i e n t m a t e r i a l could not be obtained f o r chemical c h a r a c t e r i z a t i o n . In 1974 Go et al. (56) measured the s e c r e t i o n i n various p o r t i o n s o f the g a s t r o i n t e s t i n a l t r a c t o f m a t e r i a l that i n h i b i t e d i n the assay f o r CEA. The colon secreted the l a r g e s t q u a n t i t y o f the m a t e r i a l . I t e l u t e d from a Sephadex G-200 column i n the same p o s i t i o n as CEA. The presence o f these q u a n t i t i e s of m a t e r i a l with CEA-like a c t i v i t y i n the colon lavages o f healthy i n d i v i d u a l s made i t p o s s i b l e to i s o l a t e s u f f i c i e n t m a t e r i a l f o r chemical characteri z a t i o n and d i r e c t comparison with CEA (57). The m a t e r i a l with CEA-like a c t i v i t y from c o l o n i c lavages was p u r i f i e d by g e l f i l t r a t i o n on Sepharose 6B and Sephadex G200 followed by a f f i n i t y chromatography on concanavalin A l i n k e d to Sepharose. The p u r i f i e d m a t e r i a l migrated i n polyacrylamide-sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s as a s i n g l e d i f f u s e band with m o b i l i t y i d e n t i c a l to that o f tumor CEA. I t possessed the same s p e c i f i c a c t i v i t y as CEA i n radioimmunoassay. The carbohydrate and amino a c i d compositions were s i m i l a r to those o f CEA. Moreover, methylat i o n a n a l y s i s demonstrated that the monosaccharide linkages were s i m i l a r t o those i n CEA (57), and i t s N-terminal amino a c i d sequence was homologous to CEA (j>8). Thus by a l l c r i t e r i a a p p l i e d i t was i n d i s t i n g u i s h a b l e from CEA. I t thus seems l i k e l y that CEA i s a normal t i s s u e product. This r a i s e s the question of whether the elevated serum l e v e l s observed i n malignancy r e s u l t from increased c e l l u l a r s y n t h e s i s or by r e d i r e c t i o n to the blood stream o f m a t e r i a l normally e l i m i n a t e d i n the g a s t r o i n t e s tinal tract. During the i s o l a t i o n o f CEA from l i v e r metastases o f c o l o n i c adenocarcinoma, i t was observed that a lower molecular weight component possessing CEA a c t i v i t y i n the radioimmunoassay was present i n the eluate from the Sepharose 4B column. This materi a l , which we have designated TEX, has now been i s o l a t e d and puri f i e d (59). Like CEA, TEX i s a g l y c o p r o t e i n . I t binds to E
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
19.
TODD A N D SHIVELY
Carcinoembryonic
Antigen
351
concanavalin A Sepharose from which i t can be e l u t e d by d i s p l a c e ment with methyl a-D-mannoside. By Sephadex G200 g e l f i l t r a t i o n and by sodium dodecyl s u l f a t e polyacrylamide g e l e l e c t r o p h o r e s i s i t s molecular weight was shown to be about 110,000 d a l t o n s . TEX contained 35% carbohydrate, c o n s i d e r a b l y l e s s than that present i n CEA. Linkage s t u d i e s of the carbohydrate by methylat i o n a n a l y s i s revealed that TEX contained s u b s t a n t i a l l y l e s s t e r minal g a l a c t o s e , as w e l l as l e s s 4-linked i n t r a c h a i n and 3,4branched N-acetylglucosamine than CEA. A s i n g l e treatment with periodate destroyed a l l of the s i a l i c a c i d and fucose and 25% of the N-acetylglucosamine as with CEA, but i n a d d i t i o n twice as much mannose and g a l a c t o s e are destroyed as compared with CEA (59). When the s i z e of the p r o t e i n component i s c a l c u l a t e d from the molecular weight and percent p r o t e i n , i t i s found that t h i s s i z e i s n e a r l y i d e n t i c a l to that of CEA. S i m i l a r l y the amino a c i d composition o f TEX i s n e a r l y i d e n t i c a l to that o f CEA, except f o r the presence methionine i n TEX, but no 24 N-terminal amino a c i d s of TEX d i f f e r s from CEA only i n the presence o f a l a n i n e , at p o s i t i o n 21 i n l i e u of v a l i n e i n CEA. The combined evidence, both immunological and chemical, i n d i cates that TEX i s c l o s e l y r e l a t e d to CEA, but d i f f e r s i n i t s mode and degree o f g l y c o s y l a t i o n and has at l e a s t one and probably more amino a c i d a l t e r a t i o n s i n i t s polypeptide chain (59). Since TEX, which was i s o l a t e d from a tumor, may be s i m i l a r to NCA and r e l a t e d m a t e r i a l s i s o l a t e d from normal t i s s u e , a preparation o f NCA was obtained from normal spleen t i s s u e by an immunochemical method u t i l i z i n g i n s o l u b i l i z e d a n t i b o d i e s to CEA and NCA (60). The molecular weight of the NCA obtained was found to be about 100,000 d a l t o n s . The carbohydrate composition was 30%. Unfortunately the m a t e r i a l a v a i l a b l e was i n s u f f i c i e n t f o r methylation a n a l y s i s and periodate o x i d a t i o n s t u d i e s . The sample possessed n e a r l y the same amino a c i d a n a l y s i s as CEA, but l i k e TEX contained a small but demonstrable amount of methionine. The sequence o f the f i r s t 26 N-terminal amino a c i d s was i d e n t i c a l to that o f CEA, except a t p o s i t i o n 21, where as i n TEX a l a nine i s present i n s t e a d of the v a l i n e o f CEA (60). The immunological data show that NCA and TEX are i d e n t i c a l i f tested with e i t h e r goat anti-NCA or r a b b i t anti-TEX, but TEX c r o s s r e a c t s with CEA when tested with monkey anti-CEA (59), an antiserum which was p r e v i o u s l y shown not to c r o s s r e a c t with spleen NCA (6l_). An a l t e r n a t i v e i n t e r p r e t a t i o n i s that TEX cont a i n s t r a c e amounts o f CEA]_ , r e c e n t l y described as i d e n t i c a l to CEA i n a l l respects except, perhaps, i n degree of g l y c o s y l a t i o n (62). Whatever may be the i n t e r r e l a t i o n s h i p s o f the v a r i o u s a n t i gens c r o s s r e a c t i v e with CEA (^-52,62), i t appears that from the standpoint o f primary s t r u c t u r e , i.e. sequence, of the p r o t e i n component, CEA, NCA, and TEX are c l o s e l y r e l a t e d . NCA and TEX may or may not d i f f e r from one another i n t h i s respect, but they both d i f f e r from CEA. We b e l i e v e that they a r i s e from d i s t i n c t 0W
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
352
but related genes which have a common evolutionary origin in a single gene. This primoridal gene (Figure Z) has duplicated, or replicated to more than 2 genes, which have subsequently undergone divergent evolution. A more profound understanding of the interaction of these genes and their gene products in benign and malignant disease will hopefully lead to improved clinical utility of this system. ACKNOWLEDGMENTS This review was supported in part by National Cancer Institute grants CA 16434 and CA 19163 from the National Large Bowel Cancer Program. REFERENCES 1. 2.
3.
4. 5. 6. 7. 8. 9.
Gold, P. and Freedman gens of the huma 467-481, 1965. Gold, P. and Freedman, S.O.: Demonstration of tumor specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J. Exp. Med., 121: 439-462, 1965. Thompson, D.M.P., Krupey, J., Freedman, S.O. and Gold, P.: The radioimmunoassay of circulating carcinoembryonic antigen of the human digestive system. Proc. Nat. Acad. Sci. USA, 64: 161-167, 1969. Egan, M.L., Engvall, E., Ruoslahti, E.I. and Todd, C.W.: Detection of circulating tumor antigens. Cancer, 40: 458466, 1977. Egan, M.L., Coligan, J.E. and Todd, C.W.: Radioimmunoassay for the diagnosis of human cancer. Cancer, 34: 1504-1509, 1974. Krupey, J., Gold, P. and Freedman, S.O.: Purification and characterization of carcinoembryonic antigens of the human digestive system. Nature, 215: 67-68, 1967. Coligan, J.E., Lautenschleger, J.T., Egan, M.L. and Todd, C.W.: Isolation and characterization of carcinoembryonic antigen. Immunochemistry, 9: 377-386, 1972. Egan, M.L., Lautenschleger, J.T., Coligan, J.E. and Todd, C.W.: Radioimmune assay of carcinoembryonic antigen. Imnrunochemistry, 9: 289-299, 1972. Egan, M.L., Todd, C.W. and Knight, W.S.: Co: A volume marker for the triple isotope double antibody radioimmune assay. Immunochemistry, 14: 611-613, 1977. 57
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
19.
10. 11. 12. 13. 14.
15. 16. 17. 18.
19.
20.
21. 22. 23. 24.
TODD AND SHIVELY
Carcinoembryonic Antigen
353
Slayter, H.S. and Coligan, J.E.: Electron microscopy and physical characterization of the carcinoembryonic antigen. Biochemistry, 14: 2323-2330, 1975. Slayter, H.S. and Coligan, J.E.: Characterization of carcinoembryonic antigen fractionated by concanavalin A chromatography. Cancer Res., 36: 1696-1704, 1976. Pritchard, D.G. and Todd, C.W.: Purification of carcinoembryonic antigen by removal of contaminating polysaccharides. Cancer Res., 36: 4699-4701, 1976. Krupey, J . , Gold, P. and Freedman, S.O.: Physicochemical studies of the carcinoembryonic antigens of the human digestive system. J. Exp. Med., 128: 387-398, 1968. Pauling, L., Corey, R.B. and Branson, H.R.: The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Nat. Acad. Sci. USA, 37: 205-240, 1951 Coligan, J.E., Henkart Heterogeneity o carcinoembryoni antigen chemistry, 10: 591-599, 1973. Eveleigh, J.W.: Heterogeneity of carcinoembryonic antigen. Cancer Res., 34: 2122-2124, 1974. Chism, S.E., Bell, P.M. and Warner, N.L.: Heterogeneity of CEA and 'CEA-like' preparations determined by Farr assays for lectin binding. J. Immunol. Meth., 13: 83-89, 1976. Harvey, S.R. and Chu, T.M.: Demonstration of two molecular variants of carcinoembryonic antigen by concanavalin A Sepharose affinity chromatography. Cancer Res., 35: 3001-3008, 1975. Egan, M.L., Coligan, J.E., Morris, J.E., Schnute, W.C., Jr. and Todd, C.W.: Physical characterization and structural studies of the carcinoembryonic antigen. Cancer Res., 36: 3482-3485, 1976. Clamp, J.R., Bhatti, T. and Chambers, R.E.: The examination of carbohydrate in glycoproteins by gas-liquid chromatography. In: Glycoproteins. A. Gottschalk, (ed.), Elsevier Publishing Co., 5 (part A): 300-321, 1972. Warren, L.: The thiobarbituric acid assay of sialic acids. J. Biol. Chem., 234: 1971-1975, 1959. Liu, T.-Y. and Chang, Y.H.: Hydrolysis of proteins with ptoluenesulfonic acid. J. Biol. Chem., 246: 2842-2848, 1971. Coligan, J.E. and Todd, C.W,: Structural studies on carcinoembryonic antigen: Periodate oxidation. Biochemistry, 14: 805-810, 1975. Wrann, M.M. and Todd, C.W.: Sensitive determination of sugars utilizing packed capillary columns and electron capture detection. J. Chromatog. 147: 309-316, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
354
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
25.
Coligan, J.E., Egan, M.L., Guyer, R.L., Schnute, W.C., Jr. and Todd, C.W.: Structural studies on the carcinoembryonic antigen. Ann. N.Y. Acad. Sci., 259: 355-365, 1975. 26. Spiro, R.G.: Glycoproteins. Ann. Rev. Biochem., 39: 599638, 1970. 27. Coligan, J.E., Pritchard, D.G., Schnute, W.C., Jr. and Todd, C.W.: Methylation analysis of the carbohydrate portion of carcinoembryonic antigen. Cancer Res., 36: 1915-1917, 1976. 28. Hakomori, S.: A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethylsulfoxide. J. Biochem. Tokyo, 55: 205-208, 1964. 29. Sandford, P.A. and Conrad, H.E.: The structure of the Aerobacter aerogenes A3(S1) polysaccharide. I. A reexamination using improved procedures for methylation analysis. Biochemistry, 5: 1508-1517 1971 30. Stellner, K., Saito aminosugar linkage Biochem. Biophys., 155: 464-472, 1973. 31. Björndal, H., Hellerqvist, C.G., Lindberg, B. and Svensson, S.: Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides. Angew. Chem. Intern. Ed. Engl., 9: 610-619, 1970. 32. Pritchard, D.G., Todd, C.W. and Egan, M.L.: Chemistry of carcinoembryonic antigen. Methods in Cancer Research, 14: 55-85, 1977. 33. Hammarström, S., Engvall, E., Johansson, B.G., Svensson, S., Sundblad, G. and Goldstein, I.J.: Nature of the tumor-associated determinant(s) of carcinoembryonic antigen. Proc. Nat. Acad. Sci. USA, 72: 1528-1532, 1975. 34. Heath, E.C.: Complex polysaccharides. Ann. Rev. Biochem., 40: 29-56, 1971. 35. Shively, J.E. and Todd, C.W.: Carcinoembryonic antigen. Scand. J. Immunol., in press. 36. Smith, F. and Unrau, A.M.: On the presence of l-->6 linkages in laminarin. Chem. Ind. London, 881, 1959. 37. Bessel, E.M., Thomas, P. and Westwood, J.H.: Multiple Smith degradations of carcinoembryonic antigen (CEA) and of asialo CEA. Carbohyd. Res., 45: 257-268, 1975. 38. Eisen, H.N., pp 376-377 in Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S. and Wood, W.B., Microbiology, Harper and Row, New York, 1968. 39. Morris, J.E., Egan, M.L. and Todd, C.W.: The binding of carcinoembryonic antigen by antibody and its fragments. Cancer Res., 35: 1804-1808, 1975. 40. Westwood, J.H., Bessel, E.M., Bukhari, M.A., Thomas, P. and Walker, J.M.: Studies on the structure of the carcinoembryonic antigen. I. Some deductions on the basis of chemical degradations. Immunochemistry, 11: 811-818, 1974.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
19. TODD AND SHIVELY
Carcinoembryonic Antigen
355
41. Westwood, J.H. and Thomas, P.: Studies on the structure and immunologic activity of carcinoembryonic antigen -the role of disulfide bonds. Br. J. Cancer, 32: 708-719, 1975. 42. Westwood, J.H., Thomas, P. and Foster, A.B.: The treatment of carcinoembryonic antigen with sodium metaperiodate. Biochem. Soc. Trans., 2: 1250-1251, 1974. 43. Terry, W.D., Henkart, P.A., Coligan, J.E. and Todd, C.W.: Structural studies of the major glycoprotein in a preparation with carcinoembryonic antigen activity. J. Exp. Med., 136: 200-204, 1972. 44. Terry, W.D., Henkart, P.A., Das, S., Coligan, J.E. and Todd, C.W.: Characterization of human carcinoembryonic antigens. Proceed, of Miles Symposium, pp 241-247, 1973. 45. Chu, T.M., Bhargava, A.K. and Harvey, S.R.; Structure studies of the glycoproteins associated with carcinoembryonic antige 46. Coligan, J.E., Egan and Todd, C.W.: Structural studies on the carcinoembryonic antigen. Ann. N.Y. Acad. Sci., 259: 355-365, 1975. 47. Shively, J.E., Kessler, M. and Todd, C.W.: The antigenic determinants of carcinoembryonic antigen: The formation, isolation, composition, and N-terminal sequences of its tryptic peptides. Cancer Res., submitted for publication. 48. von Kleist, S., Chavanel, G. and Burtin, P.: Identification of an antigen from normal human tissue that crossreacts with the carcinoembryonic antigen. Proc. Nat. Acad. Sci. USA, 69: 2492-2494, 1972. 49. Mach, J.-P. and Pusztaszeri, G.: Carcinoembryonic antigen (CEA): Demonstration of a partial identity between CEA and a normal glycoprotein. Immunochemistry, 9: 1031-1034, 1972. 50. Turberville, C., Darcy, D.A., Laurence, D.J.R., Jones, E.W. and Neville, A.M.: Studies on carcinoembryonic antigen (CEA) and a related glycoprotein, CCEA-2. Preparation and chemical characterisation. Immunochemistry 10: 841843, 1973. 51. Darcy, D.A., Turberville, C. and James, R.: Immunological study of carcinoembryonic antigen (CEA) and a related glycoprotein. Br. J. Cancer, 28: 147-160, 1973. 52. Ørjasaeter, H.: Study of substances related to carcinoembryonic antigens, CEA-NCA and association with α-antichymotrypsin. Acta Path. Microbiol. Scand., 84: 235-244, 1976. 53. Chu, T.M., Reynoso, G. and Hansen, H.J.: Demonstration of carcinoembryonic antigen in normal human plasma. Nature, 238: 152-153, 1972. 54. Kupchik, H.Z. and Zamcheck, N.: Carcinoembryonic antigen(s) in liver disease. Gastroenterology, 63: 95-101, 1972. 1
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
356 55. 56.
57.
58.
59.
60.
61. 62.
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Pusztaszeri, G. and Mach, J . : Carcinoembryonic antigen (CEA) in nondigestive cancerous and normal tissues. Immunoohemistry, 10: 197-204, 1973. Go, V.L.W., Ammon, H.V., Holtermuller, K.H., Krag, E. and Phillips, S.F.: Quantification of carcinoembryonic antigen-like activities in normal human gastrointestinal secretions. Cancer, 36: 2346-2350, 1975. Egan, M.L., Pritchard, D.G., Todd, C.W. and Go, V.L.W.: Isolation and immunochemical and chemical characterization of carcinoembryonic antigen-like substances in colon lavages of healthy individuals. Cancer Res., 37: 2638-2643, 1977. Shively, J.E., Todd, C.W., Go, V.L.W. and Egan, M.L.: Amino terminal sequence of a carcinoembryonic antigen-like glycoprotein isolated from the colonic lavages of healthy individuals. Canoer Res., in press. Kessler, M.J., Shively, J.E., Pritchard, D.G. and Todd, C.W.: Isolation, immunologi studies of a tumo onic antigen. Canoer Res., in press. Engvall, E., Shively, J.E. and Wrann, W.: Isolation and characterization of the normal crossreacting antigen (NCA). Homology of its N-terminal amino acid sequence with that of carcinoembryonic antigen (CEA). Proc. Nat. Acad. Sci. USA, in press. Ruoslahti, E., Engvall, E., Vuento, M. and Wigzell, H.: Monkey antisera with increased specificity to carcinoembryonic antigen (CEA). Int. J. Canoer, 17: 358-361, 1976. Hammarström, S., Svenberg, T., Hedin, A. and Sundblad, G.: Antigens related to CEA. Scand. J. Immunology, in press.
RECEIVED
December 29, 1977.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20 Status of Blood Group Carbohydrate Chains in Human Tumors WILLIAM W. YOUNG, JR. and SEN-ITIROH HAKOMORI Biochemical Oncology, Fred Hutchinson Cancer Research Center, and Departments of Pathobiology and Microbiology/Immunology, University of Washington,1124Columbia, Seattle, WA 98104 1. Introduction Clear changes of glycolipi files in cell surface membranes associated with oncogenic transformation have been described during the past ten years. Possibly the first clear documentation for this topic was the decrease of hematoside and increase of lactosylceramide in polyoma transformed BHK cells as described in 1968 (1). A number of studies carried out by different investigators agreed that two kinds of changes in carbohydrate chains may occur during oncogenic transformation (see for review ref. 2). One is the block of synthesis certain glycolipids resulting in the associated accumulation of precursor structures and the second is induction of synthesis of a unique glycolipid for the transformed cells which most probably depends on activation of a new glycosyltransferase. Since in vitro cellular systems are only models for in vivo tumors, it is important to observe the changes of membrane glycolipids in tumors, particularly in human cancer. Among the great number of carbohydrate chains in human cells and tissues, the most well-defined carbohydrate chains, in terms of their chemistry, enzymology and genetics, are the blood group determinants. For this reason, our major effort has been directed to characterizing the changes of blood group determinants in human cancer as compared to comparable normal tissue; however, it should be noted that such comparisons may not be ideal as the precursor cell of human cancer may represent only a small population of cells present in normal tissue. However, for practical purposes such as diagnosis and therapy of human tumor using a specific cell surface marker, a gross difference of carbohydrate determinants observable between tumor and normal tissue may offer useful information. The first clear change of blood group determinants in human cancer was described in the laboratory of the late Dr. Masamune, who was a good friend of the late Dr. Ward Pigman to whom this Symposium is dedicated. Oh-uti in 1949, while working in Masamune's laboratory, described the deletion of blood group activity in "blood group polysaccharide" prepared from human gastric cancer (3). A number of subsequent studies were made in 0-8412-0452-7/78/47-080-357$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
358
GLYCOPROTEINS
A N D GLYCOLIPIDS
I N DISEASE
PROCESSES
T
Masamune s l a b o r a t o r y comparing the immunological and chemical d i f f e r e n c e s o f b l o o d group g l y c o p r o t e i n s from tumors and normal mucosa (^_,J5). Independently, Davidsohn and h i s co-workers ( 6 , T _ ) , Kay and Wallace ( 8 J , Prendergast (9.), and Dabelsteen and F u l l i n g ( 1 0 ) , have s t u d i e d the s t a t u s o f b l o o d group A and B i n human tumors as compared t o normal t i s s u e . Although a few d i s c r e p e n c i e s were d e s c r i b e d , they e s s e n t i a l l y agreed that blood group A or B determinants are d e l e t e d i n v a r i o u s human tumors and t h a t the degree o f d e l e t i o n c o u l d be c o r r e l a t e d with the degree o f m a l i g nancy (6_,T_). Most o f these s t u d i e s were made b e f o r e the chemical s t r u c t u r e s o f A and B determinants were e s t a b l i s h e d . Since the s t r u c t u r a l r e l a t i o n s h i p between A, B, H, L e , Le^, I and i d e t e r minants was e s t a b l i s h e d l a t e i n the 1960 s, s t u d i e s have focused on the s t a t u s o f the p r e c u r s o r s o f ABH determinants. As seen i n Table I , accumulation o f p r e c u r s o r carbohydrate chains i s q u i t e obvious f o r some human cancers i f not a l l o f the cases. A s i m i l a r p r e c u r s o vivo transformed c e l l s an p r e c u r s o r s c o u l d be u s e f u l s u r f a c e markers o f tumor c e l l s (13). Another category o f changes i n blood group determinants that has been observed i s t h e appearance o f new b l o o d group determinants i n some human tumor t i s s u e . Examples i n c l u d e the p o s s i b l e s y n t h e s i s o f P and P]_ antigens i n the g a s t r i c tumor o f a p a t i e n t belonging t o the extremely r a r e b l o o d group p ( l ^ i ' l ^ ) and induct i o n o f A - l i k e antigen i n tumors o f host blood group 0 ( 1 6 , 1 7 ) • The A - l i k e antigen appearing i n b l o o d group 0 or B could have been the r e s u l t o f the new s y n t h e s i s o f Forssman antigen i n tumors ( l 8 , 19), These items w i l l be d i s c u s s e d e x t e n s i v e l y i n subsequent sections. a
?
2 . Accumulation o f Precursor Carbohydrates o f Blood Group Determinants Since I - , and i-determinants have been assigned as p r e cursors o f b l o o d group ABH determinants ( 2 6 ) , we have s t u d i e d the i / l r e a c t i v i t y o f w a t e r - s o l u b l e blood group g l y c o p r o t e i n s e x t r a c t e d from g a s t r o i n t e s t i n a l tumors and normal mucosal t i s s u e . The g l y c o p r o t e i n f r a c t i o n was e x t r a c t e d according t o the procedure d e s c r i b e d by Masamune et al (5) and by the p e r c h l o r i c a c i d e x t r a c t i o n method d e s c r i b e d by Krupey et al {2J). As shown i n Table I I , the p e r c h l o r i c a c i d e x t r a c t a b l e f r a c t i o n o f tumor t i s s u e showed s i g n i f i c a n t i - a c t i v i t y i n c o n t r a s t t o the normal mucosa i n h cases, whereas, 3 cases showed higher i - a c t i v i t y i n normal mucosa, and one case d i d not show any s i g n i f i c a n t d i f f e r ence (Watanabe, Hakomori and Warner, unpublished d a t a ) . Recently, the hexasaccharide s t r u c t u r e , G a i e i + 4 G l c N A c $ l + 3 G a l B l + 4 G l c N A c 3 l - * 3 Gal3l->-i+Glc was found t o be a determinant f o r i - s p e c i f i c i t i e s ( 2 8 ) . Such a s t r u c t u r e may appear o r i n c r e a s e i n some human cancer. In order t o study the s t a t u s and d i s t r i b u t i o n o f blood group c a r r i e r carbohydrate chains i n normal and cancer t i s s u e s , the r e a c t i v i t y o f g l y c o l i p i d s d i r e c t e d a g a i n s t three s t r u c t u r e s , as shown i n F i g u r e 1 , were compared u s i n g t h e i r r e s p e c t i v e a n t i bodies (2h). The complement f i x i n g r e a c t i v i t i e s o f g l y c o l i p i d s
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20.
YOUNG A N DHAKOMORI
Blood
Group
Carbohydrate
Table I. Status o f Blood Group Determinants 1.
D e l e t i o n o f AB-determinants G a s t r o i n t e s t i n a l tumors
359
i n Human Cancer.
(Oh-uti 1 9 ^ 9 ; Masamune et al 1 9 5 2 ; 1 9 5 8 ; Davidsohn et al 1 9 6 6 ; I s e k i et al 1 9 6 2 ; S t e l l n e r et al 1 9 7 3 )
C e r v i c a l cancer
(Davidsohn et al 1 9 6 9 )
Bladder cancer
(Kay and Wallace 1 9 6 1 )
Oral
(Prendergast 1 9 6 8 ; Dabelsteen and F u l l i n g 1971)
carcinoma
2 . Precursor accumulation A s s o c i a t i o n o f CEA with I (Ma) antigen
(Simmons and Perlman 1 9 7 3 )
Presence o f I (Step antigen
(Feiz
Presence o f i (Dench) antigen
(Watanabe and Hakomori unpublished)
Accumulation o f l a c t o N-triosylceramide (GlcNAcCTH) 3.
Chains
Induction o f "incompatible" determinants P i , P, P antigen i n tumor o f p - i n d i v i d u a l
1975)
(Watanabe and Hakomori 1 9 7 7 ; Karlsson 1 9 7 6 )
k
(Levine et al 1 9 5 1 )
A - l i k e antigen i n tumors of 0 - , B - i n d i v i d u a l s (Hakomori et al 1 9 6 7 ; Hakkinen 1971) k. Heterophile F-antigen as a p o s s i b l e new i s o a n t i g e n i n human and the presence o f F-antigen i n tumors o f F"-tissues
(Hakomori, Wang and Young 1 9 7 7 ; Makita 1 9 7 7 )
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
360
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE PROCESSES
Table I I . The i - a c t i v i t y o f the p e r c h l o r i c - a c i d - e x t r a c t able f r a c t i o n o f normal mucosa and tumor t i s s u e
Dench i a c t i v i t y i n Case
Blood group
MIM Tn V. His G. Gutt. A. Dalz. C. Walt. M. Hutch. Anonym.
0 A. ND 0, 0, 0, ND ND
Normal mucosa
MN Le N, L e L e , MN a
a
Tumor tissue
>50 >50
3 6
>50 6 3 >50
6 25 12 >50
A c t i v i t y i s expressed by doses o f g l y c o p r o t e i n which showed obvious hemagglutination i n h i b i t i o n i n yg. The t e s t system contained a n t i - i (Dench) serum (3 hemagglutination doses) and 1% cord e r y t h r o c y t e s K. Watanabe, S. Hakomori and G.A. Warner, unpublished data).
L-a-Fuc 1—26-Gal 1—4 B-GlcNAc1 B-Gal 1—4 B-GlcNAc 1—3B-Gal 1— 4B-Glc—Ceramide L-a-Fuc1—2B-Gal 1 - 4 B - G l c N A d '
(structure 4)_ (structure 3)
(structure 2 ) . ^structure 1)_ Journal of Experimental Medicine
Figure 1.
Structure of H glycolipid and its degradation products to which antibodies were directed 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20.
YOUNG A N D HAKOMORI
Blood
Group
Carbohydrate
Chains
361
e x t r a c t e d from normal and colon mucosa with a n t i b o d i e s d i r e c t e d against s t r u c t u r e s 1, 2 and h are shown i n Table I I I . The average r e a c t i v i t y with a n t i - s t r u c t u r e h a n t i s e r a was 1 t o l 6 0 f o r normal mucosal t i s s u e and 1 t o 750 f o r cancer t i s s u e . While the antibody d i r e c t e d against s t r u c t u r e h showed t h i s remarkable d i f f e r e n t i a l r e a c t i v i t y between normal and tumor g l y c o l i p i d s , the antibodies d i r e c t e d against s t r u c t u r e 1 and 2 showed s i m i l a r r e a c t i v i t y towards normal and tumor g l y c o l i p i d s . The g l y c o l i p i d s of s e v e r a l cases o f colon carcinoma were compared with those o f normal mucosal t i s s u e by t h i n - l a y e r chromatography (TLC). An i n t e n s i f i e d spot corresponding t o s t r u c t u r e h (GlcNAcBl^GalBl-^U Glc31-^1ceramide) was observed i n the g l y c o l i p i d f r a c t i o n o f colon carcinoma as compared t o normal mucosa. Such a remarkable accumulation o f t h i s s t r u c t u r e i n some human cancers can be regarded as a precursor accumulation. Recently, K a r l s s o n b r i e f l y mentioned the accumulation o f the same g l y c o l i p i d i n human melanoma t i s s u e (25). Sinc f o r a l l blood group ABH t i o n o f t h i s g l y c o l i p i d can be c o r r e l a t e d t o a p r e c u r s o r accumulation due t o a blocked synthesis o f these antigens. 3. Induction o f "incompatible" determinants and Forssman g l y c o lipid. The appearance o f incompatible blood group determinants o f "both the P and ABH systems have been reported (see Table I ) . Because o f the well-known c r o s s - r e a c t i o n s o f the A and Forssman antigens, there was t h e p o s s i b i l i t y t h a t the apparent "neo-A" r e a c t i v i t y t h a t was detected i n tumors o f type 0 p a t i e n t s (l6,17) could have been caused by the presence o f Forssman antigen. The heterogenetic Forssman antigen (29) i s a glycosphingol i p i d whose s t r u c t u r e has been i d e n t i f i e d as GalNAcal-*3GalNAc31"*3 Galal->UGalBl->UGlc-*ceramide (30,31). In the e a r l y l i t e r a t u r e , reviewed by Buchbinder i n 1935 1*32), the various animal species were c a t e g o r i z e d as being e i t h e r Forssman-positive or - negative. It i s now c l e a r t h a t a l l Forssman-positive animal species possess a g l y c o l i p i d with a carbohydrate s t r u c t u r e i d e n t i c a l t o that d e s c r i b e d above (33-36). In a d d i t i o n the immunodeterminant o f the Forssman antigen, namely a t e r m i n a l d i s a c c h a r i d e GalNAcal*3Gal NAc31-*-R, has been found i n other " c a r r i e r " s t r u c t u r e s as w e l l , a l l of which are Forssman a c t i v e : ( l ) a ceramide t e t r a s a c c h a r i d e o f hamster f i b r o b l a s t s , GalNAcal+3GalNAc3l+3Galal+UGal3l+ceramide (37); a p o l y s a c c h a r i d e o f Streptococcus type c (3<8); and a c e r a mide-heptasaccharide and -octasaccharide o f dog g a s t r i c mucosa (3£,U0). I t i s a l s o p o s s i b l e t h a t c e r t a i n g l y c o p r o t e i n s may be Forssman-active, s i n c e g l y c o p r o t e i n s have been r e c e n t l y detected that contain globoside and g a n g l i o s i d e r e a c t i v i t i e s (hi). In the e a r l y l i t e r a t u r e most primates were found t o be Forssman-negative, although humans o f blood groups A and AB were found t o be immunologically Forssman-reactive. These r e s u l t s could have been due t o c r o s s - r e a c t i o n s with blood group A s t r u c t u r e s . However, Forssman has been detected i n c e r t a i n human
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 80
320
1280
Cancer
160
160
160
Normal
80
160
6U0
Cancer
Case 2
80
160
320
Normal
80
160
1280
Cancer
Case 3
80
160
320
Normal
80
160
1280
Cancer
Case k
Journal of Experimental Medicine
Complement f i x i n g r e a c t i v i t i e s o f g l y c o l i p i d s e x t r a c t e d from normal colon mucosa and colon tumors with antibodies t h a t are d i r e c t e d against s t r u c t u r e 1 , 2 , and h o f F i g . 1. Numbers are r e c i p r o c a l s of the d i l u t i o n o f a n t i s e r a t h a t could f i x complement by 1 . 2 yg/50 y l o f g l y c o l i p i d antigen complexed with two times amount o f l e c i t h i n and c h o l e s t e r o l .
160
80
R e a c t i v i t y with anti-structure 2
R e a c t i v i t y with anti-structure 1 (anti-113)
320
R e a c t i v i t y with anti-structure k
Normal
Case 1
Table I I I . Complement f i x i n g r e a c t i v i t i e s o f g l y c o l i p i d s e x t r a c t e d from normal and colon mucosa.
20.
YOUNG AND HAKOMORI
Blood
Group
Carbohydrate
Chains
363
t i s s u e s i n the f o l l o w i n g recent s t u d i e s : ( l ) "by Kawanami i n a human metastatic tumor o f b i l i a r y adenocarcinoma i n l i v e r (1*2); (2) by Makita i n human lung tumor t i s s u e but not i n normal lung ( 1 8 ) ; and ( 3 ) by Hakomori et al ( 1 9 ) i n g a s t r o i n t e s t i n a l t i s s u e . In the l a t t e r r e p o r t Forssman was detected as a normal component of the g a s t r o i n t e s t i n a l mucosa ( F population) i n only 5 o f 21 cases s t u d i e d , while i n the remaining cases Forssman was not detectable i n the normal mucosa (F~ p o p u l a t i o n ) . In tumors derived from these cases the f o l l o w i n g s t r i k i n g p a t t e r n was found: ( l ) a l l tumors d e r i v e d from F~ mucosa possessed Forssman g l y c o l i p i d , while ( 2 ) none o f the tumors o r i g i n a t i n g i n F mucosa contained Forssman g l y c o l i p i d . Globos i d e (GalNAcBl->3Gaiai^l+Gal3l- UGlc- Ceramide), the immediate p r e cursor o f Forssman, was found t o be present i n l a r g e amounts i n both the normal mucosa and tumors o f a l l cases s t u d i e d . Thus, the d i s t r i b u t i o n o f Forssma i th l d t b l i k e that o f an i s o a n t i g e n Forssman i n tumors o f F represent a t u m o r - s p e c i f i c antigen, A summary o f the cases s t u d i e d t o date, a l l o f which come from Taiwan, appears i n Table IV. To f u r t h e r explore t h i s p o t e n t i a l l y e x c i t i n g area, we have begun a thorough i n v e s t i g a t i o n o f the Forssman status o f humans. Relevant aspects o f t h i s problem i n c l u d e the f o l l o w i n g : ( l ) The s u r g i c a l cases described above a l l come from Taiwan. Does such a d i s t r i b u t i o n occur i n other human populations? ( 2 ) In F~ i n d i v i d u a l s i s Forssman ab-sent i n a l l normal t i s s u e s besides the g a s t r o i n t e s t i n a l t r a c t ? This question i s c r u c i a l t o any p o s s i b l e r o l e of Forssman as a t u m o r - s p e c i f i c antigen. Related t o t h i s i s the question o f whether Forssman i s expressed as an embryonic antigen. To conduct such i n v e s t i g a t i o n s r e q u i r e s t e s t i n g t i n y amounts of t i s s u e s i n some cases. T h i s n e c e s s i t a t e s the use o f immunol o g i c a l r a t h e r than chemical methods. L i p i d antigens possess c e r t a i n immunochemical p r o p e r t i e s t h a t g r e a t l y e f f e c t t h e i r r e a c t i v i t y (see f o r reviews j+9 and _50). I n d i v i d u a l g l y c o l i p i d molecules are u n i v a l e n t and amphipathic. They do not form " t r u e " s o l u t i o n s i n water but r a t h e r form m i c e l l a r s o l u t i o n s on d i s p e r s i o n i n an aqueous s o l u t i o n by s o n i c a t i o n and h e a t i n g . M i c e l l a r aggregates o f g l y c o l i p i d s represent m u l t i v a l e n t s t r u c t u r e s which d i s p l a y minimal r e a c t i v i t y with a n t i b o d i e s . T h i s r e a c t i v i t y can be g r e a t l y stimulated by the a d d i t i o n o f " a u x i l i a r y l i p i d s " (most o f t e n a p h o s p h o l i p i d and c h o l e s t e r o l ) t o the g l y c o l i p i d s o l u t i o n p r i o r t o drying and subsequent a d d i t i o n o f b u f f e r . The l i p i d v e s i c l e s or liposomes that are produced create an obvious m u l t i v a l e n t s t r u c t u r e . However, a disadvantage o f such liposome's i s that "because o f t h e i r m u l t i - l a m e l l a r nature only those antigen molecules on the e x t e r n a l surface o f the outermost b i l a y e r (perhaps only 5$ o f the t o t a l antigen) can r e a c t with antibody. To avoid t h i s problem the liposomes can be sonicated u n t i l they reach a n e a r l y u n i l a m e l l a r s t a t e . A l t e r n a t i v e l y , the d r i e d +
+
>
v
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
364
G L Y C O P R O T E I N S A N D GLYCOLIPIDS IN
DISEASE
PROCESSES
l i p i d s can be d i s s o l v e d f i r s t i n hot MeOH or EtOH followed byb u f f e r , which appears to r e s u l t i n maximum antigen exposure as well. An a d d i t i o n a l problem of g l y c o l i p i d antigens i s t h e i r apparent "masking" when i n the presence of a l a r g e amount of other g l y c o l i p i d s . Forssman r e a c t i v i t y could not be detected i n the t o t a l n e u t r a l g l y c o l i p i d f r a c t i o n s of the s u r g i c a l cases ment i o n e d above but became q u i t e strong a f t e r p u r i f i c a t i o n o f the ceramide pentasaccharide f r a c t i o n by Iatrobeads chromatography (19). A s i m i l a r s i t u a t i o n occurred with the H - a c t i v i t y o f Hg l y c o l i p i d (U3) and I i a c t i v i t y of I i - a c t i v e g l y c o l i p i d s (unpublished o b s e r v a t i o n ) . The immunochemical methods most f r e q u e n t l y used f o r g l y c o l i p i d a n a l y s i s i n c l u d e the f o l l o w i n g . Double d i f f u s i o n i n agar g e l i s the simplest but l e a s t s e n s i t i v e method and o c c a s i o n a l l y produces spurious p r e c i p i t i n bands. I n h i b i t i o n of hemagglutinat i o n i s simple, and o f can detect about 1 nanogra able e f f o r t . Release o f trapped markers from g l y c o l i p i d - l i p o somes by antibody-complement (hk) has a s e n s i t i v i t y l i m i t o f about 50 nanograms. Recently we have developed a radioimmunoassay (RIA) f o r d e t e c t i n g g l y c o l i p i d antigens. I n i t i a l attempts t o use r a d i o l a b e l e d g l y c o l i p i d s were u n s u c c e s s f u l because of t r a n s f e r of l a b e l between "hot" and " c o l d " liposomes. The present assay u t i l i z e s a g l y c o l i p i d - p o l y m e r synthesized as shown i n F i g . 2 . F i r s t , the o l e f i n i c double bond i n the ceramide moiety o f the g l y c o l i p i d i s converted to a c a r b o x y l i c a c i d by a recent improvement (U5) of our e a r l i e r procedure (W>). Then the g l y c o l i p i d i s coupled t o a p o l y a c r y l i c hydrazide (PAH) polymer (|+7). Radiol a b e l s can be attached to both the g l y c o l i p i d and PAH moieties as shown i n F i g . 3 . I t should be noted i n passing that such a "polyg l y c o l i p i d " should prove u s e f u l not only f o r d e t e c t i o n o f g l y c o l i p i d antigens as described below but a l s o f o r q u a n t i t a t i o n of a n t i - g l y c o l i p i d s e r a and as a polymeric antigen i n immunization protocols. The RIA procedure i s summarized i n F i g . h. "Cold" g l y c o l i p i d antigen i n the form o f l i p i d v e s i c l e s i s preincubated with d i l u t e d r a b b i t anti-Forssman immunoglobulin. Then the l a b e l e d Forssman-PAH complex i s added and allowed to b i n d t o the remaining a v a i l a b l e a n t i b o d i e s . A d d i t i o n of i n t a c t , f i x e d Staphylococcus aureus, which has " P r o t e i n A" b i n d i n g s i t e s f o r immunog l o b u l i n ( U 8 ) , and c e n t r i f u g a t i o n r e s u l t s i n p r e c i p i t a t i o n of any antigen-antibody complexes. The supernatant can then be counted to determine the amount of i n h i b i t i o n of p r e c i p i t a t i o n o f l a b e l e d antigen by the unlabeled competitor. Figure 5 demonstrates t h a t t h i s assay can r e a d i l y detect 1 nanogram of Forssman, while i n h i b i t i o n by r e l a t e d s t r u c t u r e s (globoside and blood group A g l y c o l i p i d s ) i s n e g l i g i b l e . Figure 6 compares the r e a c t i v i t y of the p u r i f i e d ceramide
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20.
YOUNG AND HAKOMORI
Blood
Group
Carbohydrate
Chains
Table IV. Summary o f Forssman s t a t u s o f Taiwan tumor
Blood type
F"" cases
F
cases
cases
A
h
1
B 0
10 6
1 k
Total
20
6
SYNTHESIS OF "POLY-GLYCOLIPIDS"
Forssman g l y c o l i p i d a c e t a t e
3 ( H-labelled)
KMnO^ , crown e t h e r
"Forssman g l y c o l i p i d
Polyacrylic
acid"
hydrazide
Carbodiimide
H
t
1 4
CH0
NaBH4
Forssman-polyacrylic Figure 2.
hydrazide
Synthesis of a "poly-glycolipid" man-polyacrylic hydrazide
Forss-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
365
366
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
R NH OH 0
0 ,
[ H]GalNAcal+3GalNAc8l+3Gal 3
i H,[ C]NHNHC-CH 14
I
Forssman p o l y a c r y l i c Figure 3.
Step 1.
hydrazide
Structure of Forssman-polyacrylic
hydrazide
Glycolipid/phospholipid/cholesterol vesicles Rabbit anti-Forssman Ig d i l u t e d 1:1000 Incubate 4 ° , 2 hr 14
Step 2.
Step 3.
Figure 4.
3 C/ H 1 F o r s s m a n - p o l y a c r y l i c hydrazide (10,000 cpm per assay tube) Incubate 4 ° , 30 min [
I n t a c t Staphylococcus aureus Incubate 4 ° , 10 min C e n t r i f u g e and count supernatant Protocol for radioimmunoassay of Forssman
glycolipid
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20.
YOUNG AND HAKOMORI
Blood
Group
Carbohydrate
Chains
367
Figure 5. Inhibition of precipitation of [ C/ H]-labeled Forssman-PAH by unlabeled glycolipid antigens. Lipid vesicles of the indicated composition were incubated with anti-Forssman (10 yL, 1/1000 dilution) for 2 hr at 4°C in a total volume of 100 ixL. [ C/ H]-labeled Forssman-PAH (10,000 cpm, 100 pL) was added and incubation continued for 30 min, 4°C. S. aureus (7 mg, 700 fiL) was added and after 10 min at 4°C removed by centrifugation at 100 g for 10 min. 450 fiL of supernatant was counted, and the results are expressed as follows: 14
14
3
3
% Inhibition =
-
fl)
X
1
0
0
where A = supernatant cpm of control tube without anti-Forssman; B = supernatant cpm of control tube with anti-Forssman but no inhibitor; C = supernatant cpm of experimental tube. (X—X) Forssman/SM/CHOL; (O—O) Globoside/SM/ CHOL; and (A—A) A glycolipid/SM/CHOL. Mole ratio glycolipid/ SM/CHOL =1/250/200. h
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. Tissue dry
w e i g h t , ug
Figure 6. Forssman reactivity of purified ceramide pentasaccharide fractions of human gastrointestinal tissues as determined by radioimmunoassay. Purified ceramide pentasaccharide fractions from the normal colonic mucosa (X—X) and colon tumor tissue (O—O) from a Taiwan patient were tested for Forssman reactivity by radioimmunoassay as described in the legend to Figure 5.
100
20.
YOUNG AND HAKOMORI
Blood
Group
Carbohydrate
Chains
369
pentasaccharide f r a c t i o n from the normal mucosa and tumor t i s s u e o f one Taiwan case which by chemical a n a l y s i s had been found t o be F , as d e f i n e d above. +
k.
Concluding remarks In c o n c l u s i o n , examples have been presented i n which s t r i k i n g a l t e r a t i o n s o f b l o o d group determinants occur i n human tumor t i s s u e apparently due t o one o f two mechanisms: (a) accumulation o f p r e c u r s o r s t r u c t u r e s due t o a b l o c k i n s y n t h e s i s o f more comp l e x determinants such as A o r B; and (b) the appearance o f apparently new determinants due t o the p o s s i b l e a c t i v a t i o n o f a l l e l o m o r p h i c genes. Many examples o f the f i r s t p o s s i b i l i t y have been r e v e a l e d , not o n l y i n tumors but a l s o i n in vitro model systems as w e l l , and i s a r a t h e r w e l l - a c c e p t e d mechanism. In cont r a s t , the second process has o n l y l i m i t e d experimental evidence. In f a c t u n t i l r e c e n t l y the o n l y example was a s i n g l e case o f apparent e x p r e s s i o n o f p individual (l]+,15). Now man antigen i n F" i n d i v i d u a l s (19^) may r e s u l t from t h i s process as w e l l . Both processes may represent the e x p r e s s i o n o f o n c o - f e t a l antigens as both the " s t r u c t u r e U" ceramide t r i s a c c h a r i d e (2*0 and Forssman g l y c o l i p i d (K. Watanabe, unpublished o b s e r v a t i o n ) have been d e t e c t e d i n human f e t a l t i s s u e . In a d d i t i o n both processes generate tumor c e l l surface determinants which could serve as s p e c i f i c t a r g e t s f o r immunotherapy or f o r immunochemotherapy.
This study has been supported by N a t i o n a l Cancer I n s t i t u t e Grant CA 1 9 2 2 U , and p a r t i a l l y supported by a grant from the Cancer Research I n s t i t u t e , Inc. W.W.Y. i s r e c i p i e n t o f a f e l l o w s h i p from the Cancer Research I n s t i t u t e , Inc.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
370
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
REFERENCES 1. Hakomori, S., and Murakami, W.T. (1968) Proc. Nat. Acad. Sci. U.S.A. 59, 254. 2. Hakomori, S. (1975) Bioch. Biophys. Acta 4l7, 55. 3. Oh-uti, K. (1949) Tohoku J. Exp. Med. 51, 297. 4. Masamune, H., Yosizawa, Z., Oh-uti, K., Matuda, Y., and Masukawa, A. (1952) Tohoku J. Exp. Med. 56, 37. 5. Masamune, H., Kawasaki, H., Abe, S., Oyama, K., and Yamaguchi, Y. (1958) Tohoku J. Exp. Med. 68, 8 l . 6. Davidsohn, I., Kovarik, S., and Lee, C.L. (l966) Arch. Path. 81, 381. 7. Davidsohn, I., Kovarik, S., and Ni, Y. (1969) Arch. Path. 87, 306. 8. Kay, H.E.H., and Wallace, B.H. (1961) J. Natl. Cancer Inst. 26, 1349. 9. Prendergast, R.C., Tot P.D. d Garguilo A.W (1968) J. Dent. Res. 47, 306 10. Dabelsteen, E . , and Fulling, J.H. (1971) Scand. J. Dent. Res. 79, 387. 11. Iseki, S., Furukawa, K., and Ishihara, K. (1962) Proc. Jap. Acad. Sci. 38, 556. 12. Simmons, D.A.R., and Perlman, P. (1973) Cancer Res. 33, 313. 13. Rosenfelder, G., Young, W.W., J r . , and Hakomori, S. (1977) Cancer Res. 37, 1333. Ik. Levine, P., Bobbitt, O.B., Waller, R.K., and Kuhmichel, A. (1951) Proc. Soc. Exp. Biol. Med. 77, 403. 15. Levine, P. (1976) Ann. N.Y. Acad. Sci. 277, 428. 16. Hakomori, S., Koscielak, J., Bloch, K.J., and Jeanloz, R.W. (1967) J. Immunol. 98, 31. 17. Häkkinen, I. (1970) J. Natl. Cancer Inst. 44, 1183. 18. Makita, A. (1977) United States-Japan Joint Seminar "Structural and Functional Significance of Membrane Glycolipids", 19. Hakomori, S., Wang, S.-M., and Young, W.W., Jr. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3023. 20. Stellner, K., Hakomori, S., and Warner, G.A. (1973) Biochem. Biophys. Res. Commun. 55, 439. 21. Iseki, S., Furukawa, K., and Ishihara, K. (1962) Proc. Jap. Acad. Sci. 38, 556. 22. Cooper, A.G. (1974). Immunol. 113, 1246. 23. Feizi, T., Turberville, C., and Westwood, J.H. (1975) Lancet 1, 391. 24. Watanabe, K., and Hakomori, S. (1976) J. Exp. Med. 144, 644. 25. Karlsson, K.A. In Structure of Biological Membranes (1976), S. Abrahamsson and I. Pascher (eds.) 34th Nobel Symposium, New York, Plenum Publishing. 26. Feizi, T., Kabat, E.A., Vicar, G., Anderson, B., and Marsh, W.L. (1971) J. Immunol. 106, 1578. 27. Krupey, J., Wilson, T., Freedman, S.O., and Gold, P. (1972) Immunochemistry 9, 6l7.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
20. YOUNG AND HAKOMORI
Blood Group Carbohydrate Chains 371
28. Niemann, H., Watanabe, K., Hakomori, S., Childs, R.A., and Feizi, T. submitted for publication. 29. Forssman, J. (1911) Biochem. Z. 37, 78. 30. Siddiqui, B., and Hakomori, S. (1971) J. Biol. Chem. 246, 5766. 31. Stellner, K., S a i t o , and Hakomori, S. (1973) Arch. Biochem. Biophys. 155, 464. 32. Buchbinder, L. (1935) Arch. Pathol. 19, 841. 33. Sung, S.J., Esselman, W.J., and Sweeley, C.C. (1973) J. Biol. Chem. 248, 6528. 34. Taketomi, T., Hara, A., Kawamura, N., and Hayashi, M. (1974) J. Biochem. (Tokyo) 75., 197. 35. Ziolkowski, C.H.J., Fraser, B.A., and Mallette, M.F. (1975) Immunochemistry 12, 297. 36. Karlsson, K.-A., Pascher, I., Pimlott, W., and Samuelsson, B.E. (1974) Biomed Mass Spectrometry 1 49. 37. Gahmberg, C.G., an 2438. 38. Coligan, J . E . , Fraser, B.A., and Kindt, T.J. (1977) J. Immunol. 118, 6. 39. Slomiany, A., Slomiany, B.L., and Annese, C. (1977) FEBS Letters 81, 157. 40. Slomiany, A., and Slomiany, B.L. (1977) Eur. J. Biochem. 76, 491. 41. Tonegawa, Y., and Hakomori, S. (1977) Biochem. Biophys. Res. Commun. 76, 9. 42. Kawanami, J. (1972) J. Biochem. (Tokyo) 72, 783. 43. Stellner, K., Watanabe, K., and Hakomori, S. (1973) Biochemistry 12, 656. 44. Six, H.R., Young, W.W., J r . , Uemura, K., and Kinsky, S.C. (1974) Biochemistry 13, 4050. 45. Young, W.W., Jr., Laine, R.A., and Hakomori, S. Meth. Enzymol. in press. 46. Laine, R.A., Yogeeswaren, G., and Hakomori, S. (1974) J. Biol. Chem. 249, 4460. 47. Wilchek, M., and Miron, T. (1974) Meth. Enzymol. 34, 72. 48. Kessler, S.W. (1976) J. Immunol. 117, 1482. 49. Rapport, M.M., and Graf, L. (l969) Prog. Allergy 13, 273. 50. Marcus, D.M., and Schwarting, G.A. (1976) Advances in Immunology 23, 203. RECEIVED
March 15, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21 Role of Glycoconjugates in Expression of the Transformed Phenotype S. STEINER, D. VIA, M. KLINGER, G. LARRIBA, and S. SRAMEK Department of Virology, Baylor College of Medicine, Houston, TX 77030 R. LAINE Department of Biochemistry, University of Kentucky, Lexington, KY 40506 Recent studies hav glycolipids as being o importanc y proper ties associated with oncogenic transformation, e.g., altered antigenicity, decreased cellular adhesiveness, and altered lectin agglutinability. Moreover, in human tumors the blood group isoantigens, which are fucosyl glycolipids, are decreased (Hakomori and Jeanloz, 1964), as are the glycosyl transferases involved in the synthesis of these fucosyl glycolipids (Stellner et a l . , 1973). In contrast, the level of a simple and novel fucolipid is enhanced (Watanabe et a l . , 1976). Changes in fucoproteins in human tumors (Simmons and Pearlman, 1973) and in trypsin-sensitive surface fucopeptides in a variety of transformed cells (e.g., Buck et al. 1970; Pietropaolo et a l . , 1977) have also been observed. Furthermore, alterations in the serum level of fucosyl transferase in patients with tumors has been reported (Bauer et a l . , 1977; Kessel et a l . , 1977). These observations and related ones which suggested possible differences in the metabolism of fucosylated components in cancer cells as compared to normal cells prompted studies in this laboratory of changes in fucose metabolism in virus-transformed cells as compared to normal cells. These studies initially focused on the small molecular weight, organic-extractable fucosylated components. Murine sarcoma virus-transformed rat (MSV-NRK) cells and their nontransformed counterparts (NRK) were grown in medium supplemented with either [ H]- or [ C] fucose. The cells were extracted either with CHCl-CHOH (2:1, v/v) or 60% ethanol (v/v), and the extract chromatographed on AG50, H+ form, followed by silicic acid thin-layer chromatography (TLC). The distribution of radioactivity on the thin-layer chromatogram is shown in Figure 1. The normal cells have two prominent peaks of radioactivity, i.e., component FL4 and FL3. In contrast, in the MSV-transformed rat cells there is a marked decrease in the incorporation of labeled fucose into FL4. This decreased labeling of FL4 is approximately five-fold and is evident when either [ C]- or [ H] fucose is used. Moreover, all normal mammalian cells thus far examined 3
14
3
3
14
3
0-8412-0452-7/78/47-080-378$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
379
(human, mouse, r a t , baboon, monkey and hamster) contain FL3 and FL4. In a d d i t i o n , many d i f f e r e n t transformed c e l l s , e . g . , mouse mammary carcinoma, simian v i r u s 40-transformed c e l l s , herpes simplex virus-transformed c e l l s , manifest a decrease i n l a b e l i n g o f FL4 ( S t e i n e r e t a l . , 1973, 1974). The decreased i n c o r p o r a t i o n o f label i n t o FL4 appears to be more pronounced i n h i g h l y tumorigeni e (as compared to weakly or nontumorigenic) simian v i r u s 40transformed c e l l s , herpes simplex virus-transformed c e l l s ( S t e i ner and S t e i n e r , 1975/76), o r mouse mammary carcinoma c e l l s (unpublished o b s e r v a t i o n s ) . Based on these o b s e r v a t i o n s , s t u d i e s were undertaken to examine, i n a temporal manner, the r e l a t i o n s h i p between decreased i n c o r p o r a t i o n of l a b e l e d fucose i n t o FL4 and a l t e r a t i o n s in the metabolism o f other glycoconjugates as a f u n c t i o n o f the expression o f the transformed c e l l phenotype. We have observed that growth o f MSV-NRK c e l l s i n medium supplemented with 2 mM sodium butyrate r e s u l t s i n c e l l u l a the MSV-NRK c e l l s resembl (Figur 2). Moreover, the process i s f u l l y r e v e r s i b l e upon s h i f t o f c e l l s to standard medium. There i s a l s o an e l a b o r a t i o n o f the c y t o s k e l e t a l elements, i . e . , microfilaments and m i c r o t u b u l e s , i n these treated c e l l s (Altenburg e t a l . , 1976). Along with the morphologi c a l changes, there i s a t h r e e - f o l d increase i n the l e v e l o f P H ] fucose incorporated i n t o FL4 (Table I ) . The increased l a b e l i n g i s observed i n MSV-NRK c e l l s grown i n butyrate-supplemented medium f o r short (1 passage) and long (30 passages) periods o f time. Furthermore, s h i f t o f b u t y r a t e - t r e a t e d MSV-NRK c e l l s to c o n t r o l medium r e s u l t e d i n r e v e r s a l to the l e v e l o f c o n t r o l c e l l s . We a l s o examined the e f f e c t s o f sodium butyrate on the gangl i o s i d e composition o f the MSV-NRK c e l l s . As i n d i c a t e d i n Table I I , growth o f MSV-NRK c e l l s i n sodium butyrate-supplemented medium r e s u l t s i n an increase i n both hematoside GM3 and i n the d i s i a l o g a n g l i o s i d e G D ^ . The increased l e v e l s o f hematoside and GD-i^ were observed w i t h i n a s i n g l e passage i n sodium b u t y r a t e supplemented medium, although the maximal l e v e l o f hematoside was not observed f o r several passages. Another butyrate-induced e f f e c t i s the marked increase i n GD]/\ i n normal (NRK) c e l l s (Table II). The elevated l e v e l o f GD-|/\ i n NRK c e l l s (which are already f l a t t e n e d ) was p a r a l l e l e d by a change i n the o r g a n i z a t i o n o f the microfilaments as examined by i n d i r e c t immunofluorescent microscopy with a n t i - a c t i n . The microfilaments s h i f t e d from a random d i s t r i b u t i o n to a p a r a l l e l d i s t r i b u t i o n ( V i a and S t e i n e r , unpublished data). An increase i n the average s i z e d i s t r i b u t i o n o f t r y p s i n - s e n sitive surface fucopeptides has been observed i n a wide range o f transformed c e l l s as compared to normal c e l l s . This increase i s evident i n the MSV-NRK c e l l s as compared to NRK c e l l s (Figure 3A). There i s l i t t l e detectable s h i f t i n the molecular weight d i s t r i b u t i o n o f these components i n the f i r s t - p a s s a g e sodium b u t y r a t e t r e a t e d MSV-NRK c e l l s . By passage 2 i n sodium butyrate-supplemen-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Figure 1. Aminoacyl fucosides of NRK and MSV-NRK cells, (top) NRK cells; (bottom,) MSV-NRK cells; 3 — FL3; 4 = FL4. Cells were grown in [ H]fucose-supplemented medium for 72 hr, harvested by scraping into 60% ethanol (v/v), and extracted thrice in 60% ethanol at 100°C. The pooled extracts were subjected to column chromatography on AG50 H and the bound fraction eluted with 0.5N NHfiH. The NH^OH eluant was dried and chromatographed on plates in CHCU-CHsOH-NHfiH by volume). The distribution was determined by scraping 0.5-cm fractions from origin to solvent front. Radioactivity was quantitated with a scintillation spectrometer.
PROCESSES
3
+
0
5 10 15 FRACTION NUMBER
Figure 2. Morphological changes induced by sodium butyrate. MSV-NRK cells were cultured in control medium, i.e., untreated, and in 2mM sodium butyrate-supplemented medium for 24, 48, 72, and 96 hr and for 16 passages.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3342
0.3
0.9
0.9
0.3
1.6
FL4/FL3
[^H]fucose l a b e l i n g , h a r v e s t i n g and a n a l y s i s o f FL3a and FL4a, see Figure 1.
* Average o f two separate experiments done i n d u p l i c a t e .
For d e t a i l s of
MSV-NRK
999
3690
30
MSV-NRK
reversal, 3 passages no sodium butyrate
4334
3732
1
MSV-NRK 4167
3062
3276
FL3 cpm/ mg p r o t e i n *
5320
FL4 cpm/ mg p r o t e i n *
1006
No. o f passages i n sodium butyrate
Fucosides o f MSV-NRK C e l l s
0
line
E f f e c t o f Sodium Butyrate on the Aminoacyl
MSV-NRK
NRK
Cell
Table I .
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
382
PROCESSES
FRACTION NUMBER
FRACTION NUMBER
Figure 3. Sephadex G-50 fucopeptide profile of 2mM "butyratetreated MSV-NRK cells. Exponentially growing cells labeled with radioactive fucose for 48 hr were used in all experiments. Fucopeptides were prepared and analyzed by the general method described by Buck and co-workers (1970). (A) H-labeled (*) NRK cells vs. C-labeled (O) control MSV-NRK cells. (B) H-labeled (%) MSV-NRK cells passaged twice in butyrate medium vs. C-labeled (O) control MSV-NRK cells. 3
14
3
14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
BD
FRACTION NUMBER
(C) H-labeled (•) MSV-NRK cells passaged six times in butyrate medium vs. C-labeled (O) control MSV-NRK cells. (D) H-labeled (%) MSV-NRK cells passaged 19 times in butyrate medium vs. C-labeled (O) control MSV-NRK cells. The phenol red dye marker eluted at fractions 136-146 in column A, fractions 123-133 in column B, fractions 122-132 in column C, and fractions 125-135 in column D. Radioactivity was not detected in fractions beyond those shown in the figure. Labeling experiments with reversed radioisotopic forms of fucose, i.e., H and C, gave comparable results. 3
14
3
14
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
14
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 1
19
MSV--NRK + SB
MSV--NRK + SB
3
7.4
|16.1|
lll.2|
7.8
16.0
16.8
GM 2
6.3
6.2
6.5
6.7
3.0
2.4
GM
2
trace
trace
trace
trace
ND
ND
GMj
Ganglioside nmoles NANAV10 mg c e l l 1A
1.4
|2.4|
/2T2l
1.2
15.81
ND
GD
protein 1B
0.3
0.2
0.3
0.4
ND
ND
G D
3
1
N-acetylneuraminic a c i d detected by r e s o r c i n o l reagent, r e l a t i v e amount o f NANA in each component determined by densitometric scanning. 2 ND = Not d e t e c t e d . L i p i d s were extracted twice in CHCI3-CH3OH ( 2 : 1 , v/v) followed by two e x t r a c t i o n s i n CHCI3-CH3OH ( 1 : 2 , v / v ) . The l i p i d e x t r a c t was d r i e d , resuspended i n CHCI3-CH3OH ( 2 : 1 , v/v) and p a r t i t i o n e d according to the method o f Folch and coworkers (1957). The Folch upper phase was d r i e d , resuspended in a small volume o f H2O and d i a l y z e d . The sample was c h r o matographed on s i l i c i c a c i d t h i n - l a y e r plates (Q5, Quantum I n d u s t r i e s , F a i r f i e l d , N . J . ) i n CHC13-CH30H-H20 (60:35:8, by v o l ) . The i n d i v i d u a l g a n g l i o s i d e s were i d e n t i f i e d by comparison with authentic standards.
MSV--NRK
0
MSV -NRK
reversal (3 passages no sodium butyrate)
1
NRK + SB
No. passages in sodium butyrate
0
line
E f f e c t o f Sodium Butyrate on the Ganglioside Composition o f Murine Sarcoma Virus-Transformed NRK C e l l s as Compared with Normal NRK C e l l s
NRK
Cell
Table I I .
21. STEINER ET AL.
Glycoconjugates and the Transformed Phenotype
ted medium, a small shift can be observed (Figure 3B). By passage 6 (Figure 3C) in sodium butyrate-supplemented medium, there is a marked increase in the molecular weight distribution of the trypsin-sensitive surface fucopeptides to a distribution comparable to that of normal cells (Figure 3A). Thus, the shift in the size distribution of the trypsin-sensitive surface fucopeptides in butyrate-treated MSV-NRK cells not only appears to follow cellular flattening but also follows the increased incorporation of Ph]fucose into FL4 and the increased levels of the gangliosides GM3 (hematoside) and GDia. In order to understand the biochemical basis for the reduced level of FL4 in transformed cells, it was necessary to know the structures of FL3 and FL4. The initial examination involved indirect analyses with radioisotopically labeled FL3 and FL4 from cultured cells. These analyses indicated that these compounds had a positive charge, could be extracted with chloroform-methanol and might possess a lon ever, because of the difficultie structure by indirect means, an isolation method for the purification of sufficient quantities for direct chemical characterization was developed (Figure 4). Four- to six-week old rats were injected intraperitoneal^ with [ C]fucose. The liver, brain, intestine and kidneys were removed and extracted thrice with 60% ethanol . [ H]FL3 and [3h]FL4, purified from cell culture, were added to the extract to monitor for the fractions of interest during the purification and to evaluate the final recovery of FL3 and FL4. The ethanol-soluble fraction was chromatographed on Sephadex G-25, followed by high-voltage electrophoresis and descending paper chromatography. This last step separates FL3 from FL4, which in turn were chromatographed on silicic acid thin-layer plates and were each observed to be separable into two components, i.e., FL3a and FL3b, FL4a and FL4b. The purity of each component was examined in several TLC systems. Gas-liquid chromatography and an amino acid analyzer were used to identify the constituents of each component. The compounds were subjected to HC1methanolysis, silylated and the carbohydrates analyzed by gasliquid (GC) chromatography (Figure 5). The GC results indicated the presence of fucose in the FL3 series (as illustrated in Figure 5C for FL3b) and both fucose and glucose in the FL4 series (as illustrated in Figure 5D for FL4a). No other carbohydrate residues were observed (Figure 5A) nor did we observe the presence of long chain base (Figure 5B) or fatty acid (Larriba et a l . , 1977). Since these compounds contain a positively charged moiety, the possibility that there is an amino acid constituent was considered. Each component was subjected to strong acid hydrolysis and analyzed with an amino acid analyzer (Table III). FL3a contained threonine; FL3b, serine; FL4a, threonine; and FL4b, serine. The molar ratios of the amino acid and carbohydrate are indicated in Table IV. As can be seen, FL3a contains fucose and threonine; FL3b, fucose and serine; FL4a, glucose, fucose and threonine; and 14
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS
386
A N D GLYCOLIPIDS
I N DISEASE
PROCESSES
[ C ] f u c o s e - l a b e l e d rat tissue 14
[ H ] f u c o s e - l a b e l e d F L 3 and F L 4 f r o m cultured cells Extract Centrifugation
r
ellet
Supernatant fraction Sephadex G - 2 5 High voltage e l e c trophoresis, pH 2 . 0 (2 M acetic acid) i Descending paper chromatography (ethyl acetate-acetic a c i d - H 2 0 3:1:1, by vol) I
1
F L 3 (a and b)
F L 4 (a and b) Silicic acid T L C
(CHCI0-CH3OH-NH4OH,
40:80:25, by vol)
1
FL4a
FL4b
FL3a
FL3b
s l i c i c a c i d T L C ^ ( b u t a n o l - p y r i d i n e - H O } 6:4;3, by v o l / 0
I FL4a
I
FL4b
\
FL3a
P u r i t y examined i n a variety of T L C s y s t e m s methods: c h a r r i n g , o r c i n o l and n i n h y d r i n ) .
Figure 4.
1
FL3b (detection
Purification of aminoacyl fucosides from rat tissue
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
STEINER E T A L .
Glycoconjugates
RETENTION
TIME
and the Transformed
Phenotype
frnlnules)
Biochemical and Biophysical Research Communications
Figure 5.
Gas-liquid chromatography of FL3b and FL4a.
(A) Top profile: equimolar standard mixture of monosaccharides. (B) Second profile from top: lactosylceramide. (C) Third profile from top: FL3b. (D) Bottom profile: FL4a. Peaks correspond to the following compounds: 1, 2, 3, fucose; 4, arabinotol; 5, 6, 7, galactose; 8, 9, glucose; 10,11, mannitol; 12,13, N-acetylgalactosamine; 14, N-acetylglucosamine; 15, N-acetylneuraminic acid; and 16, dihydrosphingosine. Samples were methanolyzed, N-acetylated and silylated (Larriba et al, 1977). 1-2 fiL of each sample was then injected onto six-foot columns packed with 3% OV-1 on 80/100 Supelcoport in a Packard Becker model 419 dual column gas chromatograph withflameionization detector. N flow rate (effluent), 30 mL/min. Injector and detector temperatures, 270°C. Temperature program: temperature maintained at 160°C for 5 min, then raised 5°/min to 250°C, and maintained at 250°C for 15 min. Peak areas were integrated using a Spectra-Physics Autolab System I computing integrator. 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
388
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE PROCESSES
Table I I I . Amino Acid A n a l y s i s o f Aminoacyl Fucosides
Compound-
1
After hydrolysis
Before hydrolysis
FL3a
21.9
43.0
FL3b
23.6
46.2
FL4a
16.0
42.9
FL4b
ND2
46.3
Serine-*
--
46.8
Threonine-*
—
43.3
Samples wer 110 C f o r 24 h r , concentrated by f l a s h e v a p o r a t i o n , resuspended i n a small volume (0.05-0.1 ml) o f 0.11 M sodium c i t r a t e , pH 3 . 2 , and chromatographed on a Beckman 121 amino a c i d analyzer using a s i n g l e column, p h y s i o l o g i c a l f l u i d program. ND = Not done. 3 The amino acids with c l o s e s t r e t e n t i o n times to s e r i n e and threonine are a s p a r t i c a c i d (40.6) and p r o l i n e ( 6 5 . 9 ) . 1
2
Biochemical and Biophysical Research Communications
OH O- ^-D-glucopyranosyl-(l-**3)-0- cC-L-fucopyranosyl-L-threonine Figure 6.
Structure of O-p-D-glucopyranosylfl-*
3)-0-
«-1,-fucopyranosyl-L-ihreonine
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 0
137
53
9
FL3b
FL4a
FL4b
0
46
0
84
Threonine-*
1.1
8
1.4
1.2
1.2
0
0 0
1.1
0
1.0
0
1.0
1.0
0
1.0
0
Fuc / Glc / Thr / Ser
1.1
120
0
Serine^
!
3
Biochemical and Biophysical Research Communications
1 For d e t a i l s o f a n a l y s i s o f carbohydrate and amino a c i d s , see Figure 5 and Table I I I , respectively. 2 The nmoles o f amino a c i d and carbohydrate represent the t o t a l p u r i f i e d material r e c overed from the r a t t i s s u e s . As judged by exogenously added FL3a and FL4a from c u l tured c e l l s , recovery o f FL3a from r a t t i s s u e was approximately 16.0% and recovery o f FL4a was approximately 10.0%. The amount o f threonine and s e r i n e i s not c o r r e c t e d f o r breakdown (^10%) due to strong a c i d h y d r o l y s i s .
11
56
0
GI ucose
91
Fucose
nmoles^
FL3a
Compound
Table IV. Molar Ratios o f Carbohydrate and Amino Acids o f FL3a, FL3b, FL4a and FL4b
390
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE
PROCESSES
FL4b, g l u c o s e , fucose and s e r i n e ; a l l in an approximate r a t i o o f 1:1. Like FL4a, which i s the f u c o s e - l a b e l e d component designated as FL4 i n the e a r l i e r l a b e l i n g s t u d i e s , a u r i n a r y f u c o s i d e , which contains f u c o s e , glucose and t h r e o n i n e , has r e c e n t l y been desc r i b e d (Hallgren et a l . , 1975). This u r i n a r y f u c o s i d e has the s t r u c t u r e shown in Figure 6, i n c l u d i n g the novel feature o f an i n ternal fucose 0 - g l y c o s i d i c a l l y l i n k e d to amino a c i d . GC/mass spectrometry studies were undertaken to determine the carbohydrate sequence and linkage o f FL4a. Synthetic f u c o s i d e , o f the same s t r u c t u r e as the u r i n a r y f u c o s i d e , was most generously supplied by Dr. Svensson ( U n i v e r s i t y H o s p i t a l , Lund, Sweden; synt h e s i z e d by Drs. Garegg and Norberg, 1976) and was used as a s t a n dard. FL4a and the s y n t h e t i c fucoside were permethylated and subj e c t e d to GC/mass spectrometric a n a l y s e s . A GC/mass fragmentogram i n d i c a t e s that four fragments (m/e 393, 329, 219 and 187) are obtained from both permethylated s y n t h e t i c f u c o s i d e (Figure 7A) and permethylated FL4a fro and s y n t h e t i c fucoside ha Moreover, the fragments m/e 219 and m/e 187 are c o n s i s t e n t with the presence of terminal hexose and m/e 393 and m/e 329 are cons i s t e n t with a d i s a c c h a r i d e i n which 6-deoxyhexose (fucose) i s i n ternal and hexose i s t e r m i n a l . Neither component had an observable fragment, m/e 189, t y p i c a l of terminal f u c o s e . The s t r u c t u r e of the permethylated compounds was f u r t h e r examined f o l l o w i n g methanolysis and a c e t y l a t i o n by a n a l y s i s of the p a r t i a l l y methylated a l d i t o l a c e t a t e s . Each compound gave two peaks with major fragment m/e 262 and m/e 264, r e s p e c t i v e l y . As i n d i c a t e d in Table V I , the m/e 262 peak from d e r i v a t i z e d FL4a and s y n t h e t i c f u coside had the same r e t e n t i o n times. S i m i l a r l y , the peaks which gave a major fragment m/e 264 from d e r i v a t i z e d FL4a and s y n t h e t i c fucoside had the same r e t e n t i o n times. The mass spectra of the p a r t i a l l y methylated a l d i t o l acetates from FL4a and s y n t h e t i c f u coside were compared. The components from FL4a and the s y n t h e t i c f u c o s i d e with a major peak m/e 262 a l s o had fragments m/e 290, 118 and 131 (Figure 8 ) . These fragments are c o n s i s t e n t with an i n t e r n a l 6-deoxyhexose (Figure 9 ) . The mass spectra of the components from FL4a and s y n t h e t i c fucoside with a major fragment m/e 264 a l s o y i e l d e d fragments m/e 292, 118, 161 and 162, which i s c o n s i s t e n t with a terminal hexose (Figure 10). The combined GC, amino a c i d a n a l y s i s , and GC/mass spectrometry data i n d i c a t e that FL4a has a s i m i l a r s t r u c t u r e , i . e . , glucosyl (1—^3) f u c o s y l — • t h r e o n i n e , to that o f the u r i n a r y f u c o s i d e . The novel s t r u c t u r e of t h i s component, i . e . , with a fucose moiety which appears to be both i n t e r n a l and O - g l y c o s i d i c a l l y l i n k e d to amino a c i d , r a i s e s a question o f i t s b i o s y n t h e t i c o r i g i n . I f FL4a i s derived from f u c o p r o t e i n which has fucose 0 - g l y c o s i d i c a l l y l i n k e d to amino a c i d , then i t should be p o s s i b l e to r e l e a s e a d i s a c c h a r i d e which contains g l u c o s y l f u c i t o l by a l k a l i n e borohydride treatment o f that f u c o p r o t e i n ( s ) . An e t h a n o l - i n s o l u b l e f r a c t i o n of f u c o s e - l a b e l e d c e l l s was subjected to a l k a l i n e boro-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
391
m/e 393.2
219.1
187
i
i""""l 100 J
I""""'! I'l'l"'"""!'""""!" 200 300 400
|'i
Figure 7. Mass fragmentogram of permethylated synthetic glucosyl fucosyl threonine and FL4a. (A) Authentic glucosyl fucosyl threonine. (B) FL4a. FL4a and synthetic glucosyl fucosyl threonine were permethylated (Hakomori, 1964) and analyzed by GC/ mass spectrometry. A preliminary total mass scan was used to detect the major ions of interest. GC parameters: OV-101 column at 175°C, N carrier gas. Mass parameters: Finnagen chemical ionization; cell volts, 34.2; lens volts, —0031; EM volts, —2318; lost mass, —0640; first mass, +0099; emission mass, +00.59; collective mass, -\-00.02; ion voltage mass, +00.58; elect, energy, +0150; ion energy, +011.9, —014.6; extr. volts, +008.6. 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
392
PROCESSES
Table V. Gas-Liquid Chromatographic Retention Times o f Permethylated Compounds Compound
Time (min)
S y n t h e t i c 0-/?-D-glucopyranosyl(1—^3)-0-oC-L-fucopyranosyl FL4a
3.83 3.83
FL4a and s y n t h e t i c glucosyl fucosyl t h r e o nine were d e r i v a t i z e d and analyzed as described i n Figure 7. The permethylated compounds were detected by scanning f o r the fragments m/e 393.2, 329.1, 219.1, and 187 which may be derived as i n d i c a t e d below hexose - jfucose
threonine
219 4,
187
Table V I .
! !
393 i 329
Gas-Liquid Chromatography Retention Times o f P a r t i a l l y Methylated A l d i t o l Acetates
Parent compound
Fragment used f o r d e t e c t i o n (m/e)
Retention time (min)
FL4a Synthetic
glc-fuc-thr
262 262
3.92 3.92
FL4a Synthetic
glc-fuc-thr
264 264
4.42 4.42
P a r t i a l l y methylated a l d i t o l acetates o f FL4a and s y n t h e t i c g l u cosyl fucosyl threonine were prepared by a c i d i c methanolysis o f the methylated compound followed by treatment with t r i f l u o r o a c e t i c a c i d , reduction with sodium borodeuteride and a c e t y l a t i o n with acet i c anhydride. The p a r t i a l l y methylated a l d i t o l acetates were analyzed by GC/mass spectrometry. The major ion o f each p a r t i a l l y methylated a l d i t o l acetate was determined in a p r e l i m i n a r y mass scan and used to d e t e c t the r e s p e c t i v e p a r t i a l l y methylated a l d i t o l acetates. GC parameters: 0V-101 column at 120 C. Mass parameters: as in Figure 7.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21.
STEINER
ET AL.
Glycoconjugates
and the Transformed
Phenotype
393
262
100
r25
118 • 50
290
131 150
100
200
300
250
350
262
100
118 131 290 50
iiUJuiiuXk 100
150
I, ,1a j u , . i t . i U ,i I , , j,i,lu-i.iJi.11., m , » i 350 200 250 300
Figure 8. Total mass spectra of partially methylated alditol acetates obtained from glucosyl fucosyl threonine and from FL4a. (A) Total mass spectrum of partially methylated alditol acetate, with a major ion m / e 262 from glucosyl fucosyl threonine. (B) Total mass spectrum of partially methylated alditol acetate, with the same retention time as in (A), from FL4a. For experimental details see Table VI.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
394
I
"(total ion) MH-59
"323 129^^31 (OCH )\
? 9 CH-OC-CH,
(CH3C cb) O)
3
3
C H - O - C H ^ _ _ 118 CH-0-CH CH-O-CH CH
-o-c?c
CH -0-CH 2
3
T e r m i n a l hexose ^^ l E&3<<"-31 \ 32
D
I
O
CH-O-C-CH3 CH-O-CH3
( t o t a l
(OCHo)\ .
,
i
o
n
)
MH-59
O
(CH (X» 3
^
CH-O-C-CH. CH-0-CH
Li^l
3
CH-0-C*?CH_ I
C H
6
3
Internal 6-deoxyhexose
Figure 9. Fragmentation patterns for 2,3,4,6-tetraO-methyl-1,5 -di-O-acetyl-1-deutero-glucitol (terminal hexose) (top) and 2,4-di-0-methyl-l,3,5-tri-0acetyl-1 - deutero-fucitol (internal 6-deoxyhexose) (bottom)
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
21.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
395
264
100
rl5
I
SO
jUlill
100
rt
XOl/JL
150
Ji
1,1,1
1,1. Jl 1,111, 1,1 D U 1 200
250
300
350
Figure 10. Total mass spectra of partially methylated alditol acetates obtained from glucosyl fucosyl threonine and from FL4a. (A) Total mass spectrum of partially methylated alditol acetate with a major ion m / e 264 from glucosyl fucosyl threonine. (B) Total mass spectrum of partially methylated alditol acetate, with the same retention time as in (A), from FL4a. For experimental details see Table VI.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE
396
[ H]fucose-labeled J
PROCESSES
etha n o l - i n s o l u b l e f r a c t i o n NaOH NaBH^
Silicic acid thin-layer (CHC1 -CH 0H-H 0, 3
3
2
treatment
chromatography 60:35:8,
by vol)
1 Fucitol
Higher
molecular
+
weight
components
Disaccharidesized
(no d e t e c t a b l e
components
released
fucitol
following
m i l d acid treatment)
Cellulose T L C (butanol-ethanol-H2O, 10:1:2, Fucitol +
by vol) disaccharide-sized component (releases fucitol following m i l d a c i d treatment)
Figure 11.
Alkaline borohydride treatment of [ H]fucose-labeled tein fraction 3
glycopro-
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
Figure 12. Chromatographic comparison of "disaccharide" from alkaline borohydride-treated glycoprotein and "glucosyl fucitol" from FL4a. The disaccharide component was from alkaline horohydride-treated [ H] fucose-labeled protein. The glucosyl fucitol was obtained by mild acid hydrolysis of FL4a (0.1N HCl, 100° C, 1 hr) followed by reduction with 0.5M NaBIL, (37° C, 2 hr). These samples were chromatographed on silicic acid thinlayer plates in solvent I (CHCh-CH OH-H 0, 60:35:8, by volume), eluted, and chromatographed in solvent II (butanol-ethanol-H 0, 10:1:2, by volume), eluted, and chromatographed in solvent III (butanol—propionic acid-H 0, 6:3:4, by volume). Chromatography of reduced disaccharide (bottom) from FL4a and disaccharide from fucoprotein (top) in solvent III is shown. The two components had comparable chromatographic mobilities in all three solvents. 3
3
2
2
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
397
398
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
100
X
6 a u i
100
-
30
40 Fraction Number
Figure 13. Gel filtration analysis of "disaccharide" material from glycoprotein and from FL4a. (A) Sephadex G-10 chromatography of reduced disaccharide from [ H]fucose-labeled FL4a (see Figure 12). (B) Sephadex G-10 chromatography of disaccharide obtained from sodium borohydride-treated, [ H] fucose-labeled protein. The arrows indicate the elution volumes of the standards: S, stachyose; R, raffinose: L, lactose; and F, fucose. Sephadex G-10 chromatography was carried out on 0.8 X 70 cm columns in 0.2M ammonium acetate at pH = 7.0 and 0.8-mL fractions were collected. The position of the authentic sugars was detected by spotting a portion of each fraction on silicic acid plates followed by chromatography of the samples in butanolpyriaine-H 0 (6:4:3, by volume) and estimation of the position and relative amount of each sugar with orcinol. Radioactivity was used to locate the disaccharide components. 3
3
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
STEINER E T A L .
Glycoconjugates
and the Transformed
Phenotype
Figure 14. Acid hydrolysis of [ H]-labeled disaccharide component. Before hydrolysis: (%—•) purified [ H]-labeled disaccharidesized material from [ H]fucose-labeled protein (see Figure 8); (O—O) authentic C-fucitol. After hydrolysis: (%—•) disaccharide-sized material following acid hydrolysis (IN HCl, 100°C, 1 hr); (O—O) authentic C-fucitol added to hydrolysis mixture. Chromatography in butanol-ethanol-H 0 (10:1:2, by volume). 3
3
3
14
14
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
400
T i m e (hr) Figure 15. Pulse-labeling of NRK cells with [ H] fucose. NRK cells were pulse-labeled with [ H]fucose for 2, 4, 6, 10, and 24 hr and the radioactivity incorporated into (top) GDP-fucose (O—O) and fucoprotein (%—•) and (bottom) FL4 (O—O) and "disaccharide" from alkaline borohydride-treated protein fraction (•—•) measured. Cells were extracted in 60% ethanol (v/v). The ethanol extract was used for the analysis of FL4 (Figure 11) and GDP-fucose (descending paper chromatography, ethanol-lM ammonium acetate, pH = 7, 7:3, v/v). The radioactivity in the ethanol-insoluble fraction was used as a measure of labeling of fucoprotein. The disaccharide was obtained by alkaline borohydride treatment (see Figure 12) of a portion of the fucoprotein fraction. 3
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
STEINER E T A L .
Glycoconjugates
2
and the Transformed
4 6 Migration distance (cm)
Phenotype
8
Figure 16. Alkaline borohydride treatment of [ H]fucoselabeled proteins from normal (human embryonic lung) and transformed (A-204; human rhabdomyosarcoma) cells. Cells were labeled with [ H]fucose for 72 hr, harvested, extracted, and protein-treated with alkaline borohydride as described in Figure 12. Samples were chromatographed in solvent I. The arrow (I) indicates the position of standard glucosyl fucitol obtained from FL4a (Figure 12). The other arrow indicates the position of standard fucitol. Samples and standards also co-chromatographed in solvents II and III (Figure 12). The radioactivity in the peak labeled "disaccharide" quantitatively yielded fucitol upon mild acid hydrolysis as described in Figure 14. 3
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
402
G L Y C O P R O T E I N S A N D GLYCOLIPIDS IN DISEASE
PROCESSES
hydride treatment, i . e . , ( 3 - e l i m i n a t i o n r e a c t i o n (Figure 11). The products were analyzed, a f t e r AG50 H column chromatography, by s i l i c i c a c i d TLC and c e l l u l o s e TLC. Both f u c i t o l and a putat i v e d i s a c c h a r i d e component were observed. The [^Hlfucitol from a l k a l i n e borohydride-treated e t h a n o l - i n s o l u b l e f r a c t i o n comigrated with standard f u c i t o l in the three TLC systems employed. The higher molecular weight f u c o s y l a t e d components, which comprise the majority of the r a d i o a c t i v i t y , d i d not release detectable l e v e l s o f f u c i t o l ; the r a d i o a c t i v i t y was q u a n t i t a t i v e l y released as fucose. The p u t a t i v e d i s a c c h a r i d e had the same Rf values as reduced d i s a c c h a r i d e released from FL4 (Figure 12). On Sephadex G-10, the p u t a t i v e d i s a c c h a r i d e had a comparable e l u t i o n volume to that o f standard d i s a c c h a r i d e , i . e . , l a c t o s e (Figure 1 3 ) . Hydrolysis of the d i s a c c h a r i d e - s i z e d component (Figure 14) q u a n t i t a t i v e l y yielded f u c i t o l . The " d i s a c c h a r i d e " i c a l l y , pulse experiment to determine i f the l a b e l i n g o f the " d i s a c c h a r i d e " i s c o n s i s t e n t with i t s being a precursor of FL4 (Figure 15). The i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o GDP-fucose r a p i d l y e q u i l i b r a t e s , i . e . , by 2 hours. Incorporation o f l a b e l e d fucose i n t o f u c o p r o t e i n appears to increase s i g n i f i c a n t l y over the e n t i r e time period examined. The i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o the d i s a c c h a r i d e - s i z e d component from f u c o p r o t e i n i s maximal by. 6 hours, while there i s a 6-hour l a g i n the rapid l a b e l i n g of FL4. These r e s u l t s are cons i s t e n t with the h y p o t h e s i s , but by no means prove, that " d i s a c c h a r i d e " i s the metabolic precursor of FL4a. The d i s a c c h a r i d e s i z e d component a l s o appears to be of b i o l o g i c a l i n t e r e s t . Both t h i s component and f u c i t o l are released from normal human embryonic lung c e l l s by treatment of fucoprotein with a l k a l i n e boroh y d r i d e , while c e l l s derived from a human rhabdomyosarcoma have reduced l e v e l s of the d i s a c c h a r i d e - s i z e d component (Figure 1 6 ) . This reduction i s p a r a l l e l e d by a marked reduction i n the i n c o r poration o f r a d i o a c t i v i t y i n t o FL4a i n these c e l l s ( S t e i n e r and Melnick, 1974). In summary: (1) The aminoacyl fucosides have been observed in a l l mammalian c e l l l i n e s thus f a r examined. These components, FL3a (threonine and f u c o s e ) , FL3b ( s e r i n e and f u c o s e ) , FL4a ( t h r e o n i n e , fucose and glucose) and FL4b ( s e r i n e , fucose and g l u c o s e ) , appear to be a novel s e r i e s o f compounds with fucose O - g l y c o s i d i c a l l y l i n k e d to amino a c i d . Furthermore, glucose appears to be the terminal sugar in FL4a. (2) There i s a marked decrease in the i n c o r p o r a t i o n of r a d i o i s o t o p i c a l l y l a b e l e d fucose i n t o FL4a i n a v a r i e t y o f o n c o g e n i c a l l y transformed c e l l s . The l e v e l of FL4a a l s o appears to vary as a f u n c t i o n of the expression of the t r a n s formed morphological phenotype. (3) There i s a g l y c o p r o t e i n ( s ) in which fucose appears to be O - g l y c o s i d i c a l l y l i n k e d to amino a c i d . The r e s u l t s o f pulse studies with [ H]fucose are c o n s i s t e n t with the f u c o p r o t e i n ( s ) being a precursor of FL4a. +
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
21.
STEINER ET AL.
Glycoconjugates and the Transformed Phenotype
Acknowledgments. We wish to thank Dr. A. Beaudet and Robert Graham for running the samples on a Beckman 121 autoanalyzer, Dr. M. Steiner for helpful suggestions, and Dr. J.L. Melnick for his support during the course of this work. This work was supported by research grant CA 16,311 from the National Cancer Institute, DHEW, and by Faculty Research Award FRA-131 from the American Cancer Society, Inc. G. Larriba is a Fellow of the Program of Cultural Cooperation between Spain and the United States of America. S. Sramek is a recipient of a National Research Service Award (#1 F32 CA 05706) from the National Cancer Institute, DHEW. LITERATURE CITED Altenburg, B.A., Via, D.P. and Steiner, S. 1976. Exp. Cell Res. 102:223. Bauer, C., Kottgen, E. and Reutter, W. 1977. Biochem. Biophys. Res. Comm. 76:488. Buck, C.A., Glick, M.C 4567. Folch, K., Lees, M. and Sloane-Stanley, G.H. 1957. J. Biol. Chem. 26:497. Garegg, P.J. and Norberg, T. 1976. Carbohyd. Res. 52:235. Hakomori, S.I. 1964. J. Biochem. (Tokyo) 55:205. Hakomori, S.I. and Jeanloz, R.W. 1964. J. Biol. Chem. 239:3606. Hallgren, P., Lundblad, A. and Svensson, S. 1975. J. Biol. Chem. 250:5312. Kessel, D., Sykes, E. and Henderson, M. 1977. J . Natl. Cancer Inst. 59:29. Larriba, G., Klinger, M., Sramek, S. and Steiner, S. 1977. Biochem. Biophys. Res. Comm. 77:79. Pietropaolo, C., Yamaguchi, N., Weinstein, I.B. and Glick, M.C. 1977. Int. J. Cancer 20:738. Simmons, D.A.R. and Perlmann, P. 1973. Cancer Res. 33:313. Skelly, J., Gacto, M., Steiner, M.R. and Steiner, S. 1976. Biochem. Biophys. Res. Comm. 68:442. Steiner, S.,Brennan, P. and Melnick, J.L. 1973. Nature New Biol. 245:19. Steiner, S. and Melnick, J.L. 1974. Nature 251:717. Steiner, S., Melnick, J.L., Kit, S. and Somers, K. 1974. Nature 248:682. Steiner, S. and Steiner, M. 1975/76. Intervirology 6:32. Stellner, K., Hakomori, S.I. and Warner, G. 1973. Biochem. Biophys. Res. Comm. 55:439. Watanabe, I., Matsubara, T. and Hakomori, S.I. 1976. J. Biol. Chem. 251:2385. RECEIVED
March 31, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22 Fucosylation—A Role in Cell Function MARY CATHERINE GLICK Department of Pediatrics, University of Pennsylvania Medical School, Children's Hospital of Philadelphia, Philadelphia, PA 19104
The original observatio panied by an alteratio extended in a number of laboratories to many virus transformed and tumor cells, including human tumors (see ref. 2 and 3 for reviews). The appearance of specific glycopeptides has been directly correlated with tumorigenesis (4) and has been suggested as a diagnostic procedure for leukemia (5). The studies have progressed to the point of elucidating the partial structure of one of the major glycopeptides predominant on the surface of virus transformed cells (6). This glycopeptide is triantennary and appears to be more highly branched than the major fucose-containing glycopeptide isolated from the nontransformed counterpart. These initial results are compatible with the suggestion that the key to the alteration which leads to the formation of more highly branched oligosaccharides after virus transformation may reside around theβ-mannosecore. The partial structures were assembled from data obtained by enzyme degradation of the isolated glycopeptides with purified exoglycosidases and recovery of the released monosaccharides by gas liquid chromatography. Even though these glycopeptide differences were detected using radioactive L-fucose to mark the glycoproteins, no difference has been seen thus far between the transformed and nontransformed cell surfaces in fucose per se. In order to accent this point a variety of data from a number of cell types have been summarized (Table 1) and used to suggest that the fucosylation of membrane glycoproteins may have a special role in the functioning of the cell. The data which suggest a role for fucosylation fall into several categories: (a) more directly correlated to cell function; (b) the molecule of fucose; and (c) ancillary data. More Directly Correlated to Cell Function The first three observations listed in Table 1 fall into a category which may be more directly suggestive of a role for fucose in membrane glycoproteins. The first, the fact that most membrane glycoproteins contain fucose, has been shown with many 0-8412-0452-7/78/47-080-404$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22.
GLICK
Fucosylation—A Role in Cell Function
405
c e l l types using double l a b e l i n g experiments with r a d i o a c t i v e L-fucose and D-glucosamine or D-glucose. S i m i l a r membrane g l y c o p r o t e i n s are l a b e l e d with e i t h e r r a d i o a c t i v e L-fucose or the more general l a b e l s , D-glucosamine or D-glucose when examined by polyacrylamide g e l e l e c t r o p h o r e s i s ( 7 ) . An example of t h i s i s shown i n Figure 1 which d e p i c t s the s u r f a c e membrane glycoprot e i n s of transformed baby hamster kidney f i b r o b l a s t s marked with r a d i o a c t i v e fucose (Figure l a ) , and the same clone and the normal counterpart marked with r a d i o a c t i v e glucosamine (Figure l b ) . The only r e p r o d u c i b l e d i f f e r e n c e between the precursors was i n the high molecular weight r e g i o n of the g e l ( F r a c t i o n s 5-15). Another example i s seen i n the p a r t i a l l y p u r i f i e d membrane g l y c o peptides from hamster transformed c e l l s or t h e i r normal counterp a r t s . These c e l l s were m e t a b o l i c a l l y l a b e l e d with both D - [ ^ C ] glucose and L - [ % ] fucose (6). Again the s i m i l a r i t y of the r a d i o a c t i v e pattern f th glycopeptide suggest that a l l of these membrane glycopeptide The second p o i n t (Table 1), that fucose i s p o s i t i o n e d i n the core r e g i o n of a l l of the membrane g l y c o p r o t e i n s thus f a r examined, can be shown by the t o t a l recovery of the monosaccharide u n i t s a f t e r s e q u e n t i a l enzyme degradation of the r a d i o a c t i v e glycopeptides ( 6 ) . The r a d i o a c t i v i t y was recovered as fucose only a f t e r treatment with r a t t e s t i s a-L-fucosidase and was r e moved subsequent to the r e l e a s e of a l l the other monosaccharides with the exception of two residues of N-acetylglucosamine from the core r e g i o n . I f fucose was p o s i t i o n e d i n another l o c a t i o n , the enzymatic degradation of the o l i g o s a c c h a r i d e u n i t would not have been complete. The t h i r d p o i n t (Table 1) may be a phenomenon r e l a t e d to the g l y c o p r o t e i n s t r u c t u r e s observed. The a c t i v i t y of a-Lfucosidase was not e l e v a t e d a f t e r v i r u s transformation to the extent that the other g l y c o s i d a s e s were e l e v a t e d . The a c i d hydrolase a c t i v i t i e s of the v i r u s transformed c e l l s are expressed as percentage of the normal i n F i g u r e 2. A c i d phosphatase, which represented other lysosomal enzymes, was s i m i l a r i n both c e l l types, as were s e v e r a l exoglycosidases. The a c t i v i t i e s of $g a l a c t o s i d a s e , 3-N-acetylglucosaminidase, a-mannosidase, and a,3-N-acetylgalactosaminidase were a l l e l e v a t e d 150 to 300%. In c o n t r a s t , the a c t i v i t y of a-L-fucosidase was e l e v a t e d only 120%. In other experiments using 4-methylumbelliferyl-fucopyranoside as s u b s t r a t e , the a c t i v i t y of both c e l l types was even more s i m i l a r . Three of the four g l y c o s y l h y d r o l a s e s with e l e v a t e d a c t i v i t i e s are d i r e c t l y concerned with the degradation of the o l i g o s a c c h a r i d e u n i t s which compose the branches of the membrane g l y c o p r o t e i n s . The more branched g l y c o p r o t e i n s are those which are more predominant on the transformed c e l l s u r f a c e ( 6 ) . The f a c t that a-L-fucosidase a c t i v i t y i s s i m i l a r i n both transformed and nontransformed c e l l s suggests that even though the branching of the g l y c o p r o t e i n s i s a l t e r e d on transformation, the fucose residues and the core r e g i o n may remain unchanged.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
406
600
20
40
60
80
100
Fraction Number Figure 1. Polyacrylamide gel electrophoresis of surface membranes. Surface membranes were prepared from cells metabolically labeled with (a)-L-[ H] or -[ C] fucose and (b) d-[ H] or -[ C] glucosamine and examined by polyacrylamide gel electrophoresis. All details have been described (21). (a) C /B cells logarithmically growing (O—O) or plateu phase (•—•) and (b) C /B (O—O) and (BHK /C (•-•). 3
3
14
14
13
h
13
21
k
13
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
22.
Fucosylation—A Role in Cell Function
GLICK
407
TABLE 1 Observations which suggest a r o l e f o r f u c o s y l a t i o n i n membrane f u n c t i o n s of mammalian c e l l s . 1. 2. 3. 4. 5.
Most membrane g l y c o p r o t e i n s are f u c o s y l a t e d . Fucose i s p o s i t i o n e d near the glycopeptide core. a-L-Fucosidase a c t i v i t y i s not s i g n i f i c a n t l y elevated a f t e r v i r u s transformation. Fucose i s the only monosaccharide which occurs as an L-isomer and a deoxy sugar but never as the N-acetyl d e r i v a t i v e . Fucose i s not metabolized l i k e other monosaccharides
100
200
300
Glycosidase Activity of C /B (% of BHK /C ) 13
4
21
13
Figure 2. Acid hydrolase activities of transformed (C /B,,) and nontransformed (BHK /C ) fibroblasts. The cells, harvested when growing exponentially, were homogenized in 0.1% Triton X100. The enzyme activities were determined on the appropriate p-nitrophenyl derivatives in 3mM citrate buffer, pH = 4.5, with the exception of sialidase where fetuin in 50mM acetate buffer, pH = 4.5, served as substrate. The enzyme activities (per mg protein) of C /B cells are expressed as the percentage of the particular activity obtained with BHK /C cells. The values represent the mean obtained from three different cultures of each cell type. Acid phosphatase, which is not a glycosidase, is included for comparison. 13
21
13
13
k
21
13
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
408
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
Uniqueness o f Fucose i n Mammalian C e l l s Point 4 i n Table 1 suggests that due to i t s s t r u c t u r e alone fucose may be i n a unique p o s i t i o n . Thus f a r , fucose has been described only as the L-isomer, i n c o n t r a s t to other monosacchar i d e s which occur as D-isomers. I t i s the only deoxy sugar present i n mammalian g l y c o p r o t e i n s and thus f a r has not been described as the N - a c e t y l d e r i v a t i v e . The f a c t that i t i s not subject to the normal metabolic pathways as the other monosaccharides i s a l s o suggestive o f a unique r o l e . This i s shown by the f a c t that r a d i o a c t i v e fucose i s recovered only as fucose not only i n c e l l s i n c u l t u r e (8), but a l s o i n animals (9). Subsequent s t u d i e s may not support a l l o f these p o i n t s , and i n f a c t , recent r e p o r t s suggest that, contrary to the current dogma, fucose i s not always found i n a t e r m i n a l p o s i t i o n (10,11). A n c i l l a r y Data A d d i t i o n a l data supportin of fucose i n mammalian number o f other sources. Perhaps the most compelling i s the presence of r e l a t i v e l y l a r g e amounts o f two unusual components a s s o c i a t e d with the s u r f a c e membrane c o n t a i n i n g fucose. One o f these i s the major high molecular weight g l y c o p r o t e i n exported by human f i b r o b l a s t s i n c u l t u r e (12) . Although t h i s g l y c o p r o t e i n has been shown by others to be a component of the f i b r o b l a s t matrix, and f o r t h i s reason c a l l e d F i b r o n e c t i n (13), l i t t l e a t t e n t i o n has been given to the f a c t that i t i s r e a d i l y l a b e l e d with r a d i o a c t i v e L-fucose. The second unusual f u c o s e - c o n t a i n i n g molecule i s a low molecular weight component o f the c e l l s u r f a c e (14). Chromatography on p r e c a l i b r a t e d F r a c t o g e l PGM 2000, as w e l l as other c r i t e r i a , show that t h i s f u c o s e - c o n t a i n i n g molecule i s a p p r o x i mately 500 daltons (Figure 3). This s u r f a c e component i s not the same as the low molecular weight component o f NRK c e l l s reported by S t e i n e r (10), s i n c e fucose was r e l e a s e d by p u r i f i e d a-L-fucosidase. This enzyme was f r e e o f other d e t e c t a b l e exoglycosidases using s y n t h e t i c and n a t u r a l substrates (15), so fucose had to be i n a t e r m i n a l p o s i t i o n . A d d i t i o n a l data concerning the presence o f fucose i n membrane g l y c o p r o t e i n s i s that i n many d i f f e r e n t c e l l types exami n e d , only 30-40% o f the fucose content o f the t o t a l c e l l was found i n the membrane (7>15). This was i n c o n t r a s t to the s i a l i c a c i d content o f the membrane, which was as h i g h as 70-80% o f the t o t a l c e l l content (16). In a d d i t i o n , fucose found i n the membrane g l y c o p r o t e i n s showed two d i f f e r e n t r a t e s o f a c i d h y d r o l y s i s and two d i f f e r e n t s p e c i f i c a c t i v i t i e s ( 8 ) . Only 40% of the fucose from the membrane g l y c o p r o t e i n s o f transformed hamster f i b r o b l a s t s ( C 1 3 / B 4 ) was removed a f t e r 2 hours with 0.1 N H2SO4 a t 100°. This materi a l showed a higher s p e c i f i c a c t i v i t y than the remaining fucose which was subsequently removed by a-L-fucosidase. The reasons f o r these d i f f e r e n c e s are not apparent a t t h i s time, but the two
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22.
GLICK
Fucosylation—A
35
Role in Cell
Function
45
55
Fraction Number Figure 3. Low molecular weight fucose-containing components associated with the surface membrane (14). Radioactivity extracted from the surface of a human neuroblastoma cell line (IMR-32), metabolically labeled with L-[ H] fucose, was purified and chromographed on Fractogel PGM 2000. The column was precalibrated with BSA, bovine serum albumin; LNF, lacto-N-fucopentaose; GlcNAc-Asn, 2-acetamido-l-(\.-$-aspartamide)-l,2-di-deoxy-fi-D-glucose; Lac, lactose; Fuc, fucose. 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
409
410
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
specific activities suggest the presence of at least two fucosyltransferases. Role of Fucosylation All of the above facts can be summarized to suggest that fucose as it occurs in membrane glycoproteins has a number of unusual characteristics. It is hypothesized that because of these, fucose performs a special function for the cell membrane. It can be postulated that fucose may serve to bind proteins or polypeptides for reinsertion into membranes, or transport through the membrane, thus its position near the glycopeptide core would be an advantage. The exact role it may play, if any, remains to be shown. Cell surface variants of a number of cell types have been reported (17-20). Perhaps through use of these variants which are defective in membrane glycoproteins, the special role which fucose may have in the relationship of the surface membrane to the cell will be ascertained SUMMARY The fucose-containing glycopeptides of the membranes of virus transformed and tumor cells are clearly different from those of their normal counterparts. This difference is that the virus transformed cells express on their surfaces glycopeptides more highly branched than those found in the controls. Although fucose served as a radioactive marker to detect these differences, no difference has, as yet, been found in fucose per se. The similarities of fucose among several cell types and some unique properties of fucose in mammalian glycoproteins have been summarized and used to suggest that fucose may play a special role in the relationship of the surface membrane to the cell. ACKNOWLEDGEMENTS Supported by U.S.P.H.S. Grants CA 14037, CA 14489, and HD 08536. Literature Cited 1. 2. 3. 4. 5. 6.
Buck, C.A., Glick, M.C., and Warren, L. (1970) Biochemistry 9, 4567-4576. Glick, M.C. (1976) In Fundamental Aspects of Metastasis, L. Weiss, ed. North Holland Publishing Co., Amsterdam, pp. 9-23. Poste, G., and Nicholson, G.L., eds. (1977) Cell Surface Reviews, Vol. 4. Elsevier-North Holland Inc., Amsterdam. Glick, M.C., Rabinowitz, Z., and Sachs, L. (1974) J . Virol. 13, 967-974. Van Beek, W.P., Emmelot, P., and Homburg, C. (1977) Br. J. Cancer 36, 157-165. Glick, M.C., and Santer, U.V. (1977) In Cell Surface Carbohydrate Chemistry, Adv. Chem. Series, R.E. Harmon, ed., Academic Press, New York, pp. 13-26.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22. GLICK 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Fucosylation—A Role in Cell Function
411
Scanlin, T.F., and Glick, M.C. VII International Cystic Fibrosis Conference. In press. Fischer, A., and Glick, M.C. Manuscript in preparation. Bocci, V., and Winzler, R.J. (1969) Am. J. Physiol. 216, 1337-1342. Steiner, S., in these proceedings. Hallgren, P., Lundblod, A., and Svensson, S. (1975) J. Biol. Chem. 250, 5312-5314. Scanlin, T.F., Voynow, J.A., and Glick, M.C. Unpublished observations. Vuento, M., Wrann, M., and Ruoslahti, E. (1977) FEBS Lett. 82, 227-231. Mihalik, G.M., and Glick, M.C. Unpublished observations. Santer, U.V., and Glick, M.C. Manuscript in preparation. Glick, M.C. (1976) In Mammalian Cell Membranes, Vol. 1, G. A. Jamieson and D.M Robinson eds Butterworths London pp. 45-77. Stanley, P., Narasimhan, S., Siminovitch, L . , and Schachter, H. (1975) Proc. Natl. Acad. Sci. USA 72, 3323-3327. Meager, A., Ungkitchanukit, A., and Hughes, R.C. (1976) Biochem. J . 154, 113-124. Briles, E.B., L i , E . , and Kornfeld, S. (1977) J . Biol. Chem. 252, 1107-1116. Pouysségur, J., and Pastan, I. (1977) J. Biol. Chem. 252, 1639-1646. Greenberg, C.S., and Glick, M.C. (1972) Biochemistry 11, 3680-3685.
RECEIVED
April 17, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23 Cell-Surface Glycoproteins of Normal and Malignant Rat Liver Cells 1
2
JAMES J. STARLING, DOUGLAS C. HIXSON, SYLVIA C. CAPETILLO, EDWARD M. DAVIS, GIOVANNI NERI, and EARL F. WALBORG, JR. Research Division, University of Texas, System Cancer Science Park, Smithville, TX 78957 3
4
Malignant transformatio ations which are directly involved in many of the aberrant properties displayed by the transformed cell (1). Lectins have been used extensively as probes to study these membrane alterations because, in several in vitro cell systems, non-transformed cells are not agglutinated at lectin concentrations sufficient to agglutinate transformed or protease-treated non-transformed cells (2). Despite extensive investigation, the molecular mechanism(s) responsible for lectin-induced cytoagglutination is (are) still not clear, although several hypotheses have been proposed: exposure of cryptic lectin-binding sites (3,4); clustering and lateral mobility of lectin receptors (5-8); reduction of surface charge repulsive forces (2); increased membrane deformabi1ity (2); increased density of lectin-binding sites due to altered surface morphology (9); and structural alterations of lectin receptors (10,11). Two such lectins, concanavalin A (Con A) and wheat germ agglutinin (WGA), have been used to probe the membrane structure of normal and malignant rat liver cells. Novikoff ascites hepatoma cells were agglutinated at low concentrations of Con A and WGA (12), whereas AS-30D ascites hepatoma cells (13) were agglutinable by WGA but required high Con A concentrations for agglutination (14). Macrosialoglycopeptides isolated from the surfaces of these cells possessed potent Con A and/or WGA receptor activity suggesting that these glycopeptides may be involved in the cytoagglutination properties of the intact cells (11). Isolated adult -current address: "-current address: ""^current address: ****current address:
Dept. of Biochem., Univ. of Florida, Gainesville, FL 32610 Dept. of Pathology, Univ. of Texas System Cancer Center, Houston, TX 77030 Dept. of Biology, Yale Univ., New Haven, CT 06520 Catholic Univ. School of Medicine Rome 01168, Italy
0-8412-0452-7/78/47-080-412$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
413
r a t l i v e r c e l l s , on t h e o t h e r h a n d , were n o t a g g l u t i n a b l e by h i g h c o n c e n t r a t i o n s o f Con A o r WGA (J_l ,J_5) The p a p a i n - l a b i l e s i a l o g l y c o p e p t i d e s i s o l a t e d f r o m normal h e p a t o c y t e s e x h i b i t e d a S e p h a dex G-50 e l u t i o n p r o f i l e w h i c h was s t r i k i n g l y d i f f e r e n t f r o m t h a t o f c e l l - s u r f a c e s i a l o g l y c o p e p t i d e s i s o l a t e d f r o m N o v i k o f f and AS-30D hepatoma c e l l s and p o s s e s s e d no d e t e c t a b l e Con A o r WGA r e c e p t o r a c t i v i t y (11). These d a t a suggested major d i f f e r e n c e s i n membrane s t r u c t u r e between normal and m a l i g n a n t r a t l i v e r c e l l s . However, t h e methods employed i n t h e a b o v e s t u d i e s t o o b t a i n s i n g l e c e l l s u s p e n s i o n s from a d u l t l i v e r i n v o l v e d mechanical d i s p e r s a l a n d / o r c o l l a g e n a s e d i g e s t i o n w h i c h d i d n o t employ p e r f u s i o n techniques. The s t r u c t u r a l and m e t a b o l i c s u p e r i o r i t y o f h e p a t o c y t e s u s p e n s i o n s o b t a i n e d by c o l l a g e n a s e p e r f u s i o n o v e r t h a t o b t a i n e d by o t h e r i s o l a t i o n methods has been w e l l documented (]_6). It i s p o s s i b l e , t h e r e f o r e , t h a t t h e r e p o r t e d low a g g l u t i n a b i 1 i t y o f normal l i v e r c e l l s a n d / o of c e l l - s u r f a c e glycopeptide r e f l e c t the in v i v o p r o p e r t i e s o t h e s e c e l l s b u t may i n s t e a d be due t o t h e methods used f o r t h e i r i s o l a t i o n . The r e s e a r c h r e p o r t e d h e r e i n d e s c r i b e s t h e Con A - a n d WGAi n d u c e d a g g l u t i n a t i o n p r o p e r t i e s o f m e c h a n i c a l l y and e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s ( r e f e r s t o h e p a t o c y t e s i s o l a t e d by t h e t e c h n i q u e o f J a c o b and B h a r g a v a and by c o l l a g e n a s e p e r f u s i o n o f t h e i n t a c t l i v e r , r e s p e c t i v e l y ) and compares t h e s e p r o p e r t i e s t o t h o s e e x h i b i t e d by N o v i k o f f a s c i t e s hepatoma c e l l s . The S e p h a dex G-50 e l u t i o n p r o f i l e s and l e c t i n r e c e p t o r a c t i v i t y o f p a p a i n l a b i l e g l y c o p e p t i d e s i s o l a t e d from the s u r f a c e o f e n z y m a t i c a l l y d i s p e r s e d l i v e r c e l l s and N o v i k o f f a s c i t e s hepatoma c e l l s a r e a l s o d e s c r i b e d and an a t t e m p t i s made t o d e l i n e a t e t h o s e f a c t o r s p r i m a r i l y r e s p o n s i b l e f o r t h e i n c r e a s e d Con A - i n d u c e d a g g l u t i n a b i 1 i t y of rat hepatocytes f o l l o w i n g papain d i g e s t i o n . M a t e r i a l s and Methods Hepatoma C e l 1 s . N o v i k o f f r a t a s c i t e s hepatoma c e l l s , m a i n t a i n e d in 6-9-week-old female Sprague-Dawley r a t s (ARS/Sprague-Dawley, M a d i s o n , Wl) were c o l l e c t e d i n Medium 199 w i t h L - g l u t a m i n e , o b t a i n e d f r o m Schwarz Mann, O r a n g e b u r g , N Y ) , pH 7.k containing O . l g neomycin s u l f a t e per l i t e r (Ml99); r e s u s p e n d e d i n t h e same medium, and i n c u b a t e d 90 min i n a s h a k i n g w a t e r b a t h a t 3 7 ° in s t o p p e r e d f l a s k s g a s s e d w i t h 5% C 0 in a i r . Rat H e p a t o c y t e s . Suspensions o f e n z y m a t i c a l l y d i s p e r s e d hepat o c y t e s were p r e p a r e d by c o l l a g e n a s e p e r f u s i o n o f t h e i n t a c t l i v e r a c c o r d i n g t o t h e method o f Bonney (l_8) as d e s c r i b e d by S t a r l i n g , e_t aj_ (19). Suspensions of mechanically dispersed rat h e p a t o c y t e s were p r e p a r e d by t h e method o f J a c o b and B h a r g a v a (17) a s d e s c r i b e d by W a l b o r g et_ aj_ ( J j ) e x c e p t t h a t t h e c e l l s were i n c u b a t e d f o r o n l y 90 m i n u t e s i n M199 i n s t e a d o f 20 h . D e t e r m i n a t i o n o f c e l l v i a b i l i t y and u l t r a s t r u c t u r a l integrity. C e l l v i a b i l i t y was d e t e r m i n e d by t r y p a n b l u e s t a i n i n g (20) and u l t r a s t r u c t u r a l i n t e g r i t y was a s c e r t a i n e d by m o r p h o l o g i c a l exami n a t i o n o f the e l e c t r o n microscope. Samples were f i x e d i n IX 9
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
GLYCOPROTEINS
414
g l u t a r a l d e h y d e , post methods f o r e l e c t r o n
A N D GLYCOLIPIDS
I N DISEASE
f i x e d i n 2% OsO^, and p r o c e s s e d microscopy.
PROCESSES
by s t a n d a r d
P a p a i n d i g e s t i o n o f hepatoma c e l l s . N o v i k o f f a s c i t e s hepatoma c e l l s , washed t w i c e i n b u f f e r 1 (21) and o n c e i n b u f f e r 2 (2p a t 50g f o r 5 m i n u t e s were i n c u b a t e d w i t h p a p a i n (2 t i m e s c r y s t a l l i z e d , 666 U/ml W o r t h i n g t o n B i o c h e m i c a l C o r p . ) f o r 40 m i n u t e s a s d e s c r i b e d p r e v i o u s l y (1J_). P r o t e o l y t i c d i g e s t i o n was p e r f o r m e d u s i n g 3 U o f p a p a i n p e r ml o f c e l l s u s p e n s i o n , c o n t a i n i n g 1.5 x 10 cells/ml. These i n c u b a t i o n c o n d i t i o n s approximate t h e m i l d e s t c o n d i t i o n s w h i c h y i e l d maximal r e l e a s e o f p e p t i d e - b o u n d s i a l i c a c i d f r o m t h e s u r f a c e o f hepatoma c e l l s (l_ft) C e l l counts were p e r f o r m e d u s i n g a Model ZBI C o u l t e r C o u n t e r ( C o u l t e r Electronics, Hialeah, FL). Papain d i g e s t i o n o f l i v e r c e l l s . Mechanically dispersed l i v e r c e l l s were d i g e s t e d w i t h p a p a i n a s d e s c r i b e d p r e v i o u s l y (l_0 . Enzymatically dispersed l i v e r c e l l s i n c u b a t e d i n Ml99 + 3% f e t a l c a l f s e r u m , were washe (21) a t 20g f o r 2 m i n u t e s (21) a t a c o n c e n t r a t i o n o f 0.5 t o 1 x 10 c e l l s / m l . Ten ml a l i q u o t s o f t h e c e l l s u s p e n s i o n were p l a c e d i n s e p a r a t e 250 ml E r l e n myer f l a s k s c o n t a i n i n g 10 U o f p a p a i n p e r f l a s k and i n c u b a t e d f o r 30 m i n u t e s i n a s h a k i n g w a t e r b a t h a t 37° w h i l e b e i n g g a s s e d c o n t i n u o u s l y w i t h a 95% 0 /5% C0^. C e l l c o u n t s were p e r f o r m e d u s i n g a hemacytometer. Assay o f l e c t i n - i n d u c e d c y t o a g g l u t i n a t i o n . Lectin purification and p r e p a r a t i o n o f s e r i a l d i l u t i o n s was p e r f o r m e d a s p r e v i o u s l y described (H,l_2). Q u a n t i t a t i v e assessment o f c y t o a g g l u t i n a t i o n p r o p e r t i e s o f i n t a c t , i n c u b a t e d c o n t r o l , o r p a p a i n - t r e a t e d hepatoma and l i v e r c e l l s was a c c o m p l i s h e d u s i n g t h e p a r t i c l e c o u n t e r a g g l u t i n a t i o n a s s a y o f D a v i s et^ aj_ (22) a s d e s c r i b e d e a r l i e r (]Sj). M i c r o s c o p i c e v a l u a t i o n o f l e c t i n - i n d u c e d a g g l u t i n a t i o n o f hepatoma c e l l s and h e p a t o c y t e s was p e r f o r m e d u s i n g a m o d i f i e d v e r s i o n (19) o f t h e method o f Wray and W a l b o r g (23). Con A - i n d u c e d a g g l u t i n a b i 1 i t y o f f e m a l e S p r a g u e - D a w l e y r a t e r y t h r o c y t e s , washed 3 t i m e s i n C a - f r e e H a n k s ' medium (2*0, pH 1 .k (CFH) a t 210 g f o r 10 m i n u t e s , was d e t e r m i n e d u s i n g t h e t e s t t u b e h e m a g g l u t i n a t i o n a s s a y o f L e s e n e y et_ aj_ (25). P r e p a r a t i o n o f g l y c o p e p t i d e s c l e a v e d f r o m t h e s u r f a c e o f normal and m a l i g n a n t r a t l i v e r c e l l s by p a p a i n . A papain-labile, c e l l s u r f a c e s i a l o g l y c o p e p t i d e f r a c t i o n (C-SGP) was p r e p a r e d f r o m N o v i k o f f c e l l s u s i n g t h e p r o c e d u r e o f N e r i et_ aj_ (12), w h i l e a p a p a i n - l a b i l e , c e l l - s u r f a c e g l y c o p e p t i d e f r a c t i o n Tc-GP) was p r e p a r e d f r o m m e c h a n i c a l l y and e n z y m a t i c a l l y d i s p e r s e d l i v e r c e l l s by t h e method o f W a l b o r g e t al_ (J_0 . G l y c o g e n was removed as p r e v i o u s l y d e s c r i b e d (1J9). 7
7
2
+ 2
A n a l y s i s o f Sephadex G-50 column e f f l u e n t s . S i a l i c a c i d was d e t e r m i n e d by t h e method o f A m i n o f f (26) a f t e r h y d r o l y s i s i n 0.1N H S 0 , f o r 1 h a t 8 0 ° . N - a c e t y l n e u r a m i n i c a c i d (NANA) was used a s a s t a n d a r d . N e u t r a l s u g a r was d e t e r m i n e d by t h e method o f D u b o i s e t aj_ (27) u s i n g D - g a l a c t o s e a s a s t a n d a r d . Hexosamine 2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
415
was a s s a y e d a c c o r d i n g t o G a t t and Berman (28J a f t e r h y d r o l y s i s i n 2N HC1 f o r 8 h a t 100 d e g r e e s u s i n g 2-amino-2-deoxy-D-glucose. HC1 a s a s t a n d a r d . A s s a y o f Con A and WGA r e c e p t o r a c t i v i t y * The l e c t i n r e c e p t o r a c t i v i t i e s o f t h e c e l l - s u r f a c e g l y c o p e p t i d e f r a c t i o n s were a s s a y e d by t h e i r a b i l i t y t o i n h i b i t Con A - o r WGA-induced a g g l u t i n a t i o n o f g u i n e a p i g e r y t h r o c y t e s ( 1 4 ) . E r y t h r o c y t e s used i n t h e d e t e r m i n a t i o n o f the l e c t i n receptor a c t i v i t y of the Novikoff c e l l - s u r f a c e g l y c o p e p t i d e f r a c t i o n s were s u s p e n d e d i n Ca - a n d Mg - f r e e p h o s p h a t e b u f f e r e d s a l i n e , pH 1.k ( C M F - P B S ) , c o n t a i n i n g 0.5% BSA. P r e p a r a t i o n and p u r i f i c a t i o n o f [ ^ 1 ] Con A . l o d i n a t i o n was p e r f o r m e d a c c o r d i n g t o t h e p r o c e d u r e o f Sonoda and S c h l a m o w i t z (29). A f t e r i o d i n a t i o n , t h e [ $ \ ] Con A was p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y a t 22 d e g r e e s on Sephadex G-100 (30). Conc e n t r a t i o n o f the Con A and p r e p a r a t i o n o f s e r i a l d i l u t i o n s was p e r f o r m e d a s p r e v i o u s l y d e s c r i b e d (3 j ) • [ 5 l ] Con A b i n d i n g a s s a y to the e n z y m a t i c a l l y d i s p e r s e t h e l e c t i n b i n d i n g tube a s s e m b l y (LBTA) a s s a y (32) as d e s c r i b e d e a r l i e r (3p • ] 2
1 2
P r e p a r a t i o n and p u r i f i c a t i o n o f f e r r i t i n - c o n j u g a t e d Con A (Fer-Con A ) . F e r - C o n A was p r e p a r e d by s l o w l y a d d i n g 0.15 ml o f 0.25% g l u t a r a l d e h y d e t o CMF-PBS c o n t a i n i n g 4 . 5 mg Con A , 5^ mg m e t h y l a - D - g l u c o p y r a n o s i d e (Sigma Chem. C o . ) and 30 mg o f r e c r y s t a l l i z e d horse spleen f e r r i t i n (Pentex, M i l e s L a b o r a t o r i e s , Kankakee, 1 1 1 . ) . The c o n j u g a t i o n r e a c t i o n was a l l o w e d t o p r o c e e d f o r 6 h a t 22° and was t h e n t e r m i n a t e d by t h e a d d i t i o n o f l y s i n e t o a f i n a l c o n c e n t r a t i o n o f 2 mg/ml. The c o n j u g a t e d l e c t i n was p u r i f i e d on a 15-30% l i n e a r s u c r o s e g r a d i e n t . The F e r - C o n A was d i a l y z e d a g a i n s t CMF-PBS, c o n c e n t r a t e d t o a p p r o x i m a t e l y 3 ml by d i a l y s i s a g a i n s t p o l y e t h y l e n e g l y c o l ( a v e r a g e m o l e c u l a r w e i g h t 6000 D ) , and f u r t h e r d i a l y z e d a g a i n s t CMF-PBS. This stock s o l u t i o n o f F e r - C o n A ( a p p r o x . 4 mg/ml) a g g l u t i n a t e d r a b b i t e r y t h r o c y t e s up t o a d i l u t i o n o f 1:128 u s i n g t h e h e m a g g l u t i n a t i o n a s s a y d e s c r i b e d by S m i t h £ t aj_ ( 1 4 ) . H e p a t o c y t e l a b e l i n g by F e r - C o n A . Binding of Fer-Con A to r a t hepat o c y t e s and p r e p a r a t i o n o f s e c t i o n s f o r e l e c t r o n m i c r o s c o p y was p e r f o r m e d a s d e s c r i b e d e a r l i e r (3jJ . R e s u l t s and D i s c u s s i o n V i a b i l i t y and c y t o s t r u c t u r a l i n t e g r i t y o f h e p a t o c y t e s isolated by e n z y m a t i c and m e c h a n i c a l p r o c e d u r e s . Accurate assessment o f the s u r f a c e p r o p e r t i e s o f hepatocytes r e q u i r e s the use o f c e l l s which c l o s e l y approximate t h e i r in v i v o c o n d i t i o n . Previous r e p o r t s ( J j j on l e c t i n - i n d u c e d a g g l u t i n a t i o n o f a d u l t h e p a t o c y t e s u t i l i z e d c e l l s w h i c h were i s o l a t e d by p r o c e d u r e s w h i c h y i e l d c e l l s p o s s e s s i n g low v i a b i l i t y and poor c y t o s t r u c t u r a l integrity. We now r e p o r t t h e u s e o f h e p a t o c y t e s i s o l a t e d f r o m l i v e r s p e r fused with c o l l a g e n a s e , a technique demonstrated to y i e l d c e l l s e x h i b i t i n g h i g h v i a b i l i t y and e x c e l l e n t u l t r a s t r u c t u r a l integrity. H e p a t o c y t e s i s o l a t e d by t h i s e n z y m a t i c t e c h n i q u e were o b t a i n e d
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
416
GLYCOPROTEINS
A N D GLYCOLIPIDS
IN DISEASE
PROCESSES
i n a y i e l d o f 2-4 ml o f p a c k e d (210 g , 10 min) c e l l s / l i v e r p r i o r to i n c u b a t i o n in M l 9 9 . L e s s t h a n 20% o f t h e c e l l s were l o s t d u r i n g t h e i n c u b a t i o n i n M l 9 9 + 3% FCS. Those c e l l s s u r v i v i n g t h e s h o r t - t e r m i n c u b a t i o n were 78 + 9.5 S . D . (n=75) p e r c e n t v i a b l e a s j u d g e d by t h e i r e x c l u s i o n o f v i t a l s t a i n . Some o f t h e h e p a t o c y t e s were p r e s e n t as s m a l l a g g r e g a t e s o f 2-6 c e l l s (33); e . g . , in s e v e n d i f f e r e n t e x p e r i m e n t s an a v e r a g e o f 72% o f t h e c e l l s were p r e s e n t as s i n g l e c e l l s , 2\% i n a g g r e g a t e s o f two c e l l s and 7% i n a g g r e g a t e s o f 3~6 c e l l s . As shown i n f i g . IB these hepatocytes e x h i b i t e d e x c e l l e n t u l t r a s t r u c t u r a l morphology. They p o s s e s s e d i n t a c t p l a s m a membranes, m i t o c h o n d r i a w i t h c r i s t a e e v i d e n t and a w e l l - d e f i n e d e n d o p l a s m i c r e t i c u l u m . F o r c o m p a r a t i v e p u r p o s e s , h e p a t o c y t e s were a l s o i s o l a t e d u s i n g p e r f u s i o n w i t h c i t r a t e f o l l o w e d by m e c h a n i c a l d i s p e r s a l . T h i s p r o c e d u r e y i e l d e d an a v e r a g e o f 1.2 ml o f p a c k e d (210 g , 10 min) c e l l s / l i v e r p r i o r t o i n c u b a t i o n i n M l 9 9 Of t h e s e 70% were r e c o v e r e d a f t e r i n c u b a t i n s t a i n i n g revealed that r e p o r t e d by o t h e r s (1_6), t h e u l t r a s t r u c t u r a l i n t e g r i t y o f t h e m e c h a n i c a l l y d i s p e r s e d h e p a t o c y t e s was i n f e r i o r t o t h a t o f t h e enzymatically dispersed c e l l s ( f i g . l ) . The m e c h a n i c a l l y d i s p e r s e d h e p a t o c y t e s e x h i b i t e d d i s r u p t e d p l a s m a membranes, s w o l l e n m i t o c h o n d r i a d e v o i d o f c r i t a e , and no v i s i b l e e n d o p l a s m i c r e t i c u lum ( f i g u r e 1 A ) . L e c t i n - i n d u c e d a g g l u t i n a t i o n of hepatocytes. Lectin-induced agg l u t i n a t i o n was q u a n t i t a t e d u s i n g two c o m p l e m e n t a r y a s s a y s w h i c h m e a s u r e d t h e d e g r e e o f a g g l u t i n a t i o n as a f u n c t i o n o f t h e s i z e o f t h e c e l l a g g r e g a t e s ( m i c r o t e s t II p l a t e ) o r t h e d i s a p p e a r a n c e o f single cells (electronic particle counter). Enzymatically disp e r s e d h e p a t o c y t e s were a g g l u t i n a t e d by low c o n c e n t r a t i o n s o f Con A o r WGA, i . e . l e c t i n c o n c e n t r a t i o n s c o m p a r a b l e t o t h o s e r e q u i r e d f o r t h e a g g l u t i n a t i o n o f N o v i k o f f hepatoma c e l l s ( t a b l e I ) . Prec i s e c o r r e l a t i o n o f two c e l l t y p e s i s n o t p o s s i b l e b e c a u s e o f d i f f e r e n c e s i n c e l l s i z e and t h e p r e s e n c e o f some h e p a t o c y t e c e l l a g g r e g a t e s in the absence o f l e c t i n ( t a b l e I I ) . (The mean v o l u m e s o f r a t h e p a t o c y t e s and N o v i k o f f hepatoma c e l l s , d e t e r m i n e d u s i n g t h e Model ZBI C o u l t e r C o u n t e r , were 4850 and 1560 um, r e s p e c t i v e l y . C a l i b r a t i o n o f t h e p a r t i c l e c o u n t e r was p e r f o r m e d u s i n g m i c r o p o l y s t y r e n e s p h e r e s ( L o t n o . 1001, C o u l t e r E l e c t r o n i c s ) o f 18.04 um d i a m e t e r . A s s u m i n g t h e c e l l s t o be s p h e r i c a l , t h i s indicates t h a t h e p a t o c y t e s and N o v i k o f f c e l l s have d i a m e t e r s o f 2 1 . 0 ym and 14.4 um, r e s p e c t i v e l y ) . The p r e s e n c e o f s m a l l a g g r e g a t e s o f h e p a t o c y t e s in the absence o f l e c t i n d e s e n s i t i z e s the e l e c t r o n i c p a r t i c l e c o u n t e r a s s a y s i n c e t h e i r p a r t i c i p a t i o n in t h e a g g l u t i n a t i o n r e a c t i o n e s c a p e s measurement. No a g g r e g a t i o n o f N o v i k o f f c e l l s was p r e s e n t i n t h e a b s e n c e o f l e c t i n . The M i c r o t e s t II p l a t e a s s a y g a v e r e s u l t s s i m i l a r t o t h o s e o b t a i n e d by t h e p a r t i c l e c o u n t e r a s s a y ( t a b l e l) i n d i c a t i n g t h a t t h e d e c r e a s e in s i n g l e c e l l number measured by t h e e l e c t r o n i c p a r t i c l e c o u n t e r a t i n c r e a s i n g l e c t i n c o n c e n t r a t i o n s was due t o i n c r e a s e d a g g r e g a t e
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
Experimental Cell Research
Figure 1. Morphological comparison of mechanically and enzymatically dispersed hepatocytes. Electron micrographs of glutaraldehy de-fixed cells postfixed with OsOi,. Sections were stained with uranyl acetate and lead citrate. (A) Mechanically dispersed hepatocytes after 90 min incubation in M199 X3700; (B) enzymatically dispersed hepatocyte after papain digestion X3000. Untreated hepatocytes showed comparable cytostructural integrity.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
417
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
T y p e and
Treatment
k
a
d
0.78 0.56 0.51 0.63 0.63
3.2 0.97 0.93 1.7
1.5 4.8 6.4
3.4 0.78
1.9 2.5
(12). (22) o r Ner i e t a l d i g e s t i o n , but w i t h o u t enzyme.
M i c r o t e s t 11 p l a t e P a r t i c l e counter P a r t i c l e counter P a r t i c l e counter
M i c r o t e s t 11 p l a t e P a r t i c l e counter P a r t i c l e counter P a r t i c l e counter
OF NORMAL AND MALIGNANT RAT LIVER CELLS BY CON A AND WGA L e c t i n c o n c e n t r a t i n (ug/ml) n e c e s s a r y f o r c y t o a g g l u t i n a t i o n o f normal and m a l i g n a n t rat 1iver eel 1 s WGA Con A A s s a y System Threshold Threshold
C o n c e n t r a t i o n s a s d e f i n e d by D a v i s e t a j Incubated under the condi t i o n s o f p a p a i n
b
5
Enzymatically Dispersed Hepatocytes Intact Intact Incubated control* Papa i n - t r e a t e d N o v i k o f f Hepatoma C e l l s Intact Intact Incubated c o n t r o l Papain-treated
Cell
AGGLUTINABILITY
TABLE I
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
c
b
a
the
c
particle
375 5.6 2^5
Threshold
1/2
counter
8
assay
23 96 56
c
of
Maximal
Con A c o n e , (ug/ml)required for c y t o a g g l u t i n a t i o n of enzymatically dispersed hepatocytes
E x p r e s s e d as t h e p e r c e n t o f t o t a l c e l l s i n t h e C o n c e n t r a t i o n s as d e f i n e d by D a v i s e t a l (22).
using
time
Determined
Incubation 30 min k h 8 h
II
et
indicated
Davis
72 54 56
]
(22).
21 31 35
2
aggregate
aggregate
aj
Cell
size.
size
EFFECT OF INCUBATION TIME ON CON A-INDUCED AGGLUTI NAB ILITY AND REAGGREGATI ON OF ENZYMATICALLY DISPERSED HEPATOCYTES
TABLE
%of
9
7 15
3-6
total
b
77 70 71
% Viable cells
GLYCOPROTEINS
420
A N D GLYCOLIPIDS
IN DISEASE
PROCESSES
s i z e and t h a t t h e a g g l u t i n a b i 1 i t y o f t h e e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s was e x h i b i t e d a t w i d e l y d i f f e r e n t c e l l concentrations, e . g . 6.7 x 10 c e l l s / m l ( M i c r o t e s t II p l a t e a s s a y ) and 1.3 x 10 cells/ml ( p a r t i c l e counter assay). Lectin-induced agglutination o f t h e i n t a c t h e p a t o c y t e s was s a c c h a r i d e - s p e c i f i c s i n c e i n c l u s i o n o f 2.1 mM m e t h y l a - D - m a n n o p y r a n n o s i d e i n t h e a g g l u t i n a t i o n r e a c t i o n ( p a r t i c l e c o u n t e r assay) r e s u l t e d in a 1 . 6 - f o l d i n c r e a s e in the Con A c o n c e n t r a t i o n n e c e s s a r y f o r t h r e s h o l d and h a l f - m a x i m a l a g g l u t i n a t i o n o r i n c l u s i o n o f 4.2 mM 2 - a c e t a m i d o - 2 - d e o x y - D - g l u c o s e i n t h e a g g l u t i n a t i o n r e a c t i o n r e s u l t e d in a 2 - f o l d i n c r e a s e i n t h e WGA c o n c e n t r a t i o n n e c e s s a r y f o r t h r e s h o l d and h a l f - m a x i m a l agglutination. The s a c c h a r i d e s p e c i f i c i t y o f Con A - o r WGAi n d u c e d a g g l u t i n a t i o n o f N o v i k o f f c e l l s has been d e m o n s t r a t e d previously (12). 5
7
In a g r e e m e n t w i t h p r e v i o u s s t u d i e s (11), mechanically d i s p e r s e d h e p a t o c y t e s wer t agglutinated b concentration f Co A o r WGA as h i g h as 270 lectin-induced agglutinabi y enzymatically disperse h e p a t o c y t e s a t low c o n c e n t r a t i o n s o f WGA o r Con A ( t a b l e I) i s p r o b a b l y due t o t h e i r i n c r e a s e d v i a b i l i t y and s u p e r i o r m o r p h o l o gical integrity. Our o b s e r v a t i o n s d i f f e r f r o m t h o s e o f B e c k e r (15) who r e p o r t e d t h a t a d u l t r a t h e p a t o c y t e s , a l s o o b t a i n e d by d i s p e r s a l w i t h c o l l a g e n a s e , were n o t a g g l u t i n a t e d by h i g h c o n c e n t r a t i o n s o f Con A . However, B e c k e r (l_5) u t i l i z e d an i s o l a t i o n t e c h n i q u e w h i c h d i d n o t employ p e r f u s i o n o f t h e l i v e r i n s i t u w i t h c o l l a g e n a s e , and s u c h t e c h n i q u e s have been d e m o n s t r a t e d t o y i e l d c e l l s o f low v i a b i l i t y (16). I n v e s t i g a t i o n o f the c e l l - s u r f a c e p r o p e r t i e s o f e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s i s c o m p l i c a t e d by t h e p o s s i b l e a l t e r a t i o n o f c e l l - s u r f a c e components by c o n t a m i n a t i n g p r o t e a s e s p r e s e n t i n p r e p a r a t i o n s o f c r u d e c o l l a g e n a s e . The p r o c e d u r e f o r isolation o f h e p a t o c y t e s d e s c r i b e d h e r e i n u t i l i z e d 0.5% BSA i n t h e p e r f u s i o n medium. A c o n t r o l experiment i n d i c a t e d that t h i s c o n c e n t r a t i o n of BSA was s u f f i c i e n t t o m i n i m i z e s u r f a c e a l t e r a t i o n s by c o n t a m i n a t i n g proteases. Rat e r y t h r o c y t e s o f f e r a s e n s i t i v e s y s t e m f o r i n v e s t i g a t i o n o f t h e e f f e c t o f p r o t e a s e on Con A - i n d u c e d c y t o a g g l u t i n a t i o n (25). Rat e r y t h r o c y t e s , i n c u b a t e d a t 3 7 ° f o r 30 min i n C F H , were n o t a g g l u t i n a t e d by Con A c o n c e n t r a t i o n s as h i g h as 133 yg/ml i n t h e t e s t t u b e a g g l u t i n a t i o n a s s a y o f L e s e n e y et_ a_l_ (25). When i n c u b a t e d a t 37 f o r 30 min i n t h e p r e s e n c e o f 0.05% collagenase i n CFH o r 0.01% p a p a i n i n CFH t h e y were a g g l u t i n a t e d by 22 and 4 yg Con A / m l , r e s p e c t i v e l y . Rat e r y t h r o c y t e s , i n c u b a t e d i n t h e p r e s e n c e o f 0.05% c o l l a g e n a s e and 0.5% BSA i n CFH were n o t a g g l u t i n a t e d a t Con A c o n c e n t r a t i o n s up t o 133 y g / m l , i n d i c a t i n g t h a t t h e BSA s e r v e s as s u b s t r a t e f o r t h e p r o t e a s e p r e s e n t i n t h e c r u d e c o l l a g e n a s e p r e p a r a t i o n and p r e v e n t s t h e i r a c t i o n on t h e s u r f a c e of the rat e r y t h r o c y t e s . (34), ject
S i n c e p l a s m a membrane components u n d e r g o m e t a b o l i c t u r n o v e r p r o t e o l y t i c o r o t h e r damage t o t h e p l a s m a membrane i s s u b to r e p a i r processes. If t h e e n z y m a t i c a l l y d i s p e r s e d l i v e r
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
421
c e l l s u s e d i n t h i s s t u d y w e r e r e n d e r e d a g g l u t i n a b l e by a l t e r a t i o n s r e s u l t i n g from the c o l l a g e n a s e p e r f u s i o n , then i n c u b a t i o n o f v i a b l e l i v e r c e l l s should r e s u l t in decreasing lectin-induced a g g l u t i n a b i 1 i t y as a f u n c t i o n o f s u r f a c e r e p a i r . The d a t a i n t a b l e II show t h e e f f e c t o f i n c r e a s i n g i n c u b a t i o n t i m e on t h e Con A - i n d u c e d a g g l u t i n a b i 1 i t y o f e n z y m a t i c a l l y d i s p e r s e d liver cells. T h e r e i s a 2 - t o - 4 f o l d d e c r e a s e i n t h e Con A - i n d u c e d a g g l u t i n a b i 1 i t y o f h e p a t o c y t e s i n c u b a t e d f o r 4 h compared w i t h h e p a t o c y t e s i n c u b a t e d f o r 30 m i n ; h o w e v e r , r e a g g r e g a t i o n o f t h e h e p a t o c y t e s d u r i n g t h e k h i n c u b a t i o n may be p a r t i a l l y r e s p o n s i b l e f o r t h i s decreased a g g 1 u t i n a b i 1 i t y since aggregate format i o n w i l l d e s e n s i t i z e t h e p a r t i c l e c o u n t e r a s s a y as p r e v i o u s l y discussed. A f t e r 8 h o f i n c u b a t i o n , t h e Con A - i n d u c e d a g g l u t i n abi l i t y of e n z y m a t i c a l l y d i s p e r s e d hepatocytes is 2 - f o l d g r e a t e r t h a n h e p a t o c y t e s i n c u b a t e d f o r k h even t h o u g h t h e e x t e n t o f c e l l r e a g g r e g a t i o n was t h e same f o r h e p a t o c y t e s i n c u b a t e d k o r 8 h (table II). These of adult hepatocytes b f u n c t i o n o f r e p a i r p r o c e s s e s which a r e o p e r a t i v e d u r i n g s h o r t t e r m i n c u b a t i o n i n Ml99 + 3% F C S . E f f e c t o f p a p a i n d i g e s t i o n on Con A - o r WGA-induced a g g l u t i n a t i o n of hepatocytes. Papain treatment markedly a l t e r e d the l e c t i n induced a g g l u t i n a b i 1 i t y o f e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s . P a p a i n t r e a t m e n t was a c c o m p a n i e d by a 4 - f o l d o r 8 - f o l d r e d u c t i o n in the l e c t i n c o n c e n t r a t i o n r e q u i r e d f o r t h r e s h o l d a g g l u t i n a t i o n by Con A o r WGA, r e s p e c t i v e l y ( t a b l e I ) . Papain d i g e s t i o n d i d n o t a l t e r t h e number o r d i s t r i b u t i o n o f s m a l l a g g r e g a t e s o f h e p a t o c y t e s w h i c h were p r e s e n t i n t h e a b s e n c e o f l e c t i n , c o n sequently the increased 1 e c t i n - i n d u c e d a g g l u t i n a b i 1 i t y o f p a p a p i n - t r e a t e d c e l l s c a n n o t be a t t r i b u t e d t o an i n c r e a s e i n t h e percentage o f s i n g l e c e l l s in the c e l l s u s p e n s i o n . By c o n t r a s t , m e c h a n i c a l l y d i s p e r s e d h e p a t o c y t e s r e m a i n e d n o n - a g g l u t i n a b l e by c o n c e n t r a t i o n s o f Con A o r WGA as h i g h as 270 y g / m l , even a f t e r p a p a i n d i g e s t i o n , an o b s e r v a t i o n c o n s i s t e n t w i t h a p r e v i o u s i n v e s t i g a t i o n (]_]). In c o m p a r i s o n t o t h e e n z y m a t i c a l l y d i s persed h e p a t o c y t e s , N o v i k o f f c e l l s e x h i b i t e d o n l y minor a l t e r a t i o n s o f t h e i r Con A - o r W G A - i n d u c e d a g g l u t i n a b i 1 i t y as a r e s u l t of papain d i g e s t i o n (table l ) . Gel f i l t r a t i o n o f g l y c o p e p t i d e s c l e a v e d f r o m t h e s u r f a c e o f h e p a t o c y t e s and hepatoma c e l l s by p a p a i n . D i g e s t i o n o f normal and m a l i g n a n t l i v e r c e l l s w i t h p a p a i n d e g r a d e s t h e c e l l - s u r f a c e g l y c o p r o t e i n s , r e l e a s i n g g l y c o p e p t i d e s i n t o the e x t r a c e l l u l a r fluid ( i i ) . Papain treatment of e n z y m a t i c a l l y d i s p e r s e d hepatoc y t e s r e s u l t e d i n t h e r e l e a s e o f 0 . 0 8 y m o l e s i a l i c a c i d p e r ml o f p a c k e d (210 g , 10 min) c e l l s . A similar quantity of s i a l i c acid, 0 . 0 9 ymole/ml o f p a c k e d c e l l s , was r e l e a s e d f r o m t h e s u r f a c e o f N o v i k o f f c e l l s by d i g e s t i o n w i t h p a p a i n . The n o n - d i a l y z a b l e g l y c o p e p t i d e s c l e a v e d from the s u r f a c e o f e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s and N o v i k o f f hepatoma c e l l s were s u b m i t t e d t o g e l f i l t r a t i o n on Sephadex G-50 ( f i g . 2 ) . The e n z y m a t i c a l l y d i s -
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
422
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE
Experimental Cell Research
Figure 2. Gel filtration of the cell-surface glycopeptides from (A) Novikoff hepatoma cells; (B) enzymatically dispersed hepatocytes; and (C) mechanically dispersed liver cells. The column (2.5 X 90 cm) of Sephadex G-50 was equilibrated and eluted with 0.1 N acetic acid at a flow rate of 20 mL/hr at 22°C. Analysis of the column effluents is described in the Materials and Methods section. Sugar concentrations are expressed as fimol per fraction.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
23.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
423
p e r s e d h e p a t o c y t e c e l l - s u r f a c e g l y c o p e p t i d e s ( f i g . 2B) were r e s o l v e d i n t o two f r a c t i o n s : C - G P - A , a component e x c l u d e d f r o m t h e g e l and C - G P - B , a p r o m i n e n t s i a l o g l y c o p e p t i d e f r a c t i o n w h i c h was p a r t i a l l y a c c e s s i b l e t o t h e g e l and w h i c h c o n t a i n e d o v e r 30% o f t h e s i a l i c a c i d r e l e a s e d f r o m t h e c e l l - s u r f a c e by p a p a i n . C-GP-A i s o l a t e d f r o m e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s c o n t a i n e d a l a r g e q u a n t i t y o f n e u t r a l s u g a r w h i c h was due t o g l y c o gen r e l e a s e d d u r i n g t h e p a p a i n d i g e s t i o n , p r e s u m a b l y r e s u l t i n g f r o m t h e l y s i s o f a few h e p a t o c y t e s r i c h i n g l y c o g e n . The g l y c o g e n p r e s e n t i n C-GP-A was removed by p r e c i p i t a t i o n w i t h 50% ethanol. Comparison o f the gel f i l t r a t i o n p r o f i l e s o f the g l y c o p e p t i d e s r e l e a s e d from the s u r f a c e o f e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s and N o v i k o f f hepatoma c e l l s s u g g e s t s m a j o r q u a l i t a t i v e d i f f e r e n c e s i n t h e i r p l a s m a membrane g l y c o p r o t e i n s ( f i g . 2 ) . In c o n t r a s t to the c e l l - s u r f a c a major p o r t i o n o f the ( C - S G P - A ) was e x c l u d e d f r o m t h e g e l ( f i g . 2A). This fraction c o n t a i n e d 31% o f t h e t o t a l s i a l i c a c i d r e l e a s e d f r o m t h e c e l l surface of papain. L e c t i n r e c e p t o r a c t i v i t i e s o f c e l l - s u r f a c e g l y c o p e p t i d e s from h e p a t o c y t e s and hepatoma c e l l s . A c o m p a r i s o n o f t h e Con A and WGA r e c e p t o r a c t i v i t e s o f t h e c e l l - s u r f a c e g l y c o p e p t i d e s f r o m h e p a t o c y t e s and N o v i k o f f hepatoma c e l l s p r o v i d e d a d d i t i o n a l evid e n c e f o r q u a l i t a t i v e d i f f e r e n c e s between t h e c e l l - s u r f a c e g l y c o p r o t e i n s o f normal and m a l i g n a n t r a t l i v e r c e l l s ( t a b l e I I I ) . Since l e c t i n s bind to s p e c i f i c saccharide determinants, differences in the l e c t i n r e c e p t o r a c t i v i t e s suggest that these q u a l i t a t i v e d i f f e r e n c e s , a r e , in p a r t , s t r u c t u r a l . The g l y c o p e p t i d e s isolated from the s u r f a c e o f e n z y m a t i c a l l y d i s p e r s e d hepatocytes possessed no d e t e c t a b l e Con A o r WGA r e c e p t o r a c t i v i t y i n d i c a t i n g t h a t t h e c e l l s u r f a c e components r e s p o n s i b l e f o r t h e a g g l u t i n a t i o n o f r a t h e p a t o c y t e s a r e n o t r e l e a s e d by d i g e s t i o n w i t h p a p a i n . On t h e o t h e r hand, the c e l l - s u r f a c e g l y c o p e p t i d e f r a c t i o n s from N o v i k o f f c e l l s p o s s e s s e d p o t e n t Con A a n d / o r WGA r e c e p t o r a c t i v i t y . The m a j o r p o r t i o n o f t h e Con A and WGA r e c e p t o r a c t i v i t y r e s i d e d i n C-SGP-A a s r e p o r t e d p r e v i o u s l y (12). The l o s s o f Con A - b i n d i n g s i t e s from the s u r f a c e o f N o v i k o f f c e l l s f o l l o w i n g papain t r e a t ment has been d e m o n s t r a t e d by b i n d i n g s t u d i e s u s i n g Con A labeled with [ ^ l ] (35). These o b s e r v a t i o n s i n d i c a t e a d i f f e r e n t i a l l a b i l i t y o f t h e c e l l - s u r f a c e l e c t i n r e c e p t o r s o f normal and m a l i g n a n t r a t l i v e r c e l l s t o d e g r a d a t i o n by p a p a i n . P a p a i n - l a b i l e , c e l l - s u r f a c e g l y c o p e p t i d e s i s o l a t e d from N o v i k o f f hepatoma c e l l s r e f l e c t e d t h e c y t o a g g 1 u t i n a t i o n properties o f t h e i n t a c t c e l l s f r o m w h i c h t h e y were d e r i v e d w h i l e g l y c o p e p t i d e s i s o l a t e d from e n z y m a t i c a l l y d i s p e r s e d c e l l s d i d n o t . These d a t a s u g g e s t t h a t , c o n t r a r y t o N o v i k o f f a s c i t e s hepatoma c e l l s , the p a p a i n - l a b i l e , c e l l - s u r f a c e g l y c o p e p t i d e s i s o l a t e d from e n z y m a t i c a l l y d i s p e r s e d h e p a t o c y t e s do n o t f u n c t i o n i n t h e a g g l u t i n a t i o n of the i n t a c t c e l l s . T h i s f i n d i n g emphasizes the importance
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
e
d
c
b
a
C
C
C
d
45
29
2500 710 250 2200 + 200
<70
26
<70
17
83
S.
b
250 + 60
620 170 <70
<70 <70
D e t e r m i n e d u s i n g a s s a y o f S m i t h et^ a l ( 1 4 ) . H e m a g g l u t i n a t i o n i n h i b i t o r y u n i t s d e f i n e d by S m i t h e£ a l ( 1 4 ) . Ethanol t r e a t e d p r i o r to l e c t i n receptor a c t i v i t y assay. O v a l b u m i n g l y c o p e p t i d e p r e p a r e d as d e s c r i b e d by Montgomery and Lee Ovomucoid o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p .
6
C
Enzymatically dispersed hepatocytes C-GP-A C-GP-B Incubated N o v i k o f f hepatoma e e l 1s C-SGP-A C-GSP-B C-GSP-C Ovalbumin g l y c o p e p t i d e Ovomucoid
GIycopept ide Fractions
II I
a
2
(42),
S.D.
650 210 110
49
160
LECTIN RECEPTOR A C T I V I T I E S OF CELL-SURFACE GLYCOPEPTIDES ISOLATED FROM NORMAL AND MALIGNANT RAT LIVER CELLS Total Lectin Specific Lectin Receptor A c t i v i t y Yield Receptor A c t i v i t y HAIU/Fraction x l O " HAIU/mg mg/100 mg WGA Con A WGA Con A
TABLE
23.
STARLING E T A L .
Normal and Malignant Rat Liver Cells
425
of the p a p a i n - s t a b l e l e c t i n receptor s i t e s in determining the l e c t i n - i n d u c e d a g g l u t i n a t i o n p r o p e r t i e s o f normal l i v e r c e l l s and i n d i c a t e s t h a t t h e i n c r e a s e d a g g l u t i n a b i 1 i t y o f h e p a t o c y t e s by Con A and WGA a f t e r p a p a i n d i g e s t i o n may be due t o s e v e r a l f a c t o r s : exposure o f c r y p t i c l e c t i n receptor s i t e s ; topographical redistrib u t i o n o f l e c t i n r e c e p t o r s i t e s ; a n d / o r a c h a n g e i n membrane morphology. [ Con A b i n d i n g t o r a t h e p a t o c y t e s . Since b r i e f p r o t e o l y s i s o f n o n - t r a n s f o r m e d c e l l s r e n d e r e d them as a g g l u t i n a b l e by WGA a s t r a n s f o r m e d c e l l s , B u r g e r (3) p r o p o s e d t h a t t h e i n c r e a s e d a g g l u t i n a b i l i t y o f t r a n s f o r m e d c e l l s was due t o t h e e x p o s u r e o f c r y p t i c c e l l - s u r f a c e lectin-binding s i t e s during transformation. Since t h i s p r o p o s a l was f o r m u l a t e d , h o w e v e r , s e v e r a l investigators (36,37) h a v e r e p o r t e d t h a t normal and t r a n s f o r m e d c e l l s b i n d e q u i v a l e n t amounts o f r a d i o a c t i v e l e c t i n s u g g e s t i n g t h a t t h e i n c r e a s e d lectin-induced agglutinabi exposure o f c r y p t i c l e c t i n - b i n d i n and B u r g e r (38) r e p o r t e trypsinize non-transforme t r a n s f o r m e d 3T3 c e l l s bound 2.5"3 t i m e s more [3H] Con A t h a n n o n t r a n s f o r m e d 3T3 c e l l s u n d e r c a r e f u l l y c o n t r o l l e d c o n d i t i o n s w h i c h l i m i t e d endocytosis o f the r a d i o a c t i v e l e c t i n . These experiments again suggested the p o s s i b i l i t y that increased l e c t i n - i n d u c e d a g g l u t i n a b i 1 i t y was b r o u g h t a b o u t by e x p o s u r e o f c r y p t i c l e c t i n binding s i t e s . As shown i n t a b l e IV, i n c u b a t e d c o n t r o l and p a p a i n - d i g e s t e d h e p a t o c y t e s b i n d e q u i v a l e n t amounts o f [ ^ l ] Con A a n d e x h i b i t s i m i l a r a p p a r e n t b i n d i n g a f f i n i t y c o n s t a n t . The s i m i l a r i t y o f M 5|] Con A b i n d i n g t o i n c u b a t e d c o n t r o l and p a p a i n - d i g e s t e d h e p a t o c y t e s i s n o t due t o e n d o c y t o s i s o f t h e r a d i o a c t i v e l e c t i n because the b i n d i n g assay u t i l i z e d i n these d e t e r m i n a t i o n s m e a s u r e s o n l y t h e [ ' *>l] Con A b i n d i n g t h a t i s s p e c i f i c a l l y d i s p l a c e d by m e t h y l a - D - m a n n o p y r a n o s i d e . This i s c o n f i r m e d f u r t h e r by t h e low amount o f n o n - s p e c i f i c b i n d i n g ( l e s s t h a n 15%) o f [ 5 | ] Con A i n t h e b i n d i n g a s s a y ( t a b l e I V ) . As d i s c u s s e d e a r l i e r , h e p a t o c y t e s u s p e n s i o n s o b t a i n e d by c o l l a g e n a s e p e r f u s i o n p o s s e s s e d 28% o f t h e c e l l s i n a g g r e g a t e o f 2-6 c e l l s . The amount o f [ 5 | ] Con A nbound t o p a p a i n - d i g e s t e d h e p a t o c y t e s i s n o t e n h a n c e d by an i n c r e a s e i n t h e number o f s i n g l e c e l l s i n the hepatocyte suspension f o l l o w i n g papain d i g e s t i o n s i n c e t h e a g g r e g a t e s i z e o f h e p a t o c y t e s o b t a i n e d by c o l l a n g e a s e p e r f u s i o n i s n o t a l t e r e d by p a p a i n d i g e s t i o n . These d a t a i n d i c a t e t h a t r a t h e p a t o c y t e s do n o t e x h i b i t i n c r e a s e d Con A b i n d i n g f o l l o w i n g p a p a i n d i g e s t i o n even t h o u g h t h e h e p a t o c y t e s become s i g n i f i c a n t l y more a g g l u t i n a b l e ( t a b l e l ) . T h u s , t h e s e d a t a do n o t s u p p o r t t h e h y p o t h e s i s o f i n c r e a s e d Con A - i n d u c e d a g g l u t i n a b i 1 i t y o f p a p a i n - d i g e s t e d h e p a t o c y t e s due t o e x p r e s s i o n of c r y p t i c l e c t i n b i n d i n g s i t e s f o l l o w i n g papain d i g e s t i o n . The unaltered l e c t i n binding o f the hepatocytes f o l l o w i n g papain d i g e s t i o n i s c o n s i s t e n t with the f i n d i n g that eel 1-surface, p a p a i n - l a b i l e g l y c o p e p t i d e s from e n z y m a t i c a l l y d i s p e r s e d hepat o c y t e s p o s s e s s no d e t e c t a b l e Con A r e c e p t o r a c t i v i t y , indica2
12
12
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 8
1.8 1.1 0.96 1.3
0.80 1.1
6
Specific binding Molecules Ka bound/cel1 Li ter/moles x 10" x 10" 1 3
a
D e t e r m i n e d by b i n d i n g a s s a y o f D a v i s e t a l b Data p l o t t e d a c c o r d i n g t o t h e method ofSTeck
1ntact Incubated c o n t r o l Papain-digested
Condi t i o n s or treatment
IV
(41).
0.13
0.13 0.16
8
8
N o n - s p e c i f i c binding* Molecules/cel1 x 10"
HEPATOCYTES
and W a l l a c h
BINDING OF CON A TO RAT
TABLE
5
o o w
>
2 S
r
8
O
>
3
n o
$
to
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
Experimental Cell Research
Figure 3. Fer-Con A topographical distribution of rat hepatocytes. Electron micrograph of rat hepatocytes labeled with Fer-Con A as described in the Materials and Methods section. (A) Intact + Fer-Con A; (B) incubated control + Fer-Con A; (C) papain-digested hepatocytes + FerCon A; (D) intact hepatocytes + 0.17M methyl a-v-mannopyranoside + Fer-Con-A. X55fl00.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
428
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
Experimental Cell Research
Figure 4. Cell-surface morphology of rat hepatocytes. Electron micrograph of glutaraldehyde-fixed cells post-fixed with OsO^. Sections were stained with uranyl acetate and lead citrate. (A) Intact; (B) incubated control; (C) papain-digested hepatocytes. XH,800.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23.
STARLING E T A L .
Normal
and Malignant
Rat Liver
Cells
429
t i n g t h a t t h e s e g l y c o p e p t i d e s a r e n o t i n v o l v e d i n t h e Con A induced a g g l u t i n a t i o n o f the i n t a c t c e l l s ( t a b l e I I I ) . Con A r e c e p t o r t o p o g r a p h y . N i c o l s o n (39) o b s e r v e d t h a t F e r - C o n A r e c e p t o r s on t r a n s f o r m e d and t r y p s i n i z e d 3T3 c e l l s were i n a c l u s t e r e d t o p o g r a p h i c a l d i s t r i b u t i o n compared w i t h n o n - t r a n s f o r m e d 3T3 c e l l s . Based on t h i s o b s e r v a t i o n , N i c o l s o n p o s t u l a t e d t h a t t h e i n c r e a s e d a g g 1 u t i n a b i 1 i t y o f t r y p s i n i z e d n o n - t r a n s f o r m e d and t r a n s f o r m e d 3T3 c e l l s was due t o t h e c l u s t e r e d d i s t r i b u t i o n o f Con A r e c e p t o r s on t h e s u r f a c e o f t h e s e c e l l s w h i c h e n a b l e a g g l u t i n a t i o n t o o c c u r between c e l l - s u r f a c e a r e a s o f h i g h l e c t i n receptor density. The t o p o g r a p h i c a l d i s t r i b u t i o n s o f h e p a t o c y t e s l a b e l e d w i t h F e r - C o n A a r e shown i n F i g . 3. Altered distribution o f Con A r e c e p t o r s i t e s a f t e r p a p a i n d i g e s t i o n does n o t a p p e a r t o be r e s p o n s i b l e f o r t h e i n c r e a s e d a g g l u t i n a b i 1 i t y o f p a p a i n d i g e s t e d h e p a t o c y t e s s i n c e i n t a c t , i n c u b a t e d c o n t r o l , and p a p a i n digested hepatocytes a l Fer-Con A molecules ( f i g t o r s (5^§j7_) h a v e r e p o r t e d t h a t c l u s t e r e d d i s t r i b u t i o n s o f l e c t i n m o l e c u l e s may r e f l e c t t h e l a t e r a l m o b i l i t y o f t h e l e c t i n r e c e p t o r s r a t h e r than t h e i r i n h e r e n t t o p o g r a p h i c a l d i s t r i b u t i o n , these d a t a do n o t r u l e o u t p o s s i b l e d i f f e r e n c e s w h i c h may e x i s t i n t h e i n h e r e n t t o p o g r a p h i c a l d i s t r i b u t i o n s o f h e p a t o c y t e Con A r e c e p t o r s , a l t h o u g h s u c h d i f f e r e n c e s w o u l d n o t e x p l a i n t h e i n c r e a s e d Con A i n d u c e d a g g l u t i n a b i 1 i t y o f p a p a i n - d i g e s t e d h e p a t o c y t e s compared w i t h i n t a c t and i n c u b a t e d c o n t r o l h e p a t o c y t e s . C e l l - s u r f a c e morphology. A p l a u s i b l e e x p l a n a t i o n f o r the i n c r e a s e d Con A - i n d u c e d a g g 1 u t i n a b i 1 i t y o f p a p a i n d i g e s t e d h e p a t o c y t e s i s d e r i v e d f r o m t h e change i n s u r f a c e m o r p h o l o g y f r o m a v i l l o u s t o a smooth membrane as a r e s u l t o f p a p a i n d i g e s t i o n (fig. 4). T h i s membrane a l t e r a t i o n may a l l o w g r e a t e r a r e a s o f c o n t a c t between a g g l u t i n a t e d c e l l s t h e r e b y p e r m i t t i n g more e x tensive l e c t i n bridging to occur. T h i s m o r p h o l o g i c a l change w i l l also result i n a h i g h e r s u r f a c e d e n s i t y o f Con A bound r e c e p t o r s on p a p a i n - d i g e s t e d h e p a t o c y t e s as compared t o i n c u b a t e d c o n t r o l h e p a t o c y t e s a l t h o u g h t h i s i n c r e a s e d r e c e p t o r d e n s i t y may n o t account e n t i r e l y f o r the increased a g g l u t i n a b i 1 i t y o f papain d i g e s t e d h e p a t o c y t e s (3j0 • The a b o v e c o n c l u s i o n s a r e f u r t h e r s u p p o r t e d by t h e o b s e r v a t i o n t h a t h i g h Con A - a n d WGA-induced a g g l u t i n a t i o n o f human l y m p h o b l a s t s and normal human p e r i p h e r a l l y m p h o c y t e s was c o r r e l a t e d w i t h a smooth c e l l s u r f a c e w h e r e a s low Con A and WGA a g g l u t i n a b i 1 i t y was c o r r e l a t e d w i t h a v i l l o u s s u r f a c e morphology ( 4 0 ) . The a b o v e d a t a s u g g e s t t h a t t h e r e a r e s i g n i f i c a n t structural d i f f e r e n c e s i n t h e c e l l - s u r f a c e g l y c o p r o t e i n s o f normal and m a l i g n a n t r a t l i v e r c e l l s and t h a t v i a b l e h e p a t o c y t e s u s p e n s i o n s o f f e r a v a l u a b l e in v i v o system f o r s t u d y i n g c e l l - s u r f a c e p r o p e r t i e s a s s o c i a t e d w i t h normal c e l l s .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
430
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
ACKNOWLEDGEMENTS The authors express appreciation to Dr. Max Schlamowitz and Dr. Kay Hillman for their assistance in the Con A iodination procedure. This work was supported by grants from NCI (CA-11710), the Robert A. Welch Foundation (G-354), the Paul and Mary Haas Foundation and the George and Mary Josephine Hamman Foundation. J.J.S. was recipient of a Rosalie B. Hite predoctoral fellowship in cancer research. Portions of the tables and figures from references 19 and 31 are reproduced by permission of Academic Press. Literature Ci ted 1. Emmelot, P. (1973) Eur. J. Cancer 9, 319. 2. Nicolson, G. L. (1974) Int. Rev. Cytol. 39, 89. 3. Burger, M. M. (1969) Proc. Natl. Acad. Sci. US 62, 944. 4. Burger, M. M. (1970) Permeability and Function of Biological Membranes (ed. L. Bolis et al.), p. 107. North-Holland, Amsterdam. 5. Nicolson, G. L. (1973 6. Nicolson, G. L. (1974 Cells (ed. B. Clarkson and R. Baserga), p. 251. Cold Spring Harbor, New York. 7. Rosenblith, J. A., Ukena, T. E., Yin, H. H., Berlin, R. D., and Karnovsky, M. J. (1973) Proc. Natl. Acad. Sci. US 70, 1625. 8. Rutishauser, U. and Sachs, L. (1975) J. Cell Biol. 66, 76. 9. Collard, J. G. and Temmink, J. H. M. (1975) J. Cell Sci. 19, 21. 10. Jansons, V. K., Sakamoto, C. K., and Burger, M. M. (1973). Biochim. Biophys. Acta 291, 136. 11. Walborg, E. F., Jr., Wray, V. P., Smith, D. F., and Neri, G. (1975) Immunological Aspects of Neoplasia, p. 123. Williams and Wilkins Co., Baltimore, Md. 12. Neri, G., Smith, D. F., Gilliam, E. B., and Walborg, E. F., Jr. (1974) Arch. Biochem. Biophys. 165, 323. 13. Smith, D. F., Walborg, E. F., Jr., and Chang, J. P. (1970) Cancer Res. 30, 2306. 14. Smith, D. F., Neri, G., and Walborg, E. F., Jr. (1973) Biochem. 12, 2111. 15. Becker, F. F. (1974) Proc. Natl. Acad. Sci. US 71, 4307. 16. Schreiber, G. and Schreiber, M. (1972) Sub-cell. Biochem. 2, 321. 17. Jacob, S. T. and Bhargava, P. M. (1962) Exp. Cell Res. 27, 453. 18. Bonney, R. J. (1974) In Vitro 10, 130. 19. Starling, J. J., Capetillo, S. C., Neri, G., and Walborg, E. F., Jr. (1977) Exp. Cell Res. 104, 177. 20. Phillips, H. J. (1973) Tissue Culture: Methods and Applications (ed. P. F. Kruse and M. K. Patterson) p. 406. Academic Press, New York. 21. Walborg, E. F., Jr. Lantz, R. S., and Wray, V. P. (1969) Cancer Res. 29, 2034.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
23.
22. 23. 24. 25.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.
STARLING ET AL.
431 Normal and Malignant Rat Liver Cells
Davis, E. M., Starling, J. J., and Walborg, E. F., Jr. (1976) Exp. Cell Res. 99, 37. Wray, V. P. and Walborg, E. F.,Jr. (1971) Cancer Res. 31, 2079. Hanks, J. H. and Wallace, R. E. (1949) Proc. Soc. Exp. Biol. Med. 71, 196. Leseney, A. M., Thomas, M. W., O'Neil, P. A., and Walborg, E. F., Jr. (1974) Méthodologie de la Structure et du Métabolisme des Glycoconjugues. Actes du Colloque Internationaux n 221 due Centre National de la Recherche Scientifique, vol. 2, p. 859. Editiones du CNRS, Paris. Aminoff, D. (1961) Biochem. J. 81, 384. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350. Gatt, R. and Berman, E. R. (1966) Anal. Biochem. 15, 167. Sonoda, S. and Schlamowitz Agrawal, B. B. L 96, 23C. Starling, J. J., Hixson, D. C., Davis, E. M., and Walborg, E. F., Jr. (1977) Exp. Cell Res. 104, 165. Davis, E. M., Tsay, D. D., Schlamowitz, M., and Walborg, E. F., Jr. (1977) Anal. Biochem. 80, 416. Seglen, P. 0. (1972) Exp. Cell Res. 74, 450. Warren, L. (1969) Current Topics in Developmental Biology (ed. A. A. Moscona and A. Monroy) vol. 4, p. 197. Academic Press, New York. Walborg, E. F., Jr., Davis, E. M., Gilliam, E. B., Smith, D. F., and Neri, G. (1975) Cellular Membranes and Tumor Cell Behavior, p. 337. Williams and Wilkins, Baltimore, Md. Cline, M. J. and Livingston, D. C. (1971) Nature New Biol. 232, 155. Ozanne, B. and Sambrook, J. (1971) Nature New Biol. 232, 156. Noonan, K. D. and Burger, M. M. (1973) J. Cell Biol. 59, 134. Nicolson, G. L. (1971) Nature New Biol. 233, 244. Temmink, J. H. M., Collard, J. G., Roosien, J. and van den Bosch, J. F. (1976) J. Cell Sci. 21, 563. Steck, T. L. and Wallach, D. F. H. (1965) Biochim. Biophys. Acta 97, 510. Montgomery, R. and Lee, Y. C. (1965) Biochem. 4, 566.
RECEIVED
March 30, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24 Membrane Glycoproteins of Rat Mammary Gland and Its Metastasizing and Nonmetastasizing Tumors KERMIT L. CARRAWAY, JOHN W. HUGGINS, ANNE P. SHERBLOM, ROBERT W. CHESNUT, ROBERT L. BUCK, SUSAN P. HOWARD, CHARLOTTE L. OWNBY, and CORALIE A. CAROTHERS CARRAWAY Department of Biochemistry, Oklahoma State University, Stillwater, OK 74074
Glycoproteins are ubiquitous components of animal cell surfaces (1) and are generally believed to play important roles in neoplastic diseases (2). Glycoprotein differences between nonmalignant and malignant cells have been demonstrated by such diverse techniques as lectin agglutination (3), glycopeptide analyses (4), gel electrophoresis (5) and enzymatic cell surface labeling (6). The origin of these differences and the roles of the glycoproteins in most cellular phenomena are still unknown. One intriguing possibility is that cell surface glycoproteins may be involved in the mechanism by which tumor cells escape destruction by the immune system of the host. Two very different hypotheses have been presented to explain the role of glycoproteins in this escape from immune surveillance. The first involves the shedding of antigens from the cell surface. These antigens combine with circulating antibodies to form complexes which block antibody-mediated destruction of the tumor cells (7). Kim et al. (8) have extended this hypothesis to suggest that glycoprotein shedding in mammary tumors is related to their metastatic potential. Using a series of mouse mammary tumors selected for variations in ability to metastasize, they found an inverse correlation between metastasis and three parameters: 5'-nucleotidase activity, cell surface glycocalyx staining and immunogenicity of the tumors in rabbits. It was suggested that the loss of the cell surface constituents was due to shedding, but the mechanism was not specified. A second means by which glycoproteins might be involved in "protection" of the tumor cells was suggested by studies on the TA3 mouse mammary tumor. Two sublines of this tumor which are different in their immunological properties have been isolated. The TA3-St subline grows only in the syngeneic host, but the TA3-Ha subline is able to cross histocompatibility barriers (9). This loss of strain specificity is accompanied by a greatly increased amount of a large sialoglycoprotein (10) named 0-8412-0452-7/78/47-080-432$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24.
CARRAWAY
ETAL.
Membrane
Glycoproteins
of Rat
Gland
433
e p i g l y c a n i n . I t was hypothesized that the g l y c o p r o t e i n i n the TA3-Ha s u b l i n e "covers" the h i s t o c o m p a t i b i l i t y antigens at the c e l l surface (11), preventing t h e i r expression and thereby prev e n t i n g the r e c o g n i t i o n of these c e l l s as f o r e i g n to the host. Our own work has been concerned with the c e l l surface g l y coproteins of r a t mammary tumors and the r e l a t i o n s h i p of these g l y c o p r o t e i n s to the p r o p e r t i e s of the tumor c e l l s . In t h i s p r e s e n t a t i o n we s h a l l focus on three questions. 1) Is there a necessary r e l a t i o n s h i p between the expression of c e l l surface g l y c o p r o t e i n s and metastasis of mammary tumors as suggested by Kim et a l . (8)? 2) I s there a r e l a t i o n s h i p between the "form" or environment of the tumor and the c e l l surface g l y c o p r o t e i n s i t expresses? 3) How are the c e l l s u r f a c e p r o p e r t i e s of tumors r e l a t e d to t h e i r b i o l o g i c a l a c t i v i t i e s as tumors? Comparison of membrane g l y c o p r o t e i n s from normal mammary gland, a nonmetastasizin (R3230 tumor (13762). To determine whethe y expression of c e l l surface g l y c o p r o t e i n between nonmetastasizing and m e t a s t a s i z i n g mammary tumors, we have examined the R3230 AC and 13762 r a t mammary adenocarcinomas, both grown i n the F i s c h e r 344 r a t . The R3230 AC i s a slow-growing tumor d e r i v e d from a spontaneous carcinoma (12). I t i s hormonally responsive and e x h i b i t s l i t t l e or no m e t a s t a t i c c a p a c i t y under the c o n d i t i o n s used i n our s t u d i e s . The 13762 r a t mammary adenocarcinoma i s a methylcholanthrene-induced s o l i d tumor (13) which i s hormonally responsive and metastasizes widely and h e a v i l y . For comparison of c e l l surface g l y c o p r o t e i n s i t i s necessary to p u r i f y plasma membranes from the t i s s u e samples. P u r i f i c a t i o n of plasma membrane and other s u b c e l l u l a r t i s s u e f r a c t i o n s i s d i f f i c u l t f o r s e v e r a l reasons (14). D i s r u p t i o n of the c e l l s i n the t i s s u e almost n e c e s s a r i l y fragments the plasma membrane. Fragmentation i s not uniform and may r e s u l t i n a heterogeneous d i s t r i b u t i o n of membrane fragments, both i n terms of s i z e and of p h y s i c a l and f u n c t i o n a l a t t r i b u t e s . This complicates the s e p a r a t i o n of p a r t i c u l a r morphological u n i t s because of the v a r i a t i o n of the s i z e s , d e n s i t i e s , compositions and other f a c t o r s upon which s e p a r a t i o n of fragments i s based. Identif i c a t i o n of morphological u n i t s i s a l s o complicated by fragment a t i o n , which destroys morphological c h a r a c t e r i s t i c s and reduces membranes t o sheets or v e s i c l e s . In a d d i t i o n to morphological c r i t e r i a membrane f u n c t i o n a l a c t i v i t i e s (enzymes, antigens) must be used f o r i d e n t i f i c a t i o n of s u b c e l l u l a r components. T h i s comb i n a t i o n of heterogeneity and l o s s of morphological i d e n t i t y makes the p r e p a r a t i o n of coherent, h i g h l y p u r i f i e d s u b c e l l u l a r components very d i f f i c u l t . Most of the work i n t h i s area has been performed on l i v e r . Plasma membranes have been p u r i f i e d by a number of methods, most of which tend to give membranes derived from the b i l e c a n a l i c u l a r regions (15). Mammary t i s s u e presents i t s own p a r t i c u l a r problems.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
434
GLYCOPROTEINS
A N D GLYCOLIPIDS
I N DISEASE
PROCESSES
Because of the presence of l a r g e amounts of connective t i s s u e the e l a s t i c i t y of the t i s s u e makes homogenization d i f f i c u l t . The procedure which g i v e s the best r e s u l t s f o r normal l a c t a t i n g mammary t i s s u e i n v o l v e s vigorous homogenization i n a S o r v a l l Omni-mixer and i s o l a t i o n of a microsomal f r a c t i o n by d i f f e r e n t i a l c e n t r i f u g a t i o n (5). Microsomes are f u r t h e r f r a c t i o n a t e d by f l o t a t i o n through a sucrose d e n s i t y gradient ( F i g . 1 ) . 5'-Nucleotidase i s used as a marker f o r p r e l i m i n a r y assessment of plasma membrane p u r i f i c a t i o n , s i n c e t h i s enzyme has been shown to be present predominantly i n the plasma membrane of a number of c e l l types (14), i n c l u d i n g mammary gland and mammary tumors (16, 17). S i g n i f i c a n t p u r i f i c a t i o n of 5'-nucleotidase (>25-fold) was achieved i n both of the l i g h t e r f r a c t i o n s ( F and F ) of the discontinuous g r a d i e n t , and there i s a p a r a l l e l p u r i f i c a t i o n of Na , K -ATPase (5), another plasma membrane marker. S u c c i n i c dehydrogenase (a m i t o c h o n d r i a l enzyme) i s markedly reduced, but plasmic r e t i c u l u m marke t o s y l t r a n s f e r a s e i s s i g n i f i c a n t l y concentrated. The l a s t observation suggests G o l g i apparatus contamination (16). Further p u r i f i c a t i o n i s achieved by t r e a t i n g F i and F f r a c t i o n s s e p a r a t e l y with d i g i t o n i n and s u b j e c t i n g these to r e f l o t a t i o n on a s i m i l a r discontinuous gradient c o n t a i n i n g d i g i t o n i n . D i g i t o n i n complexes with c h o l e s t e r o l and would be expected t o s h i f t the c h o l e s t e r o l - e n r i c h e d plasma membrane f r a c t i o n to a higher d e n s i t y (18). Cholesterol-poor f r a c t i o n s such as G o l g i fragments should be unchanged i n d e n s i t y . By t h i s procedure a p o r t i o n of the d i g i t o n i n - t r e a t e d F i s s h i f t e d to F x D F , which shows a 3 - f o l d increase i n n u c l e o t i d a s e a c t i v i t y and i s devoid of g a l a c t o s y l t r a n s f e r a s e and NADPH-cytochrome c reductase (19). C l e a r l y i t has the c h a r a c t e r i s t i c s expected of a h i g h l y p u r i f i e d plasma membrane f r a c t i o n . The F f r a c t i o n behaves d i f f e r e n t l y . Part of i t r e e q u i l i b r a t e s to the Fj. p o s i t i o n upon r e f l o t a t i o n . When t r e a t e d with d i g i t o n i n , part of F i s s h i f t e d to a higher d e n s i t y (F DF ). F D F i s enriched i n 5'-nucleotidase, c h o l e s t e r o l , s i a l i c a c i d and g a l a c t o s y l t r a n s f e r a s e , p r o p e r t i e s suggesting that t h i s s u b f r a c t i o n comes from a plasma membrane c o n t a i n i n g galactosyltransferase. These procedures have been used with s i m i l a r r e s u l t s to p u r i f y plasma membranes from the s o l i d tumors. As shown i n Table I, the 5'-nucleotidase of the m e t a s t a s i z i n g 13762 tumor i s not g r e a t l y diminished when compared t o the nonmetastasizing R3230 AC. S i a l i c a c i d analyses (20) of membrane f r a c t i o n s from these two tumors a l s o f a i l to show any s i g n i f i c a n t d e p l e t i o n i n the m e t a s t a s i z i n g tumor (Table I I ) . S i a l o g l y c o p r o t e i n s a r e analyzed by polyacrylamide g e l e l e c t r o p h o r e s i s i n sodium dodecyl s u l f a t e (SDS). Whereas normal mammary membranes show three major c l a s s e s of g l y c o p r o t e i n s by t h i s technique ( F i g . 2), membranes from the tumor c e l l s have a x
2
+
+
2
x
3
2
2
2
3
2
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
CARRAWAY
Membrane Glycoproteins of Rat Gland
ETAL.
Figure 1. Schematic for membrane preparations from normal lactating mammary tissue and rat mammary tumor tissue samples
1 F.
I
A
•
A
F.DF,
i V XL
1^.
A
FJDFJ
/ MIGRATION DISTANCE (cm)
Figure 2. Membrane glycoproteins of normal lactating rat mammary tissue. Membrane fractions were prepared as previously described (5,19) and analyzed by electrophoresis on poly aery lamide gels in the presence of SDS (5). Gels were stained by the periodate-Schiff procedure and scanned on a Gilford spectrophotometer with a linear transport device.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
436
PROCESSES
TABLE I Nucleotidase A c t i v i t i e s of Mammary Tumor F r a c t i o n s Fraction
R3230 AC
13762
ymoles/hr•mg p r o t e i n Homogenate FiDF F DF 2
3
3
3.5
4.2
95
126
64
61
TABLE I I S i a l i c A c i d Content Homogenate
F
x
F
2
ug/mg p r o t e i n R3230 AC 13762
0.7 0.4
13 19
11 16
f o u r t h c l a s s of g l y c o p r o t e i n s ( F i g . 3) with an apparently lower molecular weight than the other three (5). G l y c o p r o t e i n patterns from the two tumors are very s i m i l a r , though both have patterns more complex than those of membranes from the normal gland. I t i s not p o s s i b l e to a s c e r t a i n a t present whether these s i m i l a r e l e c t r o p h o r e t i c c l a s s e s are the same molecular s p e c i e s , but i t should be f e a s i b l e to o b t a i n a t l e a s t a p a r t i a l answer to that question using immunological techniques. C l e a r l y the d e p l e t i o n of c e l l surface g l y c o p r o t e i n s i s not a u n i v e r s a l c h a r a c t e r i s t i c of plasma membranes i s o l a t e d from m e t a s t a s i z i n g mammary tumors. T h i s does not imply that these tumors do not shed t h e i r surface c o n s t i t u e n t s , nor does i t i n d i c a t e that shedding i s unimportant to metastasis. The conc e n t r a t i o n of any component i n the c e l l r e f l e c t s a balance between s y n t h e t i c and degradative pathways. Since these need to be i n v e s t i g a t e d more c a r e f u l l y , we have examined a s c i t e s c e l l s derived from the m e t a s t a t i c 13762 tumor, because these c e l l s are more e a s i l y manipulated. Glycoproteins of the a s c i t e s form of the 13762 mammary adeno care inoma. The s o l i d 13762 mammary adenocarcinoma has been converted i n t o t r a n s p l a n t a b l e , m e t a s t a t i c a s c i t e s forms MAT-A, MAT-B and
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24.
CARRAWAY
ET
AL.
Membrane Glycoproteins of Rat Gland
437
MAT-C, which we obtained from the Mason Research Laboratory, Worchester, Mass. To serve as models f o r g l y c o p r o t e i n behavior i n the s o l i d tumor, these a s c i t e s tumors must meet two c r i t e r i a . 1) The tumor s u b l i n e s and t h e i r c e l l surface p r o p e r t i e s must be s t a b l e to i n v i v o passage over the p e r i o d of experimentation. 2) The g l y c o p r o t e i n s of the a s c i t e s tumor must be the same as those of the s o l i d tumor. The question of the s t a b i l i t y of the s u b l i n e s proved to be an i n t e r e s t i n g one. During passage of MAT-B and MAT-C s u b l i n e s i n v i v o at weekly i n t e r v a l s over about s i x months, we noted that both were converted to v a r i a n t forms. We s h a l l term these MAT-B1 and MAT-C1 and w i l l discuss t h e i r a l t e r e d c e l l surface p r o p e r t i e s i n a l a t e r s e c t i o n . We suspect that the MAT-A subl i n e a l s o undergoes changes with prolonged passage i n v i v o , but we do not have an appropriate marker to demonstrate these changes. We are maintaining the MAT-B1 and MAT-C1 tumors by t r a n s p l a n t a t i o n and as comparisons. To analyze c e l l surface g l y c o p r o t e i n s of the a s c i t e s tumors, membranes were prepared by a m o d i f i c a t i o n of the method of Warren j2t a l . (21), using z i n c to " s t a b i l i z e " the c e l l surface membranes. Polyacrylamide g e l e l e c t r o p h o r e s i s of SDSs o l u b i l i z e d membranes was used f o r a n a l y s i s of s i a l o g l y c o p r o t e i n s . A l l a s c i t e s tumor s u b l i n e s showed a major band which b a r e l y penetrated the g e l s , i l l u s t r a t e d i n F i g . 4 f o r the MAT-B1 and MAT-C1 s u b l i n e s . Although SDS e l e c t r o p h o r e s i s i s u n r e l i a b l e f o r molecular weight determinations on g l y c o p r o t e i n s , g e l f i l t r a t i o n experiments a l s o suggest that t h i s s i a l o g l y c o p r o t e i n i s a l a r g e molecule. This g l y c o p r o t e i n i s observed as the major c e l l surface component when i n t a c t c e l l s are l a b e l e d with l a c t o peroxidase and I or with periodate and H-borohydride. These r e s u l t s i n d i c a t e that g l y c o p r o t e i n s i n the s o l i d tumor d i f f e r from those i n a s c i t e s l i n e s derived from i t . However, i t should be noted that the membrane preparations f o r s o l i d and a s c i t e s tumors are d i f f e r e n t . Perhaps the more s t r i n g e n t c o n d i t i o n s of c e l l d i s r u p t i o n used i n s o l i d tumor membrane p r e p a r a t i o n cause breakdown of a l a r g e g l y c o p r o t e i n to give m u l t i p l e smaller g l y c o p r o t e i n s . Two types of experiments argue against t h i s p o s s i b i l i t y . F i r s t , attempts to demonstrate a l a r g e g l y c o p r o t e i n i n the s o l i d 13762 tumor by p e r i o d a t e borohydride l a b e l i n g of the s o l u b i l i z e d t i s s u e followed by SDS e l e c t r o p h o r e s i s were negative. Second, milk f a t globule membranes contain the same three major s i a l o g l y c o p r o t e i n c l a s s e s as the normal mammary gland and s o l i d tumors. Since the milk f a t globule has not been subjected to mechanical d i s r u p t i o n , i t i s u n l i k e l y that the s i m i l a r c l a s s e s of g l y c o p r o t e i n s of normal mammary and the s o l i d tumors are products of the c e l l d i s r u p t i o n . What i s the o r i g i n of t h i s d i f f e r e n c e i n g l y c o p r o t e i n s between s o l i d and a s c i t e s forms of the tumor? Probably i t i s a response to the a l t e r e d environment of the a s c i t e s c e l l s , but 1 2 5
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
438
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Mat - Bi IM-
MIGRATION DISTANCE (cm)
Figure 4. Membrane glycoproteins of the rat mammary adenocarcinoma MAT-B1 and MAT-CI sublines. Membranes were prepared essentially as described previously (17) for MAT-A cells. Electrophoresis and staining were performed as indicated in Figure 2. About 50% more protein was applied to the gel for the MAT-B1 sample.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
PROCESSES
24.
CARRAWAY
ET AL.
Membrane
Glycoproteins
of Rat
439
Gland
the mechanism of change i s u n c l e a r . One p o s s i b i l i t y i s an a l t e r e d gene expression r e s u l t i n g i n the synthesis of d i f f e r e n t p r o t e i n s . A l t e r n a t i v e l y , the change could r e f l e c t an a l t e r e d p o s t - t r a n s l a t i o n a l event such as an a l t e r e d a d d i t i o n of carbohydrate or c r o s s l i n k i n g of the p r o t e i n s . Although we cannot answer these questions a t present, i t should be p o s s i b l e to o b t a i n answers by biochemical and immunological comparisons of g l y c o p r o t e i n s from s o l i d and a s c i t e s tumors. Surface p r o p e r t i e s of the 13762 a s c i t e s tumor c e l l s . The sublines of the 13762 a s c i t e s carcinoma d i f f e r not only from the s o l i d tumor but a l s o from each other. The d i f f e r e n c e i n membrane s i a l o g l y c o p r o t e i n content between MAT-B1 and MAT-C1 c e l l s i s i l l u s t r a t e d i n F i g . 4. The a c t u a l d i f f e r e n c e between the two i s greater than that shown because of the amounts of membranes loaded onto the g e l s . C e l l surface s i a l i c a c i d r e l e a s e d by t r y p s i n o from the MAT-C1 c e l l s tha t o t a l c e l l s i a l i c a c i d i s only about 40% g r e a t e r . Labeling s t u d i e s w i t h lactoperoxidase and I or with periodate and Hborohydride a l s o show more major s i a l o g l y c o p r o t e i n i n the MAT-C1 cells. The most s t r i k i n g d i f f e r e n c e between these s u b l i n e s i s t h e i r surface morphology, MAT-C1 c e l l s e x h i b i t i n g a very unusual surface s t r u c t u r e . T h e i r surface i s densely covered with m i c r o v i l l i extending from the c e l l body i n t o h i g h l y branched s t r u c t u r e s ( F i g . 5), whereas MAT-B1 c e l l s have a more normal appearance with fewer m i c r o v i l l i , a l l apparently unbranched (Fig. 6). MAT-B1 and CI c e l l s a l s o have d i f f e r e n t c e l l surface p r o p e r t i e s (Table I I I ) . When t r e a t e d with Concanavalin A (Con A), the MAT-B1 c e l l s r e a d i l y a g g l u t i n a t e . Under the appropriate c o n d i t i o n s of temperature and l e c t i n concentration these B l c e l l s a l s o form patches and caps. The MAT-C1 c e l l s do not respond to the l e c t i n treatment under the same c o n d i t i o n s , i n d i c a t i n g a r e s t r i c t i o n of the m o b i l i t y of the c e l l surface l e c t i n receptor s i t e s . I t i s a l s o noteworthy that the MAT-B1 and MAT-C1 c e l l s have d i f f e r e n t 5'-nucleotidase a c t i v i t i e s . This a c t i v i t y i s e s s e n t i a l l y undetectable i n B l c e l l s , even with a h i g h l y s e n s i t i v e radiochemical assay. In view of the l a r g e d i f f e r e n c e s i n c e l l surface p r o p e r t i e s one might expect s i g n i f i c a n t b i o l o g i c a l d i f f e r e n c e s between the B l and CI tumors. However, we have not found t h i s to be t r u e . The growth and k i l l i n g r a t e s of the two tumors are about the same. Tests f o r s t r a i n s p e c i f i c i t y of the tumors have not shown pronounced d i f f e r e n c e s , and p r e l i m i n a r y s t u d i e s suggest that both s u b l i n e s a r e m e t a s t a t i c . Thus i t appears that d i f f e r e n c e s i n c e l l surface p r o p e r t i e s a r e not n e c e s s a r i l y r e f l e c t e d i n the gross p r o p e r t i e s of the tumors. I t i s i n t e r e s t i n g that the p r o p e r t i e s of the MAT-B1 c e l l s f i t the c r i t e r i a o f Kim et aJL. (8) f o r a m e t a s t a t i c tumor under1 2 5
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
3
440
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
Figure 5. Scanning electron micrograph of MAT-C1 cells. Cells were removed from the animal, fixed in 3% glutaraldehyde in 0.1M sucrose-OJM cacodylate, postfixed with 2% osmium tetr oxide, dehydrated in ethanol, dried in a Polar on critical point dryer, coated with gold/palladium, and examined in a JEOL JSM-35 Scanning Electron Microscope. Note the extensive microvilli, whose branched structures can be readily observed at the periphery of the cell. Magnification, 8160.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24.
CARRAWAY E T AL.
Figure 6.
Membrane
Glycoproteins
of Rat Gland
441
Scanning electron micrograph of MAT-B1 cells prepared as noted for Figure 5. Magnification, 8840.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
442
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
PROCESSES
TABLE I I I C e l l Surface P r o p e r t i e s of MAT-B1 and MAT-C1 Mammary A s c i t e s C e l l s
Property Agglutination Patching and Capping 5 -Nucleotidase, umoles/hr*mg p r o t e i n
MAT-B1
MAT-C1
-HHH-H-H-
-
0
2.0
1
going c e l l surface shedding show s t r i k i n g s i m i l a r i t i e two s u b l i n e s , derived from the same s o l i d tumor, may represent two d i f f e r e n t modes of escaping the immune system of the host. Discussion The r e s u l t s presented permit us to supply t e n t a t i v e answers to the questions r a i s e d at the beginning of t h i s a r t i c l e . 1) There does not appear to be a necessary c o r r e l a t i o n i n mammary tumors between metastasis and g l y c o p r o t e i n shedding. However, both g l y c o p r o t e i n expression and metastasis are very complex phenomena, so t h e i r r e l a t i o n s h i p s may a l s o be complex. 2) Tumor environment apparently a f f e c t s g l y c o p r o t e i n expression, as i n d i c a t e d by the d i f f e r e n c e s between the a s c i t e s and s o l i d tumors. These r e s u l t s p o i n t out the problems i n v o l v e d i n e x t r a p o l a t i n g s t u d i e s on a s c i t e s c e l l s to conclusions about s o l i d tumors. However, the s t u d i e s on a s c i t e s c e l l s have t h e i r own inherent v a l u e . In the process of metastasis i n d i v i d u a l tumor c e l l s or small aggregates d i s s o c i a t e from the primary tumor i n t o the body f l u i d s . The s i m i l a r i t y of these d i s s o c i a t e d c e l l s to a s c i t e s c e l l s should be obvious. Since tumors l o s e many c e l l s , the c a p a c i t y of a tumor f o r metastasis must depend on the a b i l i t y of the d i s s o c i a t e d c e l l s to s u r v i v e i n the c i r c u l a t i o n and become e s t a b l i s h e d at a new s i t e . The a b i l i t y of a tumor c e l l to a l t e r i t s surface r e a d i l y i n response to a change i n environment might be a u s e f u l a t t r i b u t e f o r the m e t a s t a t i c tumor. Since the 13762 tumor has t h i s c a p a b i l i t y , f u r t h e r i n v e s t i g a t i o n of the r e l a t i o n s h i p s between the g l y c o p r o t e i n s of the s o l i d and a s c i t e s tumors may provide i n s i g h t i n t o the requirements f o r such changes. 3) The s t u d i e s on the MAT-B1 and MAT-C1 c e l l s c l e a r l y show that c e l l s with very d i f f e r e n t c e l l surface p r o p e r t i e s can be very s i m i l a r b i o l o g i c a l l y . We suspect that these r e s u l t s r e f l e c t the f a c t that d i f f e r e n t tumor c e l l s may use d i f f e r e n t ways of s u r v i v i n g the defense system of the
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24.
CARRAWAY
ET
AL.
Membrane
Glycoproteins
of
Rat
Gland
443
host. However, i t i s a l s o p o s s i b l e that the mechanisms of tumor s u r v i v a l are too s u b t l e to be detected by the methodologies used i n our s t u d i e s . The s t u d i e s presented here have r a i s e d more questions than they have answered. The d i f f e r e n c e s between the MAT-B1 and MAT-C1 c e l l s are p a r t i c u l a r l y i n t r i g u i n g . The abundance and complexity of the m i c r o v i l l i at the surface of the CI c e l l i n d i c a t e the presence of an extensive submembrane c y t o s k e l e t a l network, s i n c e the m i c r o v i l l i c o n t a i n m i c r o f i l a m e n t s . One explanation f o r the extensive, branched m i c r o v i l l i i s that there i s an imbalance i n the turnover of plasma membrane compared to c y t o s k e l e t a l m a t e r i a l . However, these c e l l s a l s o cont a i n an excess of surface s i a l o g l y c o p r o t e i n . Is t h i s a c o i n c i d e n t or a r e s u l t of some type of coupling between the submembrane c y t o s k e l e t o n and extramembrane g l y c o p r o t e i n ? This question assumes greate importanc i vie f th p o s s i b l r o l e s of c e l l surface c e l l u l a r responses. Ou preliminary s i a l o g l y c o p r o t e i n i s not s o l e l y r e s p o n s i b l e f o r i n h i b i t i o n of Con A receptor movements i n the CI c e l l s . There a l s o appears to be a c y t o s k e l e t a l involvement. Regardless of the exact mechanism by which receptor movement i s repressed, the absence of m o b i l i t y may be an important f a c e t i n escape from immune d e s t r u c t i o n . I f m o b i l i t y of the antigens of the target c e l l i s a p r e r e q u i s i t e f o r i t s d e s t r u c t i o n , the immobilization of the e n t i r e surface would be an e f f e c t i v e defense. T h i s might a l s o be considered as an a l t e r n a t i v e mechanism f o r the l a c k of s t r a i n s p e c i f i c i t y of the TA3-HA c e l l s . The decreased nucleotidase and c e l l surface g l y c o p r o t e i n s of the MAT-B1 c e l l s are a l s o i n t r i g u i n g . Can t h i s r e a l l y be explained by a "shedding" mechanism? Or has the c a p a c i t y to synthesize these molecules been l o s t ? I t i s necessary to examine the dynamics of the surfaces of these c e l l s i n order to understand t h e i r behavior. Such s t u d i e s are complex but could provide i n s i g h t i n t o the r e l a t i o n s h i p of c e l l s t r u c t u r e and n e o p l a s t i c behavior. In attempting to understand tumor c e l l behavior i n r e l a t i o n s h i p to i t s c e l l surface p r o p e r t i e s , an important concept i s that tumor c e l l s appear h i g h l y adaptable. Perhaps t h i s a d a p t a b i l i t y i s the key to the success of the tumor i n s u r v i v i n g a h o s t i l e environment and i s the cause of much of the f r u s t r a t i o n i n t r y i n g to e s t a b l i s h d e f i n i t e r e l a t i o n s h i p s between cancer and p a r t i c u l a r c e l l p r o p e r t i e s . This a d a p t a b i l i t y i s not l i m i t e d to c e l l surface p r o p e r t i e s , as evidenced by the a b i l i t y of tumors to develop drug r e s i s t a n c e . However, the c o n t r o l of the c e l l surface would a s s i s t the c e l l i n meeting the r i g o r s of i t s environment. Studies on c e l l surface v a r i a t i o n s should help us understand the r e l a t i o n s h i p of c e l l surface to tumor s u r v i v a l and the more general question of a d a p t a b i l i t y of tumor c e l l s .
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
444
GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES
Acknowledgements This is contribution P-490 of the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma. This research was conducted in cooperation with the USDA, Agricultural Research Service, Southern Region and supported by the National Cancer Institute (NO-l-CB-33910 and CA 19985), the American Cancer Society (BC-246), a Presidential Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station. Literature Cited 1. Hughes, R. C. (1976) in Membrane Glycoproteins, Butterworths, London. 2. Emmelot, P. (1973) Eur J Cancer 9 319-333 3. Nicolson, G. L. 4. Buck, C. A. Glick Biochemistry 9, 4567-4576. 5. Shin, B. C., Ebner, K. E . , Hudson, B. G., and Carraway, K. L. (1975) Cancer Res. 35, 1135-1140. 6. Hynes, R. 0. (1974) Cell 1, 147-156. 7. Hellström, K. E . , and Helström, I. (1974) Adv. Immunol. 18, 209-277. 8. Kim, U., Baumler, A., Carruthers, C., and Bielat, K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1012-1016. 9. Friberg, S., Jr. (1972) J. Natl. Cancer Inst. 48, 14631476. 10. Codington, J. F., Sanford, B. H., and Jeanloz, R. W. (1973) J . Natl. Cancer Inst. 51, 585-591. 11. Miller, S. C., Hay, E. D., and Codington, J. F. (1977) J. Cell Biol. 72, 511-529. 12. Hilf, R., Michel, I., and Bell, C. (1967) Recent Prog. Hormone Res. 23, 229-289. 13. Segaloff, A. (1966) Recent Prog. Hormone Res. 22, 351-379. 14. DePierre, J. W., and Karnovsky, M. L. (1973) J. Cell Biol. 56, 275-303. 15. Steck, T. L . , and Wallach, D. F. H. (1970) Methods in Cancer Res. 5, 93-153. 16. Keenan, T. W., Morré, D. J., and Huang, C. M. (1974) in Lactation: A Comprehensive Treatise, (B. L. Larson and U. R. Smith, eds.), Vol. 2, Academic Press, New York, pp. 191-233. 17. Carraway, K. L . , Fogle, D. D., Chesnut, R. W. Huggins, J. W., and Carraway, C. A. C. (1976) J. Biol. Chem. 251, 6173-6178. 18. Beaufay, H., and Amar-Costesec, A. (1976) Methods in Membrane Biol. 6, 1-100. 19. Huggins, J . W., and Carraway, K. L. (1976) J. Supramol. Struct. 5, 59-63.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
24. CARRAWAY ET AL. 20. 21.
Membrane Glycoproteins of Rat Gland 445
Warren, L. (1959) J. Biol. Chem. 234, 1971-1975. Warren, L . , Glick, M. C., and Nass, M. K. (1966) J. Cell. Physiol. 68, 269-287.
RECEIVED
March 13, 1978.
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR INDEX Names and numbers in bold are authors and first pages, respectively, of complete chapters. Numbers in parentheses are reference numbers which indicate that an author's work is referred to, followed by pages of that referral. Numbers in italics show the page on which the complete reference is listed.
Arbogast, B. 148 (44) 144 A r c e , A . 82 (79) 61
A Aashwell,G. 85 (168) 79 Abe, S. 370 (5) 358,359 Abelev, G . I. 339 (1) 326, (4 (5) 326, 327; 340 (40) 330 Abercrombie, M . 376 (7) 376 Ablett, S. 121 (51) 115 Achord, D . T . 149 (64) 146, (65) 146 Adair, W . L . 270 (36) 259, 260, 265 Adam, M . 254 (35) 246, (36) 246 Adelman, L . S. 149 (58) 146 Adinolfi, M . 339 (23) Agostini, R. M . , Jr. 132 (7) 124 Agrawal, B. B. L . 431 (30) 415 Aguilar, H . 225 ( 27 ) 215, 218 Akasaki, M . 40; 134 (16) 131 Akiyama, Y. 270 (32) 259 Alexander, D . 339 (20) 327 Alhadeff, J. A. 8 4 ( 1 4 1 ) 7 3 Allan, C . J . 324(17)312 Allan, D . 270 ( 41) 260, 262, 265 Allen, A. 120 (38) 113 Allen, R. C . 133 (24) 128, (25) 128 Allfrey, V . G . 199 (49) 194 Altenburg, B. A. 403,379 Amar-Costesec, A. 444 (18) 434 Ambrose, E . J . 376 (6) 375, (7) 376 Aminoff, D . 431 (26) 414 Ammon, H . V . 356 (56) 350 Anderson, B. 20 (16) 10; 370 (26) 358 Anderson, J. C . 172 (13) 165; 211, 201 Anderson, S. G . 339 (10) 326 Andersson, P. 199 (39) 188, 190 Ando, S. 80 (26) 52, (27) 52, (28) 52 Andrews, P. 40, 32; 42, 32 A n g l i n J . H . 325 (41) 322 Annese, C . 371 (39) 361 Ansell, N . 324(2)311,312 Anttinen, H . 226 (37) 218, 222, (41) 222,
190, (23) 183, 184, 187, (30) 182, 184, 191; J99 (33) 188 Ariga, K. 341 (68) 332 Arlinghaus, R. B. 180; 198 (20) 182-184, 189, 190, (21) 183, 184, (22) 183, 184, 190, (23) 183, 184, 187, (26) 182-184, 192, (27) 184, 192, (28) 184, 188-190, 192, (30) 182, 184, 191; (33) 188, (34) 188-190, 192, (55) 195, 197; 200 (62) 197 Aro, A. 147 (1) 135, 143, (23) 138 Aronson, N . N . , Jr. 147 (9) 137 Aronson, S. A . 310 (5) 295, 302 Arora,R. C . 85 (160) 77,78 Arstila, A. U . 133 (6) 130, (9) 130 Asofsky, R. 340 (47) 331 Atkinson, P. H . 102 (19) 91, 95 Atsuta, T . 83 (102) 69 Attina, D . 325 (44) 322 A u b J . C . 377 (8) 376 Auger, J. 270 (41) 260, 262, 265 August, J. T . 198 (13) 181, (16) 181 Augustyn, J. 254 (30) 243 Aula, P. 40; 133 (10) 130; 134 (15) 131, (16) 131 A u l l , F . 271 (60) 261,265 Austin, J. 148 (42) 143 Autio, S. 43; 103 (29) 98; 133 (4) 129, 130, (6) 130, (7) 130, (9) 130, (10) 130; 148 (46) 144, (47) 144 Axelrod, D . 271 (79) 268 Ayoub, E . M . 211, 202, 210 B Baeziger, J. 1 0 2 ( 8 ) 8 6 Baghdadi, S. 121 (51) 115 Baig, M . M . 211,202,210
(42) 222, (45) 222 A p f f e l , C . A . 292(16)277
American C h e n ^ I Society Library 1155 16th 8t. N. w . Washington, D. C.
20036
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
448
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Bailleul, V . 220(34) 113 Baker, A . P. 219 (22) 11, (24) 110, 111; 220 (25) 110 Baker, E . A . 84 (138) 73 Baker, J . 227 Balint, J. 158 (10) 153 Baltimore, D . 298 (7) 180, 192, (10) 181, (31) 187; 299 (39) 188, 190, (40) 190, 194 Banatwala, I. 324 (14) 312, 322 Bannikov, G . A. 341 (66) 332, (71) 332 Bargeton,E. 247 (19) 138 Barnett, D . R. 229 (3) 108, (7) 108 Barranger, J . A . 150; 258 (5) 150, 153, 157; 259 (21) 150 Barsness, L . 341 (60) 331 Bartholomew, B. A. 40, 33 Barton, M . 42, 28; 133 ( 23) 128 Baschang, G . 82 ( 46 ) 53 Bass, N . H . 249 (58) 146 Basu, M . 82 (64) 59; 82 (69 61, (87) 61, 64, 65, (88) 61, 66, (89) 61, (90) 61, (92) 64 Basu, S. 82 ( 60 ) 59, (61) 59, (64 ) 59; 82 (66) 59, 61, (67) 61, (68) 61, (69) 61, (71) 61, (72) 61, (73) 61, 66, (74) 61, (80) 61, (82) 61, 66, (84) 61, (86) 61, (87) 61, 64, 65, (88) 61, 66, (89) 61, (90) 61, (92) 64; 83 (105) 71; 84 (124)67 Bauer, C . 403, 378 Baumler, A. 444 (8) 432, 433, 439 Bearpark, T . 148 (28) 140 Beattie, G . 270 (39) 262, 266, 267 Beaufay, H . 444 (18) 434 Becker, F . F . 339 (11) 326, 328, (12) 326, (17) 333; 340 (46) 331, (47) 331; 341 (62) 332, (65) 332-334; 430 (15) 413, 420, 421 Becker, G . W . 341 (64) 332 Behrens. N . H . 40, 22, 23; 43, 23 Beiber, G . F . 84 (131) 69 Beisswenger, P. 226 ( 50 ) 223, 224 Bekesi, J. G . 292 (15) 277 Belchetz, P. 259 (19) 150 Bell, C . 444 (12) 433 Bell, J . E . 40, 32; 83 (103) 69 Bell, P . M . 353 (17) 343 Benassayag, C . 339 (16) 327, 329 Bengali, K. M . 298 (18) 181 Bennett, B. D . 84 (125) 67 Bennett, G . 40, 34, 37; 83 (120) 67 Bentvelzen, R 272 ( 63 ) 261, 265 Beranek, W . E . 43,33 Berenblum, I. 294 (50) 290 Berenson, G . S. 211, 201; 212, 201, 202; 254 (37) 246 Berezesky, I. K. 199 (56) 195
PROCESSES
Berlin, R. D . 430 ( 7 ) 412, 429 B e r m a n , E . R . 432 (28) 415 Bernard, J. 324 (16) 312, 322 Berra, B. 147 (24) 138, 140; 248 (31) 140, (32) 140; 149 (57) 145 Bertrand,I. 247 (19) 138 Bessel, E . M . 354 (37) 348, (40) 348 Beutler,E. 259 (20) 150 Beyer, T . A. 40, 32, 37; 83 (103) 69 Bhadure, S. 299 (42) 190, 194 Bhargava, A . K. 355 (45) 348 Bhargava, P. M . 430 (17) 413 Bhatti, T . 353 ( 20 ) 343 Bhavanandan, V . P. 202 (24) 98; 293 ( 25) 278 Bhoyroo, V . D . 45,22, 76; 269 (20) 258 Bielat,K. 444 ( 8 ) 433,439 Bjorklid, E . 272 (23) 167 Bjorndal H 82 ( 49 ) 56 (50 ) 56 (51) 56 Bladen, H . A. 253 ( 7 ) 227 Blair, H . 258 (18) 150 Blenkinsopp, W . K. 232 (6) 124, (10) 125 B l o b e l , G . 40,26 Bloch, K. J. 370 (16) 358, 359, 361 Bloemendal, H . 199 (41) 190, 194 Bloemers, H . P. J. 299 (41) 190, 194 Blonder, E . 85 (155) 77 Blumenfeld, O. O . 224 ( 5 ) 213 Blumenthal, H . T . 253 (4) 227, 243, (6) 227 Boat, T . F. 108; 229 (1) 108, 112, (21) 110, 111, (23) 110; 220 (26) 110, 112, (33) 113, (40) 113, 114, 116, (42) 113; 222 (49) 115, (53) 118, (55) 116, (56) 111 Butterworth, B. E . 199 (32) 187 Byers, P. H . 226(35)218 Bobbitt, O . B. 370 (14) 358, 359, 369 Bobinski,H. 29(8)6 Boccardi,V. 325 (44) 322 B o c c i , V . 422 (9) 408 Boesman, M . 339 (18) 327, 329 Bogart, B . I . 229 (19) 110 B o g e l , C . L . 339 (28) 328 Bolognesi, D . P. 297 (4) 180, 187; 298 (9) 180, 181, 187, (19) 181; 299 (50) 195 Bonney, R. J. 430 (18) 413 Boorman, K. E . 324 (5) 311 Bornstein, P. 225 (33) 218; 226 (37) 218, 222; 253 (1) 227; 254 (22) 243, 246, (23) 243, (34) 246 Bosmann, H . B. 84 (130) 69, (131) 69, (132) 69; 85 (162) 79; 298 (25) 183 Bouissou, H . 254 (25) 243 Boulding, E . T . 325 (38) 322 Bouma, B. N . 272(6) 164
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
INDEX
Bouquelet, S. 148 (28) 140 Bowman, B. H . 119 (3) 108, (7) 108, (18) 110 Boyse, E . 198 (16) 181; 199 (58) 197; 292 (5) 277 Bradfield, J. W . B. 1 3 2 ( 6 ) 1 2 4 Bradley, R. M . 310 (5) 295, 302 Brady, R. O . 83 (96) 66, (109) 71, 76; 150; 158 (1) 150, (5) 150, 153, 157, (15) 152, (16) 150, (17) 154, (18) 150; 159 ( 21) 150, (22) 150, (23) 155; 310 (5) 295, 302 Braidman, I. 159 (19) 150 Branson, H . R. 353 (14) 343, 344 Branton, D . 269 (16) 258 Braun, P. E . 8 1 ( 5 7 ) 5 9 Brennan, P 403, 379 Brennan, S. O . 132 (18) 127 Breslaw, N . 339(10) 326 Breslow, J. L . 119 (8) 108 Bretscher, M . S. 269 (6) 258 Brettschneider, I. 149 (69) 146 Brew, K. 40, 31; 42, 32; 43, 32; 44, 32; 45, 32 Briles, E . B. 411 (19) 410 Brock, D . J . 339 (25) 327 Brockerhoff, H . 211, 210 Brodbeck, U . 41, 32; 83 (123) 67 Brotz, M . 8 0 ( 1 2 ) 4 9 Brown, A. E . 8 4 ( 1 3 1 ) 6 9 Brown, B. D . 147 (1) 135, 143, (22) 138, (23) 138 Brown, J. C . 269 (9) 258; 270 ( 57 ) 261, 265 Brown, M . C . 293 (22) 277, 278, 288, 289, (37) 286, 286, (38) 280, 291, (39) 280, 282, 290; 294 (51) 290, 291, (52) 290, 291 Bruenger,E. 254 (15 ) 231 Brunngraber, E . G . 135; 147 (1) 135, 143, (3) 136, (5) 136, (8) 137, (22) 138, (23) 138, (24) 138, 140; 148 (31) 140, (32) 140, (39) 143; 149 (57) 145, (61) 146, (67) 146, (68) 146, (69) 146 Brims, G . 158(13) 153 Bruns, R. R. 293 (42) 280, 282 Brunyneel, E . 120 (46) 113 Buchbinder, L . 371 (32) 361 Buchhagen, D . L . 198 (17) 181 Buck, C . A. 310 (3) 295; 403, 378; 410 (1) 404; 444 (4) 432 Buck, R. L . 432 Buddecke, E . 211, 201, 202 Bugge, B. 41,23 Bukhari, M . A . 354 (40) 348 Bullis, C . M . 270 (57) 261, 265 Burger, M . M . 270 (56) 261; 377 (9) 376; 430 (3) 412, 425, (4) 412, (10) 412; 431 (38) 425
449
Burke, D . J . 1 0 2 ( 1 1 ) 8 6 Burnet, F . M . 84 (134) 73 Burrowes, C . E . 172 (5) 163 Burtin,P. 355 (48) 350,351 Burton, R. M . 86 (17) 49 Buscher, H . P. 44,37 Butler, W . T . 213; 225 (9) 214, (10) 214, (25) 215, (27) 215, 218, (28) 215, 218, (30) 218, (31) 218, 219; 226 (46) 222, (47) 222, (48) 222 C Calle, R. 325 (46) 319 Candler, E . L . 292 (10) 277 Canellakis, E . S. 270 (58) 261, 265 Canfield, W . M . 173 ( 35) 151 Cantrell, J. L . 325 (35) 320 Cantz M 42; 148 (53) 145; 158 (12) 153 Caputto, R. 82 (79) 61 Carlson, D . M . 20 (15) 6; 40, 36; 119 (21) 110, 111; 120 (40) 113, 114, 116; 177 Carminatti, H . 46, 28 Carraway, C . A . C . 432; 444 (17) 434, 438 Carraway, K . L . 432; 444 (5) 432, 434436, (17) 434, 438, (19) 434, 435 Carrell, R. W . 132 (18) 127, (20) 127 Carruthers, C . 444 (8) 432, 433, 439 Carson, S. D . 119 (7) 108 Carter, H . E . 85 (169) 49 Carter, W . G . 270 ( 38 ) 260, 262, 265, (59) 261, 265 Casals-Stenzel, J. 44, 37 Case, K. R. 84 (131) 69 Casjens, S. 199 (51) 195 Castellani, I. 254 (33) 243 Caterson, B. 227 Cesarini, J. P. 147 (15) 138 Chabaud, O . 84 (127) 67 Chambers, R. E . 80 ( 31) 52; 353 (20) 343 Chan, J. Y . C . 1 7 2 ( 5 ) 1 6 3 Chang, 1 147 (1) 135, 143 Chang, J.P. 430 (13) 412 Chang, Y. H . 353 (22) 343, 348 Chao, H . 83(101)69 Chatterjee, S. 83 (107) Chavanel, G . 355 (48) 350, 351 Cheetham, R. D . 83 (118) 67 Chen, J. H . 199 (45) 192 Cheng, P. 108; 119 (23) 110; 120 (26) 110, 112, (40) 113, 114, 116, (42) 113, (47) 115; 121 (49) 115, (53) 118, (54) 118, (55) 116 Chesnut, R. W . 432; 444 (17) 434, 438 Chien, J. L . 82 (69) 61; (82) 61, 66, (84) 61, (88) 61, 66, (89) 61
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
450
GLYCOPROTEINS
Childs, R. A . 371 (28) 358 Chipysheva, T . A. 341 (66) 332 Chisaka, N . 341 (67) 332 Chism, S. E . 353 (17) 343 Chou, T . H . 40,31 Chu,G.H. 225(21)215 Chu, T . M . 353 (18) 343; 355 (45) 348, (53) 350 Chung, E . 225 (22) 215 Chung, S . I . 173 ( 30) 170 Ciccarini, C . 102(19)91,95 Claeys,H. 172 (25) 169 Clamp, J. R. 80 (31) 52; 353 (20) 343 Clausen, J . E . 324 (24) 315 Cleland, W . W . 81(62)59 Cline, M . J. 431 (36) 425 Coan, M . H . 172(17)166 Cochrane, C . G . 171 (1) 163; 172 ( 4) 163, (6) 164 Codington, J . F. 277; 292 (1 291, (14) 277, (20) 277, 279 293 (21) 277, 279, (22) 277, 278, 288, 289, (27) 279, (28) 279, 280, 286, 288, (29) 279, 286, 287, (36) 279, (37) 280, 286, (38) 280, 291, (39) 280, 282, 290, (40) 280, 281, 286, 288, 290, (41) 280, 282, 286, (42) 280, 282, (43) 286, 287, (44) 286, 288, (45) 286, 287; 324 (26) 315, (27) 315; 375; 444 (10) 432, (11) 433, 442 Coffee, C . J . 148 (38) 143 Cohen, M . P. 226 (52) 223 C o h n , Z . 158(7)157 Coligan, J. E . 352 (5) 342, (7) 343, (8) 343; 353 (10) 343, (11) 343, 345, (15) 343, (19) 343, 344, (23) 343, 346, 348; 354 (25) 344, (27) 344; 355 (43) 348, (44) 348, (46) 348; 371 (38) 361 Collard, J. G . 430 ( 9 ) 412; 431 (40) 429 Colley, M . 158(14)153 Colman,R.W. 173 (33) 171 Colvin, B. 41, 32 Comb, D . G . 324 ( 21) 314 Conod, E . J. 119 (4) 108, (19) 110 Conover, J. 119 (4) 108, (19) 110 Conrad, H . E . 354 (29) 344 Cook, G . M . W . 83 (100) 69; 376 (5) 375 Cooper, A. G . 293 (22) 277, 278, 288, 289, (37) 280, 286, (38) 280, 291, (39) 280, 282, 290, (40) 280, 281, 286, 288, 290; 294 (51) 290, 291, (52) 290, 291; 370 (22) Corey, R. B. 353 (14) 343, 344 Costello, A. J. R. 149 (68) 146 Cotman, C . W . 271 (67) 266 Cowan, N . J. 40, 26 Cox, B. A . 253 (12) 228, 231, (13) 232, 233; 255 (45) 252
A N D GLYCOLIPIDS I N DISEASE PROCESSES
Cox, D . W . 132 (8) 124,125 Coyle, P. 84 (152) 75, 77; 148 (38) 143 Crawley, J. 159 (19) 150 C r e a n , E . V . 271 (64) 261 Crocker, B. F . 84 (135) 73 Crumpton, M . J. 270 (41) 260, 262, 265, (42) 260, 262, 265; 271 (62) 261, 265 Cudney, T . L . 293 (32) 279, 280 Cullen, S. E . 292 (2) 277; 376 (3) 375 Cunningham, L . W . 225 (9) 214, (10) 214, (13) 214, (27) 215, 218 Cutz, E . 41, 28; 132 (8) 124, 125 Czegledy-Nagy, E . 119 (20) 110 Czichi, U . 40, 28 D D a b a n , A . 325 (46) 319 Dabelsteen, E . 370 (10) 358, 359 Dale, G . 159 (20) 150 Dalferes, E . R. 211 D a l t o n , A . J . 200 (59) 197 Daniels, A. 148 (39) 143 Daniels, J. R. 225 (21) 215 Dankert, M . 43, 23 Danzo, B. J. 271 (61) 261 D a o u d , A . S. 254 (30) 243 Darcy, D . A. 355 (50) 350, 351, (51) 350, 351 Das, I. 120 (43) 113 Das, S. 355 (44) 348 Datta, P. 83 (113) 67 DaussetJ. 324 (16) 312,322 Davidsohn, I. 310 (6) 296; 370 (6) 358, 359, (7) 358, 359 Davidson, E . A. 102 (24) 98; 293 (25) 278 Davie, E . W . 171 (2) 163, (3) 163; 172 (7) 164, (8) 164, (9) 164, (12) 165, (14) 165, (15) 165, 166, 171, (17) 166, (18) 166, (20) 166, 167; 173 (35) 171 D a vies, D . A. L . 324 ( 8 ) 312 Davis, E . M . 276 (6) 276; 412; 431 (22) 414, 418, 419, (31) 415, 429, (32) 426, (35) 423 Davis, J. 121 (49) 115; 197 (5) 186, 195 Davis, L . G . 147 (24) 138, 140; 149 ( 67) 146, (68) 146, (69) 146 Davis, N . L . 198 (8) 180,192 Davis, P. 119 (2) 108, 109, 112 Davis, S. S. 119(11) 109 Dawson, G . 82 (85) 61; 103 (32) 98; 147 (27) 139, 140, 144; 148 (48) 144; 158 (11) 153 Dean, K. J. 84 (145) 75, (147) 75 Dearborn, D . G . 119 (6) 108; 120 (29) 112, 113
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
451
INDEX
DeBacker, M . 254 (32) 243 DeDuve, C . 147 (9) 137; 158 (2) 150 DeFries, A. 85 (155) 77 Degand, P. 120 (34) 113, (35) 113, (41) 113, (48) 115 deHaller, R. 119 (14) 109 deHarven,E. 200 (60) 197 Dekaban, A. 159 (23) 155 Del Villano, B. C . 198 (11) 181 Deman, J. 120 (46) 113 Dempo, K. N . 341 (67) 332, (70) 332 de Petris, S. 271 (68) 266, (71) 266, (73) 268 DePierre, J. W . 444 (14) 433, 434 Derbyshire, W . 121 (51) 115 Derouette, J. C . 254 (25) 243 Derouette, S. 254 (31) 243 Desai, P. R. 310 (1) 302; 311; 324 (11) 312, (12) 312, 318, (14) 312 322 (15) 312, 315, 317, 320, (19) 314 314, 320, (25) 315; 325 (28 (29) 320, 322, (35) 320, (42) 322, (43) 322 Desteno, C . V . 226 (47) 222 Deumling, B. 83 (118) 67 D i Benedetta, C . 148 (40) 143 Dicorleto, P. E . 211, 210 Dieleman, B. 46, 39 D i Palma, S. 148 (32) 140 di'Sant Agnese, P. A . 119 (2) 108, 109, 112; 120 (32) 113; 121 (50) 115 Dische, Z. 121 (50) 115; 225 (8) 213 DiScipio, R. G . 172 (8) 164 Distler, J. 149 (63) 146 Dobberstein, B. 40, 26 Dodd, B . E . 324 (5) 311 Doebber, T . 85 (165) 79 Doershuk, C . F . 119(1) 108,112 Domotor, J. J., Jr. 198 (18) 181 Dorfman, A. 148 (44) 144 Dorland, L . 45, 30 Dorval, G . 292 (18) 277 Downey, J. 172 (28) 170 Downs, F . 2 0 ( 1 4 ) 6 Dreslar, S. 198 (29) 184 Drzeniek, Z. 325 (33) 320 Dubois, M . 431 (27) 414 Duesberg, P. H . 199 (37) 188, (44) 192, (48) 194 Dugal, B. 133 (12) 130 Dumsha, B. 224 (4) 213 Durand, G . 120 (35) 113 D u r a n d , P . 45; 149 (55) 145 E Eastoe, J. E . 226 (56) 224 Ebner, K. E . 40, 32; 41, 32; 42, 32; 83 (123) 67; 444 (5) 432, 434-436
Edelman, G . M . 271 (75) 268 Edgar, G . W . F . 147 (18) 138, (19) 138 Edidin, M . 269 ( 2 ) 256 E d s a l l , J . T . 276 (4) 275 Egan, M . L . 352 (4) 342, 346, (5) 342, (7) 343, (8) 343, (9) 343; 353 (19) 343, 344; 354 (25) 344, (32) 344, (39) 348; 355 (46) 348; 356 (57) 350, (58) 350 Egan,W. 172(10)165 E g e l r u d , T . 211,204 Egge,H. 81(44)52 Ehrenreich, B. 1 5 8 ( 7 ) 1 5 7 Eisen, H . N . 354 (38) 348 Eisenberg, S. 211, 201 Elam, D . L . 293 (34) 279 Elgsaeter,A. 269 (16) 258 Emerson, W . A . 133 (21) 127; 270 (40) 260 262 Endo, Y. 340 (38) 330 Enfield, D . L . 172(16) 166 Engelhardt, N . V . 340 (40) 330 Engvall, E . 339 (13) 326; 352 (4) 342, 346; 354 (33) 344, 347, 348; 356 (60) 351, (61) 351 Epstein, J. 119(8)108 Epstein, S. 340 (55) 331 Erickson, J. S. 148 (36) 143 Ericsson, L . H . 172 (16) 166; 173 ( 35) 171 Eriksson, S. 132 (15) 126 Esselman, W . J. 81 (53) 56; 371 (33) 361 Esterly, N . R. 119 (17) 109, 110 Etchinson, J. R. 102 (17) 91 Etchison, J. 41, 24, 31 Eto, Y. 148 (41) 143,(43) 143 Evans, J. E . 80 (20) 51 Evans, L . H . 198 (29) 184 Evans, N . 293 (45) 286,287 Eveleigh, J. W . 353 ( 61) 343 Ewing, C . 132 (17) 127, (19) 127 Eylar, E . H . 41, 36; 83 (100) 69; 198 (25) 183 F Fabre, J. W . 270 (46) 260, 263 Fabre, M . T . 254 (25) 243 Fagerhol, M . 42, 28; 131 (2) 123 Fahey, J. L . 324 (9) 312 Famulare, N . G . 198 (17) 181 Fan, H . 199 (40) 190, 194, (46) 194 Farber, E . 340 (52) 331, (55) 331, (56) 331, (57) 331, (58) 331; 341 (72) 332 Farrell, P. M . 120 (30) 112 Farriaux, J. P. 45; 149 (55) 145 Federico, A. 148 (40) 143
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
452
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Feizi, T . 20 (16) 10; 310 (12 ) 302; 370 (23) 359, (26) 358; 371 (28) 358 Fernlund, P. 172 (10) 165 Ferrand, M . 84 (127) 67 Ferris, B. 270 (55) 261, 265 Fessler, J. H . 225 (34) 218 Fessler, L . I . 225 (34) 218 Fidler, I. J. 147 (15) 138 Fietzek, P. P. 225 (26) 215, 218 Filliat, M 120 (48) 115 Finch, J. E . , Jr. 225 (28) 215, 218, (30) 218, (31) 218, 219; 226 (47) 222 Findlay, J. B. C . 270 (34) 259, 260, 265 Finne, J. 147 (4) 136, (6) 136, (7) 137; 269 ( 26 ) 258 Fischer, A. 411 (8) 408 Fischer, D . 85 (166 ) 79; 149 ( 64) 146 Fischer, K. 325 (36) 322 Fisher, R. L . 132 (11) 125 Fishkin, A . F . 211, 201; 212, 201 Fishman, P. H . 83 ( 96 ) 66, (10 310(5) 295, 302 Fitzgerald, D . K. 41, 32 Fleischer, B. 84 (137) 73 Fliescher, S. 84 (137) 73 Fleisher, T . A . 133 (5) 130 Fleissner, E . 197 (1) 180, 181; 198 (8) 180, 192, (17) 181; 199 (57) 195, (58) 197 Flowers, H . 276 (1) 275 Fluharty, A . 158 (9) 153 Fogle, D . D . 444 (17 ) 434, 438 Folch, K. 403, 382 Foley, E . J . 376 (2) 375 Folk, J . E . 173 ( 30) 170 Fong, J. W . 81 (43) 52 Forbes, M . 325 (45) 322 Ford 225(13 ) 214 Forni, L . 271 (74 ) 268 Forssman, J. 371 (29) 361 Forstner, G . G . 120 (31) 112 Forstner, J . F . 120 (31) 112 Foster, A . B. 355 ( 42 ) 348 Foster, J. A . 254 (16 ) 231 Foster, L . A . 254 (15) 231 Fournet, B. 42; 45, 30; 148 (28) 140; 149 (54) 145, (55) 145 Fournier-Delpech, S. 271 (61) 261 Fowler, M . C . 149 (56) 145 Francis, G . 225 ( 31) 218, 219 Frank, H . 197 (4) 180, 187 Franke, W . W . 83(118) 67 Franzblau, C . 253 (5) 227 Fraser, B. A . 371 (35) 361, (38) 361 Fraser, I. H . 41, 32 Freedman, S. O . 352 (1) 342, (2) 342, (3) 342, (6) 342; 353 (13) 343; 370 ( 27) 358
PROCESSES
Freese, E . 83 (109) 71,76 Fry,M. 199(15)195 Friberg, S., Jr. 293 ( 30 ) 279, 290, (31) 279, (35) 279; 294 (48) 288, (49) 288; 444 ( 9 ) 432 Friedenreich, V . 325 (39) 322 Friedensen, B. 325 (34) 320 Fritz, K. E . 254 (30) 243 Fry, M . 199(15)105 F u h r e r J . P . 310(3)295 Fujikawa, K. 171 (2) 163, (3) 163; 172 (9) 164, (12) 165, (16) 166, (17) 166, (20) 166,167 Fujita,S. 341 (68) 332 Fukuda, M . 102 ( 25 ) 98; 269 ( 21) 258 Fuller, G . M . 119(7)108 Fulling, J. H . 370 (10) 358, 359 Funakoshi, I. 40; 134 (15) 131, (16) 131; 269 (27) 258 (28) 258; 293 (24) 278 75,77; 85 (161) 77,79 Furbish, F. S. 150; 158 (5) 150, 153, 157, (18) 150; 159 (21) 150 F u r i e , B . 172(11) 165 F u r i e , B . C . 172(11)165 Furthmayr, H . 42, 37; 270 (33) 259, (37) 260, 262, 265; 294 (46) 288 Furukawa, K. 370(11) 359, (21) 359 G G a c t o , M . 403,385 Gaerlan,P. 119 (4) 108 Gahmberg, C . G . 269 (10) 258; 310 (4) 295; 371 (37) 361 Gaidulus, L . 132 (17) 127, (19) 127 Gal, A . E . 150; 158 (16) 150, (17) 154; 159 ( 23) 155 Gallop, P . M . 224 (5) 213 Gan, J . C . 132(13) 126 Gardas,A. 82 (95) 64 Gardner, D . A. 82 ( 80 ) 61, (84 ) 61, (89) 61; 83 (105) 71 Garegg,P.J. 403,390 Garguilo, A. W . 370(9) 358, 359 Garner, E . F . 102 ( 22) Garver, F . 44,33 G a s i c , G . 293 (26) 279 G a s i c , T . 293 ( 26 ) 279 Gatt,R. 431 (28 ) 415 Gatt, S. 84 (138 ) 73, (153 ) 77; 85 (154) 77 Gautier, M . 102 (9) 125, (12) 125 Gaver,R.C. 80(8)48 Gazzard, B. 340 (39) 330 G e h l e r J . 148 ( 53) 145 Gelman, R. A. 120(45) 113
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
453
INDEX
Gel'Shtein, V . I. 341 (66) 332 Gentry, C . 271 (67) 266 Geren, C . R. 41,32; 42,32 Geren, L . M . 41, 32 Gersten, D . M . 84 (131) 69 Ghosh, H . P. 45, 26 Gielkens, A. L . J. 199 (41) 190,194 Gilbert, G . A. 276 (4) 275 G i l l a r d , G . C . 254 ( 38 ) 246 G i l l e s , K . A . 431 (27 ) 414 Gilliam, E . B. 430 (12) 412, 414, 418, 420, 423; 431 (35) 423 Ginsburg, V . 107 G i r e l l i , G . 325(44) 322 Giro, M . G . 254(33)243 Gitlin, D . 339 (18) 327, 329, (21) 327, 328 Glass, G . B . J . 80 (15) 49,75 G l a z e r , N . 224 (4) 213 Glew, R. H . 84 (152) 75, 77; 132 (7) 124; 148 (38) 143 Glick, M . C . 275; 276 (1) 275 404; 410 (1) 404, (2) 404, (4) 404, (6) 404, 405; 411 (7) 405, 408, (8) 408, (12) 408, (14) 408, 409, (15) 408, (16 ) 408, (21) 406; 444 (4) 432; 445 (21) 437 Glockner, W . M . 269 (25) 258 Go, V . L . W . 356 (56) 350, (58) 350 Gochman, N . 340 (35) 329 G o e b e l , H . H . 148 (33) 140 Gold, P. 339 (8) 326; 352 (1) 342, (2) 342, (3) 342, (6) 342; 353 (13) 343; 370 (27) 358 Goldberg, A. R. 377 (9) 376 Goldman, R. 271 (72) 266 Goldstein, I. J. 102 ( 22); 354 (33) 344, 347,348; 431 (30 ) 415 Goldsworthy, T . 341 (60) 331 Golovtchenko, A. M . 41, 23 Gombos, G . 270 (50) 260, 264 Goodfellow, P. 270 (43) 260, 263 Goodman, M . 225 (18) 215 G o r d , G . 339 (9) 326 Gorin,P.A.J. 81(48)56 Got, R. 83 (98) 69 G o t o h , T . 172 ( 28) 170 Gottschalk, A . 19 (6) 5, 11; 20 (22) 13; 120 (44) 113 Grabar,P. 339 (3) 326 G r a f , L . 371 (49) 363 Granatek, C . H . 292 (10) 277 Grand, R . J . 120(27) 110 Grant, M . E . 226 ( 36 ) 218 Grassman, W . 224 (1) 213, (2) 213, (3) 213 Gray, G . M . 83 (104 ) 71 G r a y , M . E . 84(125) 67 Gray, W . R. 254 (15) 231, (16) 231 Green, M . 199 (42) 190,194
Greenberg, C . S. 411(21)406 Greenspon, S. A . 225 (18 ) 215 Gregoriadis, G . 159 (19) 150 Gregory, W . 270 (31) 259 Griffin, J. H . 171 (1) 163; 172 (4) 163, (6) 164 Grimes, W . J . 83(114)67 Grimley, P. M . 199 (56) 195 Groshman, J. 293 (33) 279 Gross, J. 224 (4) 213; 226 (57) 224 Gruensteun, M . 340 (58) 331; 341 (72) 332 G u a z z i , G . C . 148 (40) 143 Guelstein, V . I. 341 (71) 332 G u g l e r , E . 120 (32) 113 G u i n t o , E . 159 (20) 150 Guminska,M. 147(17) 138 Gurd, J. M . 270 (52) 260, 264 Gurd J W 270 (49) 260 264 266
H Habal,K.M. 172(5)163 Haberland, C . 147 (22) 138; 148 (39) 143 Hadchouel, M . 132(9) 125 H a d d a d , A . 83 (120) 67 Haffenden, G . P. 132 (10) 125 Hafter,R. 224 (2) 213 Hagmar,B. 294 (53) 291 Hagopian, A. 41,36; 198 (25) 183 Haguenau,F. 200 ( 59) 197 Hakkinen, I. 370(17) 358, 359, 361 Hakomori, S. 80 ( 23 ) 51; 81 (38) 52, (47) 53, (54) 56; 82 (93) 64; 270 ( 59 ) 261, 265; 310 (4) 295, (9) 302, (15) 304; 354 (28) 344, (3) 344; 357; 370 (1) 357, (2) 357, (13) 358, (16) 358, 359, 361, (19) 358, 359, 363, 364, 369, (20) 359, (24) 358, 359, 369; 371 (28) 358, (30) 361, (31) 361, (37) 361, (41) 361, (43) 364, (45) 364, (46) 364; 403, 378 391 Halford' M . H . 225(15) 215 H a l l , T . C . 85 (162) 79 Hall, W . J . 132(5) 123 Hallgren, P. 19 (7) 6; 403, 390; 411 (11) 408 Halpern, M . S. 197 (2) 180, 181; 198 (14) 181 Haltia, M . 133 (6) 130, (7) 130 Hamers, M . N . 84 (148) 75 Hamilton, J. K. 431 (27) 414 Hamilton, R. T . 270 (45) 260, 263 Hammarstrom, S. 354 ( 33 ) 344, 347, 348; 356(62) 351 Hanafusa, H . 199 (45) 192, (49) 194 Hanafusa, T . 198 (7) 180, 192
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
454
GLYCOPROTEINS
Hanaham, D . J. 172 (26) 169 Handa,S. 80(21)51,(22)51 Hanfland,P. 81(44)52 Hanks, J . H . 431 (24 ) 414 Hanna, M . G . , Jr. 198 (19) 181; 292 (10) 277 Hansen, H . J . 355(53) 350 H a r a , A . 371 (34) 361 Hard, W., Jr. 199 (58) 197 Harmon, J. W . 340 (54) 331 Harris, H . 294 (47) 288 Harvey, S. R. 353 (18) 343; 355 (45) 348 H a s c a l l , V . C . 254 (39) 252 H a u p t , H . 173 (32) 170 Hauschka, T.S. 293 (32) 279, 280 Hauser, G . 81 (65) 59, 61, 64, 65; 82 (78) 61 Haverkamp, J. 45, 30 Havez,R. 120 (35) 113 Hawthorne, J. N . 85 (169) 49 Hay, E . D . 293 ( 41) 280, 282 (11) 433,442 Hayashi, H . 85 (164) 79 Hayashi, M . 371 (34) 361 Hay em, A. 120 (34) 113 Hayman, M . J. 370 (42) 260, 262, 265, (43) 260, 263; 271 (62) 261, 265 Haywood, W . S. 199 (47) 194 Heard, D . H . 376 (5) 375 Heath, E . C . 354 (34) 346 Hechtman,P. 85 (158) 77 Hedin, A . 356 (62) 351 Heimburger, N . 173 ( 32) 170 Heldebrant, C . M . 172 (14) 165 Hellerqvist, C . G . 81 (51) 56; 354 (31) 344 Hellstrom, K. E . 444 (7) 432 Helstrom, I. 444 ( 7 ) 432 Henderson, M . 42, 31; 403, 378 Hendricks, J. 270 (44) 260, 263, 266 Hendrix, C . F . 254 (14) 231, 234, (21) 243 Henkart, P. A . 353 (15) 343; 355 (43) 348, (44) 348 Hennigar, G . R. 133 (24) 128 Henson, L . C . 211, 210 Herberman, R. B. 324 (23) 314, 319 Hercz, A . 41, 28; 133 ( 23) 128 Herlant-Peers, M - C . 45 Hermodson, M . A . 172 (8) 164 Hernell, O . 211, 204 Herries, D . G . 42, 32; 46,32 Hers, H . G . 148 (50) 144 Herschkowitz, N . N . 148 (41) 143, (43) 143 Herscovics, A . 41, 23 Hersh, E . M . 292 (10 ) 277 Hershko, A . 199 (52) 195 Hibbert, S. 158 (16) 150; 159 (23) 155
A N D GLYCOLIPIDS I N DISEASE PROCESSES
Hickman, S. 85 (167) 79; 149 (59) 146 H i e b e r , V . 149 (63) 146 Hildebrand, J. 81 (65) 59, 61, 64, 65; 82 (78) 61 H i l f , R . 444 (12) 433 H i l l , H . D . 120 (39) 113 H i l l , H . D . J r . 41,36 H i l l , R . C . 173 (29) 170 Hill, R. L . 40, 31, 32, 37; 41, 36; 43, 33, 36, 37; 45, 32, 37; 83 (97) 64-66, (103) 69; 120 (39) 113; 172 (27) 169 Hillegass, L . M . 119 (24) 110, 111; 120 (25) 110 Hirs, C . H . W . 20 (23) 13, 14; 102 (18) 91 Hirsch, J. 120 (30) 112 Hirst, G . K . 376 (4) 375 Hixson, D . C . 276 (6) 276; 412; 431 (31) 415 429 143 H . 147 (1) 135, 143, (3) 136, (8) 137 Hoffman, U . 224 (3) 213 Holdridge, B. A . 293 (32) 279, 280 Holland, J. F . 292 (15) 277 Hollander, W . 253 ( 5 ) 227 Hollinshead, A. C . 324 (23) 314, 319 Holm, M . 8 1 ( 4 2 ) 5 2 Holtermuller, K. H . 356 (56) 350 H o m b u r g , C . 410(5)404 Hondi-Assah, T . 45; 149 (55) 145 H o r m a n n , H . 224 (2) 213 Horowitz, M . I. 19 (2) 5, 13, 14; 81 (37) 52; 84 (142) 73 Horton, R. E . 324 (7) 311, 312, 322; 325 (45 ) 322 H o s k i n s , L . C . 325 (38) 322 Howard, S. P. 432 H s i e h , T . C . Y . 85(171) 49,56 Huang, C . C 102 (6) 86, 87, 95, 98 Huang, C . M . 83 (119) 67; 444 (16) 434 Huang, I.-Y. 325 (30) 320 Hubbard, A . 270 (55) 261, 265 Hubbard, S. C . 102(12) 87 Hubbell, G . J. 212, 201, 202 Hubbell, W . L . 270 (39) 262, 266, 267 H u b e r , C . T . 269 ( 8 ) 258 Hudgin, R. L . 41, 33, 39; 44, 34; 83 (121) 67 Hudson, B. G . 42, 32; 225 (23) 215; 444 (5) 432,434-436 Huggins, J. W . 432; 444 (17) 434, 438, (19 ) 434, 435 Hughes, L . E . 292(17) 277 Hughes, R. C . 19 (5) 5; 276 ( 2 ) 275; 411 (18) 410; 444(1) 432 H u i j i n g , F . 158 (4) 153
Hof,
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
INDEX
455
Hunsmann, G . 298 (15) 181, (19) 181 Hunt, L . M . 42,24, 31 Hunt, R. C . 269 (9) 258; 270 (57) 261,' 265 H u p e r , G . 198 (19) 181 Huprikar, S. V . 325 (37) 312, 322 Hurley, N . E . 298 (8) 180,192 H y d e , R . W . 232 (5) 123 Hynes, R. O. 269 (13) 258; 444 (6) 432 I Iammarino, R. M . 341 (73) Ide, H . 270 (48) 260, 263, 264, 266 IhleJ.N. 298 (18) 181,(19) 181 Ikeda, H . 298 (16) 181; 199 (58) 197 Inbar, M . 272 (76) 268, (77) 268 Irimura, T . 293 (29) 279, 286, 287 Iseki, S. 370 (11) 359,(21) 359 Isemura, M . 42, 33 Isenberg, J . N . 122; 133 (3) (8) 130, 131 Ishibashi, T . 82 (70) 61, (83) 61; 83 (102) 69 Ishihara, K. 370 (11) 359, (21) 359 Ishii, M . 222, 201 Ishizuka, H . 342 ( 68 ) 332 Ishizuka,I. 80 (14) 49 Issacs, R. 320 (7) 296, 298, (8) 300, 308 Isselbacher, K. J. 83 (116) 67, 69; 84 (128) 69 I t o , N . 340 (55) 331 Ito, S. 42, 30; 45, 30; 203 (216) 98 Iwamori, M . 80 (19) 51, (24) 51, (25) 51 Iwata, M . 270 (48) 260, 263, 264, 266 Iyer,R. 220 (40) 113,114,116 J Jabbal, I. 42,31; 44,34; 83 (121) 67 Jackson, C . M . 272 (24) 167 Jackson, R. L . 20 (23) 13, 14; 269 (7) 258 Jacob, J. C . 247 (20) 138 Jacob, S . T . 430(17) 413 Jacobs, H . G . 225 (27) 215, 218 Jacobs, M . 255 (45) 252 Jacobsen, A. 272 (28) 170 Jacobson, K. 272 (78) 268 Jacques, P. 258 (13) 153 Jagarlamoody, S. M . 292 (8) 277 Jakstys, M . M . 324 (10) 312 James, A . M . 376 (6) 375 James, R. 355(51) 350,351 Jamieson, J. C . 42,26, 28 Jamjoom, G . A . 298 (20) 182^184, 189, 190, (22) 183, 184, 190, (26) 182-184, 192, (27) 184, 192; 299 (55) 195, 197; 200(62) 197 Jansons, V . K. 270 (56) 261; 430 (10) 412 Jansson,P.E. 82 ( 55 ) 56
Jarasch,E. 83(118)67 Jarlfors,U. 44,32 JarmolychJ. 254 (30) 243 Javaid, J. I. 147 (3) 136, (8) 137, (24) 138, 140; 149 (67) 146, (68) 146 Jayle,M.F. 339 (16) 327,329 Jeanloz, R. W . 3, 42, 23; 220 (41) 113; 225 (15) 215; 292 (14) 277, (20) 277, 279, 280, 288; 293 (21) 277, 279, (22) 277, 278, 288, 289, (27) 279, (28) 279, 280, 286, 288, (29) 279, 286, 287, (36) 279, (38) 280, 291, (40) 280, 281, 286, 288, 290, (43) 286, 287, (45) 286, 287; 294 (51) 290, 291; 324 (26) 315, (27) 315; 370 (16) 358, 359, 361; 403, 378; 444 (10) 432 Jeffery,P.K. 222 (52) 115 Jenner, F . A . 43; 133 (1) 129, 130 Jentoft,N 222 (54) 118 Ji, T . H . 269 (5) 256, 258, 259, 266, 268, (15) 258 Jirgensons, B. 133 (22) 127 Tohansson, B. G . 354 (33) 344, 347, 348 Joklik,W. 199 (43) 190,194 Jolly, R. D . 103 (28) 98; 248 (49) 144 Jones, E . W . 355 (50) 350, 351 Jones, J. 259 (21) 150 Jourdian, G . W . 40, 33, 36; 249 (63) 146 K K a b a t , D . 298 (29) 184 Kabat, E . A. 20 (16) 10, (17) 9; 370 (26) 358 Kadar,A. 254 (27) 243 Kagan, H . M . 253 ( 8 ) 227 Kahan, B. D . 292 (1) 277; 324 (10) 312 Kahane, I. 270 ( 37 ) 260, 262, 265 Kahne,I. 269 (7) 258 Kamimura,N. 342 (68) 332 Kanai,K. 340(38) 330 K a n e d a , H . 342 ( 68 ) 332 Kaneko, A. 342 (67) 332, (70) 332 Kanfer, J. N . 248 (34) 143 Kang, A. H . 225 ( 29 ) 215, 218 Kaplan, A . 85 (166) 79; 249 (64) 146, (65) 146 Kaplan, H . S . 298 (12) 181 Karlsson, K. A . 79 (5) 48, 53; 80 (9) 48; 82 (39) 52, (40) 52, (41) 52, (45) 52; 370 ( 25 ) 359, 361; 371 (36) 361 Karnovsky, M . J. 430 (7) 412, 429 Karnovsky, M . L . 444 (14) 433, 434 Karshin, W . L . 180; 298 (20) 182-184, 189, 190, (22) 183, 184, 190, (23) 183, 184,187,(30) 182,184,191 Kartenbeck, T. 83 (118) 67
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
456
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE
PROCESSES
Kato,H. 172(12)165 Kiyosawa, I. 42, 32 Katona, E. 42, 28 Klaiber, M. 342 (72) 332 Katz, F. 46, 26 Klebe,R.J. 229 (3) 108 Katzman,R.L. 225 (15) 215 Klein, E. 292 (12) 277, (18) 277; 376 (1) Kaufman, B. 81 (60) 59, (61) 59; 82 (67) 375 61, (68) 61, (71) 61, (72) 61, (73) 61, Klein, G. 292 ( 7 ) 277, (12 ) 277; 293 (22) 66, (74) 61 277, 278, 288, 289; 294 (47) 288, (48) Kaul, K. 85 (170) 49, (171) 49, 56 288, (49) 288; 376 (1) 375 Kawamura, N. 80 (10) 48; 371 (34) 361 Klein, K. M. 340 (46) 331 KawanamiJ. 371 (42) 363 Kleinerman, J. I. 229 (21) 110, 111; 222 Kawasaki, H. 370 ( 5 ) 358, 359 (56) 111 Kawasaki, T. 85 (168) 79 Klemer,A. 222,202 Kay, H. E. H. 370 (8) 358, 359 Klenk,H.D. 298 (17) 181 Kaye,C.I. 324 (10) 312 Klibansky,C. 85 (155) 77 Kean, E. L. 324 (22) 314 Klimmerer, T. W. 84 (131) 69 Keegstra,K. 202 (11) 86 Kline, I. 293 ( 34 ) 279 Keenan, T. W. 83 (111) 67, (119) 67, Klinger, M . 378; 403, 385, 387 Knight, W.S. 352(9) 343 (122) 67; 84 (124) 67; 444 (16 ) 434 Kefalides, N. A. 224 ( 6 ) 213, 215; 225 Knox,W.E. 340 (59) 331 (12) 214, (19) 215, 216, (20 (36) 218, (53) 224 Kellenberg, E. 299 (54) 195 95; 203 (26) 98 Kelly, P. 272 (67) 266 KohnJ. 340 (39) 330 Kempf,S.F. 82(81)61 Kohno,M. 247(14) 137 Kenne, L. 82 (55) 56 Koide,N. 202 (14) 87,91,98 Kennedy, E. P. 82 (62) 59 Konigsberg, W. H. 82 ( 56 ) 59 Kennel, S. H. 298 (11) 181 Koono,M. 85 (164) 79 Kerr, I. M. 299 (35) 188 Koonstra, W. 272 ( 63 ) 261, 265 Kessel, D. 40,31; 42, 31; 403, 378 Kopchick, J. J. 298 (27) 184, 192, (28) Kessler, M. 355 (47) 348, 350; 356 (59) 184,188-190,192 350, 351 Koplitz, M. 342 ( 61) 331 Kessler, S. W. 372 (48) 364 Koppel,D.E. 272 (79) 268 Khalifa, A. 226(52) 223 KoppelJ.L. 84 (135) 73 Khan, M. A. 229 (13) 109; 220 (37) 113 Korant, B. D. 299 (53) 195 Khatra, B. S. 42,32 Kornblatt, M. J. 80(13)49 Khramkova, N. I. 339 (1) 326 Kornfeld, R. 29 (3) 5, 6, 8, 12; 202 (10) Kiely, M. L. 42,26 86; 269 (23) 258, (29) 259, (30) 259 Kihara,H. 258 (9) 153 Kornfeld, S. 29 (3) 5, 6, 8, 12; 45, 24, 26, Kin,N. 203 (30) 98, (31) 98 31; 202 (8) 86, (10) 86, (13) 87; 269 Kindt, T.J. 372 (38) 361 (23) 258, (29) 259, (30) 259; 270 ( 31) King,F. 259 (22) 150 259, (36) 259, 260, 265, (40) 260, 262; King, J. 299 (51) 195 422 (19) 410 King, M. 220 (45) 113 Koscielak, J. 82 (91) 64-66, (95) 64; 370 Kinsky, S.C. 372 (44) 364 (16) 358, 359, 361 Kijimoto, S. 82 (70) 61, (83) 61 Koshland, D. E. 273 (31) 170 Kim,U. 444 (8) 432,433,439 Kosjakov, P. N. 324 (4) 311 Kim, Y. S. 295; 310 (1) 295, 306, (7) 296, Kottgen, E. 403, 378 298, (8) 300, 308, (10) 302, 304, 305, Kovarik, S. 320 (6) 296; 370 (6) 358, 359, (16) 304,305,307, 308 (7) 358, 359 Kishimoto, Y. 80 (19) 51 Kraemer,P. 298 (24) 183 Kisiel, W . 160; 272 (18) 166, (20) 166, Krag,E. 356(56) 350 167, (21) 166,167; 173 (35) 171 Krakower, C. A. 225 (18 ) 215 Kit, S. 403,379 Kramer, R. H. 270 (58) 261, 265 Kitagawa,T. 341 (60) 331 Kramsch, D. M. 253 (5) 227 Kitchen, B. J. 42, 32 Kreese, H. 249 (62) 146; 258 (12) 153 Kivimaki,T. 233 (9) 130 Krupey, J. 352 (3) 342, (6) 342; 353 (13) Kivirikko, K. I. 225 (32) 218, 222; 226 343; 370(27)358 (37) 218, 222, (38) 222, (41) 22, (42) Krusius, T. 247 (2) 136, (4) 136, (6) 136, 222, (43) 222, (44) 222, (45) 222
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
INDEX
457
Kuhl,W. 159 ( 20) 150 Lemm, J. 229 (12) 109 Kuhlenschmidt, M. S. 248 (38) 143 Lennarz, W. J. 40, 28; 43, 23, 24; 45, 23; Kuhmichel, A. 370 (14) 358, 359, 369 46, 21-23 Kiihn, K. 224 (3) 213; 225 ( 26 ) 215, 218LeQuire,V. S. 84 (125) 67 Kuhn,R. 84(143 ) 75 Lerch, R. M. 253 ( 8 ) 227 Kundo,S.K. 82 (48) 56 Lerner, M.P. 325 (41) 322 Kundu,S.K. 82 ( 43 ) 52 Lerner,R.A. 298 (11) 181 Kupchik, H. Z. 355 (54) 350 Leseney, A. M. 432 ( 25 ) 414, 420 Kurachi, K. 272 (3) 163, (7) 164 Lessof, M. H. 339 (23) Kurosky, A. 229 (7) 108 Lester, R. L. 85 (170) 49, (171) 49, 56 Letoublon, R. 83 (98) 69 Kusiak, J. 259 (5) 150,153,157 Letts, P. J. 42,34 Kutty,K.N. 247 (20) 138 Lev, R. 276 ( 5 ) 275 Kyriakides, E. 258 (10) 153 Levi, R.I. 222,201 Levin, J. G. 299 (56) 195 Levine, P. 324 (1) 311, 322; 370 (14) 358, LafitteJ.J. 220 (48) 115 359, 369, (15) 358, 369 Lagunoff,D. 258 (3) 153 Levitt, M. H. 342 ( 74 ) 334 Levitzki,A 273 (31) 170 Laine, R. 378 Laine, R. A. 82 (93) 64; 85 372 (45) 364,(46) 364 Lhermitte, M. 220 (48) 115 Laipio, M. L. 233 (10) 130 Lamblin, G. 220 (35) 113, (41) 113, (48) Li, E. 422 (19) 410 Li,S. 85 (159) 77 115 Li,S.C. 80 (34) 52,73 LaMontJ.T. 84 (128) 69 Li, Y. 258 (11) 153 LeMount, J. T. 83 (116) 67, 69 Li, Y. T. 80 (34) 52, 73, (35) 52; 85 (159) Lamport, D. T. A. 29 (10) 6, (11) 6 77 Landsteiner, K. 324 (1) 311, 322 Liang-Tang, L. K. 339 (15) 327 Landy, M. 292 (11) 277 Lieberman, J. 132 (17) 127, (19) 127 Lang,S. 258 (6) 153 Liedgren,H. 82 (55) 56 Langlois, A. J. 298 (9) 180,181,187 Light, D. 85(156) 77 Lankester, A. 377 (8) 376 Lankf ord, B.J. 229 (3) 108, (7) 108 Lillford, P. J. 222 (51) 115 Lantz,R. S. 430 ( 21) 414 Lilliehook, B. 293 ( 35 ) 279 Larriba, G . 3 7 8; 403, 385, 387 Lindberg, B. 82 (49) 56, (50) 56, (51) Larsson, C. 232 (15) 126 56, (52) 56, (55) 56; 354 (31) 344 Laurell, C. B. 42,28; 232 (14) 126 Linsley, K. B. 293 (29) 279, 286, 287 Laurence, D. J. R. 355 (50) 350, 351 Lione,A. 84(131)69 Lautenschleger, J. T. 352 (7) 343, (8) 343 Lippman, M. 292 (19) 277; 293 (34) 279 Lawford, G. R. 42, 26 Lis, H. 269 (17) 258 Lazareva, M. N. 340 (40) 330 Lisowska, E. 325 (33) 320 Leamnson, R. N. 297 (2) 180, 181; 298 Litt, M. 229 (13) 109; 220 (37) 113 (14)181 Liu,T.-Y. 353 ( 22 ) 343,348 Livingston, D. C. 431 (36) 425 Leblond, C. P. 40, 34, 37; 83 (120) 67 Lockhart, L. H. 229 (18) 110 Lee, C. L. 320 (6) 296; 370 (6) 358, 359 Lockney,M. 83 (108) 71 Lee, D. 42, 32 Lodish, H. F. 43, 26; 44, 26, 28; 46, 26; Lee, N. 293 (22) 277, 278, 288, 289 299 (35) 188 Lee, Y. C. 29 (13) 6; 202 (21); 148 (38) Lombardi,B. 342 (73) 143; 431 (42) 424 Long,C. 297 (5) 180,195 Ledeen, R. W. 80 (33) 52; 82 (43) 52 Long, M . M . 227; 253 (10 ) 228, 230; 254 (48) 56 ' (14) 231, 234, (17) 231, 235-237, 240Lees, M. 403,382 242, (18) 237-239, (19) 243, (20) 243, Leffert, H. L. 339 (12) 326; 340 (33), 244, (21) 243; 255 (41) 248, 252, (45) (34), (37) 329 252, (47 ) 247 Legaz, M. E. 272 (9) 164, (12) 165, (14) Lonngren, J. 81 (55) 56 165,(17) 166 Lehman, E. D. 42,32 Loor,F. 272 (74) 268 LeKim, D. 82 (58) 59 Lopez-Vidriero, M. T. 220 (43) 113 Leloir, L. F. 40,22, 23; 43,23 Lorand,L. 172 (28) 170 V
1
V
1
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
458
GLYCOPROTEINS
Lotan, R. 256; 270 (39) 262, 266, 267; 271 (72) 266 Lote, C . J. 19 (8) 6, (9) 6 Louisot, R. S3 (98) 69 Lowick, J. H . B. 376 (6) 375 Luftig, R. B. 200 (61) 197 Lundblad, A . 29 (7) 6; 42; 43; 103 (28) 98, (29) 98; 148 (46) 144, (47) 144, (49) 144; 403,390; 422 (11) 408 Luner, S.J. 325 (40) 322 M Maccioni, H . J. 82 (79) 61 McClean, R. J. 84 (132) 69 McCluer, R. H . 80 (19) 51, (20) 51 McConnell, H . M . 270 ( 35 ) 259, 260 McCreaJ.F. 84 (134) 73 McCullagh, K. G . 254 (31) 243 McGuire, E . J. 40, 36; 42, 36, 37 37; 83 (112) 67,(121) 67 Mach, J. 355 (49) 350, 351; 356 (55) 350 Macher, B. A . 47; 80 (18) 49; 83 (108) 71 Mclntire, K. R. 339 (27) 328, 332, (28) 328, (29) 328 McKee, P. A. 272 (13) 165, (27) 169; 273 (29) 170 McKhann, C . F . 292 (8) 277 McKibbin, J. M . 82 (41) 52 McKnight, G . S. 42,26 McKusick, V . A. 226 (55) 224 McLellan, W . 298 (13) 181 MacPherson, I. 310 (14) 302 McQuiston, D . T . 324 (17) 312 McSherry, N . R. 229 (5) 108 Magee, S. C . 42, 32; 42, 32 Magnusson, S. 272 (25) 169 Mahler, H . R. 270 ( 49 ) 260, 264, 266; (52) 260, 264 Mahley, R . W . 84(125) 67 Maier, V . 222, 201, 202 Makita, A. 82 (70) 61, (83) 61; 83 (102) 69; 370 (18) 358, 359, 363 Maley, F. 86; 202 (1) 86, 95, (2) 86, 88, 93, 98, (3) 86, (4) 86, (5) 86, (7) 86, 90, 94, (9) 86, 97, (15) 87-89, 98, (16) 87-89, 98, (16) 87, 89, 90, (23) 95, 100; 103 (33) 98, 101, (34) 101; 247 (11) 137,(12) 137,(13) 137 Mallette, M . F . 372 (35) 361 Mallucci, L . 272 (71) 266 Maloney, W . H . 229 (21) 110, 111 Mangos, J. A. 229 (5) 108 Manley, G . 222, 201 Mann, D . L . 324(9) 312 Mann, K. G . 272 (11) 165 Mansson, J. E . 80 (35) 52
A N D GLYCOLIPIDS I N DISEASE PROCESSES
Marchesi, V. T . 20 (21) 13, 18, 19; 42, 37; 269 ( 24 ) 258, 259; 270 (33) 259, (37) 260, 262, 265; 294 (46) 288 Marciani, D . J. 199 (38) 188, 194 Marcus, D . M . 371 (50) 363 Mareel, M . 220 (46) 113 MarIer,E. 272 (27) 169 Maroteaux, P. 45; 149 (55) 145 Marsh, W . L . 20 (16) 10; 370 (26) 358 Marshall, R. D . 42, 23, 24, 36 Martens, P. 226 (56) 224 Martensson, E . 80(11) 48 Martin, C . L . 229 (10) 108 Martin, G . R. 226 (35) 218; 253 (7) 227 Martin, G . S. 199 (36) 188 Martin, I. 84 (142) 73 Martin, J. P. 232 (12) 125 Masamune, H . 370 ( 4 ) 358, 359, (5 ) 358, 359 Masseyeff,R. 339 (26) 328 Masson, P. K. 42 Masubuchi, M . 44, 33 Masukawa, A. 370 (4) 358, 359 Matalon, R. 248 (44) 144, (48) 144; 258 (11) 153 Matsubara, T . 403, 378 Matsumura,G. 202 (25) 98 Matthews, L . 229 (12) 109, (21) 110, 111 Mattsson, K. 133 (2) 129 Matuda,Y. 370 (4) 358,359 Matukas,V.J. 225 (16) 215 Mavligit, G . M . 292(10 ) 277 Mawal,R. 42, 32; 42, 32 Mawhinney, T . P. 254 (30) 243 Mayer, H . - E . , Jr. 102(6) 86, 87, 95, 98 Mayo, B . J . 229 (3) 108 Meager, A . 422 (18) 410 Meezan,E. 320 (2) 295 Meier, C . 248 (41) 143 Mellan,P. 299 (44) 192 M e l l a r s , R . C . 298 (16) 181 Mellon, P. 299 (48) 194 Melnick, J. L . 403, 379, 402 Mendicino, J. 44, 33 Merkow, L . 340 (55) 331 Merlin, L . M . 83 (122 ) 67 M e r s k y , H . 233 (1) 129,130 Meyer, F . A. 220 (45) 113 Meyer, R. G . 272 ( 9) 164 Michael, A. F . 226(54) 224 Michaelson, M . 340 (51) 331 Michalski, J. C . 42; 149 (54) 145, (55) 145 Michel, I. 444 (12 ) 433 Migita, S. 293 ( 23 ) 278, 288 Mihalev,A. 339 (30) 328 Mihalik, G . M . 411 (14) 408, 409
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
INDEX
459
Miller, D.H. 19 (10) 6 Murakami, W. T. 310 (13) 302; 370 (1) Miller, D. K. 293 (39) 280, 282, 290, (40) 357 280, 281, 286, 288, 290; 294 (51) 290, Muramatsu, T. 42, 30; 43, 30; 45, 33; 202 291,(52 ) 290, 291 (14) 87, 91, 98,(19) 91, 95 Miller, E.C. 340(54) 331 Murgita, R. A. 340 (41) 330, (42) 330 Miller, E. J. 225 (16 ) 215, (17 ) 215, (22)Murphy, C. 40, 31 215, (28) 215, 218, (30) 218 Murphy, E . C , Jr. 180; 198 (28) 184, Miller, J. A. 340 (54) 331 188-190,192; 299 (34) 188-190,192 Miller, M. 19(10)6 Murray, R. K. 80 (13) 49 Miller, S. C, 293 (41) 280, 282, 286; 444 Murray-Lyon, A. M. 340 (39) 330 (11) 433, 442 Murthy, M . S. 320 (11) 302; 311; 324 Minase,T. 341 (70) 332 (15) 312, 315, 317, 320; 325 (29) 320, Miron,T. 371 (47) 364 322, (43) 322 Mitchell, L. W. 255 (41) 248, 252, (45) Myerowitz, R. 249 (63) 146 252,(47) 247 Myllyla, R. 226 (42) 222, (43) 222, (44) Miyazaki, M. 340 ( 36 ) 329 222 Moennig, V. 298 (15) 181 Moertel, C. G. 339 ( 29 ) 328 N Molnar, J. 43, 26; 83 (101) 69 Momoi,T. 80 (27) 52 Montelaro, R. C. 197 (4) 180 (50) 195 Nadler, H. L. 229 (9) 108, (10) 108 Montgomery, P. C. 147 (15) 138 Nagai, Y. 80 (24) 51, (25) 51, (26) 52, Montgomery, R. 20 (20) 11; 202 (6) 86, (27) 52; 324 (13) 312, 314 87, 95, 98, (21), (22); 432 (42) 424 Nager,C. 222,202 Montreuil, J. 29 (4) 5; 43, 21, 24, 26, 33; Nakada, H. 269 (27) 258, (28) 258; 293 45, 30; 248 (28) 140; 249 (55) 145 (24) 278 Mookerjea, S. 42, 32 Nakomori, S. 320 (13) 302 Moreau, R. 324 (16) 312, 322 Nant6,V. 233 (10) 130 Morell,P. 82 (63) 59 Narasimhan, S. 21; 43, 29-31, 34; 45, 29; Morgan, I. G. 270 (50) 260, 264 324 (19) 314, 320; 422 (17) 410 Morgan, W. T. J. 20(18)9 Naso, R. B. 180; 298 (20) 182-184, 189, Morgello, S. 294 (52) 290, 291 190, (21) 183, 184, (22) 183, 184, 190, Moro,S.P. 232 (8) 124,125 (23) 183, 184, 187, (26) 182-184, 192, Morre, D. J. 83 (111) 67, (118) 67, (119) (30) 182, 184, 191; 299 (33) 188, (55) 67, (122) 67; 84 (124) 67, (125) 67; 195 197 444 (16) 434 Nass, M'.K. 445 (21) 437 Morris, H. P. 340 (45) 331 Nathenson, S. G. 202 (19) 91, 95; 292 (2) Morris, J. E. 353 (19) 343, 344; 354 (39) 277; 324 (9) 312; 376 (3) 375 348 Natsu-ume, S. 293(23) 278, 288 Morris, N. P. 225 (34) 218 Nelson, M. M. 339 ( 25 ) 327 Morris, N. R. 229 (3) 108 Nemerson, Y. 172 (19) 166, (22) 166, 167 Morrison, M. 269 (8) 258 Neri, G . 276 (6) 276; 412; 430 (11) 412Morrison, T. G. 43, 26 415, 420, 421, (12) 412, 414, 418, 420, Moscatelli, E. A. 79 (7) 48 423, (14) 412, 414, 415, 424, (19) 413, Moschetto, Y. 254 (32) 243 414; 431 (35) 423 Moscona, A. A. 292 (3) 277, (4) 277 Neufeld, E. F. 85 (167) 79; 249 (59) 146, Moser,H.W. 80(19)51 (60) 146, (66) 146 Moskal, J. R. 47; 82 (80) 61, (84) 61, Neufeld, L. 258 (8) 153 (89) 61; 83 (105) 71, (108) 71 Neurath,H. 272 (16) 166 MoullecJ. 324 (16) 312,322 Neutra, M. 120 (27) 110 Movat,H.Z. 272 (5) 163 Neville, A. M. 355 (50) 350, 351 Mudholkar, G. C. 232 (5) 123 Newman, C. H. H. 147 (21) 138 Mueller, T.J. 269 ( 8 ) 258 Newman, R.A. 269 ( 25 ) 258 Mueller-Lantsch, N. 299 (46) 194 Newton, W. A. 119 (15) 109 Muir, L. 29 (13) 6; 254 (34) 246 Ng, V. L. 200 (62) 197 Muller-Spreer, H. C. 222, 201 Ng Ying Kin, N. M. K. 43; 46; 147 (25) Mullinger, R. N. 222, 201 139, (26) 139; 148 (29) 140, (30) 140 Munro, J. R. 43, 31, 34 Ni,Y. 370(7) 358,359
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
460
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Nichols, M. 339 (11) 326, 328 Nickel, R. 340 (48) 331, (50) 331 Nicol,D. 158(3)153
PROCESSES
Orgebincrist, M. C. 272 (61) 261 0rjasaeter, H. 355 (52) 350, 351 Orr,A.H. 340 (39) 330 Orr,T.W. 340 (53) 331 Nicolson, G . L . 20 (24) 16, (25) 16, 17, (27) 19; 256; 269 (1) 256, 258, (4) Osawa, T. 269 (21) 258; 270 ( 32 ) 259; 256, 258, 268, (5) 256, 258, 259, 266, 293 (29) 279, 286, 287 268, (11) 258, (14) 258, (15) 258, (18) Overkerk, C. H. 248 (45) 144 258, 266, 268; 271 (65) 266, (66) 266, Owens, M. C. 132 (18) 127, (20) 127 (70) 266; 410 (3) 404; 430 ( 2 ) 412, Ownby, C . L . 432 (5) 412, 429, (6) 412, 429; 431 (39) Oyama,K. 370 ( 5 ) 358,359 429; 444 (3) 432 Oyasu,R. 324(10) 312 Ozanne,B. 432 (37) 425 Niemann, H. 372 (28) 358 Nilsen-Hamilton, M. 270 (45) 260, 263 Nilsson, B. 103 (28) 98 P Nilsson, S. F. 270 (47) 260, 263, 265 Pacuszka,T. 82 (91) 64-66 Noonan, K. D. 432 ( 38 ) 425 Pain, P. H. 220 (38) 113 Norberg, T. 403, 390 Norden, N. E. 42; 43; 103 (28) 98, (29) Painter, R.G. 269 (14 ) 258 Pallavicini, C. 222 (50) 115 98; 248 (46) 144, (47) 144 (49) 144 NordinJ.H. 29(12)6 Nordquist, R. E. 325 (41) 32 , Norgaard-Pederson, B. 339 (24) Norman, R. M. 147 (21) 138; 249 (56) 145 Palo, J. 133 (2) 129, (6) 130, (7) 130, (9) 130, (13) 130, 131; 248 (51) 145, (52) Norton, W. T. 80 (12) 49 145 Nosslin, B. 232(14) 126 Panet,A. 198 (7) 180,192 Nowotny, A. 293 (33) 279 Papas, T.S. 199 (38) 188,194 Nunez, E. 339 (16) 327, 329 Parker, S. 340 (58) 331 Nunez, H . A . 47 Parodi, A. J. 40,22, 23; 43,23 Nylen,M. 253 (7) 227 Partridge, S. M. 253 (2) 227 Pascher, I. 81 (40) 52, (41) 52, (42) 52; O 371 (36) 361 Oakes, D. D. 339 (8) 326 Pastan,I. 411(20)410 O'Brien, J. S. 84 (141) 73; 248 ( 35) 143, Patel,V. 148 (33) 140 (36) 143 Patt,L.M. 83(114)67 Ockerman, P. A. 42; 103 (28) 98; 248 (46) Paul,B. 158 (10) 153 Pauling, L. 353 (14) 343, 344 144, (49) 144 Paulson, J. C. 43,33; 44,36, 37 Oda,T. 340(38) 330 Pawson,T. 199 (36) 188 O'Donnell, P. 199-200 (58) 197 Paz, M.A. 224 ( 5 ) 213 Ogata, S. 43, 30 Pearlstein, E. S. 269 (13) 258 Ogawa,K. 342 (70) 332 Peers, M.C. 149(55) 145 Ohashi, M. 80 (29) 52 Ohnishi, T. 253 (11) 228, 237, 240; 254 Pentchev, P. 150; 158 (5) 150, 153, 157, (18) 237-239; 255 (45) 252 (16) 150, (17) 154, (18) 150; 159 (23) Ohno, S. 293 (23) 278,288 155 Oh-uti, K. 370 (3) 357, 359, (4) 358, 359 Perdome, J. M. 310 (7) 296, 298 Oikarinen, A. 226 (41) 222, (45) 222 Perlman, P. 370 (12 ) 359; 403, 378 Okamoto, K. 254 (19) 243, (20) 243, 244, Pernis, B. 271 (74) 268 (21) 243 Perova, S. D. 339 (1) 326 Okita,K. 342 (72) 332 Perret,V. 276 (5) 275 Old, L.J. 292(5) 277 Perrotto, J. L. 84(128 ) 69 Olivecrona, T. 222, 204; 222, 204, 207-210 Peters, J.H. 292(16) 277 Olshevsky, U. 299 (35) 188, (39) 188, 190 Peters, S. P. 84 (152) 75, 77; 148 (38) 143 Onda,H. 342 ( 69 ) 332 Peterson, T. 172 ( 25) 169 O'Neil, P. A. 431 (25) 414, 420 Petterson, B. M. 199 (38) 188, 194 Onoe, T. 341 (67) 332, (70) 332 Philipson, L. 199 (39) 188,190 Opheim, D. J. 84 (139) 73; 247 (10) 137 Phillips, H. J. 430 (20) 413 Oppeheim, J. D. 272 (60) 261, 265 Phillips, S. F. 356 ( 56 ) 350 Oppenheimer, E. H. 229 (17) 109,110 Pieringer, R. A. 84(136) 73
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
461
INDEX
Pietropaolo, C. 403, 373 Pietrzykowa, B. 247 (17) 138 Piez,K.A. 226(35)218 Pigman, W. 29 (2) 5, 13, 14; 20 (14) 6; 276 (5) 275 Pilotti, A. 82 (52) 56 Pimlott,W. 372 (36) 361 Pincus, T. 298 (16) 181; 299 (58) 197 Pinter, A. 297 (1) 180,181 Pinteric, L. 42, 34; 44,34; 83 (121) 67 Pinto da Silva, P. 269 (11) 258 Pister,L. 298 (15) 181 Pitlick, F. A. 270 (53) 261, 264 Pitot,H. C. 342 ( 60 ) 331 Pizieux,0. 254 ( 32 ) 243 Pizzo,S. V. 273 (29) 170 Planinsek, J. A. 293 (32) 279,280 Plantner, J. J. 20(15)6 Pless, D. D. 43,23, 24 Plummer, T. H., Jr. 202 (1) 86 (4) 86,(5) 86,(9) 86, 97 Podolsky, D. K. 83 (116) 67, 69; 84 (129) 69 Podrazky, V. 254 (35) 246, (36) 246 Poler, M. 340 (44) 330 Polim,G. 232 (12) 125 Pollitt, R. J. 43; 133 (1) 129, 130, (13) 130,131, (14) 131 Polony, I. 220 (40) 113,114,116 Porter, C.J. 84 (135) 73 Porter, M. 258 (9) 153 Poschmann, A. 325 (36) 322 Poste, G. 269 (5) 256, 258, 259, 266, 268; 272 (78) 268; 420 (3) 404 Potter, J.L. 229 (12) 109 Pouyssegur, J. 422 ( 20 ) 410 Powell, J. T. 43,32; 44,32 Prehn, R. T. 292 (6) 277, (9) 277 Prendergast, R. C. 370 (9) 358, 359 Presper, K. A. 82 (90) 61, (92) 64 Prestegard, J. H. 82 (56) 59 Pretty, K. M. 43; 133 (13) 130, 131, (14) 131 Price, H. C. 82 (43) 52 Price, J. M. 340 (54) 331 Prieels, J. P. 40, 37 Prilzl,P. 258 (3) 153 Princler, G. L. 339 (28) 328 Pritchard, D. G. 353 (12 ) 343; 354 (27) 344, (32) 344; 356 (57) 350, (59) 350, 351 Prockop, D. J. 226 (36) 218, (37) 218, 222, (49) 223 Pusztaszeri, G. 355 ( 49 ) 350, 351; 356 (55) 350 R
Rabb,M.C. 272 (68) 266 Rabinowitz, Z. 410 (4) 404
Radcliffe, R. 272 (19) 166, (22) 166, 167 Radhakrishnamurthy, B. 222; 222, 201, 202; 254 (37) 246 Radin, N. S. 82 (63) 59; 85 (160) 77, 78; 248 (36) 143 Raff, M.C. 272 (71) 266 Raghupathy, E. 83 (99) 69 Ramseur, J. M. 299 (56) 195 Rao, A. K. 44, 33 Rao, G.J. S. 229 (9) 108 Rapaka, R. S. 254 (20) 243, 244 Rapoport, S. 258 (5) 150,153,157 Rapport, M. M. 85 (154) 77; 371 (49) 363 Rauvala, H. 84 (144) 75; 247 (4) 136, (7) 137 Read, R. C. 222, 201-204, 210 Rearick, J. I. 43, 33; 44, 36, 37 Rebers,P.A. 432 (27) 414 Redi K B M 225(24) 215
112, (43) 113; 222 (52) 115 Reinhold, V. N. 225 (15) 215 Reisfeld, R. A. 292 (1) 277 Rekosh,D. 298 (31) 187 Renton,P.H. 324 (18) 312 Reutter, W. 403, 378 Reynolds, J. A. 220 (39) 113 Reynoso,G. 355 (53) 350 Rhodes, R. K. 225 (22) 215 Richard, M. 83 (98) 69 Richards, F. M. 269 (12) 258 Richer, C. 220 (34) 113 Riekkinen, P. 233 (6) 130, (9) 130 Riordan, J. 43, 31, 34 Ripamonte, A. 254 (19) 243 Risteli, L. 225 ( 32 ) 218, 222; 226 (42) 222, (43) 222, (44) 222 Robbins, P. W. 44, 24; 46, 24; 83 (106) 71; 202 (12) 87; 320 (2) 295, (14) 302 Robert, B. 254 (25) 243, (27) 243 Robert, L. 254 (25) 243, (26) 243, (28) 243, (31) 243 Roberts, B. I. 222, 201-204, 207, 210 Roberts, G. P. 220 (36) 113; 292 (17) 277 Robertson, J. 42, 24, 31; 202 (17) 91 Robertson, M. 42, 24, 31 Robinson, D. S. 222, 201 Robinson, G. B. 40, 26 RobozJ.P. 292 (15) 277 Roden, L. 44, 21, 33, 34 Rodman, J. 85 (165) 79; 158(6) 153 Roepstorff, P. 272 (10) 165 Rogentine, G. N., Jr. 324 (9) 312 Rogers, J. 270 ( 31) 259 Rogers, L. A. 272 (27) 169 Roghmann, K. J. 232 (5) 123 Rome, L.H. 249 ( 60) 146 Ronin,C. 84 (127) 67 Ronzio, R. A. 84(126) 67
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
462
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE
Roosien, J. 431 (40 ) 429 Roseman, S. 40, 33, 36; 44, 34, 36, 37; 81 (60 ) 59, (61) 59, (64 ) 59; 82 (68) 61, (71) 61, (72) 61, (73) 61, 66, (74) 61, (75) 61, 67; 83 (112) 67, (121) 67; 324 (21) 314 Rosenblith, J. 271 (69) 266; 430 (7) 412, 429 Rosenfeld,L. 148 (38) 143 Rosenfelder, G. 370 (13) 358 Rosenthal, A. L. 19 (12) 6 Ross, A. H. 270 (35) 259,260 Ross, R. 253 (1) 227; 254 (22) 243, 246, (23) 243, (24) 243, (26) 243, 246, (29) 243, (34) 246 Rossomando, E. F. 271 (64) 261 Roth, S. 83 (110) 67, (112) 67, (117) 67; 84(133) 69 Rothman, J. E. 44, 26, 28 Roukema, P. A. 148 (45) 14 Roussel, P. 120 (35) 113, (41 115 Roveri,N. 254(19 ) 243 Rubin, D. 226 (57) 224 Rueckert, R. R. 298 (8) 180, 192; 299 (32) 187 Ruizitt,B. 254 (37) 246 Ruoslahti, E. 276 (3) 275; 339 (7) 326, (13) 326, (14) 327; 352 (4) 342, 346; 356 (61) 351; 422 (13) 408 Rutishauser, U. 430 (8) 412 Rutzky, L. P. 324 (10) 312 Ryd,W. 294 (53) 291 Ryd,W. 294 (53) 291 Ryhanen, L. 226 (37) 218, 222, (38) 222, (39), (40) 218 Ryman, B. 258 (14) 153
PROCESSES
Santer, U. V. 420 ( 6 ) 404, 405; 422 (15) 408 Sarvin,A.J. 249 (50) 146 Sass-Kortsak, A. 232 (8) 124,125 Sato T. 44 43 Saunders, T\E. 299 (33) 188 Savolainen, H. 133 (11) 130, (13) 130, 131; 248 (51) 145, (52) 145 Scanlin, T. F. 422 ( 7 ) 405, 408, (12 ) 408 Scanlon, E . F.
311; 324 (12) 312, 318,
(15) 312, 315, 317, 320; 325 (29) 320, 322, (43) 322 Schacter, H .
21; 42, 31, 33, 39; 42, 26, 34;
43, 29-31, 34; 44, 21, 22, 26, 33, 34, 36, 37; 45, 29; 46, 31; 80 (13) 49; 83 (121) 67; 324 (19) 314, 320; 422 (17) 410 Schafer,D.D. 82 (56) 59 Schafer, W. 297 (4) 180, 187; 298 (9) 180,181,187,(15) 181,(19) 181
Scheraga, H. A. 276 (4) 275 Scherrer, M. 297 (5) 180,195 Schiffman,E. 253 (7) 227 Schimke, R. T. 42, 26 Schiphorst, W. C. M. 46, 33, 39 Schlamowitz, M. 431 (29) 415, (32) 426 Schleich, J. 224 (1) 213 Schlesinger, J. 272 (79) 268 Schlesinger, P. 258 (6) 153 Schlesinger, P. H. 85 (165) 79 Schlesinger, S. 45, 24, 26, 31; 202 (13) 87 Schmickel, R. D. 249 (63) 146 Schmid,K. 42,33 Schmidt-Ullrich, R. 270 (44) 260, 263, 266 Schneider, M. 325 (46) 319 Schnute, W. C., Jr. 353 (19) 343, 344; 354 (25) 344, (27) 344; 355 (46) 348 S Schofield, D. J. 226 ( 36 ) 218 Schotz, M. C. 222,210 Saccomani, G. 255 (44) 252 Sachs, L. 272 (76) 268, (77) 268; 420 (4) Schreiber, G. 430 (16 ) 413, 416, 420 Schreiber, M. 430 (16) 413, 416, 420 404; 430 (8) 412 Schultz,A. 82 ( 64 ) 59 Sadler, J.E. 44,36,37 Saito, H. 82 (54) 56; 354 (30) 344; 372 Schut, B. L. 45, 30 Schwarting, G. A. 371 (50) 363 (31) 361 Schwartz, M. L. 173 ( 29) 170 Sajdera,S.W. 254 (39) 252 Schwartz, R. H. 232 (5) 123 Sakamoto, C. K. 430 (10) 412 Schwick,H. 273 (32) 170 Salvaterra, P. M. 270 (52) 260, 264 Schwyzer, M. 42, 36; 44, 37; 45, 37; 83 Sambrook, J. 431 (37 ) 425 Samuelsson, B. E. 82 ( 40 ) 52, (41) 52, (97) 64-66 Scribney, M. 82 (59) 59 (42) 52; 372 ( 36 ) 361 Scrimgeour, J. B. 339 (25) 327 Sandberg, L. B. 254 (15) 231, (16) 231 Seaman, F. 376(5) 375 Sandford, P. A. 354 (29) 344 Segaloff,A. 444(13 ) 433 Sando, G. N. 249 (60) 146, (66) 146 Sanford, B. H. 292 (20) 277, 279, 280, 288; Seglen,P.O. 432 (33) 416 293 (21) 277, 279, (27) 279, (28) 279, Segrest, J. P. 20 (26) 19; 269 (7) 258 280, 286, 288; 324 (26) 315; 444 (10) Seifert,E. 298 (15) 181 Seifter, S. 224(5) 213 432
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
463
AUTHOR
INDEX
Sell, S.
326; 339 (6) 326, (9) 326, (11)
326, 328, (12) 326, (17) 333, (19) 327329, (20) 327, (31) 328, 330, 331, 334; 340 (32) 328, (34), (35) 329, (43) 330, (44) 330, (45) 331, (48) 331, (49) 331, (50) 331, (51) 331; 341 (61) 331, (62) 332, (65) 332-334, (73), (75) 334 Sen, A. 198 (6) 180,195 Senior, R. G. 46; 103 (30) 98; 148 (29) 140 Seppala, M. 339 (7) 326, (22) 327 Seyer, J. M. 225 ( 29 ) 215, 218 Shander, M. H. M. 197 (2) 180,181 Shanmugam, G. 199 (42) 190,194 Shapira,E. 119 (10) 108 Shapiro, D. 158 (16) 150 Shapiro, L. 85 (167) 79; 158(8) 153 Sharon, N. 269 (17 ) 258; 270 (38) 260 262, 265; 271 (72) 266 Sharp, H. L. 131 (1) 122-125 233 (3) 129,(8) 130, 131 Sheppard, H. W., Jr. 340 (43) 330, (44) 330, (50) 331 Sherblom, A. P.
432
Sherlock, S. 132 (11) 125 Sherr, C. J. 45, 26; 198 (6) 180,195 Shih, C. K. 220 (37) 113 Shiloach, J. 158 (18) 150 Shin, B. C. 444 (5) 432, 434-436 Shincarial, A. 199 (43) 190,194 Shinitzky, M. 272 (76) 268, (77) 268 Shinozuka,H. 342 (73) Shively, J. E .
342; 354 (35) 346; 355 (47)
348, 350; 356 (58) 350, (59) 350, 351, (60) 351 Shotten, D. M. 269 (16) 258 Showe, M.H. 299 (54) 195 Shur, B. 83 (110) 67; 84 (133) 69 Shuster, J. 339 (8) 326
Slomiany, A. 80 (15) 49, 75, (36) 52; 82 (37) 52; 82 (94) 64; 372 ( 39 ) 361, (40) 361 Slomiany, B. L. 80 (15) 49, 75, (36) 52; 82 (37) 52; 82 (94) 64; 371 (39) 361, (40) 361 Sly, W. S. 85 (166) 79; 249 (64) 146, (65) 146 Small, B. 46,26 Smith, A. E. 299 (36) 188 Smith, C. A. 45, 32 Smith, C M . 272 (26) 169 Smith, D. F. 430 (11) 412-415, 420, 421, (12) 412, 414, 418, 420, 423, (13) 412, (14) 412, 414, 415, 424; 431 (35) 423 Smith, E.L. 82(41)52 Smith, F. 202 (22); 354 (36) 348; 431 (27) 414 Smith R.T 292 (11) 277 Snell,E.E. 82 (57) 59 Soloff, M. S. 339 (15) 327 Somers, K. 403, 379 Sonoda,S. 431 (29) 415 Sottrup-Jensen, L. 272 (25) 169 Spector,S. 229 (12) 109 Spielvogel, C. 248 (34) 143 Spik, G. 45, 30; 248 (28) 140; 249 (55) 145 Spiro,M.J. 45,22,76 Spiro, R. G. 29 (1) 5-7, 13, 15; 45, 22, 76; 224 (7) 213; 225 (11) 214, 215, (14) 215, (23) 215; 226 (50) 223, 224, (51) 223; 269 (19) 258, (20) 258; 354 (26) 344 SprangerJ. 42; 248 (53) 145 Springer, G . F. 293 (36) 279; 310 (11) 302; 311; 324 (2) 311, 312, (3) 311,
322, (6) 311, (7) 311, 312, 322, (11) 312, (12) 312, 318, (13) 312, 314, (14) Siddiqui, B. 79 ( 6 ) 48, 49; 295; 320 (10) 312, 322, (15) 312, 315, 317, 320, (19) 302, 304, 305; 371 (30) 361 314, 320, (20) 314, 320, (25) 315, (27) Sillerud, L. O. 82 (56) 59 315; 325 (28) 314, 317, (29) 320, 322, Siminovitch, L. 45, 29; 411(17) 410 (30) 320, (31) 320, (32) 320, (34) Simmons, D. A. R. 370 (12) 359; 403, 378 320, (35) 320, (37) 312, 322, (40) Simmons, J. L. 83 (109) 71, 76 322, (42 ) 322, (43 ) 322, (45 ) 322 Singer, S. J. 20 (24) 16; 269 (1) 256, 258, Squire, R.A. 341 (74) 334 (3) 256 Sramek, S. 378; 403, 385, 387 Sirakov,L. M. 339 (30) 328 Srinivasan, S. R. 211 Six,H.R. 372 (44) 364 Stacey,D. W. 199 (49) 194 Sizaret, P. 339 (10) 326 Stahl, P. 85 (165) 79; 158 (5) 153 Skehel,J.J. 272 ( 62 ) 261,265 Stalder,K. 324 (6) 311 Skelly, H. 339 (19) 327-329; 340 (34) Stallcup, W. B. 173(31)170 Skelly, J. 403, 385 Stanislawski-Birencwajg, M. 339 (3) 326 Slayter, H. S. 293 (40) 280, 281, 286, 288, Stanley, P. 43, 29, 30, 31; 45, 29; 411 (17) 410 290, (44) 286, 288; 353 (10) 343, (11) Starcher, B. C. 253 (10) 228, 230, (12) 343, 345 228, 231; 255 (43) 252 Sloane-Stanley, G. H. 403, 382
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
464
GLYCOPROTEINS
Starling, J. J.
276 (6) 276; 412; 430 (19)
413, 414; 431 (22) 414, 418, 419, (31) 415, 429 Steck, T. L. 431 (41) 426; 444 (15 ) 433 Steer, C. 159 (21) 150 Stefanko,St. 147 (17) 138 Stegner, H. E. 325 (36) 322 Steigerwald, J. C. 82 (72) 61 Stein, M. 148 (34) 143 Steineman, A. 270 (54) 261, 265 Steiner, D .
378
Steiner, M. 403,379,385 Steiner, S. 403, 379, 385, 387, 402; 411 (10) 408 Steinman, H. M. 41, 36 Stellner, K. 81 (54) 56; 320 (9) 302; 354 (30) 344; 370 (20) 359; 372 (31) 361, (43) 364; 403, 378 Stenflo, J. 272 (10) 165; 173 ( 34) 1718 Stern, R. C. 222 (49) 115 Stetson, B. 46, 24 Stewart, T. H. M. 324 (23) 314, 319 Sticht, G. 81 (58) 59 Stillman, D. 340 (35) 329, (51) 331 Stirling, J. 148 (28) 140 Stockert,E. 199 (58) 197 Stoffel, W. 81 (58) 59 Stoffyn, A. 45, 33; 46, 33 Stoffyn, P. 45,33; 46, 33; 82 (78) 61 Stone, J. D. 84 (134) 73 Stoolmiller, A. C. 82(81) 61 Storm, E. 272 (23) 167 Strand, M. 198 (16) 181 Stratton,F. 324 (18) 312 Strecker, G. 42; 45, 30; 248 (28) 140; 149 (54) 145, (55) 145 Strobach, D. R. 85 (169) 49 Stromberg, K. 298 (8) 180,192 Str0mme, J. 133 (12) 130 Struck, D. K. 45, 23 Stryer,L. 270 (54) 261,265 Sturgess, J. 229 (20) 110; 220 (28) 112 Sturgeon, P. 324 (17) 312 Subba,R.K. 84 (136) 73 Sugahara, K. 40; 134 (15) 131, (16) 131 Sukeno,T. 202 (3) 86 Sullivan, S.J. 299 (50) 195 Summers, D. F. 202 (17) 91 Summers, P. F. 42, 24, 31 Sundblad, G. 354 (33) 344, 347, 348; 356 (62) 351 Sung, S.J. 371 (33) 361 Susz, J. 247 (1) 135,143, (3) 136 SuttieJ. W. 272 (24) 167 Sung, S.-S.J. 80 (32) 52; 82 (53) 56; 84 (140) 73, (146) 75, (147) 75 Suzuki, A. 82 (21) 51,(22) 51 Suzuki, K. 84 (149) 75, (150) 75; 147 (20) 138
A N D GLYCOLIPIDS
I N DISEASE
PROCESSES
Suzuki, M. 80(14)49 Suzuki, Y. 247 (20) 138 Sveger, T. 232 (3) 123,124,128 Svenberg,T. 356(62) 351 Svennerholm, L. 80 (35) 52 Svensson, S. 29 (7) 6; 42; 43; 82 ( 49 ) 56, (50) 56, (51) 56, (52) 56; 203 (28) 98, (29) 98; 248 (46) 144, (47) 144; 354 (31) 344, (33) 344, 347, 348; 403, 390; 422 (11) 408 Sweeley, C . C .
47; 79 (6) 48, 49, (7) 48;
80 (8) 48, (18) 49, (30) 52, (32) 52; 82 (53) 56; 82 (85) 61; 83 (107), (108) 71; 84 (140) 73, (145) 75, (146) 75, 77, (147) 75; 85 (161) 77, 79; 372 (33) 361 Swerdlow, A. 220 (32) 113 Switzer, M.E. 272 (13) 165 Sy,D 43,26 Szigetti, M.
254 ( 25 ) 243 T
Tabas, I. 45, 24, 26, 31; 202 (13) 87 Taber, R. 298 (31) 187 Tager, J. M. 84(148) 75 Tai, T. 45, 33; 202 (20) 95; 103 (26) 98 Takashi,N. 203 (27) 98 Taket,K. 340 ( 36 ) 329 Taketomi, T. 80 (10) 48; 372 (34) 361 Tanaka,H. 84 (150) 75 Tarentino, A. L .
86; 202 (1) 86, 95, (2)
86, 88, 93, 98, (3) 86, (4) 86, (5) 86, 97; (15) 87, 88, 89, 98, (23) 95, 100; 203 (33) 98, 101, (34) 101; 247 (11) 137, (12) 137 Taswell,H.F. 324(17) 312 Tatarinov, Y. 339 (2) 326 Taylor, L. 232 (11) 125 Tchipysheva, T. A. 341 (71) 332 Teasdale, C. 292 (17) 277 Teebor,G.W. 342 (64) 332 Tegtmeyer, H. 324 (13) 312, 314; 325 (37) 312, 322 Temmink, J. H. M. 430 ( 9 ) 412; 431 (40) 429 Terao, T. 270 ( 48 ) 260, 263, 264, 266 Terry, W. D. 339 (14) 327; 353 (15 ) 343; 355 (43) 348,(44) 48 Testas,M. 83 (101) 69 Thatcher, J. 292(17) 277 Thieffry,S. 247 (19) 138 Thistlewaithe, W. 84 (125) 67 Thomas, D. B. 269 (22) 258 Thomas, M. A. W. 20 (22) 13; 220 (44) 113 Thomas, M. W. 432 ( 25 ) 414, 420
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
465
INDEX
Thomas, N. R. 226 (56) 224 Thomas, P. 354 (37) 348, (40) 348; 355 (41) 348, (42) 348 Thompson, A. R. 172(9) 164 Thompson, D. M. P. 352 (3) 342 Thompson, W. D. 254 (17) 231, 235-237, 240—242 Tieslau,C. 377 (8) 376 Tingey, A. H. 147 (16) 138, 145, (21) 138; 149 (56) 145 Titani,K. 172 (16) 166 Todaro, G. J. 198 (6) 180, 195; 310 (5) 295 302 Todd, C . W .
342; 352 (4) 342, 346, (5)
342, (7) 343, (8) 343, (9) 343; 353 (12) 343, (15) 343, (19) 343, 344, (23) 343, 346, 348, (24) 344; 354 (25) 344, (27) 344, (32) 344, (35) 346, (39) 348; 355 (43) 348, (44) 48 (46) 348 (47) 348, 350; 356 (57) 350 (59 ) 350,351 Tokura,S. 272 (28) 170 Tom, B. H. 324 (10) 312 Tomasi, T. B., Jr. 340 (41) 330, (42) 330 Tomita, M. 42, 37; 269 (24) 258, 259; 270 (33) 259 Tonegawa,Y. 372 (41) 361 Tonneguzzo, F. 45, 26 Tot, P. D. 370 (9) 358,359 Touster, O. 84 (139) 73; 247 (10) 137 Toyoshima, S. 269 (21) 258 Trayer, I. P. 45, 32 Trefts,P. 340 (43) 330 Trelstad, R. L. 226 (57) 224 Tress, E. 299 (57) 195, (58) 197 Tribulev, G. P. 324 (4) 311 Trier, J. S. 220 (27) 110 Trimble, R. B.
86;
202 (7) 86, 90, 94,
(16) 87, 89, 90, (23) 95, 100; 147 (13) 137 Trowbridge, I. S. 270 ( 45 ) 260, 263 Tsai,W.P. 297 (5) 180,195
Uhlenbruck, G. G. 269 ( 25 ) 258 UhrJ.W. 45,26 UittoJ. 226 (49) 223 Ukena, T. E. 271 (69) 266; 430 ( 7 ) 412, 429 Umenoto, J. 202 (24) 98 Ungkitchanukit, A. 422 (18) 410 Unrau, A. M. 354 (36) 348 Uriel, J. 339 (3) 326 U r r y , D . W . 227; 253 (3) 249-252, (9) 227, (10) 228, 230, (11) 228, 237, 240, (12) 228, 231; 254 (14) 231, 234, (17) 231, 235-237, 240-242, (18 ) 237-239, (19) 243, (20) 243, 244, (21) 243; 255 (40) 252, (41) 248, 252, (42) 249, 252, (44) 252, (45) 252, (46) 229, (47) 247 Ushijima,K. 85 (164) 79 Usui,N 272 (66) 266
Vallette, G. 339 (16 ) 327, 329 Van Beek, W. P. 420 ( 5 ) 404 Van Den Berg, G. 248 (45) 144 van den Bosch, J. F. 431 (40) 429 Van den Eijnden, D. H. 46, 33, 39; 293 (43) 286, 287, (45) 286, 287 VanHoff,F. 248 (50) 144 Van Zaane, D. 299 (41) 190,194 Vanaman, T. C. 40, 31 Vance, D. E. 80 (30) 52 Vance, J. C. 232(5) 123 Vargas, V.I. 46,28 Vehar, G. 272 (15) 165, 166, 171; 173 (36) 171 Velicer, L. F. 83 (107) Venditti, J. M. 293 (34) 279 Via,D.
378; 403, 379
Vicar, G. 370 (26) 358 Vicari,G. 20 (16) 10 Vincendon, G. 270 ( 51) 260, 264 Tsao, D . 295; 320 (1) 295, 306, (16 ) 304, Vitetta,E. 299 (58) 197 Vliegenthart, J. F. G. 45, 30 305, 307, 308 Vogt,P.K. 299 (44) 192 Tsay, D. D. 432 (32) 426 Tsay, G. C. 103 (32) 98; 247 (27) 139, Voight,K.D. 222,201 Volpin, D. 254 (19) 243, (33) 243 140,144; 148 ( 48) 144 Von Den Helm, K. 299 (37) 188 Tsopanakis, A. D. 46, 32 VonFigura,K. 249 (62) 146 Tsukamoto-Adey, A. 297 (3) 180,181 von Kleist, S. 355 (48) 350, 351 Tung, J. S. 299 (58) 197 Turberville, C. 310 (12 ) 302; 355 (50) Voynow,J.A. 422 (12 ) 408 350, 351, (51) 350, 351; 370 ( 23 ) 359 Vuento, M. 276 (3) 275; 356 (61) 351; 422 (13) 408 Turco, S.J. 46,24; 202 (12) 87 Tzingilev, D. 339 (30) 328 W
U
Uemura, K. 371 (44) 364 Ueno,K. 80 (28) 52
Waechter, C. J. 46,21,22, 23 Wagh, P. V . 201; 222, 201-210 Walborg, E. F. 276 (6) 276
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
466
GLYCOPROTEINS
A N D GLYCOLIPIDS I N DISEASE PROCESSES
Walborg, E . F., Jr.
5; 412; 430 (11) 412Wetmore, S. 43, 31, 34 415, 420, 421, (12) 412, 414, 418, 420, Whelan,W. 258 (4) 153 423, (13) 412, (14) 412, 414, 415, 424, White, D. 83(117)67 (19) 413, 414, (21) 414; 431 (22) 414, White, H. J. 222, 201-204, 210 418, 419, (23 ) 414, (25 ) 414, 420, (31)Whitehead, J. 320 (1) 295, 306, (10) 302, 415,429, (32) 426, (35) 423 304, 305 Waldmann, T. A. 339 (27) 328, 332, (29) Whitehead, J. S. 42, 36; 295 328 Whitehead, R. H. 292 (17) 277 Walker, J. M. 354 (40) 348 Wiegandt, H. 82 (46) 53; 84 (143) 75 Walker-Nasir, E. 120 (41) 113 Wiener, F. 294 (48) 288, (49) 288 Wallace, B. H. 370 (8) 358, 359 Wiener, G. 294 (47) 288 Wallace, R. E. 431 (24) 414 Wiesman,U.N. 220 (33) 113 Wallach, D. F. H. 270 (44) 260, 263, 266; Wiesmann,U. 248 (43) 143 431 (41) 426; 444 (15) 433 Wiggins, R.C. 272 (6) 164 Waller, R. K. 370 (14) 358,359, 369 Wigzell, H. 292 (18) 277; 356 (61) 351 Walsh, K. A. 171 (2) 163; 172 (16) 166 Wilchek, M. 372 (47) 364 Waltuck,B. 158 (4) 153 Wile, A. G. 325 (40) 322 Wang,F.F.C. 102(18)91 Williams, A. F. 270 (46) 260, 263 Wang,K. 269 (12) 258 Williams, D. 46, 31 Wang, L. H. 199 (44) 192 Wang, S.-M. 370 (19) 358, 359 369 Wilson, J. R. 21; 42, 28; 46, 31 Wang, S.W. 310(15)304 Wilson, T. 370 (27) 358 Wanner,A. 120 (30) 112 Winzler, R. J. 20 (19) 9; 224 (6) 213, 215; Ward, S.R 247 (21) 138 269 (22) 258; 411 (9)408 Warner, G. 370 ( 20 ) 359; 403, 378 Wirth, D. F. 46, 26; 202 (12) 87 Warner, N. L. 353 (17) 343 Witte, O. N. 297 (3) 180, 181; 298 (12) Warren, C. D. 42, 23 181 Warren, L. 320 (3) 295; 353 (21) 343; Witting, L. A. 248 (39) 143 403, 378; 420 (1) 404; 431 (34) 420; Witz,I.P. 292 (18) 277 444 ( 4 ) 432 : 445 ( 20 ) 434, (21) 437 Wold,F. 259 (25) 157 Wasniowska, K. 325 (33) 320 Wolf, A. 83(106) 71 Watabe,H. 341 (63) 332 Wolf, D. P. 229 (13) 109; 220 (37) 113 Watanabe, A. 340 (36) 329 Wolfe, L. S. 43; 46; 103 (30) 98, (31) 98; Watanabe, I. 148(33) 140; 403,378 247 (25) 139, (26) 139; 248 (29) 140, Watanabe, K. 80 (23) 51; 82 (93) 64; 370 (30) 140 (24) 358, 359, 369; 371 (28) 358, (43) Wolman, S. R. 340 (46) 331, (47) 331 364 Wong,C.G. 80(32) 52 Watkins, W. M. 46,34 Wood, R. E. 229 (1) 108, 112, (23) 110; Watson, K. F. 298 (27) 184, 192, (28) 220 (30) 112 184, 188-190,192 Wood,T.G. 299 (33) 188 Waxdal, M. J. 270 (47) 260, 263, 265 Wrann, M. 276 (3) 275; 353 ( 24 ) 344; Weinberg, R. 299 (39) 188,190 422 (13) 408 Weinstein, I. B. 403, 378 Wrann, W. 356(60) 351 Weinstein, M. J. 272 (14) 165 Wray, V. P. 430 (11) 412-415, 420, 421, Weiser, M. M. 83 (115) 67, (116) 67, 69; (21) 414; 432 (23) 414 84 (128) 69,(129) 69 Wu,E.-S. 272 (78) 268 Weiss, J. B. 29(8)6,(9)6 Wu,H.C. 320 (2) 295 Weiss, L. 293 (32) 279,280 Wu,Y.-C. 202(21) Weissman, I. L. 297 (3) 180, 181; 298 Wusteman, F. S. 254 (38) 246 (12) 181 Wepsic, H. T. 339 (6) 326; 340 (48) 331 Werber, U. 222, 201 Y Wessels, M. 232 (19) 127 Westall,F. C. 42,36 Yahara,I. 272 (75) 268 Westberg, N. A. 226 (54) 224 Yamaguchi, N. 403, 378 Westenbrink, F. 272 ( 63 ) 261, 265 Westwood, J. H. 320 (12) 302; 354 (37) Yamaguchi, Y. 370 (5) 358,359 348, (40) 348; 355 (41) 348, (42) 348; Yamakawa, T. 80 (14) 49, (21) 51, (22) 51, (29) 52 370 (23) 359
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
AUTHOR
INDEX
Yamashina, I. 40; 134 (15) 131, (16) 131; 147 (14) 137; 269 (27) 258, (28) 258; 293 (24) 278 Yamashita, K. 45, 33; 102 (20) 95; 103 (26) 98 Yamauchi, F. 44,33 Yanagimachi, R. 271 (66) 266 Yanjg, H. J. 310 (11) 302; 325 (30) 320, (31) 320,(32) 320,(34) 320 Yates, S.G. 172 (8) 164 Yeung,K.K. 82(84)61 Yin, H. H. 271 (69) 266; 430 (7) 412, 429 Yip, M.C. M. 82 (76) 61,(77) 61 Yogeeswaren, G. 371 (46) 364 Yoshida, A. J32 (17) 127, (19) 127 Yoshida,Y. 341 (67) 332 Yoshiki,T. 198 (16) 181 Yoshinaka, Y. 200 (61) 197 Yosizawa, Z. 44, 33; 370 ( 4 ) 358 359 YoulosJ. 121(50)115 Young, E. 149 ( 58) 146
467 Young, W . W . , Jr. 310 (15) 304; 357; 370 (13) 358, (19) 358, 359, 363, 364,
369; 371 (44) 364,(45) 364 Yu, R. 80 (28) 52, (33) 52; 81 (56) 59 Yu,S.D. 132(13)126 Yu,S.V. 253 (6) 227 Yu,S.Y. 253 (4) 227,243 Yunis,E.J. 132(7)124 Z
Zambotti, V. 148 (31) 140; 149 ( 57) 145 Zamcheck, N. 355 (54) 350 Zanetta, J. P. 270 ( 50 ) 260, 264, (51) 260, 264 Zeman,W. 148 (33) 140 Zentgraf,H. 83 (118) 67 Zilversmit, D. B. 211, 210 Zinn, A. B. 20(15)6 Ziolkowski C H J 371 (35) 361
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
SUBJECT INDEX
Abrus precatorius 262 Acceptor specificity of glycosyltransferases 64 Acetates, partially methylated alditol 392 total mass spectra of 393, 395 N-Acetyl derivative 408 N-Acetylgalactosamine 28 4-sulfatase 14 N-Acetyl-D-galactosamine-type oligo saccharides, biosynthesis of serine (threonine)34 a, /8-N-Acetylgalactosaminidase 405 N-Acetylgalactosaminitol 287 N-Acetylglucosamine 287 a-N-Acetylglucosaminidase 144-146 /?-N-Acetylglucosaminidase 137,405 endo137 N-Acetyllactosamine type 26 Acid hydrolase activities of transformed and nontransformed fibroblasts 407 hydrolysis 52,408 phosphatase 405 sialic 177,279 Acidic components, tracheobronchial glycoprotein in sparsely 116 Adenocarcinoma cells, cell surface glycoproteins of human colonic .. 304 Adenocarcinoma, T A 3 , mammary 278 A F P (alphafetaprotein) 335,336 in abnormal pregnancy, increased amniotic fluid 328 and albumin, properties of rat 327 concentrations in rats, serum 333 during fetal development, some proposed functions of 330 as an indicator of early events in chemical carcinogenesis 326 properties of 326 Agaricus bisporus 262 Agglutinability, effect of incubation time on Con A-induced 419 Agglutination, Con A - and W G A induced 413 Agglutination, effect of papain digestion on Con A - or W G A induced 421 Agglutinin titer score, anti-T 318
Agglutinins with human breast gland membrane-cytoplasm preparations, colon carcinomata and controls, anti-blood group M , N , and precursor Albumin, properties of rat A F P and .. Alkaline borohydride treatment of H fucose-labeled gylcoprotein
313 327
3
Alpha-l-antitrypsin 122 Alphafetaprotein (see A F P ) Amides, fatty acid 78 Amidohydrolase, l-aspartamido-/?-Nacetylglucosamine 145 Amidohydrolase, 4-L-aspartylglycosylamine 137 Amino acid(s) analysis of aminoacyl fucosides 388 compositions of epiglycanin, carbohydrate and 281 molar ratios of carbohydrate and . . . 389 sequence(s) 15,217,287 and domanis of glycophorin 18 at N-terminus of C E A 349 Ammonium sulfate fraction, chromatography of 208 Ancillary data 408 Antigen(s) of cancer cells, secreted glycoproteins and cell-surface 275 carcinoembryonic ( C E A ) , a marker of human colonic cancer 342 by ectoglycoproteins, masking of cell-surface 277 glycolipid 367 from human blood group O , M N red cells, preparation of T 3 human carcinoma-associated precursors of the blood group M N 311 major histocompatibility 278 masking 278 tumor specific 277 Antigenic activities 277 Antisera, absorption of 312 ai-Antitrypsin deficiency 28 Aortae, concentration of component sugars of normal and artherosclerotic 203
469
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
470
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Aortic intima, glycoprotein inhibitor of lipoprotein lipase from 201 Ascites cells, cell surface properties of M A T - B 1 and M A T - C 1 mammary 442 Asn-associated glycopeptides 86 Asn-GlcNAc initiation, subcellular site of 26 oligosaccharides, biosynthesis of .... 25 type oligosaccharides, initiation of .. 25 Asparagine -N-acetyl-D-glucosamine linkage 21 glucosaminyl 98 glycopeptides linked to 12 residue 23 structure of heterosaccharides linked JV-glycosically to 11 1-aspartamido-^-IV-acetylglucosamine and amidohydrolase 145 Aspartylglucosaminuria 129,145 4-L-Aspartylglycosylamine amido hydrolase 13 Atherosclerosis 201
Blood group (continued) M N antigens, human carcinomaassociated precursors of the .. M and N glycoproteins, human . . . M , N , and precursor agglutinins with human breast gland membrane-cytoplasm preparations O, M N red cells, preparation of T antigen from human Borohydride treatment of H fucoselabeled proteins, alkaline Bovine milk enzyme Brain Breast gland membrane-cytoplasm preparations, colon carcinomata and controls, anti-blood group M , N , and precursor agglutinins with human
311 316
313 312
3
401 32 135
313
C
B Bacteria and vaccines Bacterium tularensis Battens disease Biosynthesis of acedic glycosphingolipids of blood-group-related glycosphingolipids and catabolism of glycoproteins of neutral glycosphingolipids of sulfo-glycosphingolipids Blood clotting, basic mechanisms involved in coagulation biochemistry of extrinsic pathway of intrinsic pathway of molecular events in proteins of zymogen activation mechanisms in collection and serologic procedures group activities in colonic mucosa, glycolipid carbohydrate chains in human tumors, status of determinants, accumulation of precursor carbohydrates of .. determinants in human cancer, status of -related glycosphingolipids, biosynthesis of
PROCESSES
311 323 146 62 63 21 62 62 160 160 160 166 163 162 161 160 312 305 357 358 359 63
C. parvum C thymidine CEA composition of proposed genetic evolution for protein chains of sequence of amino acids at Nterminus of structural units of polysaccharide portion of C a C l and the pentapeptide, P M R titration data of Calcium ion binding to the elastin matrix, neutral-site Cancer carcinoembryonic antigen ( C E A ) , a marker of human colonic cells, secreted glycoproteins and cell-surface antigens of glycoprotein-associated A B O ( H ) glycoproteins in colonic of masking by endogenous glycoproteins, relevance to human .. status of blood group determinants in human Carbohydrate(s) and amino acid compositions of epiglycanin and amino acids, molar ratios of .... of blood group determinants, accumulation of precursor chains, O-glycosyl-linked chains in human tumors, status of blood group 1 4
323 72 345 349 349 347
2
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
241 229 342 275 303 288 359 281 389 358 279 357
SUBJECT
Carbohydrate (s) (continued) composition of the membrane fraction of normal and cancerous colonic mucosa 298 content of galactoprotein I 307 content of invertase preparations... 90 diseases affecting the levels of collagen-associated 223 material, N-glycosyl-linked 287 metabolism, genetic disorders of complex 107 moieties of the collagens in health and disease 213 moiety, structure of the 214 relative to mannose, molar ratio of .. 299 residues, terminal 76 Carcinogenesis, alphafetaprotein as an indicator of early events in chemical 326 Carcinogens, hypothesized target chemical toxins and chemica Catabolism, glycoprotein 39,135 Catabolism, glycosphingolipid 71 Cell(s) agglutinability of normal and malignant rat liver 418 aminoacyl fucosides of N R K and MSV-NRK 380 cell-surface glycoproteins of normal and malignant rat liver 412 cell surface properties of M A T - B 1 and M A T - C 1 mammary ascites 442 components, lectin affinity chromatography of nerve 264 disease, I136 electron micrograph of T A 3 - H a ascites 283,284 -free product, gel electrophoresis of the 191 -free synthesis of an unglycosylated precursor 188 hepatoma 413 with H fucose, pulse-labeling of NRK 400 -mediated immune response, measurement of 314 membrane components, lectin affinity purification of lymphoid 262 membranes, sialic acid contents of tumor 436 papain digestion of 414 preparation of T antigen from human blood group O, M N red 312 role for fucosylation in membrane functions of mammalian 407 surface (s) antigens of cancer cells, secreted glycoproteins and 275 3
471
INDEX
Cell (s) (continued) surface(s) (continued) antigens by ectoglycoproteins, masking of electron micrograph of T A 3 - H a ascites epiglycanin on glycoconjugates, synthesis of glycoproteins of human colonic adenocarcinoma cells lectin receptors, dynamics of properties of the 13762 ascites tumor properties of M A T - B 1 and M A T C l mammary ascites cells .... effect of sodium butyrate on the aminoacyl fucosides of M S V N R K cells effect f sodiu butyrat
277 285 289 68 304 266 439 442 381
uniqueness of fucose in mammalian 408 viability 413 Ceramide, synthesis of 59 Ceroid lipofuscinoses, neuronal146 Chitobiosyl, di-N-acetyl 86 Cholesterol 145 Chromatographic comparison of "disaccharide" and "glucosyl fucitol" 397 Chromatographic retention times of permethylated compounds, gasliquid 392 Chromatography affinity 343 of ammonium sulfate fraction 208 gas-liquid 387,404 gel filtration 286 ion exchange 343 of tryptic digests 186,189,193 lectin affinity 258 of components from other cell types 264 of nerve cell components 264 Chromobacterium violaceum 322 Cirrhosis 122 Clostridium perfringens 75 Clotting, basic mechanisms involved in blood 160 Coacervate, electron micrographs of tropoelastin 249 Coacervation blocked a-elastin temperature profiles of 250 polypentapeptide temperature profiles of 251 process of 247
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
472
GLYCOPROTEINS
Coagulation blood biochemistry of extrinsic pathway of intrinsic pathway of molecular events in proteins of zymogen activation mechanis ns in disorders mechanism involving proteolysis in the termination of Collagen(s) -associated carbohydrate, diseases affecting the levels of enzymatic steps in the biosynthetic attachment of hexose to in health and disease, carbohydrate moieties of structures and characteristics interstitial types, occurrence in different Colon carcinomata and controls, antiblood group M , N , and precursor agglutinins with human breast gland membrane-cytoplasm preparations Colon, glycoconjugates alterations in malignant and inflammatory disease of Colonic cancer, carcinoembryonic antigen ( C E A ) marker of human Concanavalin A Core, complex oligosaccharide Core sequence Corynebacterium parvum Covalent linkage of saccharides to peptide chain Cystic fibrosis, mucous glycoproteins in Cytoagglutination lectin-induced assay of Cytoplasm preparations, colon carcinomata and controls, anti-blood groups M , N , and precursor agglutinins with human breast gland membraneCytostructural integrity
160 160 166 163 162 161
PROCESSES
Di-N-acetyl chitobiosyl 86 Difucohexaose, mass spectrum of permethylated 55 Digestion, lysosomal 151 Digests, ion-exchange chromatography of tryptic 186,189,193 Disaccharide component, acid hydrolysis of H-labeled 399 "Disaccharide" and "glucosyl fucitol," chromatographic comparison of .. 397 Disease(s) affecting the levels of collagenassociated carbohydrate 223 Battens 146 carbohydrate moieties of the collagens in health and 213 of the colon, glycoconjugates alterations in malignant and inflammatory 295 3
160 160 170 223 221 213 21 215
313 295 342 136 87 86 323 6 108 412 412 414
313 415
D Dansylated substrates Deficiency, multiple sulfatase 6-Deoxyhexose, internal Detergents on the glycoprotein binding efficiencies, effect of increasing concentrations of
A N D GLYCOLIPIDS I N DISEASE
98 143 394 286 267
glycoprotein storage Gaucher's I-cell infection and degenerative liver lung lysosomal enzyme deficiency Maroteaux-Lamy Niemann-Pick's Sandhoff's Tay-Sachs Disorders, coagulation Dixon plot Dolichol phosphate sugars Dolichol pyrophosphate oligosaccharide Dolichos biflorus
122 143,152 136 177 123 123 135 143 145 138-140 139,140 160 206 22
E Eccrine glands, electrolyte composition of Ectoglycoproteins, endogenous Ectoglycoproteins, masking of cellsurface antigens by 4-Eicosasphingenine Elastin and elastin repeat peptides, proteoglycan interaction with ion titration of the repeat hexapeptide of pentapeptide of a-Elastin calcium elemental maps C D spectra of secondary ion image temperature profiles of coacervation, blocked X-ray spectrum
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
22 262
109 278 277 48 253 238 235 232 248 232 250 232
SUBJECT
473
INDEX
Electrolyte composition of eccrine sweat glands 109 Electron micrograph of polypentapeptide 249 Electron micrographs of tropoelastin coacervate 249 Electrophoresis, gel of the cell-free product 191 of mature viral proteins 182 of native invertase 92 polyacrylamide 405 of surface membranes 406 of R-MuLV specific glycoproteins .. 185 Elongation with the Golgi apparatus .. 29 Emphysema 122 Endo-D 87 Endo-H, composition of compounds tested as substrates for 97 Endo-H, substrate speficity of 99 Endo-L 8 substrate speficity of 9 Endo-y8-N-acetylglucosaminidase-H (endo-H) 86,93,137 Endo-y8-glucosaminidase 136 Endocytosis of lysosomal glycosidases 78 Endoglycosidase, hen oviduct 100 Endoglycosidase treatment 90 Enterobacteriaceae
323
Enzyme(s) bovine milk 32 deficiency diseases, lysosomal 135 degradation 404 into lysosomes, incorporation of exogenous 150 replacement 150 trials in Gaucher's disease 154 Enzymatic steps in the biosynthetic attachment of hexose to collagen 221 Epiglycanin 278, 282,287 carbohydrate and amino acid compositions of 281 on cell surfaces 289 isolation of 286 physicochemical characterization of 287 Escherichia coli
73, 322
Ester groups, sulfate143 Eukaryotic organisms, glycolipids of .. 47 Exocytosis 146 of lysosomal glycosidases 78 Exoglycosidases 71, 74, 404 F
Fatty acid amides Ferritin, polycationic Fetal development, some proposed functions of AFP during Fiber, molecular pathology of vascular elastic
78 282 330 227
Fibrin formation of cross-linked Fibroblasts acid hydrolase activities of transformed and nontransformed ... human Fibronectin Filtration analysis of "disaccharide" material from glycoprotein and from FL4a, gel FL4a gel filtration analysis of "disaccharide" material from Fluid mosaic model of membrane structure Focusing, isoelectric Forssman glycolipid, induction of "incompatible" determinants and
160 169 146 407 408 408
398 391 398 17 343
361
-polyacrylic hydrazide, synthesis of "poly-glycolipid" 365 reactivity of purified ceramide pentasaccharide fractions 368 status of Taiwan tumor cases 365 Fucopeptide profile, Sephadex G-50 383,384 Fucose -containing components, low molecular weight 409 -labeled glycoprotein fraction, alkaline borohydride treatment of H 396 -labeled proteins, alkaline borohydride treatment of H 401 in mammalian cells, uniqueness of .. 408 pulse-labeling of NRK cells with H 400 L-Fucose 404 a-Fucosidase 137,144 a-L-Fucosidase, rat testis 405 Fucosides, aminoacyl amino acid analysis of 388 of MSV-NRK cells, effect of sodium butyrate on 381 of NRK and MSV-NRK cells 380 from rat tissue, purification of 386 Fucosidosis 136,137,144 a-Fucosidosis 98 Fucosylation 404 in membrane functions of mammalian cells, role for 407 role of 410 3
3
3
G
Galactoprotein I carbohydrate content of Galactose -containing unit, structure of
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
307 287 217
474
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE
Galactosidase 405 £-Galactosidase 137,140,146 Galactosyltransferase 31 Ganglioside(s) 138-140,145, 304 composition of murine sarcoma virus-transformed N R K cells, effect of sodium butyrate on .... 382 Gangliosidosis, G M 1 98,136-140,146 Gangliosidosis, G M 2 98,136-138 Gaucher patients, glycolipid accumulation in livers of 156 Gaucher's disease 143,152 enzyme replacement trials in 154 Gene order of the proteins 187 Gene products by tumors, oncodevelopmental 329 Genetic disorders of complex carbohydrate metabolism 107 Genetic evolution for the protein chains of C E A , proposed 34 G l c N A c residue 2 Globopentaglycosylceramide 61 Globotetraglycosylceramide 61 C N M R spectrum of 60 Globotriglycosylceramide 61 Glucocerebrosidase 143 Glucocerebroside 155 0-/?-D-Glucopyranosyl (1 -> 3 ) - 0 - « - L fucopyranosyl-L-threonine, structure of 388 D-Glucosamine 405 Glucosaminyl asparagine 98 Glucose containing unit, structure of .. 217 D-Glucose 405 ^-Glucuronidase 146 Glycoasparagine sequences 131 Glycoconjugates alterations in malignant and inflammatory disease of the colon 295 in expression of transformed phenotype, role of 378 synthesis of cell surface 68 Glycoglycerolipids 47-49 Glycolipid(s) accumulation in livers of Gaucher patients 156 antigens 367 blood group activities in colonic mucosa 305 of eukaryotic organisms 47 extracted from normal and colon mucosa, complement fixing reactivities of 362 induction of "incompatible" determinants and Forssman 361 macro64 membrane-associated 375 methods of analysis of 49 1 3
PROCESSES
Glycolipid (s) (continued) radioimmunoassay of Forssman 366 structure of H 360 structure and metabolism of 47 Glycopeptide(s) 86,135,139,412 accumulation of 38 Asn-associated 86 bonds 7 cell-surface 413 gel filtration of 422 lectin receptor activities of 423,424 gel filtration of 421 linked to asparagine 12 linked to serine or threonine, structures of 8 of the normal and cancerous mucosa, hemagglutination inhibition by the membrane 298 papain-labile 413 3
and domains of 18 Glycoprotein^) 138 -associated A B O ( H ) glycoproteins in colonic cancer 303 associated with the celle matrix, high molecular weight 276 binding efficiencies, effect of increasing concentrations of detergents on 267 biosynthesis 21 of a membrane-bound 27 catabolism 21,39,135 and cell-surface antigens of cancer cells, secreted 275 into cellular membranes, integration of 16 comparison of membrane 433 discharge of H-labeled Ill discharge of S0 -labeled Ill fraction, alkaline borohydride treatment of H fucose-labeled 396 gel electrophoresis of R - M u L V specific 185 gel filtration analysis of "disaccharide" material from 398 human blood group M and N 316 of human colonic adenocarcinoma cells, cell surface 304 using immobilized lectins, isolation and characterization of membrane 260 inhibitor of lipoprotein lipace from aortic intima 201 intracellular viral specific 183 lectin affinity purification of R B C .. 259 during malignant transformations .. 375 membrane 256, 404, 405, 435 3
35
4
3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
SUBJECT
475
INDEX
Glycoprotein (s) (continued) molecules, endogenous 277 mucous 14 abnormal milieu for 112 altered physiochemical properties of 113 in cystic fibrosis 108 of normal and malignant rat liver cells, cell-surface 412 porcine intimal 203 (lipolipin) sutdies on 202 of rat mammary gland, membrane .. 432 of rat mammary tumors, membrane 438 relevance to human cancer of masking by endogenous 288 secretory 26 in sparsely acidic components, tracheobronchial 116 storage disease 122 structure, current concepts o studies on human intimal 20 Glyccsaminoglycans 138 Glycosidase(s) 297 activities in normal and cancerous colonic mucosa 303 endocytosis of lysosomal 78 exocytosis of lysosomal 78 physiological significance of 79 purification of several 73 substrate specificities of 73 Glycosphingolipid(s) 47-49,74 biosynthesis 59 of acidic 62 of blood group-related 63 of neutral 62 of sulfo62 catabolism 71 Glycosyl asparagine derivatives 96 O-Glycosyl-linked carbohydrate chains 279 N-Glycosyl-linked carbohydrate material 287 Glycosylation, factors controlling hydroxylysine formation and subsequent 222 Glycosylation, intercellular 67 Glycosylhydrolases 405 Glycosyltransferase (s) 297 acceptor specificity of 64 activities in human colonic mucosa 299 activities in normal and cancerous colonic mucosa 301 localization 67 regulation of 69 Goblet cells 34 Golgi apparatus 146 elongation within the 29
H H-labeled glycoproteins, discharge of Health and disease, carbohydrate moieties of the collagens in Hemagglutination inhibition by the membrane glycopeptides of the normal and cancerous mucosa Hen oviduct endoglycosidase Heparan-N-S-sulfatase Hepatocytes lectin-induced agglutination of adult mechanically and enzymatically dispersed morphological comparison of rat binding of Con A to cell-surfac morpholog f
3
111 213 286 298 100 144 415 413 417 413 426 428
Hepatoma cells, papain digestion of .. 414 Heterosaccharide (s) information content of 9 linked N-gylcosically to asparagine, structure of 11 linked glycosically to serine or threonine, structure of 6 moieties on peptide, distribution of 13 Hexapeptide 239 of elastin, ion titration of the repeat 238 Hexosaminidase 140 /^-Hexosaminidase 138,146 Hexose to collagen, enzymatic steps in the biosynthetic attachment of 221 to procollagen a chains, biosynthetic attachment of 218 terminal 394 Human carcinoma-associated precursors of the blood group M N antigens 311 colonic adenocarcinoma cells, cell surface glycoproteins of 304 colonic adenocarcinomatous tisues, studies in 296 fibroblasts 408 Hunter 144 cells 143 Hurler 144 Hydrazide, structure of Forssmanpolyacrylic 366 Hydrazide, synthesis of "poly-glycolipid," Forssman polyacrylic 365 Hydrolysis, acid 52 of H-labeled disaccharide component 399 3
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
476
GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE
Hydrolysis of oligosaccharides, rates of Hydroxylsine formation and subsequent glycosylation, factors controlling Hydroxylysines and lysines, sequences around 4-D-Hydroxysphinganine Hyperplasia atypical of liver Hypersecretion, mucous Hypersensitivity, measurement of delayed-type "recair Hypertrophy
88 222 219 48 109 334 336 109 314 109
I a-Iduronidase a-L-Iduronidase Immune response, measuremen cell-mediated Immunogenic Immunoresistant Incubation time on Con A-induced agglutinability, effect of Infected cells, pulse-chase experiment in R - M u L V Infection and degenerative diseases .. Intracellular steps in the biosynthesis of procollagen Intracellular viral specific glycoproteins Invertase electrophoresis of native preparations, carbohydrate content of stability of native and carbohydrate-depleted Ion complexes, water titration of strontium, calcium, and magnesium L-Isomer
144 14 31 279 279 419 186 177 220 183 91 92 90 92 239 408
L Lacto-N-tetraose, mass spectrum of permethylated Lamy disease, MaroteauxLectin(s) affinity chromatography of components from other cell types of nerve cell components purification of lymphoid cell membrane components purification of R B C glycoproteins -binding sites, cryptic cell-surface ..
54 143 412 258 264 264 262 259 425
PROCESSES
Lectin (s) (continued) concanavalin A (Con A ) and wheat germ agglutination ( W G A ) .... 412 -induced agglutination of adult hepatocytes 415 -induced cytoagglutination, assay of 414 isolation and characterization of membrane glycoproteins using immobilized, 260 of oligosaccharide side chains, possible binding region for 267 receptor activity 413 of cell-surface glycopeptides .423,424 receptors, dynamics of cell surface .. 266 Vicia graminea 280 Leukemia 404 virus membrane proteins, biosynthesis of murine 177 Leukodystrophy, Metachromatic 143 Lipofuscinoses, neuronal-ceroid 146 Lipopolysaccharides 321 Lipoprotein lipase from aortic intima, glycoprotein inhibitor of 201 Liver(s) 335 atypical hyperplasia of 336 cells agglutinability of normal and malignant rat 418 cell surface glycoproteins and normal and malignant 412 normal 413 papain digestion of 414 disease 123 of Gaucher patients, glycolipid accumulation 156 injury, hepatotoxic 329 storage material 125 L u n g disease 123 Lysines, sequences around hydroxylysines and 219 Lysosomal accumulation 153 blockage 150 digestion 151 enzyme deficiency diseases 135 uptake 153 Lysosome (s) 137,146 incorporation of exogenous enzymes into 150 M Macrosialoglycopeptides Malignant transformation glycoproteins during Mammalian cells Mannoglycopeptides
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
412 412 375 277 138
SUBJECT
477
INDEX
Mannose 287 molar ratio of carbohydrate relative to 299 /3-Mannose core 404 a-Mannosidase 98,144, 405 Mannosidosis 144 Mannosyl oligosaccharides 98 Mannosyl residues 98 Maroteaux-Lamy disease 143 Masking 364 antigen 278 of cell-surface antigens by ectoglycoproteins 277 by endogenous glycoproteins, relevance to human cancer of .. 288 in the T A 3 tumor, further evidence for 288 Matrix, high molecular weight glycoproteins associated with the cell 276 Matrix, neutral-site calcium io ing to the elastin 22 Meconium ileus 108 Megalosaccharide 10 Membrane alterations 412 -bound glycoprotein, biosynthesis of 27 components, lectin affinity purification of lymphoid cell 262 -cytoplasm preparations, colon carcinomata and controls, antiblood group M , N , and precursor agglutinins with human breast gland 313 flow, hypothesis 68 functions of mammalian cells, role for fucosylation in 407 glycoproteins 256, 404,405 using immobilized lectins, isolation and characterization of 260 hypothetical structure of plasma 257 polyacrylamide gel electrophoresis of surface 406 structure, fluid mosaic model of 17 Metabolism, genetic disorders of complex carbohydrate 107 Metachromatic Leukodystrophy 143 Metastasis 432 Methanolysis 52 iV-Methyl-N-acetyl-3,6-di-0-methyl1,4,5-tri-O-acetygalactosaminitol, mass spectrum of 57 N-Methyl-N-acetyl-4,6-di-0-methyl1,3,5-tri-O-acetylgalactosaminitol, mass spectrum of 57 N-Methyl-N-acetyl-3,4,6-tri-0-methyl1,5-di-O-acetylgalactosaminitol, spectrum of 57 Methylation analysis 349 Mice, pneumonia virus of 282
Mice, strain A 288 Microbes, T-specific substances in .... 321 Micrograph, electron 440,441 of rat hepatocytes 427 of T A 3 - H a ascites cell 283 surface 285 of TA3-St ascites cell 284 Microheterogeneity 128 Microscopy, dark field light 282 Microscopy, electron 282 Morphology, surface 439 Mouse, A . C A 288 Mucins, ovine submaxillary 35 Mucins, porcine submaxillary 35 Mucolipidoses 145 Mucopolysaccharidoses 144 Mucosa, colonic carbohydrate composition of the membrane fraction of 298 glycolipid blood group activities in glycosidase activities in glycosyltransferase activities in human hemagglutination inhibition by the membrane glycopeptides of .... sialytransferases of Mucosa and tumor tissue, perchloricacid extractable fraction of normal Mucous glands glycoproteins abnormal milieu for altered physiochemical properties of hypersecretion Murine sarcoma virus-transformed rat ( M S V - N R K ) cells Multiple sulfatase deficiency
305 303 301 299 298 301 360 34 14 112 113 109 378 143
N Nascent peptide 26 Neuraminidase 73,286 Neuronal-ceroid lipofuscinoses 146 Neutralization theory, neutral site/ charge 228 Niemann-Pick's Disease 145 Nodule, neoplastic 335 5'-Nucleotidase 434 Nucleotides, sugar 177 O Oligomannoside type 26-29 Oligosaccharides 135 accumulation of 38 biosynthesis of serine (threonine )N-acetyl-D-galactosamine type 34
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
478
Oligosaccharides (continued) core, complex 87 dolichol pyrophosphate 22 initiation of 21 Asn-GlcNAc type 25 mannosyl 98 processing 24-28 rates of hydrolysis 88 side chains, possible binding region for lectins of 267 Olive oil 209 Oncogenic transformation 357 Osmium tetroxide 53 Ovine submaxillary mucins 35 Ozonolysis 53
P Papain digestion on con A - or WGA-induced agglutination, effect of of hepatoma cells of liver cells Papain-labile glycopeptides Pathology of vascular elastic fiber, molecular Pentaglycosylceramide Pentapeptide of elastin P M R titration data of C a C l and .... Pentasaccharide fractions, Forssman reactivity of purified ceramide .... Peptide chain, covalent linkage of saccharides to distribution of heterosaccharide moieties on maps of the envelope-related protein nascent proteoglycan interaction with elastin and elastin repeat Perchloric acid extractable fraction of normal mucosa and tumor tissue Perfusion, collagenase Permethylated compounds, gas-liquid chromatographic retention times of Permethylation Phenotype, role of glycoconjugates in expression of transformed Phosphoglycoprotein Phosphohexosyl Phospholipid Physiocochemical properties of mucous glycoproteins, altered .... 2
421 414 414 413 227 58 236 235 241 368 6 13 190 26 253 286 360 43 392 52 378 146 146 145
PROCESSES
Pick's Disease, Niemann145 Pioncytotic 146 Plasma membrane 146, 434 hypothetical structure of a 257 Pneumonia 280 "Poly-glycolipid," Forssman-polyacrylic hydrazide, synthesis of .... 365 Polyhexapeptide, cross-linked 245 Polypentapeptide cross-linked 244 electron micrograph of 249 temperature profiles of coacervation 251 Polysaccharide portion of C E A , structural units of 347 Porcine intimal glycoprotein 203 (lipolipin), studies on 202 pancreatic ribonuclease 14 A F P in abnormal 328 Procollagen a chains, biosynthetic attachment of hexose to 218 Procollagen, intracellular steps in the biosynthesis of 220 Protein(s) alkaline borohydride treatment of H fucose-labeled 401 biosynthesis of murine leukemia virus membrane 180 of blood coagulation 161 -bound oligosaccharides, processing of 24 chains of C E A , proposed genetic evolution for 348 gel electrophoresis of mature viral 182 gene order of 187 interactions, l i p i d 77 model for synthesis and processing of R - M u L V 190,193 model for synthesis and processing of viral envelope 194 peptide maps of envelope-related .. 190 of Rauscher murine leukemia virus, virion 181 viral 187 Proteoglycan interaction with elastin and elastin repeat peptides 253 Proteolysis 160,286 in the termination of coagulation, mechanism involving 170 Proteolytic activity, residual 94 Prothrombin activation 167 Pseudomonas vaccine 323 Pulse-chase experiment in R - M u L V infected cells 186 Pulse-labeling of N R K cells with H fucose 400 3
3
113
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
SUBJECT
479
INDEX
Purification of R B C glycoproteins, lectin affinity Purification of glycosidases
259 73
Rabbit tracheal explants 117 Radioimmunoassay of Forssman glycolipid 366 Rat(s) A F P and albumin, properties of 327 hepatocytes 413 binding of Con A to 426 cell-surface morphology of 428 electron micrograph of 427 liver cells, agglutinability of normal and malignant 418 liver cells, cell-surface glycoproteins of normal and malignan mammary gland, membrane proteins of 432 mammary tumor, tissue 435 membrane glycoproteins 438 ( M S V - N R K ) cells, murine sarcoma virus-transformed 378 serum alphafetaprotein concentrations in 333 testis a-L-fucosidase 405 tissue, purification of aminoacyl fucosides from 386 Rauscher murine leukemia viral proteins, synthesis and process of 193 virus 282 envelope proteins, synthesis of .... 196 virion proteins of 181 "Recall" hypersensitivity, measurement of delayed-type 314 Recall skin reactivity, delayed-type .... 319 Receptor activity 412 assay of Con A and W G A 415 Ribonuclease, porcine pancreatic 14 Ricinus communis 296, 306 R N A , translation of fractionated R - M u L V genomic 190
S. abortus equi 322 S. typhi 320 Saccharides to peptide chain, covalent linkage of 6 Sachs disease, T a y 138-140 Salmonella milwaukee 322 Sandhoff's disease 138-140 Sanfilippo A and B 143-156 Secretory glycoproteins 26
Sephadex G-50 column effluents, analysis of 414 elution profile 413 fucopeptide profile 383, 384 Serine structures of glycopeptides linked to 8 structure of heterosaccharides linked glycosidically to 6 (threonine) -N-acetyl-D-galactosamine-type oligosaccharides, biosynthesis of 34 Sialic acid 177,279,287 contents of tumor cell membranes .. 436 residue 33 Sialoglycopeptide (s) 135,140,146 cell-surface 413 papain-labile 413 Sialtransferases 33 Skin reactivity, delayed-type recall .... 319 S0 -labeled glycoproteins, discharge of HI Sodium butyrate on the aminoacyl fucosides of M S V - N R K cells, effect of 381 Sodium butyrate on ganglioside composition of murine sarcoma virustransformed N R K cells, effect of 382 Spectrum (a) of a-elastin, C D 248 of globotetraglycosylceramide, C NMR 60 IR 230 of N-methyl-N-acetyl-3,6-di-0methyl-l,4,5-tri-0-acetylgalactosaminitol, mass 57 of N-methyl-N-acetyl-4,6-di-0methyl-l,3,5-tri-0-acetylgalactosaminitol, mass 57 of N-methyl-IV-acetyl-3,4,6-tri-0methyl-l,5-di-0-acetylgalactosaminitol 57 of partially methylated alditol acetate, total mass 393, 395 of permethylated difucohexaose, mass 55 of permethylated lacto-N-tetraose, mass 54 Sphinganine 48 4-Sphingenine 48 Sphingomyelin 145 Sphingomyelinase 145 Staphylococcus aureus 364 Storage disorders 154 Strain specificity 290 Streptococcus 361 Streptomyces plicatus 86-88, 93
35
4
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
1 3
480
G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE
Substrate specificities of glycosidases 73 Sugar(s) deoxy 408 dolichol phosphate 22 of normal and atherosclerotic aortae, concentration of component 203 nucleotides 177 Sulfatase deficiency, multiple 143 heparan-N-S144 sulfoiduronate 144 Sulfatase A 143 Sulfatase B 143,144 4-Sulfatase, ^-acetylgalactosamine .... 144 Sulfate-ester groups 143 Sulfatides 143 Sulfoiduronate sulfatase 144 Surface morphology 429 T Tay-Sachs disease 138-140 Temperature profiles of coacervation, blocked-elastin 250 Temperature profiles of coacervation, polypentapeptide 251 Tetrahymena pyriformis 101 Therapy 128 Threonine permethylated synthetic glucosyl fucosyl 391 structures of glycopeptides linked to 8 structure of heterosaccharides linked glycosidically to 6 Thrombin 160 Tissue carcinomatous and control 311 normal and lactating mammary 435 rat mammary tumor 435 studies in human colonic adenocarcinomatous 296 Titer score, anti-T agglutinin 318 Titration data of C a C l and the pentapeptide, PMR 241 data, C M R 242 of the repeat hexapeptide of elastin, ion 238 of strontium, calcium, and magnesium ion complexes, water 239 Torpedo californica 264 Toxins and chemical carcinogens, hypothesized targets for chemical 337 Tracheal explants, rabbit 117 Tracheobronchial secretions 113 Transformation, oncogenic 357 Triantennary 404 2
PROCESSES
Tropoelastin coacervate, electron micrographs of tropoelastin Tumor cases, Forssman status of Taiwan .. cell(s) membranes, sialic acid contents of metastatic surface properties of the 13762 ascites development fractions, nucleotidase activities of mammary gastric growth membrane glycoproteins of rat mammary specific antigens status of blood group carbohydrate further evidence for masking in .. tissue, perchloric-acid extractable fraction of normal mucosa and
249 365 404 436 277 439 331 436 358 331 438 277
288 360
U Unglycosylated precursor, cell-free synthesis of an
188
V Vaccines, bacteria and 311 Vaccines, microbial 321 Vibrio cholerae 75, 312, 320 Vicia graminea 279 lectin 280 Virion proteins of Rauscher murine leukemia virus 181 Virus assembly 195 mammary tumor 282 membrane proteins, biosynthesis of murine leukemia 180 particles 180 Rauscher leukemia 282 transformation 404 -transformed N R K cells, effect of sodium butyrate on ganglioside composition of murine sarcoma 382 -transformed rat ( M S V - N R K ) cells, murine sarcoma 378 virion proteins of Rauscher murine leukemia virus 181 Z
Zymogen activation mechanisms i n blood coagulation
In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.
160