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THE COMPLEMENT FactsBook

Other books in the FactsBook Series: Robin Callard and Andy Gearing The Cytokine FactsBook Steve Watson and Steve Arkinstall The G'Protein Linked Receptor FactsBook Rod Pigott and Christine Power The Adhesion Molecule FactsBook Shirley Ay ad, Ray Boot-Handford, Martin J. Humphries, Karl E. Kadler and C. Adrian Shuttle worth The Extracellular Matrix FactsBook, 2nd edn Grahame Hardie and Steven Hanks The Protein Kinase FactsBook The Protein Kinase FactsBook CD-Rom Edward C. Conley The Ion Channel FactsBook I: Extracellular Ligand-Gated Channels Edward C. Conley The Ion Channel FactsBook II: Intracellular Ligand-Gated Channels Edward C. Conley and William J. Brammar The Ion Channel FactsBook rV: Voltage-gated Channels Kris Vaddi, Margaret Keller and Robert Newton The Chemokine FactsBook Marion E. Reid and Christine Lomas-Francis The Blood Group Antigen FactsBook A. Neil Barclay, Marion H. Brown, S.K. Alex Law, Andrew J. McKnight, Michael G. Tomlinson and P. Anton van der Merwe The Leucocyte Antigen FactsBook, 2nd edn Robin Hesketh The Oncogene and Tumour Suppressor Gene FactsBook, 2nd edn Jeffrey K. Griffith and Clare E. Sansom The Transporter FactsBook Tak W. Mak, Josef Penninger, John Rader, Janet Rossant and Mary Saunders The Gene Knockout FactsBook Steven G.E. Marsh, Peter Parham and Linda D. Barber The HLA FactsBook

THE COMPLEMENT FactsBook Bernard J. Morley Mark J. Walport Imperial College School of Medicine Hammersmith Campus, London, UK

ACADEMIC PRESS A Harcourt Science and Technology Company

San Diego San Francisco New York Boston London Sydney Tokyo

This book is printed on acid-free paper. Copyright © 2000 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press A division of Harcourt Science and Technology Company 24-28 Oval Road, London NWl 7DX, UK http://www.hbuk.co.uk/ap/ Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.apnet.com ISBN 0-12-733360-6 Library of Congress Catalog Card Number: 99-65744 A catalogue for this book is available from the British Library

Typeset by Mackreth Media Services, Hemel Hempstead, UK Printed in Great Britain by Redwood Books, Trowbridge, Wiltshire 00 01 02 03 04 RB 9 8 7 6 5 4 3 2 1

Contents Abbreviations

Vll

Preface

Vlll

Section I THE INTRODUCTORY CHAPTERS Chapter 1 Introduction Bernard J. Morley and Mark J. Walport Chapter 2 The Complement System Bernard J. Morley and Mark J. Walport

Section II THE COMPLEMENT PROTEINS Part 1 C l q and the Collectins Clq Franz Petry and Michael Loos Mannose-binding lectin Peter Lawson and K.B.M. Reid Bovine conglutinin Peter Lawson and K.B.M. Reid SP-A Robert B. Sim SP-D Robert B. Sim Part 2 Serine Proteases Clr Nicole Thielens and Gerard J. Arlaud Cls Nicole Thielens and Gerard J. Arlaud MASP-1 Teizo Fnjita, Yuichi Endo and Misao Matsushita MASP-2 Steen V. Petersen and Jens C. Jensenius

16 31 36 41 46

52

56

61

65

Factor D 69 Jurg Schifferli and Sylvie Miot C2 73 Yuanyuan Xu and John E. Volanakis Factor B 78 Antonella Circolo and Harvey R. Cohen Factor I 83 Bernard f. Morley Part 3 C3 Family C3 Marina Botto C4 David E. Isenman C5 Rick A. Wetsel Part 4 Terminal Pathway Components C6 Michael Hobart C7 Michael Hobart

88 95 104

112 117

Contents

C8 Francesco Tedesco, Mnason E. Plumb and fames M. Sodetz C9 B. Paul Morgan

123 131

Part 5 Regulators of Complement Activation (RCA) CRl 136 Lloyd B. Klickstein and Joann M. Moulds CR2 146 Joel M. Guthridge and V. Michael Holers Decay-accelerating factor 152 L Kuttner-Kondo, W.G. Brodbeck and M.E. Medof Membrane cofactor protein 156 M. Kathryn Liszewski and John P. Atkinson C4b-binding protein 161 Santiago Rodriguez de Cordoba, Olga Criado Garcia and Pilar Sanchez-Corral Factor H 168 Richard G. DiScipio

Part 6 Cell Surface Receptors ClqRp Andrea J. Tenner C3a receptor Robert S. Ames C5a receptor Andreas Klos and Wilfried Bautsch CR3 Yu Xia and Gordon D. Ross CR4 Alex Law

176 180 184 188 198

Part 7 Miscellaneous Complement Components CI inhibitor 206 Rana Zahedi and Alvin E. Davis III Apolipoprotein J (clusterin) 210 Mark E. Rosenberg Properdin 215 Timothy Parries CD59 219 B. Paul Morgan 223 Index

Abbreviations ClINH C4BP CRD CRP DAF EBV EGF FGF fMLP GPI HIV IFNy Ig IL-1 LAD LPS MAC MBL MCP MHC MIDAS M,(K) NK PDGF PMA PMN PTK RaRF RFLP SAP SDS-PAGE SLE TGF^ TNFa VNTR VWF

CI inhibitor C4b-binding protein carbohydrate-recognition domain C-reactive protein decay-accelerating factor Epstein-Barr virus epidermal growth factor fibroblast growth factor formyl-methionyl-leucyl-phenylalanine glycosylphosphatidylinositol human immunodeficiency virus interferon y immunoglobulin interleukin 1 leukocyte adhesion deficiency lipopolysaccharide membrane attack complex mannose-binding lectin membrane cofactor protein major histocompatibility complex metal ion-dependant adhesion site relative molecular mass natural killer platelet-derived growth factor phorbol myristate acetate polymorphonuclear leukocyte protein tyrosine kinase Ra-reactive factor restriction fragment length polymorphism serum amyloid protein polyacrylamide gel electrophoresis in sodium dodecyl sulfate systemic lupus erythematosus transforming growth factor ^ tumour necrosis factor a variable number tandem repeat von Willebrand factor

Preface The authors wish to thank all those who contributed entries for this volume and for their comments and suggestions. In addition, we are indebted to a number of contributors for additional information they provided. Dr Robert Sim for Figure 2 in Chapter 2, Dr David Isenman for the C3 and C4 catabolism diagrams and Dr Robert Ames for the C3a and C5a receptor diagrams. We would also like to thank Dr James Sodetz for advice on nomenclature, and Dr Alex Law for providing much of the information used in the CR3 chapter on deficiency and polymorphism, including unpublished data. We would like to thank Dr Robert Sim for critical reading of the introduction and Jane Rose for prolific proofreading. Finally, we would like to thank Dr Lilian Leung for her encouragement in the final stages of the preparation of this book. The field of complement is rapidly changing with the constant addition of new data. In light of this, we would be grateful if readers could point out any errors, omissions or indeed new information which could then be incorporated into future editions of this book. Please send these to the Editor, The Complement FactsBook, Academic Press, 24-28 Oval Road, London NWl 7DX, UK.

Bernard J. Morley

Mark f. Walport

Section I

THE INTRODUCTORY CHAPTERS

This Page Intentionally Left Blank

1 Introduction AIMS AND SCOPE OF THE BOOK The aim of this book is to present concise biochemical information about the proteins of the complement system. A novel aspect of this book compared with others in the FactsBook series is the inclusion of cDNA structure and intron-exon boundary details. This enables the design of primers for DNA amplification by the polymerase chain reaction, facilitating both functional mutation studies and the design of probes for expression work. The focus of the book is on the human system, though accession numbers have been included for other species. In the case of conglutinin, where no human homologue has been identified, the bovine molecule has been described. The complement proteins are largely built up from protein modules and it is therefore quite easy to divide them into families of structurally related molecules. This is the basis for the separate chapters. A few proteins escape such simple classification (CI inhibitor, apolipoprotein } (clusterin), properdin and CD59) and these have been grouped together in a separate chapter.

ORGANIZATION OF THE DATA Entries are classified into the following sections, each of which is briefly described.

Other names Entries are identified by the accepted nomenclature for the complement system as described^'^. More recently characterized components are entered under their most commonly used name. Historically, many of the complement proteins have been known by alternative names, or were identified as members of other protein families. Hence different researchers may know them by different designations. All of these alternatives have been included.

Physicochemical properties This section includes data on the number of amino acids in the mature protein and leader peptide (if present); the pi; the molecular weight, both observed under reduced and non-reduced conditions, and predicted based on amino acid composition; the number and location of putative N-glycosylation sites, and if known, whether the sites are occupied; and the number and location of interchain disulfide bonds. Intrachain disulfide bonds are not listed, nor are O-linked glycosylation sites, though the latter are mentioned in the structure section.

Structure Details of the three-dimensional structure where known are included in this section together with any other significant features.

Function The mechanism of activation of the molecule is detailed in this section, together with a brief description of its role in the complement pathway. Other functional activity, outside the complement pathway is also mentioned. The modular structure of each protein is illustrated and the functional importance of each

Introduction

Table 1. Key to the schematic diagrams. All diagrams show modules to scale, with the key illustrating average sizes. SYMBOL

PROTEIN MODULE

ABBREVIATION

Complement control protein repeat

CCP

Serine protease domain

—

Factor I/membrane attack complex C6/7 module

FIMAC

0

Epidermal growth factor-like repeat

EGF

I

Calcium-binding epidermal growth factor-like repeat

Ca^+ EGF

iiiiiiiiiiiiiiiii

Von Willebrand factor type A

VWFA

Thrombospondin type 1 repeat

TSPl

0

Low density lipoprotein receptor class A repeat

LDLRA

mm

CUB domain (first identified in Clr/Cls, uEGF, bone morphogenic protein) Membrane attack complex proteins/perforin-like segment

CUB

Collagen-like domain

—

Carbohydrate-recognition domain

CRD

Alpha-helical coiled-coil "neck'' region

—

•

MACPF

Serine, threonine, proline-rich mucin-like domain STP

I

Cytoplasmic domain

—

Transmembrane domain ( Q for C3aR and C5aR) — Glycosylphosphatidylinositol anchor

GPI anchor

Other domains (see individual sections) Scale: 200 amino acids module noted. A key for the common protein modules is provided in Table 1, together with their full names and the abbreviations^ used throughout the text. Modules which are only present in a single protein in this book, are indicated by a white box and the nature of that module is indicated in the protein modules

Introduction

section of the particular entry. For non-modular proteins such as the C3a and C5a receptors, a diagram has been included only if this helps to illustrate important structural features. In the case of C3 and C4, a diagram has been included to show the degradation pathways of these proteins since this is pertinent to their function.

Tissue distribution For the secreted proteins, the typical serum concentration is provided and other biological fluids known to contain the protein are indicated. The primary site of synthesis is given, together with secondary sites. These are not meant to be exhaustive lists of cells expressing a given protein. In many cases, C3 for example, a large number of cell types have been assayed for expression. However, the absence of a cell or tissue from the list should not be taken as evidence that there is no expression from that cell type. For cell surface proteins, cell types which have been clearly demonstrated to express the molecule are listed.

Regulation of expression Stimuli which alter protein expression are described. Mechanisms, if known, are detailed.

Protein sequence The sequence is shown in the single letter amino acid code. Numbering starts with the initiator methionine residue. The leader sequence is underlined, as are cleavage sites between chains and any special features of specific molecules, for example the residues which form the thioester bond in C3/C4 and the transmembrane domains of the C3a and C5a receptors. Putative and known N-linked glycosylated sites are indicated by N. Sites known not to be occupied are not indicated.

Protein modules For the protein modules listed in Table 1, the leader sequence and some important binding regions, the amino acid boundaries and exons are indicated. For C3 and C4, the thioester domain is indicated, while for the serine proteases, the position of the catalytic triad of the active site (Fi-D-S) is listed.

Chromosomal location The chromosomal location of the gene in both human and mouse, where known, is given. Closely linked genes are also indicated.

cDNA sequence The cDNA sequence is given. Where known, the sequence starts with the 5' end of the message. Otherwise, the most 5' sequence is given. All possible exons are included in the sequence. Where alternative splicing removes an exon from the mature message, this is noted. The initiation codon, termination codon and the putative polyadenylation signals are all indicated. In addition, exon-intron boundaries are shown by underlining the first five nucleotides in each exon. No

Introduction

intronic sequences are included. Where there are discrepancies in published sequences, these are indicated.

Genomic structure where the structure of the human gene is known (with the exception of conglutinin, for which the bovine gene structure is given), this is drawn to scale. The gene is represented by a single horizontal line while the exons are indicated by vertical bars, also to scale. Only the first and last exons are numbered, together with a central exon for the larger genes.

Accession numbers Only the GenBank/EMBL accession numbers are included. These are listed as cDNA or genomic depending on the sequences they contain.

Deficiency The mode of inheritance of deficiency in humans is stated together with the functional effects of deficiency and any clinical correlates. The molecular basis is stated, for example in factor I: A1282 to T, H418 to L; three chromosomes/patients/families where A is the normal nucleotide 1282 is the position in the presented cDNA sequence T is the mutant nucleotide H is the normal amino acid 418 is the position in the presented protein sequence L is the mutant, non- or aberrantly functional amino acid and 'three chromosomes/patients/families' represents the number of times this mutation has been described.

Polymorphic variants Polymorphic variants at the protein level, at the level of restriction fragment length polymorphisms (RFLPs) or where the molecular basis is fully described are listed. Alleles are named A/B where A is the nucleotide/amino acid to the left of the numbering.

References A fully comprehensive list of references is not compatible with the format. However, each entry includes the major references, while key references are highlighted in bold. These represent either important work in the field or key reviews which will link to further references.

References ^ World Health Organization. (1968) Bull. WHO 39, 935-938. 2 lUIS-WHO Nomenclature Committee (1981) J. Immunol. 127, 1261-1262. ^ Bork, P. and Bairoch, A. (1995) Trends Biochem. Sci. 20, Suppl. March C03.

2 The Complement System HISTORICAL PERSPECTIVE In the late nineteenth century, much scientific interest was focused on the mechanisms involved in protecting the body from attack by microorganisms. Two apparently contradictory theories of bacteriolysis emerged during this time. The first, the ''cellular theory'', stemmed from the work of Elie Metchnikoff who demonstrated the existence of blood cells which could ingest invading bacteria. The second, the "humoral theory" of bacteriolysis, was based on work from Fodor, Nuttall and Buchner who identified a heat-labile component of fresh, cell-free serum which was capable of bacteriolysis^. Buchner termed this activity "alexin", from the Greek "without a name". In 1894, Pfeiffer observed that cholera vibrios injected into the peritoneum of immune guinea pigs were lysed^. Towards the end of the nineteenth century, Bordet working at the Pasteur Institute, extended this work by demonstrating that serum from immune animals lost its lytic activity after heating but that activity could be fully restored by the addition of non-immune serum. Bordet surmised that two factors were involved, one of which was heat-labile and the other was a stable substance present in immune serum^. The former he assumed was alexin while the latter he termed the "sensitizer". Meanwhile, Ehrlich and Morgenroth, examining erythrocyte haemolysis by immune serum, confirmed the idea that two "principles" were required for lysis. The first principle, which was present in a thermostable form in immune serum, they termed "amboreceptors" or "immune bodies". The second, a heat-labile substance present in the "body juices", they called "complement" due to the fact that it "complemented" the activity of the amboreceptors. However, it was Bordet and Gengou who described the first complement fixation test, thereby establishing the quantitative role played by complement in cell lysis and dispelling the idea that it was merely an accessory factor as implied by Ehrlich's name. For this reason, Bordet is generally credited with the discovery of the complement system. In the absence of robust biochemical techniques, elucidation of the proteinaceous nature of complement and of the multiple components proceeded fairly slowly over the next 40 years. However, by the late 1920s due to the work of Ferrata initially, and Coca and Gordon subsequently, four individual components were recognized. By 1941, Pillemer and co-workers had confirmed the proteinaceous nature of complement^. During the 1960s, Nelson characterized at least six components from guinea pig serum that were necessary for haemolytic activity^, while MiillerEberhard and colleagues focused on the purification and characterization of each of these components^. Also in the 1960s, Ueno and later Mayer used a reconstitution assay, adding partially purified components to antibody-sensitized sheep red blood cells, to unravel the reaction sequence of the classical pathway. The identification of the alternative pathway involved many of the same investigators in another complex challenge. Pillemer described the depletion of C3 from serum by zymosan in the absence of any effect on CI, C2 and C4 levels in 1953. He also identified properdin as an activating factor in what he termed the properdin pathway^. Nelson offered an alternative explanation for these data in 1958*. He proposed that the properdin system was actually the classical pathway, but activated via antibodies to zymosan. In 1971, Miiller-Eberhard purified C3 proactivator and proposed the C3 activator system as an alternative method of complement activation^, thus supporting Pillemer's original hypothesis.

The Complement System

MODULAR STRUCTURE OF COMPONENTS The cloning and sequencing of the complement components in the last 20 years has augmented the extensive protein sequence already in existence and enabled protein structures to be identified. This has revealed the modular nature of the complement proteins and allowed their classification into five functional groups based on common structural motifs.

Clq and the coUectins (Figure 1) SP-D

I_J4^^^

SP-A C1q chains Conglutinin MBL Figure 1. Modular structure of Clq and the coUectins. See Table 1 for key. Additional domains are the globular region for Clq fCI^J; ^^<^ for conglutinin and MBL, the N-terminal cysteine-rich region f[]j.

The structure of Clq is unusual and was originally described^^, supported by electron micrographs, as resembling a ''bunch of tulips''. Clq has 18 subunit chains formed into collagen-like triple helices with globular heads, through which Clq interacts with immunoglobulin. The serum lectin molecules, MBL and conglutinin, together with the lung surfactant proteins, SP-A and SP-D, share a marked similarity to Clq. They also have 12-18 polypeptide chains organized into the collagen-like domain with a globular C-terminus; however, in contrast to Clq, the globular domains of the coUectins bind a range of sugar moieties (Figure 2).

Serine proteases (Figure 3) The enzymes of the complement system are serine proteases of the chymotrypsin family and trypsin subfamily. The distinguishing feature of this group is the serine protease domain containing the catalytic triad of histidine, aspartic acid and serine^^ The remaining domains of the enzymes such as the CCPs of C2 and factor B, are probably involved in binding and substrate specificity.

SHRS (SP-A a3)

2x a2 + 4x a3

SH6R (SP-A a2 and a3)

S H20126

SH

a2 ril u3

a3 a?a2

Dimer

Hexamer

Figure 2. Chain association i n the collectins: the proposed disulfide pattern, oligomerization, and overall morphology o f S P - A . The disulfide pattern is based on the presence of both the 02 and a3 chains i n each S P - A molecule, which allows the optimum pairing of all the cysteine residues involved (based on reference 27).

The Complement System

FD

C1r

C1s

MASP-1

Hi

MASP-2

FB

C2 Fl Figure 3. Modular structure of the serine proteases. See Table 1 for key. Additional domain is the scavenger receptor cysteine-rich or CDS domain of FI (\ \).

a chain

C3

P chain P chain a chain

C4

y chain

C5

°n^^^^

Key: Disulphide bonds E3 ll^iiiiiiiii^iill

family.

a chain p chain

1 A Thioester site

igure 4. The C3

•••

j_

C3a/C4a/ C5a C3d/C4d

400 aa

1

The Complement System

C3 family (Figure 4) The C3 family of proteins, C3, C4 and C5 together with the non-complement protein a2"i^^croglobulin, are thought to have evolved from a common ancestral gene. An approximate pairwise identity of 25% has been identified between the molecules and a 75% identity between the same protein from different species^^. They are synthesized as single chains, but post-translational processing involving proteolytic modification, glycosylation and sulfation results in the disulfide-linked multichain structure. The most important feature of these molecules, though lacking in C5, is the internal thioester^^. This is formed in the a chain between the cysteine and glutamic acid of the tetrapeptide ''cysteine-glycine-glutamic acidglutamine'' and allows the activated C3b* and C4b* molecules to bind covalently to other molecules (Figure 5). Glycine H COO-

../™xCO NH CO -

NH

^

. \

\

CO

/ /

/

,CH2

CHc

NH

CH Glutamic acid

Cysteine CH

I

I

CHp

CO

SH

NH

I ^

I

CH

/

Glutamine

\

0 = C ' - C H 2 -CH ;2 NHo

CO - - NH

Glycine H

/

NH

/ CO -

NH

^

. \

Cysteine CH CH

Thioester bond

CH

COO"

\

/ CH2 CH2

NH

CO

/

CH Glutamic acid

I

CO

2 \

I

NH

\

CH C -CH2 -CH 2

Glutamine CO

NH

Figure 5. Formation of the highly reactive thioester bond in C3 and C4. The thioester bond is in bold.

The Complement System

Terminal pathway (Figure 6) The terminal components, from C6 to C9, are composite molecules, built on a framework of four distinct modules,- a thrombospondin type 1 repeat, a lowdensity lipoprotein receptor class A repeat, a perforin-like segment and an epidermal growth factor-like repeat. The most crucial of these is the perforin or lytic module. Perforin is the molecule released from intracellular granules by cytotoxic T cells. It requires calcium ions alone to generate cylindrical structures penetrating cell membranes which have a marked similarity to the membrane attack complex (MAC)^^. Although C9 in vivo requires C6-8 to perform the same function, under certain conditions it can polymerize to form similar cylindrical structures. The function of the remaining modules in the proteins remains unclear.

C6

C7

K. \ K • \ j'

A

y

fe>^^<^^
C8a C8p C9

10

Figure 6. Modular structure of terminal pathway components. See Table 1 for key to modules.

Regulators of complement activation (RCA) (Figure 7) Proteins in this group share varying numbers of the repeating motif termed the complement control protein (CCP) repeat, and also known as the short consensus repeat (SCR), first identified in factor B^^. These globular units contain approximately 60 amino acids, built around a backbone of four disulfide-bonded cysteine residues; first to third and second to fourth. A framework of residues are conserved around these cysteines. The number of CCPs varies from two in C2 and factor B to 30 in CRl and they are thought to play a role in binding to C3, C4 and their breakdown products. The genes encoding these regulatory molecules are closely linked on chromosome lq32 in humans and are probably derived from a single ancestral gene.

The Complement System

CR1 CR2

DAF

MCP C4BPa C4BP(3

—mj

FH Figure 7. Regulators of complement activation. See Table 1 for key. Additional domain is the C-terminal oligomerization domain for C4BP ([Z\). CRl is represented as four long homologous repeats (bracketed) of seven CCP domains each, while CR2 has four repeats containing four CCP modules each. In CR2 the alternatively spliced eleventh CCP domain is represented as (9)±.

PATHWAYS The complement system is composed of four pathways (reviewed in ref. 16). Three of these (the activation pathways) are involved in the generation of C3 and C5 convertases which feed into the fourth, terminal or lytic, pathway (Figure 8). Different activators initiate each of the pathways. Thus, the classical pathway, which links innate and adaptive immunity, is activated by the binding to Clq of antibodies complexed with antigens. It is also activated in an antibody-independent manner by the binding of C-reactive protein, complexed with ligand, and by many pathogens including gram-negative bacteria. The alternative pathway is antibodyindependent. It is activated by the direct covalent binding of C3 to pathogens and ''altered self'. The lectin pathway, though structurally very similar to the classical pathway and utilizing many of the same components, is activated by the binding to MBL of the sugar mannose, present on the surface of many pathogens. Each pathway is highly regulated to avoid inappropriate activation and consumption of the component molecules. This is particularly true for the alternative pathway, where the C3 convertase incorporates activated C3 into the enzyme complex, resulting in an amplification loop. Without precise control, alternative pathway components would be rapidly utilized. Control is provided by both serum and cell surface molecules.

The Complement System

Classical Activation Pathway

Mannose-binding lectin Activation Pathway

Alternative Activation Pathway

C1q

MBL

C3 + H2O

CIrg-CISg

MASP-1,2

C3(H20)

D \AA/^^- C3(l-^)B

C4

C3(H20)Bb C4b

+ cis \ A A / ^ C2 -^AAA; MASP I C3 Convertase

C4b2a

C5 Convertase

C4b2a3b

C3

r"^C3a

C2b

C3bBb

Terminal Pathway

Membrane Attack Complex L_

M-C5b678:poly9

Figure 8. Overview of the activation of the complement system. Open arrows represent activation via changes in conformation while \AA/W represents an enzymatic cleavage step. Overlined components (Cls) are activated enzymes, derived from zymogen precursors.

The Complement System

Classical pathway The CI complex, which initiates activation of the classical pathway, is a multimolecular complex containing one molecule of Clq with two molecules each of Clr and Cls. The binding of the globular domains of Clq to the Fc regions of immunoglobulins, specifically IgG and IgM, initiates a series of conformational and enzymatic changes within the complex^ ^. Initiation is also brought about by a variety of pathogens in the absence of specific antibodies. First, CI inhibitor (Cl-INH) is displaced from the complex, allowing autoactivation of the proenzyme Clr. This in turn cleaves and activates the second Clr molecule. The two molecules of Cls are then rapidly cleaved by Clr to form the active CI serine protease. At this point, ClINH acts as a second stage inhibitor, binding to the Clr and Cls active sites, causing their inactivation and dissociation from the complex. Activated Cls cleaves C4, resulting in the production of the anaphylatoxin C4a and C4b. Exposed within the C4b fragment is a highly labile internal thioester bond (C4b*). Most of this C4b* is hydrolysed in the fluid phase. However, in the presence of an activating surface such as a microorganism or an immune complex, C4b* reacts with hydroxyl or amino groups via its reactive acyl group, resulting in deposition in clusters close to the activating CI complex. Bound C4b acts as an acceptor for binding of C2, which is then cleaved by Cls. The small C2b fragment is released while the C2a, containing a serine protease domain, remains bound to C4b and forms the classical pathway C3 convertase, C4b2a. The convertase then cleaves C3 at a single point in the a chain, releasing the anaphylatoxin C3a and the highly labile C3b*. As with C4b*, cleavage of C3 to C3b* exposes the reactive thioester which is now available to bind covalently to the activating surface. Binding of C3 in the vicinity of the C3 convertase results in the formation of the classical pathway C5 convertase, C4b2a3b. The exposed thioester, as with C4b*, is extremely labile and consequently only C3b in the immediate vicinity of the convertase will escape hydrolysis and inactivation. Considerable amplification occurs during activation, with approximately 240 C3b molecules deposited on the activating cell surface for each Clq molecule^*. It is this amplification which would result in rapid depletion of complement if it were not carefully regulated by a number of cell surface and serum proteins. The role of ClINH has already been discussed. The remainder of the control proteins function either to inhibit association of the convertases or to promote their dissociation and catabolism. Factor I is a member of the serine protease family and inactivates both C3b and C4b in the presence of a cofactor, either CRl and MCP (both membrane-bound) or for C4, C4BP (fluid phase). Factor H, which is a fluidphase cofactor for C3 degradation by factor I in the alternative pathway, may also function in the classical pathway. C4BP, along with CRl and DAF, another membrane-bound protein, accelerate dissociation of the C3 and C5 convertases^^.

Mannose-binding lectin pathway The lectin pathway is highly analogous to the classical pathway and shares C2, C3 and C4 with it. However, initiation of the pathway is antibody-independent with the difference lying in the initiation complex. In the lectin pathway, the CI complex is replaced by a homologous complex, containing MBL and MASP-1 and -2. MBL is activated by binding to mannose-containing proteins or carbohydrates

The Complement System

on bacterial or viral surfaces^^. The MASP-1 and -2 enzymes function in an analogous manner to Clr and Cls and the result is cleavage of C4. The pathway then follows the classical pathway.

Alternative pathway This pathway is also antibody-independent and provides an initial line of innate immune defence. It is activated by a host of diverse substances including zymosan, lipopolysaccharide from gram-negative bacteria, cell wall teichoic acid from grampositive bacteria, whole microorganisms, lymphoblastoid cells and certain mammalian cell surfaces such as rabbit erythrocytes^^. The exact processes involved in alternative pathway activation are not fully defined, however, the most likely explanation remains the ''tickover'' hypothesis^^. This is based on the continuous low-grade hydrolysis of circulating serum C3 to produce a molecule, C3(H20), which is structurally and functionally similar to C3b* (Figure 9). The

C3

3(HP)

Continuous low grade hydrolysis

B

C3(HP)B -^VAAAD

1

C3(H,0)Bb -i- Ba Carboxypeptidase N

y

C3-

C3b* + C3a

C3bBbP DAF--3 CR1—3

Properdin

Ba +C3bBb-^

(J.

B

Fl + cofactor (FH orMCPorCRI depending on local environment)

t -C3bB

Figure 9. The formation and control of the alternative pathway amplification loop. Dashed arrows represent inhibition or dissociation, while v\A/w represents an enzymatic cleavage step. Overlined components (C3hBb) are activated enzymes, derived from zymogen precursors, and C3b* represents C3 with a reactive thioester.

The Complement System

C3(H20) can then bind factor B. Factor B, bound to C3(H20), is a substrate for factor D, which, unlike other complement serine proteases that are present as zymogens in the serum, circulates in its active form. The C3(H20)Bb complex so formed is an unstable fluid-phase C3 convertase and can cleave C3 to yield C3a and the labile C3b* component. If an activating surface is present then the C3b* will bind and initiate the positive feedback loop characteristic of the alternative pathway. The activated C3b* is rapidly hydrolysed and catabolized in the absence of an activating surface. In contrast, C3b bound to an activating surface will bind factor B. The C3bB is a substrate for further cleavage by factor D, resulting in the formation of the C3 convertase C3bBb. The stability of this convertase is enhanced by the binding of properdin. Bb is the serine protease element of this convertase, playing an analogous role that of the homologous C2a fragment in the classical C3 convertase, C4b2a3b. Since C3 is a component of its own convertase, this loop of activation creates considerable C3b binding in the vicinity of the initiator convertase molecule. The addition of a second C3b molecule to the C3 convertase results in the formation of the alternative pathway C5 convertase C3bBb3b. The fate of C3b is critical to the regulation of the amplification loop. Persistence of C3b allows further binding of factor B, leading to the formation of more C3bBb convertase enzyme and hence, amplified C3 cleavage. Catabolism of C3b prevents formation of the convertase enzyme and inhibits amplification of C3 cleavage. The key enzyme that catabolizes C3b is factor I, and this requires one of a number of cofactors to bind to C3b. In plasma, C3b is rapidly catabolized by factor 1 using factor H as a cof actor. On cell surfaces, MCP or CRl are the major intrinsic cell membrane cofactors for factor I. Factor H may also bind as an extrinsic cofactor from plasma. There is competition for binding to C3b between factor B and these cofactors of factor I. If the binding of factor B is favoured over cofactor binding, then C3 convertase formation results, leading to amplified C3 cleavage. If factor H, CRl or MCP binding are favoured, then C3b catabolism by factor I occurs, which downregulates complement activation. Autologous cell surfaces are highly protected from C3 convertase formation by several mechanisms. The first is the presence of intrinsic cofactors for factor I such as MCP and C R l . The second is their sialic acid content, which favours factor H binding to C3b compared with factor B. The third mechanism is the presence of other cell membrane molecules such as DAF, which inhibit C3 convertase assembly and promote convertase dissociation. In contrast, bacteria and other pathogens lack the host cofactors for C3b cleavage by factor I, though some have evolved similar molecules. The carbohydrate composition of many pathogens, poor in sialic acid, also favours the binding of factor B rather than factor H, promoting convertase assembly and C3 activation^^.

Terminal pathway The three distinct activation pathways of complement converge with the formation of a C5 convertase and it is cleavage of C5 by these convertases that initiates the lytic or terminal pathway. This first step releases the powerful anaphylatoxin C5a and leaves the metastable C5b*. Unlike the closely related C3 and C4 molecules, C5 does not contain an internal thioester bond. Despite this, binding of C5b to the surface-bound C3b in the convertase causes the transient exposure of a hydrophobic acceptor site for C62^. In contrast to the activation

The Complement System

pathways, which require enzymatic cleavage for activation, the terminal pathway relies on conformational changes induced by binding. Thus, binding of C6 facilitates binding of C7 which alters the conformation of the complex. First, C5b67 is released from the C5 convertase and secondly, exposure of a hydrophobic region of C7 allows penetration into the lipid bilayer of the cell membrane. Binding of C8 to the C5b67 complex occurs via the P subunit, resulting in a conformational change and insertion of the a chain of C8 into the membrane. A variable number of C9 molecules associate with the C5b678 complex, resulting in the mature membrane attack complex (MACj^^^. During activation of complement, some of the forming C5b67 complexes may deposit on neighbouring cells, which could result in MAC formation and lysis, socalled ''bystander lysis''. However, this phenomenon is prevented by clusterin in the fluid phase and also by CSp, which binds to C5b67 close to the site of membrane attachment, preventing membrane insertion. Autologous cell surfaces are also protected by the important regulatory protein CD59, which inhibits the incorporation of C8 and C9 into the MAC. The formation of the MAC in a cell or bacterial membrane has a number of possible consequences. The first of these is lysis. This results from membrane disruption caused by the formation of MAC-induced ion-permeable channels^^ and/or leaky patches^^. The second is the triggering of a variety of cellular metabolic pathways. Activation of these pathways may result in the synthesis and release of inflammatory mediators such as leukotrienes and prostaglandins. However, injury does not always result from the insertion of MAC into cell membranes. Cells may release fragments containing clustered MACs as part of the defence mechanism against self-injury by autologous complement activation.

FUNCTION Detailed consideration of the physiology and pathology of the complement system is outside the scope of this book. The reader is referred to several recent reviews and books describing the complement system^<^'27-29 Figure 10 summarizes the different physiological activities of complement. Complement is a major component of the innate immune system and the two main pathways of activation of complement, as an innate host defence mechanism, are by the mannose-binding lectin pathway and the alternative pathway. However, the classical pathway is also activated, in the absence of specific antibody, by gram-negative bacteria and a number of viruses. The links between the adaptive immune system and complement are also extremely important and may be divided into two categories. The first is the activity of the complement system as one of the major effector pathways of host defence following engagement of the adaptive immune system. The binding of antigen by the majority of antibody classes causes activation of the classical pathway of complement. This leads to deposition of the complement opsonins, C4b and C3b, on the immune complexes, release of the anaphylatoxic peptides, C3a and C5a, and formation of the membrane attack complex. This pathway enables and enhances the killing of bacteria which have bound specific antibody. The second link between adaptive immunity and complement is the role of complement in enhancing and amplifying adaptive immune responses-^^^^.

The Complement System

antigen / antibody immune complex

microbes witti terminal mannose groups

bacteria, fungi, virus or tumour cells

MBL - MASP1 - MASP2

C3(H20)

< H . ^ f ^ anaphylatoxin ne complex ^ ^ -'^ 8 immune c a C f ^ - ^opsonin [ modification fi lymphocyte activation clearance of apoptotic cells

C5

*-anaphylatoxin

C6 C7 C8

^ - «• lysis poly-CQ-V. ^ ^

I, *•

membrane perturbation Figure 10. The effector mechanisms of the complement system. Dashed arrows indicate the functions of pathway components. Examples in this category include the reduction of B cell thresholds for activation by ligation of C3 receptors on B lymphocytes; the role of C3 receptors on follicular dendritic cells in germinal centres in maintaining immunological memory; and the role of immune complexes triggering complement in the induction phase of contact hypersensitivity reactions^^.

Complement and disease Complement plays an important role in the pathogenesis of many diseases. Complement deficiency, though extremely rare, is illuminating because it illustrates the unique roles of the complement system in the context of the milieu

The Complement System

of the Hving organism^^. Much more common are diseases in which activation of complement plays an important role in tissue injury and also in the resolution of inflammation.

Complement deficiency Much has been learnt about the physiological activities of the complement system by studying patients with spontaneous inherited deficiencies of individual complement proteins^^. The molecular basis of complement deficiency is described in each of the relevant entries. Three categories of disease are associated with inherited deficiencies of complement proteins: infectious diseases, systemic lupus erythematosus (SLE)-like diseases and CI inhibitor deficiency-linked diseases. Infectious diseases illustrate the role of complement in host defence against infection. Three types of infection may be seen. (1) Pyogenic infections associated with C3 deficiency can be due to mutations either in the C3 gene, or in the control proteins, factor I or factor H, deficiency of which cause unregulated C3 consumption. These demonstrate the role of complement as opsonin. (2) Pyogenic infections associated with partial MBL deficiency^^ occur predominantly early in life, during the period between loss of maternally transferred immunity and the development of a mature antibody system. These illustrate the activity of the MBL pathway in vivo. (3) Infections associated with Neisseria occur in patients lacking MAC proteins or proteins of the precursor activation pathways of the MAC. This association demonstrates the role of complement-mediated lysis in host defence against neisserial infection^^. Systemic lupus erythematosus-like diseases, the second category of disease associated with complement deficiency, are linked to deficiencies of the classical pathway proteins Clq, Clr and Cls, C4 and C2. The precise mechanism of this association is not established but recent evidence suggests that it may illustrate the role of complement in the normal process of resolution of inflammation, especially in the scavenging and clearance of immune complexes and apoptotic cells-^''. In diseases associated with CI inhibitor deficiency^*, heterozygous deficiency of a regulatory protein leads to loss of control of the activation of Clr, Cls, plasmin, factor Xlla and kallikrein. Attacks of angioedema follow and are thought to be mediated by kinins, including a kinin derived from C2, and bradykinin. Much has been learnt by the study of those rare and unfortunate humans with spontaneous inherited complement deficiencies. A number of spontaneous models of complement deficiencies have also been characterized in other species, including pigs, rabbits, guinea-pigs, rats and mice. The ability to target specific genes in mice has resulted in the development of a range of animals with individual and multiple complement deficiencies. Investigation of these models is likely to add significantly to our knowledge of the physiological activities of the complement system.

Complement and tissue injury The role of complement in contributing to the pathogenesis of disease may be considered in three categories: systemic disease, local tissue injury and the resolution of inflammatory injury. An important example of the role of complement in causing systemic injury is in bacterial sepsis. Large-scale activation of the complement cascade leads to substantial anaphylatoxin production, which in turn activates neutrophils and other leukocytes, all of which

The Complement System

contribute to severe morbidity. One of the consequences of extracorporeal circulation of blood over artificial surfaces in haemodialysis machines and heart-lung bypass machines is large-scale activation of complement. There has consequently been great effort to develop artificial membranes and surfaces which minimize complement activation. Local complement activation also plays a role in augmenting tissue injury in many diseases. Infarcted tissue activates complement, for example, following myocardial infarction. The potential clinical importance of complement activation in myocardial infarction has been shown in experimental models in which inhibitors of complement activation were found to reduce infarct size-^^. In autoimmunity, complement is activated and deposited at sites of tissue injury in many different diseases, such as rheumatoid arthritis, systemic vasculitis and myasthenia gravis. Evidence that complement contributes to tissue injury has come from experimental models of membranous nephritis, where deficiency of the membrane attack complex reduces the degree of proteinuria. On the other hand, evidence is beginning to emerge that, in some circumstances, complement may be protective against the development of tissue injury, which would fit with the role of complement in promoting the clearance of immune complexes and, possibly, apoptotic cells'^^. There is an emerging body of evidence indicating that Fc receptors may play a dominant role in transducing proinflammatory signals, rather than complement. This underlines a protective role for complement against inflammatory disease''^.

References ' Buchner, H. (1889) Zbl. Bakt. (Naturwiss.) 5, 817; 6, 1. 2 Pfeiffer, R. and Issaeff, R. (1894) Z. Hyg. Infektionskr. 17, 355. ^ Bordet, J. (1909) In Studies in Immunity. J. Wfley &L Sons, New York. ^ Pillemer, L. et al. (1941) J. Exp. Med. 74, 927-934. ^ Nelson, R.A. et al. (1966) Immunochemistry 3, 111. ^ Mufler-Eberhard, H.J. (1975) Annu. Rev. Biochem. 44, 697-724. ^ Pillemer, L. et al. (1954) Science 120, 279-285. « Nelson, R.A. (1968) J. Exp. Med. 108, 515-535. ^ Gotze, O. and Miiller-Eberhard, H.J. (1971) J. Exp. Med. 134, 90s-108s. ^« Reid, K.B.M. and Porter, R.R. (1976) Biochem. J. 155, 19-23. '' Perona, J.J. and Craik, C.S. (1995) Protein Sci. 4, 337-360. ^2 Campbell, R.D. et al. (1988) Annu. Rev. Immunol. 6, 161-195. '' Tack, B.F. et al. (1980) Proc. Natl Acad. Sci. USA 77, 5764-5768. ^^ Liu, C.C. et al. (1995) Immunol. Today 16, 194-201. ^5 Morley, B.J. and Campbell, R.D. (1984) EMBO J. 3, 153-157. ^^ Volanakis, J.E. and Frank, M.M. eds (1998) The Human Complement System in Health and Disease, Marcel Dekker, New York. '' Sim, R.B. and Reid, K.B.M. (1991) Immunol. Today 12, 307-311. ^« Ollert, M.W. et al. (1994) J. Immunol. 153, 2213-2221. ^^ Liszewski, M.K. and Atkinson, J.P. (1998) In The Human Complement System in Health and Disease (Volanakis, J.E. and Frank, M.M. eds). Marcel Dekker, New York, pp. 149-165. 20 Epstein, J. et al. (1996) Curr. Opin. Immunol. 8, 29-35. 2^ Pangburn, M.K. and Miiller-Eberhard, H.J. (1984) Semin. Immunopathol. 7, 163-192.

The Complement System

22 Nicol, A.E. and Lachmann, P.J. (1973) 24, 259-275. 23 Miiller-Eberhard, H.J. (1986) Annu. Rev. Immunol. 4, 503-528. 2^ Mold, C. (1998) In The Human Complement System in Health and Disease (Volanakis, J.E. and Frank, M.M. eds). Marcel Dekker, New York, pp. 309-325. ^^ Bhakdi, S. and Tranum-Jensen, J. (1991) Immunol. Today 12, 318-320. 26 Esser, A.F. (1991) Immunol. Today 12, 316-318. 27 Lu, J. and Sim, R.B. (1994) In New Aspects of Complement Structure and Function (Erdei, A. ed.), R.G. Landes Co., Austin, TX, pp. 85-106. 2* Davies, K.A. et al. (1994) Springer Semin. Immunopathol. 15, 397-416. 29 Janeway, C.A., Travers, P., Walport, M. and Capra J.D. eds (1999) Immunobiology: The Immune System in Health and Disease, 4th edn. Current Biology and Garland Publishing, New York. ^^ Fearon, D.T. and Carter, R.H. (1995) Annu. Rev. Immunol. 13, 127-149. 3^ Fearon, D.T. and Locksley, R.M. (1996) Science 272, 50-53. 32 Carroll, M.C. (1998) Annu. Rev. Immunol. 16, 545-568. 33 Tsuji, R.F. (1997) J. Exp. Med. 186, 1015-1026. 3^ Colten, H.R. and Rosen, F.S. (1992) Annu. Rev. Immunol. 10, 809-834. 35 Turner, M.W. (1996) Immunol. Today 17, 532-540. 36 Figueroa, J. et al. (1993) Immunol. Res. 12, 295-311. 37 Botto, M. et al. (1998) Nature Genet. 19, 56-59. 3« Agostoni, A. and Cicardi, M. (1992) Medicine Baltimore 71, 206-215. 39 Kalli, K.R. et al. (1994) Springer Semin. Immunopathol. 15, 417-431. ^» Ravetch, J.V. and Clynes, R.A. (1998) Annu. Rev. Immunol. 16, 421-432.

Section II

THE COMPLEMENT PROTEINS

This Page Intentionally Left Blank

Parti Clq and the CoUectins

i|

l i s protein Franz Petry and Michael Loos, University of Mainz, Mainz, Germany

Physicochemical properties C l q is made up of three individual polypeptide chains, A, B and C, which are synthesized as pre-molecules with 22, 25 and 28 amino acid leader sequences, respectively.

pP M, (K)

9.3 459.32

M,(K) N-linked glycosylation site Interchain disulfide bonds

A chain 27.5 1 (146) 26

B chain 25.2

C chain 23.8

29

32 (to 32 in second C chain)

Structure C l q has a characteristic '^bunch of tulips'' appearance under the electron microscope, with six globular heads connected by six collagen-like stalks forming a central fibril stem. Glucosylgalactosyl disaccharide units are linked to certain hydroxylysine residues in the collagen regions of all three chains.

Function C l q has a critical function in host defence and clearance of i m m u n e complexes. Antibody-independent activation of the classical pathway via C l q has been demonstrated for certain viruses, gram-negative bacteria, D N A and poly anions. The binding sites for various proteins including endotoxin, CRP, serum amyloid A, D N A and heparin have been mapped to the A chain of C l q . C l q binds to IgG- and IgM-bearing i m m u n e complexes via its globular heads. Other Ig classes do not activate the classical pathway via C l q . Binding of C l q to i m m u n e complexes leads to activation of the CI subcomponent C l r and then C l s . Two molecules of each, C l r and C l s , are assembled with C l q at the collagen-like region to form macromolecular C I . Activation of CI initiates the activation of classical pathway of complement. After activation, the CI complex is dissociated by the CI esterase inhibitor (Cl-INH). Clq-bound i m m u n e complexes bind to C l q receptors on various cell types.

Tissue distribution^'^'^ Primary site of synthesis: macrophages. Secondary sites: follicular dendritic cells, interdigitating cells and further cells of the monocyte-macrophage lineage.

D

Regulation of expression Unknown.

Protein sequences^^ A chain MEGPRGWLVL QGEPGAPGIR GTKGSPGNIK FVCTVPGYYY VSGGMVLQLQ

CVLAISLASM TGIQGLKGDQ DQPRPAFSAI FTFQVLSQWE QGDQVWVEKD

VTEDLCRAPD GEPGPSGNPG RRNPPMGGNV ICLSIVSSSR PKKGHIYQGS

GKKGEAGRPG KVGYPGPSGP VIFDTVITNQ GQVRRSLGFC EADSVFSGFL

RRGRPGLKGE 50 LGARGIPGIK 100 EEPYQNHSGR 150 DTTNKGLFQV 2 00 IFPSA

B chain MKIPWGSIPV PGTPGIKGEK APGAPGPKGE YEPRSGKFTC NTFQVTTGGM A

LMLLLLLGLI GLPGLAGDHG SGDYKATQKI KVPGLYYFTY VLKLEQGENV

DISQAQLSCT EFGEKGDPGI AFSATRTINV HASSRGNLCV FLQATDKNSL

GPPAIPGIPG PGNPGKVGPK PLRRDQTIRF NLMRGRERAQ LGMEGANSIF

IPGTPGPDGQ GPMGPKGGPG DHVITNMNNN KWTFCDYAY SGFLLFPDME

LGLKLLLLLL GIPAIPGIRG EGRYKQKFQS KVPGLYYFVY LQVGEEVWLA

LLALRGQANT PKGQKGEPGL VFTVTRQTHQ HASHTANLCV VNDYYDMVGI

GCYGIPGMPG PGHPGKNGPM PPAPNSLIRF LLYRSGVKW QGSDSVFSGF

LPGAPGKDGY 50 GPPGMPGVPG 100 NAVLTNPQGD 150 TFCGHTSKTN 2 00 LLFPD

C chain MDVGPSSLPH DGLPGPKGEP PMGIPGEPGE YDTSTGKFTC QVNSGGVLLR

50 10 0 150 2 00 250

The leader sequences are underlined and the N-linked glycosylation site in the A chain is indicated (N).

Protein modules A chain 1-22 31-109 110-245

Leader peptide Collagen-like region Globular region

exon 1 exon 1/2 exon 2

Leader peptide Collagen-like region Globular region

exon 1 exon 1/2 exon 2

Leader peptide Collagen-like region Globular region

exon 1 exon 1/2 exon 2

B chain 1-25 31-114 115-251

C chain 1-28 31-114 115-245

Chromosomal location Human«: Ip34-lp36.3. Mouse^: chromosome 4, 66.1 cM.

cDNA sequences ClqA (manual entry from ref. 7) TGTGATGTCC CGGGGATGGC TTGTGCCGAG CCAGGCCTCA GGCCTTAAAG CCAGGGCCCA AGCCCAGGAA CCAATGGGGG CAGAACCACT GTGCTGTCCC CGCTCCCTGG ATGGTGCTTC CACATTTACC GCCTGAGCCA TAAAAAGGGG GGCTGCCCGT

AACCTGCCCA TGGTGCTCTG CACCAGACGG AGGGGGAGCA GAGACCAGGG GCGGCCCCCT ACATCAAGGA GCAACGTGGT CCGGCCGATT AGTGGGAAAT GCTTCTGTGA AGCTGCAGCA AGGGCTCTGA GGGAAGGACC GCGCTATTGC GACACATGCT

GGCCCTCCCG TGTGCTGGCC GAAGAAAGGG AGGGGAGCCC GGAACCTGGG CGGGGCCCGT CCAGCCGAGG CATCTTCGAC CGTCTGCACT CTGCCTGTCC CACCACCAAC GGGTGACCAG GGCCGACAGC CCCTCCCCCA TTCAGCTGCT CTAAGAAGCT

TGTCTCCACA ATATCGCTGG GAGGCAGGAA GGGGCCCCTG CCCTCTGGAA GGCATCCCGG CCAGCCTTCT ACGGTCATCA GTACCCGGCT ATCGTCTCCT AAGGGGCTCT GTCTGGGTTG GTCTTCAGCG CCCACCTCTC GAAGGAGGGC CGTTTCTTAG

GAGGCATCAT CCTCTATGGT GACCTGGCAG GCATCCGGAC ACCCCGGCAA GAATTAAAGG CCGCCATTCG CCAACCAGGA ACTACTACTT CCTCAAGGGG TCCAGGTGGT AAAAAGACCC GCTTCCTCAT TGGCTTCCAT ATGCTCTGAG ACCTCTTCCT

GGAGGGTCCC GACCGAGGAC ACGGGGGCGG AGGCATCCAA GGTGGGCTAC CACCAAGGGC GCGGAACCCC AGAACCGTAC CACCTTCCAG CCAGGTCCGA GTCAGGGGGC CAAAAAGGGT CTTCCCATCT GCTCCGCCTG AGCCCAGACT GGAATAAA

CTGATGTTGC GGGCCCCCAG CCTGGGACCC GAGTTCGGAG GGCCCCATGG TCGGGAGACT CCCCTGCGCC TATGAGCCCC CACGCCAGCT AAGGTGGTCA GTCCTCAAGC CTGGGCATGG GCCTGACCTG ACCCCCAACA AGTGAATGAG CCCAGCACTG

TCCTGCTCCT CCATCCCTGG CAGGGATAAA AGAAGGGAGA GCCCTAAAGG ACAAGGCCAC GGGACCAGAC GCAGTGGCAA CTCGAGGGAA GCTTCTGTGA TGGAGCAGGG AGGGTGCCAA TGGGCTGCTT CCACCCCTTG TAAATAAACT GCACACCAGA

GGGCCTAATC 60 CATCCCGGGT 120 AGGAGAGAAA 180 CCCAGGGATT 240 TGGCCCAGGG 300 CCAGAAAATC 360 CATCCGCTTC 420 GTTCACCTGC 480 CCTGTGCGTG 540 CTATGCCTAC 600 GGAGAACGTC 660 CAGCATCTTT 720 CACATCCACC 780 CCCAGCCAAT 840 CTTCAAGGCC 900 AGTGCCATGC 1020

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

ClqB (manual entry from ref. 6 ATGAAGATCC GATATCTCCC ATCCCTGGGA GGGCTTCCAG CCTGGGAATC GCCCCTGGAG GCCTTCTCTG GACCACGTGA AAGGTGCCCG AACCTCATGC AACACCTTCC TTCCTGCAGG TCCGGGTTCC CCGGCTCCCC GGACACAGTA AAGGAACAGT TCAGAAATGT

CATGGGGCAG AGGCCCAGCT CACCTGGCCC GGCTGGCTGG CAGGAAAAGT CCCCAGGCCC CCACAAGAAC TCACCAACAT GTCTCTACTA GTGGCCGGGA AGGTCACCAC CCACCGACAA TGCTCTTTCC CTGCCAGCAA GGGCTTGGTG GGTCTAATTC TGGTTACATG

CATCCCAGTA CAGCTGCACC CGATGGCCAA AGACCATGGT CGGCCCCAAG CAAAGGTGAA CATCAACGTC GAACAACAAT CTTCACCTAC GCGTGCACAG CGGTGGCATG GAACTCACTA AGATATGGAG CGCTCACTCT AATGCTGCTG AACTCTGTGT AATGAA

ClqC (manual entry from ref. 7) TCTCTCCCTC GGGCTGAAGC TGCTACGGGA GGACTGCCGG AAAGGGCAGA CCCCCTGGGA GGCAGATACA CCTGCACCCA GACACGAGCA GCGTCGCATA TTCTGTGGCC CAGGTGGGCG GGCTCTGACA TCGAGACCCA CTGCATCCTT CACCCCCTCC GGTTCCTGGG AGTATTGGAA

CCAGTTCCTT TGCTGCTGCT TCCCAGGGAT GGCCCAAGGG AGGGAGAACC TGCCAGGGGT AGCAGAAATT ACAGCCTGAT CTGGCAAGTT CAGCCAACCT ACACGTCCAA AGGAGGTGTG GCGTCTTCTC CGGGCCTTCC GCCTAGACCA CCATGGGTTC ACACTTAACC GGGGTGGGGA

CTCCGGGATG TCTGCTGCTG GCCCGGCCTG GGAGCCAGGA CGGCTTACCC GCCCGGCCCC CCAGTCAGTG CAGATTCAAC CACCTGCAAA GTGCGTGCTG AACCAATCAG GCTGGCTGTC CGGCTTCCTG ACCTCCTCAG TTCTCCCCTC TCTCCTTCCT AATGCCTTCT GATATATAAA

GACGTGGGGC CTCGCCCTCA CCTGGGGCAC ATCCCAGCCA GGCCATCCTG ATGGGCATCC TTCACGGTCA GCGGTACTTA GTCCCCGGCC CTGTACCGCA GTCAACTCGG AATGACTACT CTCTTCCCCG CTTCTGCTAG CAGGGAGCCC CTGAACTTCT GGTACTGCCA TAAA

CCAGCTCCCT GGGGCCAAGC CAGGGAAGGA TTCCCGGGAT GGAAAAATGG CTGGAGAGCC CTCGGCAGAC CCAACCCGCA TCTACTACTT GCGGCGTCAA GCGGTGTGCT ACGACATGGT ACTAGGGCGG GACCCACCTT ACCCTGACCC TTAGGAGTCA TTCTTTTTTT

GCCCCACCTT 60 CAACACAGGC 120 TGGGTACGAC 180 CCGAGGACCC 240 CCCCATGGGA 300 AGGTGAGGAG 360 CCACCAGCCC 420 GGGAGATTAT 480 TGTCTACCAC 540 AGTGGTCACC 600 GCTGAGGTTG 660 GGGCATCCAG 720 GCAGATGCGC 780 ACTGGCCAGT 840 ACCCCCACTG 900 CTGCTTGTGT 960 TTTTTTTTCA 1020

The first five nucleotides of the second exon in each cDNA are underlined to indicate the intron-exon boundaries. The putative polyadenylation sites, (AATAAA, A and C chains,- AATGAA, B chain), the initiation (ATG) and termination codons (TGA, A and B chains,- TAG C chain) are indicated.

Genomic structure^'^^^ The Clq gene cluster is located on a 24kb stretch of DNA and the genes are aligned in the order A-C-B with the same 5'-3' orientation. Each gene consists of two exons separated by one intron. Intergenic distances are indicated in the graphical representation below. The mouse Clq gene cluster shows the same organization, order and orientation with slight differences in the intron sizes and the intergenic distances. A 1kb C B

1 •

2 •

1 •

1

2

1

2

1 1

•

•

1 •

1 •

Accessior1 numbers Human^ iviouse^'^ ^^ BALB/c 129 BALB/c BALB/c Rat5

B chain A A B C B

chain chain chain chain chain

cDNA X03084 X58861 M22531 X66295 X71127

Genomic K03430 X92958 X92959 X92960

Deficiency Human Autosomal recessive. Lack of Clq leads to a loss of activation of the classical pathway. Patients suffer from severe recurrent viral and bacterial

infections and from immune complex deposition leading to glomerulonephritis and systemic lupus erythematosus (SLE)-like symptoms. Mutations identified (reviewed in refs. 12 and 13): A gene C670 to T; Q208 to stop six families; Turkey, Slovak Republic B gene Gl 19 to A; G40 to D one family; Morocco C523 to T; R175 to stop one patient; Mexico C gene G127 to A; G43 to R three families; Germany, India, Saudi Arabia C232 to T; R69 to stop one family; Yugoslavia del C239; frameshift - S136 to stop one patient; England Mouse A Clq knockout mouse has been generated by targeted gene disruption^''.

Polymorphic variants None described.

References ^ 2 3 ^ 5 6 7 « ^ '0 " 12 13 ^^

Heinz, H.P. (1989) Behring Inst. Mitt. 84, 20-31. Reid, K.B.M. (1989) Behring Inst. Mitt. 84, 8-19. Antes U. et al. (1984) J. Immunol. Meth. 74, 299-306. Petry, F. et al. (1991) J. Immunol. 147, 3988-3993. Schwaeble, W. et al. (1995) J. Immunol. 155, 4971-4978. Reid, K.B.M. (1985) Biochem. J. 231, 729-735. Sellar, G.C. et al. (1991) Biochem. J. 274, 481-490. Sellar, G.C. et al. (1992) Immunogenetics 35, 214-216. Petry, F. et al. (1996) Immunogenetics 43, 370-376. Petry, F. et al. (1989) FEBS Lett. 258, 89-93. Petry, F. et al. (1992) Eur. J. Biochem. 209, 129-134. Walport, M.J. et al. (1998) Immunobiology 199, 265-285. Petry, F. (1998) Immunobiology 199, 286-294. Botto, M. et al. (1998) Nature Genet. 19, 56-59.

Mannose-binding lectin

MBL

Peter Lawson and K.B.M. Reid, Department of Biochemistry, University of Oxford, Oxford, UK Other names Mannose-binding protein, mannan-binding lectin, mannan-binding protein, core-specific lectin. Ra-reactive factor (RaRF) corresponds to MBL and its associated serine proteases (MASP-1 and MASP-2). In mice, RaRF subcomponents P28a and P28b refer to MBL-C and MBL-A, respectively.

Physicochemical properties Mannose-binding lectin is a 248 amino acid molecule including a 20 amino acid leader sequence. Mature protein: pi predicted 5.3 M, (K) predicted 24 observed 32 (reduced) -600 (non-denatured) N-linked glycosylation sites none Interchain disulfide bonds 25, 32, 38 (arrangement unknown)

Structure MBL forms a trimer of a single polypeptide chain of 32kDa. The polypeptide chain is composed of an N-terminal cysteine-rich region, a collagen-like region, an a-helical neck region and a carbohydraterecognition domain. Formation of the triple helical collagen-like region may be initiated by the neck region. MBL associates to form higher order oligomers of 2-6 trimers, with the hexameric form resembling the ''bunch of tulips'' structure used to describe the structure of Clq, as judged by electron microscopy. An interruption in the collagen-like sequence results in a kink in the collagen stalks^. The crystal structure of the carbohydraterecognition domain of MBL, as well as a trimeric structure have been determined^-^.

Function MBL interacts with highest affinity to the simple carbohydrates Nacetylglucosamine and mannose. The functions of MBL are mediated by binding carbohydrate on the surface of pathogens, viruses, yeast, fungi and bacteria^. The effector functions are mediated through the Clq receptors, the most recently described is by Nepomuceno and colleagues^, or through the activation of complement^, via the associated serine proteases MASP-1* and MASP-2^. MBL is an important component of innate immune defence and aids the development of the adaptive immune system^'^. MBL has also been shown to act as a direct opsonin, leading to attachment, uptake and killing of Salmonella montevideo by phagocytes^^. MBL can inhibit influenza A virus haemagglutination^^^ the binding of MBL to different viral strains is carbohydrate dependent and leads to complement

Mannose-binding lectin

activation^-^. MBL also inhibits HIV infection of CD4* lymphocytes^^, acts as an acute-phase protein^^ and low levels of MBL have been associated with a defect in opsonization and increased risk of infection during childhood^'^^.

Tissue distribution Serum protein: 1 ^ag/ml (average) sera concentration in Caucasian populations. There is a wide range of MBL concentrations in individuals (O-SjUg/ml) due to the presence of MBL structural and promoter region alleles in the population (see below). Primary site of synthesis: liver^^. Secondary sites: kidney^^.

Regulation of expression MBL is an acute-phase protein, a modest 1.5- to 3-fold increase of serum MBL concentration is seen after malaria infection and trauma (surgery)^*. MBL expression in vitro is enhanced by IL-6, dexamethasone and heat shock but inhibited by IL-P^. The promoter region contains the consensus sequences for three glucocorticoid response elements and a heat shock element, consistent with MBL being an acute-phase protein^^.

Protein sequence^^^^^ MSLFPSLPLL GRDGTKGEKG PDGDSSLAAS VKALCVKFQA NRLTYTNWNE

LLSMVAASYS EPGQGLRGLQ ERKALQTEMA SVATPRNAAE GEPNNAGSDE

ETVTCEDAQK GPPGKLGPPG RIKKWLTFSL NGAIQNLIKE DCVLLLKNGQ

TCPAVIACSS NPGPSGSPGP GKQVGNKFFL EAFLGITDEK WNDVPCSTSH

PGINGFPGKD 5 0 KGQKGDPGKS 10 0 TNGEIMTFEK 150 TEGQFVDLTG 2 00 LAVCEFPI

The leader sequence is underlined, there are no potential N-linked glycosylation sites. The collagen-like region is interrupted. Seven G-X-Y repeats are followed by a G-Q-G interruption, followed by 12 collagen triplets. Some of the prolines in the Y position in the collagen-like region are hydroxylated (shown in bold), P82 is hydroxylated in MBL purified from the liver but not when purified from the serum^^.

Protein modules 1-20 21-41 42-100 101-124 125-248

Leader peptide N-terminal cysteine rich region Collagen-like domain Alpha-helical coiled-coil ''neck'' region Carbohydrate-recognition domain (CRD)

exon 1 exon 1 exon 1/2 exon 3 exon 4

Residues important for calcium-dependent recognition of carbohydrate are E212, N214, E220, N232 and D2332.

Mannose-bindine lectin

Chromosomal location Human^^ H u m a n MBL, the orthologue of rhesus monkey MBL-C, is on chromosome 10, 10qll.2-q21, 74-75 cM. Centromere ... JNK-46 ... MBL ... ANK-3 ... Telomere A mannose-binding lectin mRNA which encodes for a truncated protein has been identified in a liver cDNA library, this expressed pseudogene, the rhesus monkey MBL-A orthologue, is localized on chromosome 10 at 10q22.2-q22.323.

Mouse^^ Mbll, mannose-binding lectin A, serum chromosome 14, 15 cM. Mbl2, mannose-binding lectin C, liver chromosome 19, 25 cM.

cDNA sequence^^ 20 GCTCGGTAAA GTGAGGACCA TCTTACTCAG TGTAGCTCTC GAAAAGGGGG CCTCCAGGAA GGAAAAAGTC GAAATGGCAC TTCTTCCTGA TTCCAGGCCT ATCAAGGAGG CTGACAGGAA TCTGATGAAG ACCTCCCATC TTGTCTTTTT TTCCTCATAT CCAACAAAGC ATATAATATT TAGTTTAATT CGGATTTATT GGGTAGAGGG TCAGGTATTA GAGATATTAA

TATGTGTTCA TGTCCCTGTT AAACTGTGAC CAGGCATCAA AACCAGGCCA ATCCAGGGCC CGGATGGTGA GTATCAAAAA CCAATGGTGA CTGTGGCCAC AAGCCTTCCT ATAGACTGAC ATTGTGTATT TGGCCGTCTG ACTGCAACCC CCAGCATTGT AATAATAGTA TTTAATATAT AATCTGTAAT TTTCCATTTA CTCCCCTAAT AGAAAATCTA ACCATGTA

TTAACTGAGA TCCATCACTC CTGTGAGGAT CGGCTTCCCA AGGGCTCAGA TTCTGGGTCA TAGTAGCCTG GTGGCTGACC AATAATGACC CCCCAGGAAT GGGCATCACT CTACACAAAC GCTACTGAAA TGAGTTCCCT ACAGGCCCAC TCCTTTTGTG GTAGTAGTAG ACTATGAGGC GCTTTCGATA CAACAAACAC GACATCACCA TTTTTGTAAC

TTAACCTTCC CCTCTCCTTC GCCCAAAAGA GGCAAAGATG GGCTTACAGG CCAGGACCAA GCTGCCTCAG TTCTCTCTGG TTTGAAAAAG GCTGCAGAGA GATGAGAAGA TGGAACGAGG AATGGCCAGT ATCTGAAGGG AGTATGCTTG GGCAATCACT TTAGCAGCAG CCTATCTTTT GTGTTAACTT CTGTGCTCTG CAGTTTAATA TTTCTCTATG

CTGAGTTTTC TCCTGAGTAT CCTGCCCTGC GGCGTGATGG GCCCCCCTGG AGGGCCAAAA AAAGAAAAGC GCAAACAAGT TGAAGGCCTT ATGGAGCCAT CAGAAGGGCA GTGAACCCAA GGAATGACGT TCATATCACT AAAAGATAAA AAAAATGATC CAGTAGTAGT GCATCCTACA GCTGCAGTAT TTGAGCCTTC CCACAGCTTT AACTCTGTTT

TCACACCAAG GGTGGCAGCG AGTGATTGCC CACCAAGGGA AAAGTTGGGG AGGAGACCCT TCTGCAAACA TGGGAACAAG GTGTGTCAAG TCAGAATCTC GTTTGTGGAT CAATGCTGGT CCCCTGCTCC CAGGCCCTCC TTATATCAAT ACTAACAGCA CATGCTAATT ATTAATTATC GAAAATAAGA CTTTCTGTTT TTACCAAGTT TCTTTCTAAT

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320

The methionine initiation codon (ATGJ and the termination codon (TGAj are indicated, no potential polyadenylation signals (AAT AAA) are seen. The first five nucleotides of each exon are underlined to indicate intron-exon boundaries.

Genomic structure^^^^ The gene spans 3.5 kb and is encoded by four exons. 1kb

H—h

Mannose-binding lectin

Accession numbers Human Mouse BALB/c CBA/J BALB/c CBA/J Rat

Bovine Rhesus monkey Rabbit Chicken

MBL-C Pseudogene MBL-A(MW:Z) MBL-C iMbl2) MBL-C MBL-A Pseudogene MBL-C MBL-A MBL-C MBL-C MBL-C

cDNA X15422^5

Genomic X15954-X1595720.22 AFO1938223

D l 144125 S4229226 D1144025 S4229426 M1410327 X0502328 See ref. 27

U09006-U090112^ U09012-U0901724

M14104-M1410529 M1410629

D73408^o L439123^ L4391P^ D84293^2 AF022226

Deficiency C223 to T; R52 to C G230 to A; G54 to D G239 to A; G57 to E Homozygotes for these variants have undetectable or trace amounts of MBL in their serum (see below for heterozygotes).

Polymorphic variants Three variants in exon 1 have been linked to increased susceptibility to recurrent infections in infants^^'^^ and adults^^'^*^. Variant alleles of MBL predisposes individuals to enhanced susceptibility to HIV and progression to AIDS^^'^^. Individuals with mutant MBL alleles have low molecular weight forms of MBL, 100-200 kDa, compared with the wild-type MBL of 600 kDa. Variants identified^'^^'^^^: C223 to T; R52C. Gene frequency of 0.05 in African and European populations. Heterozygotes have a 50% reduction in the level of serum MBL. Linked to a promoter haplotype (HY) which is associated with high MBL concentrations. G230 to A; G54 to D. Gene frequencies of 0.03 in African populations and 0.13 in European and Eskimo populations. 12.5% level of serum MBL. Linked to a promoter haplotype (LY) which is associated with median MBL concentrations. This variant causes an interruption in the collagenlike sequence and prevents complement activation

Maniiose-bindine lectin

G239 to A; G57 to E. Gene frequencies of 0.23 in African and 0.02 in European populations. 12.5% level of serum MBL. Linked to a promoter haplotype (LY) which is associated with median MBL concentrations. This variant causes an interruption in the collagen-like sequence. Complement activation not determined.

References ' 2 3 4 5 6 7 s 9 0

20 2^ 22 23 24 25 26 27 28 29 30 3^ 32 ^3 34 35 36 37 3« 39 40

Thiel, S. and Reid, K.B. (1989) FEBS Lett. 250, 78-84. Weis, W.L et al. (1992) Nature 360, 127-134. Weis, W.I. and Drickamer, K. (1994) Structure 2,1227-1240. Sheriff, S. et aL (1994) Nature Struct. BioL 1, 789-794. Turner, M.W. (1996) Immunol. Today 17, 532-540. Nepomuceno, R.R. et al. (1997) Immunity 6, 119-129. Ikeda, K. et al. (1987) J. Biol. Chem. 262, 7451-7454. Matsushita, M. and Fujita, T. (1992) J. Exp. Med. 176, 1497-1502. Thiel, S. et al. (1997) Nature 386, 506-510. Carroll, M.C. and Prodeus, A.P. (1998) Curr. Opin. Immunol. 10, 36-40. Kuhlman, M. et al. (1989) J. Exp. Med. 169, 1733-1745. Anders, E.M. et al. (1990) Proc. Natl Acad. Sci. USA 87, 4485-4489. Anders, E.M. et al. (1994) J. Gen. Virol. 75, 615-622. Ezekowitz, R.A. et al. (1989) J. Exp. Med. 169, 185-196. Ezekowitz, R.A. et al. (1988) J. Exp. Med. 167, 1034-1046. Super, M. et al. (1989) Lancet 2, 1236-1239. Morio, H. et al. (1997) Eur. J. Biochem. 243, 770-774. Thiel, S. et al. (1992) Clin. Exp. Immunol. 90, 31-35. Arai, T. et al. (1993) Q. J. Med. 86, 575-582. Taylor, M.E. et al. (1989) Biochem. J. 262, 763-771. Kurata, H. et al. (1994) J. Biochem. Tokyo 115, 1148-1154. Sastiy, K. et al. (1989) J. Exp. Med. 170, 1175-1189. Guo, N. et al. (1998) Mamm. Genome 9, 246-249. Sastry, R. et al. (1995) Mamm. Genome 6, 103-110. Kuge, S. et al. (1992) Biochemistry 31, 6943-6950. Sastry, K. et al. (1991) J. Immunol 147, 692-697. Drickamer, K. et al. (1986) J. Biol. Chem. 261, 6878-6887. Oka, S. et al. (1987) J. Biochem. Tokyo 101, 135-144. Drickamer, K. and McCreary, V. (1987) J. Biol. Chem. 262, 2582-2589. Kawai, T. et al. (1997) Gene 186, 161-165. Mogues, T. et al. (1996) Glycobiology 6, 543-550. Kawai, T. et al. (1998) Glycobiology 8, 237-244. Sumiya, M. et al. (1991) Lancet 337, 1569-1570. Summerfield, J.A. et ai. (1997) BMJ 314, 1229-1232. Summerfield, J.A. et al. (1995) Lancet 345, 886-889. Garred, P. et al. (1995) Lancet 346, 941-943. Pastinen, T. et al. (1998) AIDS Res. Hum. Retroviruses 14, 695-698. Garred, P. et al. (1997) Lancet 349, 236-240. Madsen, H.O. et aL (1995) J. ImmunoL 155, 3013-3020. Madsen, H.O. et aL (1994) Immunogenetics 40, 37-44.

Bovine conglutinin Peter Lawson and K.B.M. Reid, Department of Biochemistry, University of Oxford, Oxford, UK Note: Conglutinin is believed to be restricted to bovidae. Despite extensive searching at the protein, cDNA and genomic levels, this gene product or gene appears not to be present in humans or mice.

Physicochemical properties Bovine conglutinin is a 391 amino acid polypeptide chain including a 20 amino acid leader sequence. pP predicted 5.9 four isoforms 5.3-6.1 Mr (K) predicted monomer 35.7 observed 43 (reduced) -1000 (non-denatured) N-linked glycosylation site (predicted) 1 (337) Interchain disulfide bonds 20, 25, 58 (arrangement unknown)

Structure Conglutinin, as with all members of the collectin family, is composed of three polypeptide chains which associate together to form a trimeric subunit. Each polypeptide chain consists of a collagen-like region with a C-terminal Ca^+dependent carbohydrate-recognition domain (C-type lectin). In conglutinin, four of the trimeric subunits, 12 chains, are linked by cysteine bonds at the Nterminus to form a cruciform-type structure. Electron microscopy confirms the quaternary structural arrangement, with each collagen rod in the cruciform structure having a length of 38 nm^. The carbohydrate-recognition domain is believed to form a structure analogous to the X-ray crystal model for the C-type lectin domain of mannose-binding lectin.

Function The functions of conglutinin are mediated through its affinity for carbohydrate, showing greatest specificity for iV-acetylglucosamine amongst simple sugars. Conglutinin interacts with the complement system by binding to one of the carbohydrate chains of C3, only when presented on iC3b, a breakdown product of activated C3^. This results in agglutination of complement-fixed surfaces, known as conglutination. Conglutinin interacts with various microorganisms. It inhibits haemagglutination and acts as an opsonin for influenza A virus'^'^. Conglutinin displays antibacterial activity against Salmonella typhimurium and E. coli^, binds to gpl60, the coat protein of HIV, inhibiting the attachment to CD4^. Conglutinin also enhances complement-dependent respiratory burst of phagocytes induced by E. colP. Low levels of conglutinin are associated with increased infections in cattle^. Conversely, conglutinin can also enhance herpes simplex virus type 2 infection in mice^^.

Bovine conulutinin

Tissue distribution Serum protein: 40-70 ^g/ml approx.^^. Primary site of synthesis: liver^^ Secondary sites: not known. However, immunohistochemical localization of conglutinin to the follicular dendritic cells of the spleen, tonsils and lymph nodes as well as endothelial cells of blood vessels, especially in the glomeruli of the kidney has been noted^^. This probably does not reflect de novo synthesis.

Regulation of expression The regulation of conglutinin has not been well characterized. However, it appears to be developmentally regulated, since serum levels increase with age^^. Conglutinin levels are affected by infection, since serum levels drop in cows with mastitis and experimentally induced bovine leukaemia virus infection^^. The promoter region of the gene has been sequenced and contains transcription factor-binding sites for PEAS, NF-IL6, H-APF-1, SP-1, AP-1 and LF-Al, a liver-specific transcription factor^^.

Protein sequence (bovine)^^ MLLLPLSVLL DGQDGRECPH DTGPRGPPGM GFPGPSGLKG RGDPGETGAK GQAVGEKIFK QEKNAYLSMN FPDGKWNDVP

LLTQPWRSLG GEKGDPGSPG PGPAGREGPS EKGAPGETGA GESGLAEVNA TAGAVKSYSD DISTEGRFTY CSKQLLVICE

AEMTTFSQKI PAGRAGRPGW GKQGSMGPPG PGRAGVTGPS LKQRVTILDG AEQLCREAKG PTGEILVYSN F

LANACTLVMC VGPIGPKGDN TPGPKGETGP GAIGPQGPSG HLRRFQNAFS QLASPRSSAE WADGEPNNSD

SPLESGLPGH GFVGEPGPKG KGGVGAPGIQ ARGPPGLKGD QYKKAVLFPD NEAVTQMVRA EGQPENCVEI

50 10 0 150 2 00 2 50 3 00 3 50

This protein sequence is deduced from a cDNA sequence^^. There are four differences (underlined single amino acids) when this sequence is compared with two other cDNA sequences^^'^^ and one sequence derived by amino acid sequencing^* (see Polymorphic variants section). The leader sequence is underlined. The potential N-linked glycosylation site is indicated (N); however, this involves N337 which is one of the residues considered to be involved in carbohydrate recognition. Carbohydrate analysis reveals the presence of O-linked glycans of GalNAc and alpha (2-3) linked sialic acid type. No N-linked glycans were demonstrated^^. Hydroxylated lysines and prolines are marked in bold; probably all eight hydroxylysines are glycosylated with a disaccharide^*.

Protein modules 1-20 21-45 46-216 217-244 245-371

Leader peptide N-terminal cysteine region Collagen-like sequence Neck region CRD

exon exon exon exon exon

3 3 3-7 8 9

Residues believed to be important for calcium-dependent recognition of carbohydrate are E335, N337, E345, N357 and D358.

Bovine conglutinin

Chromosomal location Bovine^^^: chromosome 28ql8, domestic cow (Bos taurus). Centromere ... IDVGA43 ... CGNl ... IDVGA8 (D28S10) ... Telomere River buffalo^^: chromosome 4pl6.

cDNA sequence (bovine)^^';,i7 ATTTGTGTGG CTCAACTTGC AGCAAGTGAG TGCTGCTCCT AGAAAATACT CTGGTCATGA CACCAGGACC GAGACAATGG CAGGTATGCC CTCCAGGCAC GCATACAGGG CTGGAGCCCC CTTCAGGTGC GAGCAAAGGG TAGATGGACA TCCCTGATGG ATTCAGATGC CAGCCGAGAA GCATGAATGA ATTCCAACTG TGGAAATCTT TCTGCGAGTT CTGCCAACAT GGGATGAGGC TGGCTTC

AGGTTCTGAA TTTCCTGTGT AGGAAACAAG GCTCACACAG GGCCAATGCC TGGACAAGAT TGCAGGACGA CTTTGTTGGA TGGACCAGCT ACCAGGCCCC CTTCCCAGGC TGGACGTGCT CAGGGGCCCC GGAGAGTGGG TCTGCGACGC CCAGGCTGTC AGAGCAGCTC CGAGGCCGTG CATCTCCACG GGCCGATGGG TCCTGATGGC TTGAGCTCTC CCCAATAAAA CACCAGGCAG

ACCCTAGAGG CCAATAGCAC CCAGCATTGT CCCTGGAGAT TGTACCCTGG GGGAGAGAAT GCAGGAAGGC GAACCTGGAC GGAAGAGAAG AAAGGAGAGA CCCTCGGGTC GGGGTGACAG CCAGGACTGA CTTGCAGAGG TTCCAAAATG GGGGAGAAGA TGCAGAGAGG ACACAGATGG GAGGGGAGGT GAGCCCAACA AAGTGGAATG CCACCCACCC AGGTGACCCT GCCTCCTATG

ACGCAACTGT TGCAGACTCC AAGAGGACAT CCCTGGGAGC TTATGTGTAG GTCCCCATGG CTGGATGGGT CAAAGGGAGA GCCCCTCAGG CTGGGCCCAA TCAAAGGAGA GGCCTTCTGG AGGGGGACAG TCAATGCTCT CCTTCAGTCA TCTTCAAGAC CTAAGGGACA TCAGAGCCCA TCACTTACCC ACAGTGATGA ACGTACCCTG CAGGGAAGGG CTGCTGCTCA GAACTCCTCC

GAGTGTCTCT AGTACTAGCC GCTTCTCCTC AGAAATGACA CCCCCTGGAG AGAGAAGGGG TGGCCCTATT CACTGGGCCA GAAGCAGGGG AGGAGGAGTG GAAAGGTGCC AGCCATAGGT AGGTGATCCT GAAGCAGGGG GTATAAGAAA AGCAGGTGCT GCTGGCCTCC GGAAAAGAAT CACTGGGGAA GGGACAACCA CAGTAAGCAA GCAGTGCCCA GGGCTTCCCC CTCAGAATAA

GTCCTAGCCT TGTCCAGAGC CCTCTCTCCG ACCTTTTCTC AGTGGCTTGC GATCCAGGTT GGGCCGAAAG CGTGGGCCTC AGCATGGGAC GGTGCCCCAG CCCGGGGAGA CCACAGGGCC GGAGAAACAG GTGACAATCT GCGGTGCTCT GTAAAGTCAT CCACGCTCTT GCTTACCTGA ATACTGGTCT GAGAACTGTG CTCCTTGTGA GAGCTGTGAG ACTGAGCCAC AGTTTGAAAC

60 12 0 18 0 240 3 00 3 60 42 0 480 540 600 6 60 720 7 80 840 900 9 60 102 0 108 0 1140 12 00 12 60 132 0 13 80 1440

The cDNA show^n is a hybrid of the deduced cDNA from a genomic sequence^^ (nucleotides 1-1337) and a partial cDNA^^ (nucleotides 1338-1447). The initiation codon (ATG), the termination codon (TGAI and the polyadenylation signal (AATAAAI are indicated. The first five nucleotides in each exon are underlined to indicate the intron/exon boundaries.

Genomic structure (bovine)^^^^^ The gene spans approximately 11 kb and is encoded by nine exons. The collagen region is encoded in five exons. Collagen encoding exons 4-7 probably arose from a single exon by gene duplication (see the genomic structure for mannose-binding lectin for comparison).

m 1

I "-^ I '

"

m^—

9

Bovine con^lutinin

Accession numbers ill

cDNA 014085^^ L1887P7 X71774^2

Bovine

Genomic D25294-D25302^5 U06852-U0686022

Deficiency None identified.

Polymorphic variants Potential polymorphic variants identified from the three cDNA sequences, two genomic sequences and one protein sequence are shown here. Position 1 4-5 14 17 18 380 521 557 676 704 749 786-788 811 833 836 845 854 857 860 890 973 1154 1310 1315

Genomic^^ starts at 19

A G C G(R) C G AAG (K) T(V) G A T G A C A A(E) C A T

Genomic^^ A T T G A G C G(R) C G AAG (K) T(V) G A T G A C A A(E) C A T

cDNA^2

cDNA^7

cDNA^6

starts at 12 C starts at 60 C insertionT G T C A G T A A A G G C G C A(H) A(H) G(R) C C A A G G AAG (K) AAG (K) AAG (K) C(A) T(V) T(V) G G A A A C T C T G G A A A C C G C A A G T(V) A(E) A(E) T C C T A A T C T

Protein^* starts at 219

-

R

S V

E

-

Updated and modified from ref. 22. Numbering based on the cDNA shown above. The amino acids shown on the far right are the differences seen in the full-length protein sequence, obtained by amino acid sequencing^*, when compared with all the cDNA sequences. Base changes shown in the table are mostly silent except where the translated amino acids are shown in parenthesis. The protein is translated from base 219 and finishes at base 1271. Dashes indicate where sequence is not available for comparison.

References i Holmskov, U. et al. (1995) Biochem. J. 305, 889-896. 2 Strang, C.J. et al. (1986) Biochem. J. 234, 381-389. 3 Laursen, S.B. et al. (1994) Immunology 81, 648-654.

Bovine conglutinin

^ 5 6 7 « 9 ^0 ^^ ^2 " ^^ ^5 ^6 ^7 ^s ^9 20 2^ 22

Hartshorn, K.L. et al. (1993) J. Immunol. 151, 6265-6273. Hartley, C.A. et al. (1992) J. Virol. 66, 4358-4363. Friis Christiansen, P. et al. (1990) Scand. J. Immunol. 31, 453-460. Andersen, O. et al. (1991) Scand.}. Immunol. 33, 81-88. Friis, P. et al. (1991) Immunology 74, 680-684. Holmskov, U. et al. (1998) Immunology 93, 431-436. Fischer, P.B. et al. (1994) Scand. J. Immunol. 39, 439-445. Akiyama, K. et al. (1992) Am. J. Vet. Res. 53, 2102-2104. Lu, J. et al. (1993) Biochem. J. 292, 157-162. Holmskov, U. et al. (1992) Immunology 1^, 169-173. Akiyama, K. et al. (1992) J. Vet. Med. Sci. 54, 977-981. Kawasaki, N. et al. (1994) Biochem. Biophys. Res. Commun. 198, 597-604. Suzuki, Y. et al. (1993) Biochem. Biophys. Res. Commun. 191, 335-342. Liou, L.S. et al. (1994) Gene 141, 277-281. Lee, Y.M. et al. (1991) J. Biol. Chem. 266, 2715-2723. Andersen, O. et al. (1992) J. Struct. Biol. 109, 201-207. Gallagher, D.S., Jr. et al. (1993) Mamm. Genome 4, 716-719. lannuzzi, L. et al. (1994) Hereditas 120, 283-286. Liou, L.S. et al. (1994) J. Immunol. 153, 173-180.

SP-A Robert B. Sim, MRC Immunochemistry Unit, Oxford, UK

D

Other names Surfactant protein A, pulmonary surfactant (glyco)protein A, PSP-A.

Physicochemical properties SP-A is a member of the collectin family, which are typically oligomers of short (Mr (K) 25-45) polypeptide chains, each containing collagenous sequence at the N-terminus and a C-type lectin domain at the C-terminus^ SP-A is made up of up to 18 polypeptide chains of two types, named a2 and oc3, thought to be in the ratio 1:2^. The two polypeptide types are ~98% identical, but are products of separate genes. Each polypeptide is 248 amino acids long, including a 20 amino acid leader sequence. There is variability in the position of cleavage of the leader sequence in rat SP-A^, and this may be so in human SP-A, such that some mature polypeptides may contain C(20). Mature protein: pl M, (K)

predicted observed N-linked glycosylation sites (occupied) Interchain disulfide bonds

-4.4-4.8 -4.5-5.0 (deglycosylated) 24.2 (without glycosylation) 30-33 (secreted form)

1 (207) a2 chain 26, 68 20 Potential Disulfide bridging pattern not established.

a3 chain 26, 68, 85 20 Potential

Structure SP-A has a quaternary structure similar to Clq. Three polypeptide chains (thought to be, optimally, two a3 and one a2) fold to form a heterotrimer. Trimerization involves formation of a coiled-coil at the neck regions and a collagen triple helix"^. Interchain disulfide bridges are likely to form between C26, 68, 85 and possibly C20^. The three-chain subunits then associate covalently or non-covalently to form a structure with up to six subunits, and a total of 18 lectin domains, very similar in shape to Clq. The six-subunit form has M^ (K) approx. 540, Stokes' radius 9.S nm,S2o, w -14^. Analysis of SP-A in lung surfactant fluid and synovial fluid indicates that 40% or more of the SP-A present is not fully assembled into the sixsubunit form, but exists as smaller oligomers^. There is O-linkage of GalGlc disaccharides in the collagen-like region, but the number is unknown.

Function SP-A is the major protein of lung surfactant, where it is found associated with the surfactant lipids, with only -10% of SP-A in solution in the aqueous phase. It is thought to have two major functions, namely innate

immune defence and involvement in surfactant metabolism^'*. It is considered to have a major role in surfactant recycling, i.e. secretion and uptake by alveolar type II cells, and to have a structural role in the organization of tubular myelin. SP-A knockout mice, however, do not appear to have gross abnormalities in surfactant metabolism^. The knockout mice, however, have been shown to be more susceptible to some bacterial infections, consistent with an important role of SP-A in innate immunity. SP-A binds to a wide range of bacteria, viruses, fungi and particulate materials such as pollen grains by interaction of the lectin domains with surface saccharides (mainly mannose, glucosamine, fucose residues p'*'^^. The smaller oligomeric forms of SP-A show much poorer binding than is seen with the fully assembled six-subunit form^'". SP-A acts as an opsonin, and to some extent as an agglutinin. SP-A has no known enzymic activity, and is not known to interact with any complement system proteins, although it interacts {in vitro) with the same receptor(s) as Clq. SP-A has specific interactions with a number of cell types, including macrophage, neutrophils and specialized epithelia. There is controversy as to the receptors involved. Several candidate SP-A receptors have been reported. These include: (a) the collectin receptor (cClqR, cell surface calreticulin)^^'^^ which has been shown to mediate uptake of SP-A-coated Staphylococcus aureus by monocytes^^; (b) ClqRp^^, which is implicated in phagocytic uptake,- (c) SP-A-binding protein (M^jK) 210), involved in uptake of mycobacteria and BCG by rat macrophage and human monocytes^^'^^ and (d) the SP-AR [M, (K) 30)^«. It has also been shown that cell surface oligosaccharide may bind SP-A via its lectin domains^^ or that phospholipids may bridge between SP-A and a cell surface structure^^, such as CD14, the M,(K) 55 LPS receptor^^.

Tissue distribution SP-A is found in lung surfactant up to 5% of the total weight of the surfactant lipid/protein assembly. It is also present in amniotic fluid^^ (~5 mg/litre at term) and synovial fluid. SP-A is undetectable in normal blood plasma, but may be found in plasma in certain lung diseases, e.g. ARDS^^. Primary site of synthesis: alveolar type II cells. Secondary sites: Clara cells, serous glands of the proximal trachea, lachrymal and salivary epithelia, pulmonary parenchyma, mesothelium and synoviocytes*.

Regulation of expression Dexamethasone, IL-la, and raised oxygen concentration all increase mRNA and protein synthesis.

Protein sequence MWLCPLALNL KGDPGPPGPM AHLDEELQAT IQEACARAGG SDGTPVNYTN

ILMAASGAAC GPPGEMPCPP LHDFRHQILQ RIAVPRNPEE WYRGEPAGRG

EVKDVCVGSP GNDGLPGAPG TRGALSLQGS NEAIASFVKK KEQCVEMYTD

GIPGTPGSHG IPGECGEKGE IMTVGEKVFS YNTYAYVGLT GQWNDRNCLY

LPGRDGRDGL 50 PGERGPPGLP 100 SNGQSITFDA 150 EGPSPGDFRY 2 00 SRLTICEF

This is the sequence of the a3 allele 6A . The leader sequence is underlined, and the N-linked glycosylation site is indicated (N). There are six variants of a3 sequence reported, and four a2 variants^^. a3 sequences are characterized by the residues M, D, I, C at positions 66, 73, 81, 85 respectively, while a2 sequences have T, N, V, R at these positions.

Protein modules^^-^^ 1-20 28-100 101-152 153-248

Leader sequence (may vary) Collagen-like domain Alpha-helical coiled-coil ''neck'' region CRD

exon exon exon exon

4 4-6 6/7 7

Chromosomal location Human25: 10q21-24. Centromere ... SFTPD ... SFTPA2 ... Pseudogene ... SFTPAl ... Telomere^^ Gene names for SP-A are SFTPAl and 2 or SFTPl or PSAP and for SP-D are SFTPD, SFTP4 or PSPD. This region of the chromosome also contains the gene for MBL. Mouse: chromosome 14, 14 cM.

cDNA sequence^^ CGGAGACCCA AGAAAGAGCA CTTGATGGCA TATCCCCGGC AGGAGACCCT AAATGATGGG TGGCGAGAGG CCACGACTTT AATGACGGTA TCAGGAGGCA TGAGGCCATT GGGTCCCAGC GTACCGAGGG GCAGTGGAAT ATTTAGGCCA CTTGGTCTGT

AGCAGCTGGA GCGACTGGAC GCCTCTGGTG ACTCCTGGAT GGCCCTCCAG CTGCCTGGAG GGCCCTCCAG AGACATCAAA GGAGAGAAGG TGTGCCAGAG GCAAGCTTCG CCTGGAGACT GAGCCCGCAG GACAGGAACT TGGGACAGGG GAGATGCTAG

GGCTCTGTGT CCAGAGCCAT CTGCGTGCGA CCCACGGCCT GCCCCATGGG CCCCTGGTAT GGCTTCCAGC TCCTGCAGAC TCTTCTCCAG CAGGCGGCCG TGAAGAAGTA TCCGCTACTC GTCGGGGAAA GCCTGTACTC AGGACGCTCT AACTCCCTTT

GTGGGTCGCT GTGGCTGTGC AGTGAAGGAC GCCAGGCAGG TCCGCCTGGA CCCTGGAGAG TCATCTAGAT AAGGGGAGCC CAATGGGCAG CATTGCTGTC CAACACATAT AGACGGGACC AGAGCAGTGT CCGACTGACC CTGGCCTTCG CAACA

GAGTTTCTTG CCTCTGGCCC GTTTGTGTTG GACGGGAGAG GAAATGCCAT TGTGGAGAGA GAGGAGCTCC CTCAGTCTGC TCCATCACTT CCAAGGAATC GCCTATGTAG CCTGTAAACT GTGGAGATGT ATCTGTGAGT GCCTCCATCC

GAGCCTGAAA TCAACCTCAT GAAGCCCTGG ATGGTCTCAA GTCCTCCTGG AGGGGGAGCC AAGCCACACT AGGGCTCCAT TTGATGCCAT CAGAGGAAAA GCCTGACTGA ACACCAACTG ACACAGATGG TCTGAGAGGC TGAGGCTCCA

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

This is the sequence of an (x3 allele. The initiation codon (ATG) and termination codon (TGA) are indicated. The first five nucleotides of exons 4-7 are underlined to indicate intron-exon boundaries. There is considerable variation in the length and sequence composition of both the 5' and 3' untranslated regions.

Genomic structure The genomic organization of each SP-A gene is similar. The SP-A genes consist of seven exons^^'^^. 1 kb 1

•-

. 1

H-H Accession numbers Human (a3) Mouse Rat Dog Guinea-pig Pig Rabbit

M13686 S48768 M33201 Ml 1769 U40869 L41350 J03542

Deficiency No clearly defined genetic deficiency of SP-A has been reported, nor have any allelic variants been reported to have grossly altered function. Large variations in the degree of polymerization of SP-A have been observed between individuals, and since lower polymers have impaired carbohydratebinding function, there may be functional deficiency of SP-A while the total SP-A concentration appears normal^'^^. It is not currently known what factors control the degree of polymerization. SP-A knockout mice do not have grossly altered surfactant function^. Lowered concentration of SP-A in the lung has been reported in association with asthma^^.

Polymorphic variants Both SP-A genes exhibit variability: splicing variability in the 5' untranslated region and sequence variability within the coding and the 3' untranslated regions. All four untranslated regions can vary in length. This genetic variability affects the level of SP-A mRNA and the ratio of SFPTA1:SFPTA2 mRNA. Variants in coding sequence of the SFTPAl gene (protein a3) are named 6A (sequences of which are shown above), and 6Ao-6A/'2^-27^29 Variants in coding sequence of the SFTPA2 gene (protein a2) are named lA, and lAg-lAj. It is thought that specific SP-A alleles are associated with increased risk of respiratory distress syndrome^^.

References ^ Malhotra, R. et al. (1994) Clin. Exp. Immunol. 97, suppl. 2, 4-9. 2 Voss, T. et al. (1991) Am. J. Respir. Cell Mol. Biol. 4, 88-94. 3 Elhalwagi, B.M. et al. (1997) Biochemistry 36, 7018-7025.

^ Hoppe, H.J. and Reid, K.B.M. (1994) Protein Sci. 3, 1143-1158. ^ Lu, J and Sim, R.B. (1994) In New Aspects of Complement Structure and Function (Erdei, A. ed.), R.G. Landes Co., Austin, TX, pp. 85-106. 6 Hickling, T.P. et al. (1998) Mol. Med. 4, 265-276. ^ Weaver, T.E. and Whitsett, J.A. (1991) Biochem.}. 273, 249-264. « McCormack F.X. and Whitsett, J.A. (1996) In Collectins and Innate Immunity (Sastry, K.N. et al. eds) R.G. Landes Co., Austin, TX, pp. 9-50. ^ Korfhagen, T.R. et al. (1996) Proc. Natl Acad. Sci. USA 93, 9594-9599. 0 Holmskov, U. et al. (1994) Immunol. Today 15, 67-74. Stuart, G.R. et al. (1996) Exp. Lung Res. 22, 467-487. Malhotra, R. et al. (1992) Eur. J. Immunol. 22, 1437-1445. Sim, R.B. et al. (1998) Immunobiology 199, 208-224. Geertsma, M.F. et al. (1994) Am. J. Physiol. 267, L578-L584. Nepomuceno, R.R. et al. (1997) Immunity 6, 119-129. Chroneos, Z.C. et al. (1996) J. Biol. Chem. 271, 16375-16383. Weikert, L.F. et al. (1997) Am. J. Physiol. 272, L989-L995. Strayer, D.S. et al. (1993) J. Biol. Chem. 268, 18679-18684. McCormack, F.X. et al. (1994)}. Biol. Chem. 269, 29801-29807. 20 Mcintosh, J.C. et al. (1996) A m . } . Respir. Cell. Mol. Biol. 15, 509-519. 2^ Sano, H. et al. (1998) Abstr. 5th Marburg Surfactant Symposium on Surfactant and Alveolar Biology. 22 Miyamura, K. et al. (1994) Biochim. Biophys. Acta 1210, 303-307. 23 Doyle, I.R. et al. (1995) Am. J. Respir. Crit. Care Med. 152, 307-317. 2^ Karinch, A.M. et al. (1997) Biochem. J. 321, 39-47. 25 Hoover, R.R. and Floros, J. (1998) Am. J. Resp. Cell. Mol. Biol. 18, 353-362. 26 White R.T. et al. (1985) Nature 317, 361-363. 27 Karinch, A.M. and Floros, J. (1995) Am. J. Resp. Cell. Mol. Biol. 12, 77-88. 28 Floros, J. et al. (1985) J. Biol. Chem. 260, 495-500. 29 Floros J. et al. (1986) J. Biol. Chem. 261, 9029-9033. 30 van de Graaf, E.A. et al. (1992) J. Lab. Clin. Med. 120, 252-263. 3^ Kala, P. et al. (1998) Pediatr. Res. 43, 169-177.

SP-D Robert B. Sim, MRC Immunochemistry Unit, Oxford, UK

Other names Surfactant protein D, pulmonary surfactant (glyco)protein D, PSP-D, SFTP4, CP4.

Physicochemical properties SP-D is a member of the coUectin family, which are typically oligomers of short Mj. (K) 25-45 polypeptide chains, each containing collagenous sequence at the N-terminus and a C-type lectin domain at the C-terminus^. The most commonly observed form of SP-D is made up of 12 identical polypeptide chains. Each polypeptide is 375 amino acids long, including a 20 amino acid leader sequence. The arrangement of domains or modules in the polypeptide is very similar to that in SP-A, but the collagenous region is much longer (177 residues instead of 73p'^. Mature protein: pi predicted M,(K)

predicted predicted observed

7.0 (theoretical, deglycosylated) 35.5 43.6 (secreted formp 40-44

In SDS-PAGE the unreduced protein behaves as a M,{K) 130-150 species, consistent with a disulfide-linked trimer^. N-linked glycosylation sites (occupied)

1 (90)

Interchain disulfide bonds 35, 40 Disulfide bridging pattern not definitively established.

Structure SP-D has a quaternary structure similar to bovine conglutinin, and its assembly is similar to that of Clq or SP-A'*. Three polypeptide chains fold to form a homotrimer. Trimerization involves formation of a coiled-coil at the neck regions and a collagen triple helix^. Interchain disulfide bridges are likely to form between C35 and C40. The three-chain subunits then associate non-covalently at their N-terminal ends to form a very extended cross-shaped structure, commonly with four subunits, and a total of 12 lectin domains. The four-subunit form has M^ (K) approx. 510-520, and a span of 92 nm. Electron microscopy and hydrodynamic studies of SP-D (both native and recombinant) show that a proportion of SP-D occurs as smaller oligomers with 1, 2 or 3 subunits, while some occurs as very large assemblies (fuzzy balls) with up to 32 subunits/96 lectin domains'*'^. Olinkage of Gal-Glc disaccharides in the collagen region is likely, but the number is unknown.

Function SP-D is a major protein of lung surfactant, where, unlike SP-A, it is found mainly (50-90%) in the aqueous phase^. Although it does interact with certain phospholipids^, it was not thought to have a major role in surfactant metabolism. However, recent work on SP-D knock-out mice does suggest a role in surfactant homeostasis. The mice showed accumulation of surfactant lipid, SP-A and SP-B in the alveolar space as well as abnormalities in alveolar type II cells and alveolar macrophage*. SP-D appears to have an important role in innate immunity^'^. SP-D binds to a wide range of bacteria, viruses, fungi and particulate materials by interaction of the lectin domains with surface saccharides (mainly glucose, glucose oligomers, mannose, inositol in phosphatidylinositol). SP-D agglutinates such targets, and stimulates neutrophil respiratory burst, and is reported to have chemotactic activity for monocytes and neutrophils. SP-D has no known enzymic activity, and is not known to interact with any complement system proteins. Unlike SP-A, it does not interact {in vitro) with the same receptor(s) as Clq^^. SP-D has specific interactions with a number of cell types, including alveolar macrophage, neutrophils and monocytes. It does not interact with the same candidate receptors as those discussed for SP-A. It appears to bind to certain cell types via a calcium ion-dependent interaction of its own lectin domains with cell surface carbohydrate^'^. gp340, a member of the scavenger receptor family, is expressed on alveolar macrophage, and is a candidate receptor for SP-D. SP-D lectin domains interact with gp340 in a calcium ion-dependent protein-protein interaction^^

Tissue distribution SP-D is found in lung surfactant at a concentration several-fold lower than SP-A. It is detectable at very low level (<100ng/ml) in normal blood plasma, and at higher levels in plasma from patients with some forms of interstitial lung disease^'^. It is present in amniotic fluid^^, where it may originate from the fetal lung. The concentration rises to ~2 mg/litre at term. Primary site of synthesis: alveolar type II cells, non-ciliated bronchiolar epithelial cells. Secondary sites: also found associated with lachrymal and salivary epithelia, and is made by mucous cells in the stomach.

Regulation of expression Regulation of expression of SP-D has not been investigated extensively. Glucocorticoids increase expression in rat and human fetal lung tissue^.

Protein sequence^-'^ MLLFLLSALV DGRDGREGPR DTGPSGPPGP GMQGSAGARG PGLKGDKGIP VELFPNGQSV QQLWAKNEA CVEIFTNGKW

LLTQPLGYLE GEKGDPGLPG PGVPGPAGRE LAGPKGERGV GDKGAKGESG GEKIFKTAGF AFLSMTDSKT NDRACGEKRL

AEMKTYSHRT AAGQAGMPGQ GALGKQGNIG PGERGVPGNT LPDVASLRQQ VKPFTEAQLL EGKFTYPTGE WCEF

MPSACTLVMC AGPVGPKGDN PQGKPGPKGE GAAGSAGAMG VEALQGQVQH CTQAGGQLAS SLVYSNWAPG

SSVESGLPGR GSVGEPGPKG AGPKGEVGAP PQGSPGARGP LQAAFSQYKK PRSAAENAAL EPNDDGGSED

50 100 150 2 00 250 3 00 3 50

The leader sequence is underlined and the N-linked glycosylation site is indicated (N).

Protein modules^^^'^^ 1-20 46-222 223-261 262-375

Leader sequence Collagen-like domain Alpha-helical coiled-coil ''neck'' region CRD

exon 1 exon 1-5 exon Gjl exon 7

Chromosomal location^^^ Human: 10q22.2-23.1. Near the genes for SP-A and MBL. See SP-A section. Mouse: chromosome 14, 14 cM.

cDNA sequence^ CGGAATTAGG TACTTGTGCA GAGGAGGTCT TTCCTCCTCT AAGACCTACT GAGAGTGGCC GGGGACCCAG GTTGGGCCCA CCAAGTGGAC GGGAAGCAGG AAAGGAGAAG CCTAAGGGAG GGGTCTGCTG AAGGGGGACA GTTGCTTCTC GCTTTCTCTC ATTTTCAAGA GCTGGTGGAC GTCGTAGCTA TTCACCTACC GATGATGGCG GCTTGTGGAG AGTGCTTGGC CTAATAAAAA

GAGATAGTTG ATGATGGTAA GCGGCTTGGA CTGCACTGGT CCCACAGAAC TGCCTGGTCG GTTTGCCAGG AAGGGGACAA CTCCAGGACC GGAACATAGG TAGGTGCCCC AGCGAGGTGT GAGCCATGGG AAGGCATTCC TGAGGCAGCA AGTATAAGAA CAGCAGGCTT AGTTGGCCTC AGAACGAGGC CCACAGGAGA GGTCAGAGGA AAAAGCGTCT CCAGGAGTTT GGTGACCATC

GTATTAGGAT AAGGGTAGCT GCTCCTGGGG CCTACTCACA AACGCCCAGT CGATGGACGG AGCTGCAGGG TGGCTCTGTT TCCCGGTGTG ACCTCAGGGC AGGCATGCAG CCCTGGTGAG TCCCCAGGGA TGGAGACAAA GGTTGAGGCC AGTTGAGCTC TGTAAAACCA TCCACGCTCT TGCTTTCCTG GTCCCTGGTC CTGTGTGGAG TGTGGTCTGC GGCCAGAAGT AAAAAAAAAA

TAGGATTGTT TACTGGTTGT CCTAACAAAA CAGCCCCTGG GCTTGCACCC GATGGGAGAG CAAGCAGGGA GGAGAACCTG CCTGGTCCAG AAGCCAGGCC GGCTCGGCAG CGTGGAGTCC AGTCCAGGTG GGAGCAAAGG TTACAGGGAC TTCCCAAATG TTTACGGAGG GCCGCTGAGA AGCATGACTG TATTCCAACT ATCTTCACCA GAGTTCTGAG CAAGGCTTAG

GTGAAGTATA CCTCCGATTC AGAAACCTGC GCTACCTGGA TGGTCATGTG AGGGCCCTCG TGCCTGGACA GACCAAAGGG CTGGAAGAGA CAAAAGGAGA GGGCAAGAGG CTGGAAACGC CCAGGGGACC GAGAAAGTGG AAGTACAGCA GCCAAAGTGT CACAGCTGCT ATGCCGCCTT ATTCCAAGAC GGGCCCCAGG ATGGCAAGTG CCAACTGGGG ACCCTCATGC

GTACGGATGC AGGTTAGAAT CATGCTGCTC AGCAGAAATG TAGCTCAGTG GGGCGAGAAG AGCTGGCCCA AGACACTGGG AGGTCCCCTG AGCTGGGCCC CCTCGCAGGC AGGGGCAGCA CCCGGGATTG GCTTCCAGAT CCTCCAGGCT CGGGGAGAAG GTGCACACAG GCAACAGCTG AGAGGGCAAG GGAGCCCAAC GAATGACAGG TGGGTGGGGC TGCCAATATC

60 12 0 180 240 3 00 3 60 42 0 48 0 540 600 6 60 72 0 780 840 900 9 60 102 0 1080 114 0 12 0 0 12 60 132 0 13 8 0

I

The initiation codon (ATG), termination codon (TGA) and polyadenylation signal (AATAAA) are underlined, as are the first five nucleotides of each exon to indicate exon/intron boundaries. There is at least one further exon upstream of exon 1, encoding 5' UTR sequence^.

Genomic structure^^^ The SP-D gene consists of at least seven exons.

. 1

2kb I

1

iim

Accession numbers Human Mouse Rat Bovine

X65018 L40156 M81231 X75911

Deficiency No clearly defined genetic deficiency of SP-D has been reported, nor have any allelic variants been reported to have grossly altered function. There may be variation in the degree of polymerization of SP-D between individuals, but this has not been extensively investigated. Lower polymers have impaired agglutination activity^. As for SP-A, it is not currently known what factors control the degree of polymerization. SP-D knockout mice have altered surfactant homeostasis^. Lowered concentration of SP-D in the lung has been reported in association with asthma^"^.

Polymorphic variants There are no reports of genetic variation in SP-D. There are minor discrepancies between D N A sequences^'^.

References ^ 2 3 ^ 5 ^

Malhotra, R. et al. (1994) Clin. Exp. Immunol. 97, suppl. 2, 4-9. Crouch, E. et al. (1993) J. Biol. Chem. 268, 2976-2983. Lu, J. et al. (1992) Biochem. J. 284, 795-802. Holmskov, U. et al. (1995) Biochem. J. 305, 889-896. Hoppe, H.J. and Reid, K.B.M. (1994) Protein Sci. 3, 1143-1158. Crouch, E. and Hartshorn, K. (1996) In Collectins and Innate Immunity (Ezekowitz, R.A.B. et al. eds), R.G. Landes, Austin, Texas, pp. 640-645. 7 Taneva, S. et al. (1997) Biochemistry 36, 8173-8179. « Botas, C. et al. (1998) Proc. Natl Acad. Sci. USA 95, 11869-11874. 9 Reid, K.B.M. (1998) Biochim. Biophys. Acta 1408, 290-295. 10 Miyamura, K. et al. (1994) Biochem. J. 300, 237-242.

^^ ^2 ^3 ^^

Holmskov, U. et al. (1997) J. Biol. Chem. 272, 13743-13749. Miyamura, K. et al. (1994) Biochim. Biophys. Acta 1210, 303-307. Rust, K. et al. (1991) Arch. Biochem. Biophys. 290, 116-126. Kishore, U. and Reid, K.B.M. (1998) In Human Protein Data (Haeberli, A. ed.) Wiley-VCH Verlag, Weinheim.

Part 2 Serine Proteases

Clr

EC 3.4.21.41

Nicole Thielens and Gerard J. Arlaud, Jean-Pierre Ebel Institute of Structural Biology, Grenoble, France

Physicochemical properties

Illlii

•III ••li liiiiill liliill

lllllli

Human Clr is a non-covalent homodimer of M, (K) 173. Each monomer is synthesized as a single-chain proenzyme molecule of 705 amino acids including a 17 amino acid leader sequence. Activation occurs through cleavage of a single bond (R463-I464), yielding two disulfide-linked chains A and B. Mature protein: 4.9 Pl 173.0 MAK] A chain B chain Amino acids 18-463 464-705 31.2 predicted 55.3 M,(K) N-linked glycosylation sites 2(125,221) 2(514,581

^^m ^^^li^^^^il^^^^^^ll Interchain disulfide bonds

^m

1A-B

451

577

Structure Each monomer comprises an interaction region [a] derived from the Nterminal half of the A chain, and a catalytic region [y-B] comprising two CCP modules and the serine protease B domain^'^. Assembly of the Clr-Clr dimer occurs through the catalytic regions-^. The interaction regions are located at each end of the dimer and mediate calcium-dependent association with Cls within the Cls-Clr-Clr-Cls tetramer'^. A threedimensional homology model of the activated form of the Clr catalytic regions is available^.

Function Clr is a serine protease with very narrow trypsin-like specificity that is responsible for activation of the CI complex, a two-step process involving (1) Clr autoactivation, and (2) Cls cleavage by activated Clr^^^. Both reactions occur through cleavage of a single Arg-lle bond that generates two-chain active proteases. Ca'*

Tissue distribution Serum protein: 34 A^g/ml in plasma. Primary site of synthesis: hepatocytes. Secondary sites: monocytes, epithelial and endothelial cells^, cells of the central nervous system*.

Regulation of expression Unknown, but Clr and Cls are coordinately expressed.

Protein sequence 9-12 MWLLYLLVPA PTGYRVKLVF GKKEFMSQGN KLGEEDPQPQ ASGYISSLEY PYDQLQIYAN RYTTEIIKCP LHSFTAVCQD RIQYYCHEPY CGKPVNPVEQ AHTLYPKEHE SYNFEGDIAL EKIAHDLRFV QGDSGGVFAV MEEED

LFCRAGGSIP QQFDLEPSEG KMLLTFHTDF CQHLCHNYVG PRSYPPDLRC GKNIGEFCGK QPKTLDEFTI DGTWHRAMPR YKMQTRAGSR RQRIIGGQKA AQSNASLDVF LELENSVTLG RLPVANPQAC RDPNTDRWVA

IPQKLFGEVT CFYDYVKISA SNEENGTIMF GYFCSCRPGY NYSIRVERGL QRPPDLDTSS IQNLQPQYQF CKIKDCGQPR ESEQGVYTCT KMGNFPWQVF LGHTNVEELM PNLLPICLPD ENWLRGKNRM TGIVSWGIGC

SPLFPKPYPN DKKSLGRFCG YKGFLAYYQA ELQEDRHSCQ TLHLKFLEPF NAVDLLFFTD RDYFIATCKQ NLPNGDFRYT AQGIWKNEQK TNIHGRGGGA KLGNHPIRRV NDTFYDLGLM DVFSQNMFCA SRGYGFYTKV

NFETTTVITV QLGSPLGNPP VDLDECASRS AECSSELYTE DIDDHQQVHC ESGDSRGWKL GYQLIEGNQV TTMGVNTYKA GEKIPRCLPV LLGDRWILTA SVHPDYRQDE GYVSGFGVME GHPSLKQDAC LNYVDWIKKE

50 100 15 0 2 00 250 3 00 3 50 400 45 0 50 0 55 0 600 650 700

The leader sequence and the cleavage site (RI) between the A (N-terminal) and B (C-terminal) chains are underlined. The iV-linked glycosylation sites (all occupied) are indicated (N). N167 undergoes full post-translational hydroxylation.

Protein modules 1-17 18-139 140-192 193-304 305-372 375-447 448-463 464-705

Leader sequence CUB EGF-Ca2CUB CCP CCP Connecting segment Serine protease domain

Catalytic triad: H502, D557, S654.

Chromosomal location Human^^: 12pl3. The Clr and Cls genes lie in a tail-to-tail orientation, with a distance of about 9.5 kb between their 3' ends^^.

cDNA sequence^^ TGCACGAAGA TTGTACCTCC AAGTTATTTG ACAACCACTG GACCTGGAGC AGCCTGGGGA GAATTTATGT GAGAATGGGA GATGAATGTG CTGTGTCACA GAAGACAGGC TACATCTCCA ATCCGGGTGG GACCACCAGC ATTGGCGAGT GATCTGCTGT ACCGAGATCA CTGCAGCCTC CTCATAGAGG TGGCATCGTG AATGGTGACT TACTACTGCC CAAGGGGTGT ATTCCTCGGT ATCATCGGAG CACGGGCGCG CTGTATCCCA ACAAATGTGG CCGGACTACC GAAAATAGTG TTCTACGACC GCTCATGACC CTCCGGGGAA TCTCTAAAGC AACACTGATC TATGGCTTCT GAGGACTGAG CAAAAAACAA AGAAAGACCG AACCAAAGGG

CGCTGTCGGG TGGTGCCGGC GGGAGGTGAC TGATCACAGT CTTCTGAAGG GGTTCTGTGG CCCAAGGGAA CCATCATGTT CTTCCCGGAG ACTACGTTGG ATTCCTGCCA GCCTGGAGTA AGCGGGGCCT AAGTACACTG TCTGTGGGAA TCTTCACAGA TCAAGTGCCC AGTACCAGTT GGAACCAGGT CCATGCCCAG TCCGTTACAC ATGAGCCATA ACACCTGCAC GCTTGCCAGT GGCAAAAAGC GGGGCGGGGC AGGAACACGA AAGAGCTCAT GTCAGGATGA TCACCCTGGG TGGGCTTGAT TCAGGTTTGT AGAATAGGAT AGGACGCCTG GCTGGGTGGC ACACCAAAGT CCCAGAATTC CTGACCAGTT TGTGTGAAAT CCCCTTTCTT

AGAGCCCAGG CCTGTTCTGC TTCCCCTCTG CCCCACGGGA CTGCTTCTAT GCAACTGGGT CAAGATGCTG CTACAAGGGC CAAATTAGGG AGGCTACTTC GGCTGAGTGC CCCTCGGTCC CACCCTGCAC CCCCTATGAC GCAAAGGCCC TGAGTCGGGG CCAGCCCAAG CCGTGACTAC GCTGCATTCC ATGCAAGATC CACCACAATG TTACAAGATG AGCACAGGGC GTGTGGGAAG CAAGATGGGC CCTGCTGGGC AGCGCAAAGC GAAGCTAGGA GTCCTACAAT TCCCAACCTC GGGCTATGTC CCGTCTGCCC GGATGTGTTC CCAGGGGGAT CACGGGCATC GCTCAACTAC ACTAGGTTCG GTTGATAACC TCTCTTTCCT TCTTCTGAGG

ATTCAACACG AGGGCAGGAG TTCCCCAAGC TACAGGGTGA GATTATGTCA TCTCCACTGG CTGACCTTCC TTCCTGGCCT GAGGAGGATC TGTTCCTGCC AGCAGCGAGC TACCCCCCTG CTCAAGTTCC CAGCTACAGA CCCGACCTCG GACAGCCGGG ACCCTAGACG TTCATTGCTA TTCACAGCTG AAGGACTGTG GGAGTGAACA CAGACCAGAG ATTTGGAAGA CCCGTGAACC AACTTCCCCT GACCGCTGGA AACGCCTCTT AATCACCCCA TTTGAGGGGG CTCCCCATCT AGTGGCTTCG GTAGCTAATC TCTCAAAACA AGTGGGGGCG GTGTCCTGGG GTGGACTGGA AATCCAGAGA ACTAAGAGTC GTAGTCCCAT ATTGCAGAGG

GGCCTTGAGA GCTCCATTCC CTTACCCCAA AGCTCGTCTT AGATCTCTGC GCAACCCCCC ACACAGACTT ACTACCAAGC CCCAGCCCCA GTCCAGGCTA TGTACACGGA ACCTGCGCTG TGGAGCCTTT TCTATGCCAA ACACCAGCAG GCTGGAAGCT AGTTCACCAT CCTGCAAGCA TCTGCCAGGA GGCAGCCCCG CCTACAAGGC CTGGCAGCAG ATGAACAGAA CCGTGGAACA GGCAGGTGTT TCCTCACAGC TGGATGTGTT TCCGCAGGGT ACATCGCCCT GCCTCCCTGA GGGTCATGGA CACAGGCCTG TGTTCTGTGC TTTTTGCAGT GCATCGGGTG TCAAGAAAGA GCAGTGTGGA TCTATTAAAA TGATGTACTT ATATAG

AATGTGGCTC CATCCCTCAG CAACTTTGAA CCAGCAGTTT TGATAAGAAA GGGAAAGAAG CTCCAACGAG TGTGGACCTT GTGCCAGCAC TGAGCTTCAG GGCATCAGGC CAACTACAGC TGATATTGAT CGGGAAGAAC CAATGCTGTG GCGCTACACC CATCCAGAAC AGGCTACCAG TGATGGCACG AAACCTGCCT CCGTATCCAG GGAGTCTGAG GGGAGAGAAG GAGGCAGCGC CACCAACATC TGCCCACACC CCTGGGCCAC CAGCGTCCAC GCTGGAGCTG CAACGATACC GGAGAAGATT TGAGAACTGG TGGACACCCA AAGGGACCCG CAGCAGGGGC GATGGAGGAG AAAAAAAAAA TTACTGATGC TACCTGAAAC

60 12 0 180 240 3 00 3 60 42 0 48 0 540 600 660 720 7 80 840 900 9 60 102 0 1080 1140 12 00 12 60 132 0 13 8 0 1440 1500 1560 162 0 1680 1740 1800 1860 192 0 1980 2 040 2100 2160 222 0 22 80 2 340

Only the position of the final intron-exon boundary is known and the first five nucleotides of the final exon are underlined (1400-1404). The methionine initiation codon (ATG), the termination codon (TGAI, and the probable polyadenylation signal (ATTAAA) are indicated.

Genomic structure The gene structure is unknown but the serine protease domain is encoded by a single exon^^.

Accession numbers Human9-^2

X04701 M14058

Deficiency Cases of Clr deficiencies are rare, and usually combined with a Cls deficiency. They are associated with an increased susceptibility to pyogenic infection and immune complex disease such as systemic lupus erythematosus^^.

Polymorphic variants T506C; L152S^^-^2. Two common variants and one rare have been identified by isoelectric focusing of plasma samples^'''^^.

References ' Villiers, C.L. et al. (1985) Proc. Natl Acad. Sci. USA 82, 4477-4481. 2 Busby, T.F. and Ingham, K.C. (1987) Biochemistry 26, 5564-5571. 3 Lacroix, M. et al. (1997) Biochemistry 36, 6270-6282. ^ Thielens, N.M. et al. (1990) J. Biol. Chem. 265, 14469-14475. 5 Cooper, N.R. (1985) Adv. Immunol. 37, 151-216. 6 Arlaud, G.J. and Thielens, N.M. (1993) Methods Enzymol. 223, 61-82. 7 Strunk, R.C. and Colten, H.R. (1993) In Complement in Health and Disease, 2nd edition (Whaley, K., Loos, M. and Weiler, }.M. eds), Kluwer Academic Publications, Dordrecht, pp. 127-158. « Morgan, B.P. and Casque, P. (1996) Immunol. Today 17, 461-466. 9 Arlaud, C.J. and Cagnon, J. (1983) Biochemistry 22, 1758-1764. 0 Arlaud, C.J. et al. (1987) Biochem. J. 241, 711-720. ^ Journet, A. and Tosi, M. (1986) Biochem. J. 240, 783-787. 2 Leytus, S.P. et al. (1986) Biochemistry 25, 4855-4863. 3 Van Cong, N. et al. (1988) Hum. Cenet. 78, 363-368. ^ Kusumoto, H. et al. (1988) Proc. Natl Acad. Sci. USA 85, 7307-7311. 5 Endo, Y. et al. (1996) Int. Immunol. 9, 1355-1358. 6 Morgan, B.P. and Walport, M.J. (1991) Immunol. Today 12, 301-306. 7 Kamboh, M.I. and Ferrel, R.E. (1986) Am. J. Hum. Cenet. 39, 826-831. s Kamboh, M.I. et al. (1988) Hum. Cenet. 81, 93-94.

Cls

EC 3.4.21.42 Nicole Thielens and Gerard J. Arlaud, Jean-Pierre Ebel Institute of Structural Biology, Grenoble, France

Physicochemical properties Human Cls is a glycoprotein of M^ (K) 79.8. It is synthesized as a singlechain proenzyme molecule of 688 amino acids including a 15 amino acid leader sequence. Activation occurs through cleavage of a single bond (R437-I438), yielding two disulfide-linked chains A and B. Mature protein: 4.5 pi MAK] 79.8 A chain B chain Amino acids 438-688 16-437 52.2 Mr (K) observed 27.7 AT-linked glycosylation sites 2(174,406) Interchain disulfide bonds 1 A-B 425 549

Structure Cls comprises an interaction region [a] corresponding to the N-terminal half of the A chain, and a catalytic region [y-B] comprising two CCP modules and the serine protease B domain^-^. A three-dimensional homology model of the activated form of the Cls catalytic region is available^.

Function Cls is a highly specific serine protease that mediates proteolytic activity of the CI complex towards its substrates C4 and C2. It cleaves a single Arg-Ala bond in C4 to yield C4a and C4b, and a single Arg-Lys bond in C2 to yield C2a and C2b^«.

Tissue distribution Serum protein: 31 ^g/ml in plasma. Primary site of synthesis: hepatocytes. Secondary sites: monocytes, epithelial and endothelial cells^, cells of the central nervous system^*^.

Regulation of expression Unknown, but Clr and Cls are coordinately expressed.

Protein sequence^^-^^ MWCIVLFSLL HLYFTHLDIE VPYNKLQVIF IGGYFCSCPP RCEYQIRLEK CGHGFPGPLN NSVWEPAKAK KLKCQPVDCG YHCAGNGSWV WQVFFDNPWA KMLTPEHVFI PGTSSDYNLM EKPTADAEAY GLVSWGPQCG

AWVYAEPTMY LSENCAYDSV KSDFSNEERF EYFLHDDMKN GFQWVTLRR lETKSNALDI YVFRDWQIT IPESIENGKV NEVLGPELPK GGALINEYWV HPGWKLLEVP DGDLGLISGW VFTPNMICAG TYGLYTRVKN

GEILSPNYPQ QIISGDTEEG TGFAAYYVAT CGVNCSGDVF EDFDVEAADS IFQTDLTGQK CLDGFEWEG EDPESTLFGS CVPVCGVPRE LTAAHWEGN EGRTNFDNDI GRTEKRDRAV GEKGMDSCKG YVDWIMKTMQ

AYPSEVEKSW RLCGQRSSNN DINECTDFVD TALIGEIASP AGNCLDSLVF KGWKLRYHGD RVGATSFYST VIRYTCEEPY PFEEKQRIIG REPTMYVGST ALVRLKDPVK RLKAARLPVA DSGGAFAVQD ENSTPRED

DIEVPEGYGI PHSPIVEEFQ VPCSHFCNNF NYPKPYPENS VAGDRQFGPY PMPCPKEDTP CQSNGKWSNS YYMENGGGGE GSDADIKNFP SVQTSRLAKS MGPTVSPICL PLRKCKEVKV PNDKTKFYAA

50 100 15 0 2 00 2 50 3 00 3 50 400 45 0 5 00 550 60 0 650

The leader sequence and the cleavage site (RI) between the A (N-terminal) and B (C-terminal) chains are underlined. N-linked glycosylation sites (all occupied) are indicated (N). N149 undergoes partial post-translational hydroxylation^^.

Protein modules 1-15 Leader sequence CUB 16-128 129-174 EGF-Ca2CUB 175-289 CCP 290-355 358-422 CCP Connecting segment 423-437 438-688 Serine protease domain Catalytic triad: H475, D529, S632.

exon 2/3 exon 3/4 exon 5 exon 617 exon 8/9 exon 10/11 exon 12 exon 12

Chromosomal location Human^5. i2pi3. The Clr and Cls genes lie in a tail-to-tail orientation, with a distance of about 9.5 kb between their 3' ends^^.

I

cDNA sequence^^ GGGCCGGAGT GGGGTTGCCC CTTCTGAAGG CAGGGTGGAC TTATGCTGAG CAGTGAGGTA CTTCACCCAT CTCAGGAGAC TCCAATTGTG CTTTTCCAAT TGAATGCACA TTACTTCTGC TAATTGCAGT CAAACCATAT AGTGGTGGTG CTGCCTTGAC TGGATTCCCT AACTGATCTA CTGCCCTAAG TAGAGATGTG TGCAACATCT ATGTCAACCT AGAGAGCACT GGAAAATGGA GCTGGGCCCG AGAAAAACAG CTTCTTTGAC TGCTCATGTT GACCTCACGG ATGGAAGCTG GCGGCTGAAA CTCTTCCGAC AGAGAAGAGA AAAATGCAAA TCCTAACATG TGGGGCCTTT GTCCTGGGGG CTGGATAATG CCCACCAGCC CATTATTTCA TGAGACGCCT TCTGACTCCT ACTCCTTTCT CAAAATTCCA TATAAGC

TCCTGCAGAG AGCATGCCCA CTCCAAAGTC AAATCGCCAG CCTACCATGT GAGAAATCTT CTGGACATTG ACTGAAGAAG GAAGAGTTCC GAAGAGCGTT GATTTTGTAG TCCTGCCCCC GGGGATGTAT CCAGAGAACT ACCTTGCGGA AGTTTAGTTT GGGCCTCTAA ACAGGGCAAA GAAGACACTC GTGCAGATAA TTCTATTCGA GTGGACTGTG TTGTTTGGTT GGAGGTGGGG GAGCTGCCGA AGGATAATTG AACCCATGGG GTGGAGGGAA CTGGCAAAAT CTGGAAGTCC GACCCAGTGA TACAACCTCA GATCGTGCTG GAAGTGAAAG ATCTGTGCTG GCTGTACAGG CCCCAGTGTG AAGACTATGC TCTCCAAGGG TCATGACTGA TGCTAGAGGT TGGGGTCCTT TGCACTATTC TTTACTTGAT

GGAGCGTCAA CTGGCAGGAG CGGAGTGCAG AGATGTGGTG ATGGGGAGAT GGGACATAGA AGCTGTCAGA GGAGGCTCTG AAGTCCCATA TTACGGGGTT ATGTCCCTTG CGGAATATTT TCACTGCACT CAAGGTGTGA GAGAAGATTT TTGTTGCAGG ATATTGAAAC AAAAGGGCTG CCAATTCTGT CCTGTCTGGA CTTGTCAAAG GCATTCCTGA CTGTCATCCG AGTATCACTG AATGTGTTCC GAGGATCCGA CTGGTGGAGC ACAGGGAGCC CCAAGATGCT CAGAAGGACG AAATGGGACC TGGATGGGGA TTCGCCTCAA TGGAGAAACC GAGGAGAGAA ATCCCAATGA GGACCTATGG AGGAAAATAG TGGTGACCAA AAGAAGACAC AGAGTTTGAT TCCCCGGAGT CACAGGGATA CATTCTCAGT

GGCCCTGTGC AGAGGGAACT AAAGCCAGGA CATTGTCCTG CCTGTCCCCT AGTTCCTGAA GAACTGTGCG TGGACAGAGG CAACAAACTC TGCTGCATAC TAGCCACTTC CCTCCATGAT GATTGGGGAG ATACCAGATC TGATGTGGAA AGATCGGCAA CAAGAGTAAT GAAACTTCGC TTGGGAGCCT TGGGTTTGAA CAATGGAAAG ATCCATTGAG CTACACTTGT TGCTGGTAAC AGTCTGTGGA TGCAGATATT GCTCATTAAT AACAATGTAT CACTCCTGAG AACCAATTTT CACCGTCTCT CCTGGGACTG GGCGGCAAGG CACAGCAGAT GGGCATGGAT CAAGACCAAA GCTCTACACA CACCCCCCGT TGCATTACCT GAGCGAATGA CATAGAATTG ACCTATTGTA CCTTAATTCT ATCCACTGTC

TGCTGTCCCT GACCCACTTG CCAAGAGACA TTTTCACTTT AACTATCCTC GGGTATGGGA TATGACTCAG AGCAGTAACA CAGGTGATCT TATGTTGCCA TGCAACAATT GACATGAAGA ATTGCAAGTC CGGTTGGAGA GCAGCTGACT TTTGGTCCTT GCTCTTGATA TATCATGGAG GCGAAGGCAA GTTGTGGAGG TGGAGTAATT AATGGTAAAG GAGGAGCCAT GGGAGCTGGG GTCCCCAGAG AAAAACTTCC GAGTACTGGG GTTGGGTCCA CATGTGTTTA GATAATGACA CCCATCTGCC ATCTCAGGCT TTACCTGTAG GCAGAGGCCT AGCTGTAAAG TTCTACGCAG CGGGTAAAGA GAGGACTAAT TCTGTTCCTT TTTAAATAGA TGCTGGTCAT GATAACACTA TTGTTTCCTC TATGTACAAT

GGGGGCCAGA CTCCTACCAG GGCAGCTCAC TGGCATGGGT AGGCATATCC TTCACCTCTA TGCAGATAAT ATCCCCACTC TTAAGTCAGA CAGACATAAA TCATTGGTGG ATTGCGGAGT CCAATTATCC AAGGGTTCCA CAGCGGGAAA ACTGTGGTCA TCATCTTCCA ATCCAATGCC AATATGTCTT GACGTGTTGG CCAAACTGAA TTGAAGACCC ATTACTACAT TGAATGAGGT AACCCTTTGA CCTGGCAAGT TGCTGACGGC CCTCAGTGCA TTCATCCGGG TTGCACTGGT TACCAGGCAC GGGGCCGAAC CTCCTTTAAG ATGTTTTCAC GGGACAGTGG CTGGCCTGGT ACTATGTTGA CCAGATACAT ATGATATTCT ACTTGATTGT ACATTTGTGG TGGGTGGGGC TTTACCTGTT AAAGGATGTT

60 12 0 180 240 3 00 3 60 42 0 4 80 540 6 00 660 72 0 780 840 9 00 9 60 102 0 10 8 0 114 0 12 0 0 12 60 13 2 0 13 8 0 144 0 150 0 15 60 162 0 1680 17 4 0 18 0 0 18 6 0 192 0 19 8 0 2 04 0 2100 216 0 222 0 22 8 0 23 40 24 0 0 24 60 2 52 0 2 58 0 2 64 0

The first five nucleotides in each exon are underHned to indicate intron-exon boundaries. Exon 2 is only five nucleotides long (203-207) and contains the intiation codon. To avoid confusion, this exon is not indicated with additional underlining, but is indicated by the double-underlined methionine initiation codon (ATG). The termination codon (TAA) and the polyadenylation signal (AATAAA) are indicated.

Genomic structure The Cls gene spans about 10.5 kb, and contains 12 exons. The whole serine protease domain and the preceding short connecting segment are encoded by a single exon^*^'^^.

i III

I I III I I 1kb

Accession numbers Human^^-^^ Hamster^*

X06596 M18767 J04080 X16160

Deficiency Cases of Cls deficiencies are rare, usually partial, and combined with a Clr deficiency. They are associated with an increased susceptibility to pyogenic infection and immune complex disease such as systemic lupus erythematosus^^.

Polymorphic variants No polymorphism has been identified at the DNA or protein level. N406 in human Cls is linked to either biantennary or triantennary complex-type oligosaccharides, giving rise to three major types of Cls molecules of M, (K) 79.3, 79.9, and 80. P*^. One common and two uncommon variants have been identified by isoelectric focusing of plasma samples^^.

References ' 2 3 4 5 6 7 « ^

Villiers, C.L. et al. (1985) Proc. Natl Acad. Sci. USA 82, 4477-4481. Busby, T.F. and Ingham, K.C. (1988) Biochemistry 27, 6127-6135. Medved, L.V. et al. (1989) Biochemistry 28, 5408-5414. Thielens, N.M. et al. (1990) J. Biol. Chem. 265,14469-14475. Rossi, V. et al. (1995) Biochemistry 34, 7311-7321. Cooper, N.R. (1985) Adv. Immunol. 37, 151-216. Arlaud, G.J. and Thielens, N.M. (1993) Methods Enzymol. 223, 61-82. Arlaud, G.J. et al. (1998) Adv. Immunol. 69, 249-307. Strunk, R.C. and Colten, H.R. (1993) In Complement in Health and Disease, 2nd edition (Whaley, K., Loos, M. and Weiler, J.M. eds), Kluwer Academic Publications, Dordrecht, pp. 127-158.

0 Morgan, B.P. and Gasque, P. (1996) Immunol. Today 17, 461-466. Mackinnon, CM. et al. (1987) Eur. J. Biochem. 169, 547-553. Tosi, M. et al. (1987) Biochemistry 26, 8516-8524. Kusumoto, H. et al. (1988) Proc. Natl Acad. Sci. USA 85, 7307-7311. Thielens, N.M. et al. (1990) Biochemistry 29, 3570-3578. Van Cong, N. et al. (1988) Hum. Genet. 78, 363-368. Tosi, M. et al. (1989) J. Mol. Biol. 208, 709-714. Endo, Y. et al. (1998) J. Immunol. 161, 4924-4930. Kinoshita, H. et al. (1989) FEES Lett. 250, 411-415. Morgan, B.P. and Walport, M.J. (1991) Immunol. Today 12, 301-306. Petillot, Y. et al. (1995) FEES Lett. 358, 323-328. Kamboh, M.I. and Ferrell, R.E. (1987) J. Immunogenet. 14, 231-238.

MASP-1

•

Teizo Fujita, Yuichi Endo and Misao Matsushita, Department of Biochemistry, Fukushima Medical University, 1-Hikariga-oka, Fukushima 960-1295, Japan Other names Mannose (mannan)-binding lectin (MBL)-associated serine protease 1, PI00.

Physicochemical properties MASP-l is synthesized as a single-chain polypeptide of 699 amino acids including a 19 amino acid leader sequence. In the unactivated proenzyme form, MASP-1 is a single polypeptide of Mr (K) 93, while in the active form, it consists of two polypeptides (heavy and light chains) linked by a disulfide bond. Mature protein: pi (predicted) Mr(K) Amino acids Mr (K) predicted observed iV-linked glycosylation sites Potential Interchain disulfide bond 1 heavy-light

•

5.18 93 Heavy chain 20-448 49 66

Light chain 449-699 28 31

4(49,178,385,407)

0

436

572

Structure None known.

Function Proenzyme MASP-1 forms a complex with MASP-2 and MBL and circulates in blood*. Upon binding of the MBL-MASP complex to appropriate carbohydrates via MBL, MASP-1 is cleaved between R448 and 1449 converting to the active serine protease form consisting of two polypeptides (heavy and light chains)2. In addition to MASP-1, MASP-2 is also converted to the active form and the MBL-MASP complex then activates C4 and C2 resulting in the formation of the C3 convertase, C4b2a. Although the precise functional roles of MASP-1 in the complex remain to be elucidated it is able to cleave C2 in the fluid phase. MASP-1 can also cleave C3 to give C3a and C3b, the latter of which participates in the formation of the C3 convertase, C3bBb3.

Tissue distribution Serum protein: Gjng/ml in serum^. (MASP-1 exists as MBL-bound and MBLunbound forms in serum.) Primary site of synthesis: liver^.

Regulation of expression MASP-l is upregulated by IL-6 and by dexamethasone in the rat hepatocytes^.

Protein sequence^'^ MRWLLLYYAL TVPDGFRIKL TPGQEWLSP LSCDHYCHNY DFPNPYPKSS VGPKVLGPFC CPELQPPVHG KDGTWSNKIP YYKMLNNNTG NGRPAQKGTT PTLRDSDLLS NDVALVELLE LMEIEIPIVD VTLNRERGQW

CFSLSKASAH YFMHFNLESS GSFMSITFRS IGGYYCSCRF ECLYTIELEE GEKAPEPIST KIEPSQAKYF TCKIVDCRAP lYTCSAQGVW PWIAMLSHLN PSDFKIILGK SPVLNAFVMP HSTCQKAYAP YLVGTVSWGD

TVELNNMFGQ YLCEYDYVKV DFSNEERFTG GYILHTDNRT GFMVNLQFED QSHSVLILFH FKDQVLVSCD GELEHGLITF MNKVLGRSLP GQPFCGGSLL HWRLRSDENE ICLPEGPQQE LKKKVTRDMI DCGKKDRYGV

IQSPGYPDSY ETEDQVLATF FDAHYMAVDV CRVECSDNLF IFDIQDHPEV SDNSAENRGW TGYKVLKDNV STRNNLTTYK TCLPVCGLPK GSSWIVTAAH QHLGVKHTTL GAMVIVSGWG CAGEKEGGKD YSYIHHNKDW

PSDSEVTWNI CGRETTDTEQ DECKEREDEE TQRTGVITSP PCPYDYIKIK RLSYRAAGNE EMDTFQIECL SEIKYSCQEP FSRKLMARIF CLHQSLDPGD HPQYDPNTFE KQFLQRFPET ACAGDSGGPM IQRVTGVRN

50 100 150 2 00 2 50 3 00 3 50 400 45 0 500 55 0 60 0 65 0

T h e leader sequence and the cleavage site (RI) to generate the heavy and light chains are underlined. Potential N-linked glycosylation sites are indicated (N).

Protein modules Leader sequence 1-19 20-139 CUB 140-183 EGF-Ca2CUB 184-297 298-363 CCP 364-434 CCP Serine protease domain 435-699 Catalytic triad: H490, D552, S646.

D

Chromosomal location Human^: 3q27-28.

exon exon exon exon exon exon exon

1/2 2/3 4 5/6 7/8 9/10 11-16

I

cDNA sequence5,7 ATTCCGGCAC CGGGCAGCCG TGCTTCTCCC ATCCAGTCGC ACTGTCCCAG TACCTTTGTG TGTGGCAGGG GGCTCCTTCA TTTGATGCCC CTGTCCTGTG GGCTACATCC ACTCAAAGGA GAATGCCTGT ATATTTGACA GTTGGTCCAA CAGAGCCACA AGGCTCTCAT AAAATCGAGC ACAGGCTACA AAGGATGGGA GGAGAGCTGG TCTGAGATCA ATATATACCT ACCTGCCTTC AATGGACGCC GGGCAGCCCT TGCCTCCACC CCTTCTGAGT CAGCATCTCG AATGACGTGG ATCTGTCTGC AAGCAGTTCT CACAGCACCT TGTGCTGGGG GTGACCCTGA GACTGTGGGA ATCCAGAGGG TGTGGGCAGT CCTCCCATCC CCCTTTCACT GGCTCTATCA GTTTCTTCAT AGATTAAGTA ATGCACCTTA GCCTGGGTCT TGCCCTTAAA GCTCATCCCT

AGGGACACAA GGAGCACCCA TGTCAAAGGC CTGGTTATCC ATGGGTTTCG AATATGACTA AGACCACAGA TGTCCATCAC ACTACATGGC ACCACTACTG TCCACACAGA CTGGGGTGAT ATACCATCGA TTCAGGACCA AAGTTTTGGG GTGTCCTGAT ACAGGGCTGC CCTCCCAAGC AAGTGCTGAA CGTGGAGTAA AACACGGGCT AATACTCCTG GTTCTGCCCA CAGTGTGTGG CAGCCCAGAA TCTGCGGAGG AGTCACTCGA TCAAAATCAT GCGTCAAACA CTCTGGTGGA CTGAGGGACC TGCAAAGGTT GCCAGAAGGC AGAAGGAAGG ATAGAGAAAG AGAAGGACCG TCACCGGAGT CAGTAGCAGA CCCCTCCTTC CTCTTTAAAG CCTTACTAGT AAGATGGAAA ATAGATGCAT GCAGAAGGTC TAGCATTGAT TGCTGTATGC AAAAAAGTAA

ACAAGCTCAC AGGCAGGAAA TTCAGCCCAC AGACTCCTAT GATCAAGCTT TGTGAAGGTA CACAGAGCAG TTTCCGGTCA TGTGGATGTG CCACAACTAC CAACAGGACC CACCAGCCCT GCTGGAGGAG TCCTGAGGTG GCCTTTCTGT CCTGTTCCAT AGGAAATGAG CAAGTATTTC GGATAATGTG CAAGATTCCC GATCACCTTC TCAGGAGCCC AGGAGTCTGG GCTCCCCAAG AGGCACCACT CTCCCTTCTA TCCGGGAGAT CCTGGGCAAG CACCACTCTC GCTGTTGGAG GCAGGAGGAA CCCAGAGACC TTATGCCCCG GGGAAAGGAC AGGCCAGTGG CTACGGAGTA GAGGAACTGA GGACGATCCT GGCCTATCCA AGATGGAGCA TTGGAGTGCT TGCTATACCT AGCACTTAAC GATGTGTCTA CAGTGACACA TTTTTTGCCA AACAGACAAA

CCAACAAAGC ATGAGGTGGC ACCGTGGAGC CCCAGTGATT TACTTCATGC GAAACTGAGG ACTCCCGGCC GATTTCTCCA GACGAGTGCA ATTGGCGGCT TGCCGAGTGG GACTTCCCAA GGTTTCATGG CCCTGCCCCT GGAGAGAAAG AGTGACAACT TGCCCAGAGC TTCAAAGACC GAGATGGACA ACCTGTAAAA TCTACAAGGA TATTACAAGA ATGAATAAAG TTCTCCCGGA CCCTGGATTG GGCTCCAGCT CCGACCCTAC CATTGGAGGC CACCCCCAGT AGGCCAGTGG GGAGCCATGG CTGATGGAGA CTGAAGAAGA GCCTGTGCGG TACCTGGTGG TACTCTTACA ATTTGGCTCC CCGATGAAAG TTACTGGGCA AGAGAGTGGT GGGCAGGTGA TACCTACCTC AGAGTGCATA CCAGGCAGCG CCTCTCCCCT CCGTGCAACT AAAAAAAAAA

CAAGCTGGGA TGCTTCTCTA TAAACAATAT CAGAGGTGAC ACTTCAACTT ACCAGGTGCT AGGAGGTGGT ATGAGGAGCG AGGAGAGGGA ACTACTGCTC AGTGCAGTGA ACCCTTACCC TCAACCTGCA ATGACTACAT CCCCAGAACC CGGCAGAGAA TACAGCCTCC AAGTGCTCGT CATTCCAGAT TTGTAGACTG ACAAGCTCAC TGCTCAACAA TATTGGGGAG AGCTGATGGC CCATGCTGTC GGATCGTGAC GTGATTCAGA TCCGGTCAGA ATGATCCCAA TGAATGCCTT TCATCGTCAG TTGAAATCCC AAGTGACCAG GTGACTCTGG GCACTGTGTC TCCACCACAA TCAGCCCCAG CAGCCATTTC ATAGAGCAGG CAGAACACAG CTTCATCTCT GTAAAAGTCT GCATACACTG AAGCTCTCTT CAACCTTGAC TGCCCAACAT AAAAAAAAA

GGACCAAGGC TTATGCTCTG GTTTGGCCAG TTGGAATATC GGAATCCTCC GGCAACCTTC CCTCTCCCCT TTTCACAGGC GGACGAGGAG CTGCCGCTTC CAACCTCTTC CAAGAGCTCT GTTTGAGGAC CAAGATCAAA CATCAGCACC CCGGGGCTGG TGTCCATGGG CAGCTGTGAC TGAGTGTCTG TAGAGCCCCA CACATACAAG TAACACAGGT AAGCCTACCC CAGGATCTTC ACACCTGAAT CGCCGCACAC CTTGCTCAGC TGAAAATGAA CACATTCGAG CGTGATGCCC CGGCTGGGGG GATTGTTGAC GGACATGATC AGGCCCCATG CTGGGGTGAT CAAGGACTGG CACCACCAGC TCCTTTCCTT TATCTTCACC GCCGAATCCA TCGAACTTCA GATGAGGAAA TTTTCAATAA ACAAACCCCT CATCTCCATC CAATCTTCAC

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760

The first five nucleotides in each exon are underHned to indicate intron-exon boundaries. The methionine initiation codon (ATGI and the termination codon (TGA) are indicated.

Genomic structure^^^ The gene spans more than 50 kb and contains at least 16 exons as illustrated below. 2kb

16

I

I

h

I I II

"hf

I II I II

Accession numbers Human ^.7,8,9 Mouse^^ Rat (partial)^ Xenopus^

D

cDNA D28593 D17525 D16492 AF004661 D83276

Genomic D61690-5 AB10813-22

Deficiency No known deficiencies.

Polymorphic variants No known polymorphisms.

References 1 Matsushita, M. et al. (1998) Immunobiology 199, 340-347. 2 Matsushita, M. and Fujita, T. (1992) J. Exp. Med. 176, 1497-1502. 3 Matsushita, M. and Fujita, T. (1995) Immunobiology 194, 443-448. ^ Terai, I. et al. (1997) Clin. Exp. Immunol. 110, 317-323. 5 Sato, T. et al. (1994) Int. Immunol. 6, 665-669. 6 Knittel, T. et al. (1997) Lab. Invest. 77, 221-230. ^ Takada, F. et al. (1993) Biochem. Biophys. Res. Commun. 196, 1003-1009. 8 Endo, Y. et al. (1996) Int. Immunol. 8, 1355-1358. 9 Endo, Y. et al. (1998) J. Immunol. 161, 4924-4930. 10 Takayama, Y. et al. (1994) J. Immunol. 152, 2308-2316.

MASP-2 Steen V. Petersen and Jens C. Jensenius, Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark Other n a m e s Mannose (mannan)-binding lectin (MBL)-associated serine protease component of the mouse RaRF complex^

2,

Physicochemical properties MASP-2 is synthesized as a single-chain molecule of 686 amino acids including a leader sequence of 15 amino acids yielding a mature protein of 671 residues of M^ (K) 74.2. The sequence presents a peptide bond which is likely to be cleaved upon activation (predicted in analogy to C l r and Cls). The resulting A chain (N-terminal 429 residues) and B chain (C-terminal 242 residues) are predicted to be held together with a disulfide bond.

Mature protein: pP M, (K) predicted observed Amino acids M, (K) predicted observed N-linked glycosylation sites Interchain disulfide bonds 1 A-B

5.43 (calculated) 74.2 76.0 A chain 16-444 47.7 52 none

B chain 445-68( 26.5 31

434

552

Structure The structure of MASP-2 has not been determined.

Function MASP-2 is a trypsin-like serine protease^ and presumably plays an important role in the initiation of the MBL complement activation pathway. It is activated upon binding of the MBL-MASP complex to oligosaccharides (unpublished observation) and subsequently cleaves C4, generating C4a and C4b. No direct evidence of MASP-2 being a C2-cleaving protease has yet been published, but it seems most likely that this is the case since activated MASP-2 blotted onto a PVDF (polyvinylidene difluoride) membrane is capable of generating a C3 convertase upon incubation with serum (unpublished observation). While MASP-1 reportedly is capable of directly cleaving C3, MASP-2 is considered to be the C4b2b-generating protease of the lectin pathway. The function of the N-terminal part has not been assessed. It might be speculated that this part of the molecule participates in intermolecular interactions.

Tissue distribution Serum protein: concentration unknown. Primary site of synthesis: likely to be the liver (unpublished observation). Secondary sites: MASP-2 RNA was not identified in tissues from heart, brain, placenta, lung, kidney and pancreas by northern blotting (unpublished observation).

Regulation of expression This has not been investigated.

I

Protein sequence^ MRLLTLLGLL APPGYRLRLY PGKDTFYSLG TCDHHCHNHL YPRPYPKLSS DREEHGPFCG PYPMAPPNGH DGSWDRPMPA YTMKVNDGKY AKPGDFPWQV TLKRLSPHYT ICLPRKEAES AYEKPPYPRG GIVSWGSMNC

CGSVATPLGP FTHFDLELSH SSLDITFRSD GGFYCSCRAG CTYSISLEEG KTLPHRIETK VSPVQAKYIL CSIVDCGPPD VCEADGFWTS LILGGTTAAG QAWSEAVFIH FMRTDDIGTA SVTANMLCAG GEAGQYGVYT

KWPEPVFGRL LCEYDFVKLS YSNEKPFTGF YVLHRNKRTC FSVILDFVES SNTVTITFVT KDSFSIFCET DLPSGRVEYI SKGEKSLPVC ALLYDNWVLT EGYTHDAGFD SGWGLTQRGF LESGGKDSCR KVINYIPWIE

ASPGFPGEYA SGAKVLATLC EAFYAAEDID SALCSGQVFT FDVETHPETL DESGDHTGWK GYELLQGHLP TGPGVTTYKA EPVCGLSART AAHAVYEQKH NDIALIKLNN LARNLMYVDI GDSGGALVFL NIISDF

NDQERRWTLT GQESTDTERA ECQVAPGEAP QRSGELSSPE CPYDFLKIQT IHYTSTAQPC LKSFTAVCQK VIQYSCEETF TGGRIYGGQK DASALDIRMG KWINSNITP PIVDHQKCTA DSETERWFVG

50 10 0 150 2 00 2 50 3 00 3 50 40 0 450 500 55 0 60 0 65 0

The leader sequence and the activation peptide bond (RI) are underlined.

Protein modules^

Leader peptide 1-15 CUB 16-135 136-182 EGF-Ca2183-292 CUB CCP 293-362 CCP 363-431 432-444 Linker peptide 445-686 Serine protease domain Catalytic triad: H483, D532, S633.

Chromosomal location Human^: chromosome 1.

cDNA sequence^ CTCGTGCAAT CTTCTGTGTG CGCCTGGCAT CTGACTGCAC TCCCACCTCT CTGTGCGGGC CTGGGCTCCA GGGTTCGAGG GCGCCCACCT GCAGGCTACG TTCACCCAGA TCCAGTTGCA GAGTCCTTCG CAAACAGACA ACAAAAAGCA TGGAAGATCC GGCCACGTTT GAGACTGGCT CAGAAAGATG CCTGATGATC AAAGCTGTGA AAATATGTGT GTCTGTGAGC CAAAAGGCAA GCAGGTGCAC AAACATGATG TATACACAAG TTTGACAATG ACGCCTATTT ACTGCATCTG GACATACCGA AGGGGAAGTG TGCAGAGGTG GTGGGAGGAA TACACAAAAG GCGTGTCTGC CGTGGCTCGA AACAGACTCC CCATTGACTC ATGTCACTGC GTTGGCATTT AAAAAAAAAA

TCGGCACGAG GCTCGGTGGC CCCCCGGCTT CCCCCGGCTA GCGAGTACGA AGGAGAGCAC GCCTGGACAT CCTTCTATGC GCGACCACCA TCCTGCACCG GGTCTGGGGA CTTACAGCAT ATGTGGAGAC GAGAAGAACA ACACGGTGAC ACTACACGAG CACCTGTGCA ATGAGCTTCT GATCTTGGGA TACCCAGTGG TTCAGTACAG GTGAGGCTGA CTGTTTGTGG AACCTGGTGA TTTTATATGA CATCCGCCCT CCTGGTCTGA ACATAGCACT GTCTGCCAAG GATGGGGATT TTGTTGACCA TAACTGCTAA ACAGCGGAGG TAGTGTCCTG TTATTAACTA AGTCAAGGAT GAAGCATTCA AGGTGAGGCT AAGGGGACAT TCAAATTACA CTGTAAACTG AAAAA

GCTGGACGGG CACCCCCTTG TCCAGGGGAG CCGCCTGCGC CTTCGTCAAG AGACACGGAG TACCTTCCGC AGCCGAGGAC CTGCCACAAC TAACAAGCGC GCTCAGCAGC CAGCCTGGAG ACACCCTGAA TGGCCCATTC CATCACCTTT CACAGCGCAG AGCCAAATAC GCAAGGTCAC CCGGCCAATG CCGAGTGGAG CTGTGAAGAG TGGATTCTGG ACTATCAGCC TTTTCCTTGG CAACTGGGTC GGACATTCGA AGCTGTTTTT GATTAAATTG AAAAGAAGCT AACCCAAAGG TCAAAAATGT CATGCTTTGT GGCACTGGTG GGGTTCCATG TATTCCCTGG TCTTCATTTT TCATTACTGT GCTGTCATTT AAACCACGAG TTTCATTACC CCTGTCCATG

CACACCATGA GGCCCGAAGT TATGCCAATG CTCTACTTCA CTGAGCTCGG CGGGCCCCTG TCCGACTACT ATTGACGAGT CACCTGGGCG ACCTGCTCAG CCTGAATACC GAGGGGTTCA ACCCTGTGTC TGTGGGAAGA GTCACAGATG CCTTGCCCTT ATCCTGAAAG TTGCCCCTGA CCCGCGTGCA TACATCACAG ACCTTCTACA ACGAGCTCCA CGCACAACAG CAAGTCCTGA CTAACAGCTG ATGGGCACCC ATACATGAAG AATAACAAAG GAATCCTTTA GGTTTTCTTG ACTGCTGCAT GCTGGCTTAG TTTCTAGATA AATTGTGGGG ATCGAGAACA TAGAAATGCC GGACATGGCA CTCCACTTGC AGTGACAGTC TTAAAAAGCC CTCTTTGTTT

GGCTGCTGAC GGCCTGAACC ACCAGGAGCG CCCACTTCGA GGGCCAAGGT GCAAGGACAC CCAACGAGAA GCCAGGTGGC GTTTCTACTG CCCTGTGCTC CACGGCCGTA GTGTCATTCT CCTACGACTT CATTGCCCCA AATCAGGAGA ATCCGATGGC ACAGCTTCTC AATCCTTTAC GCATTGTTGA GTCCTGGAGT CAATGAAAGT AAGGAGAAAA GAGGGCGTAT TATTAGGTGG CTCATGCCGT TGAAAAGACT GTTATACTCA TTGTAATCAA TGAGGACAGA CTAGAAATCT ATGAAAAGCC AAAGTGGGGG GTGAAACAGA AAGCAGGTCA TAATTAGTGA TGTGAAGACC GTTGTTGCTC CAGTTTAATT ATCTTTGCCC AGTCTCTTTT TTAAACTTGT

CCTCCTGGGC TGTGTTCGGG GCGCTGGACC CCTGGAGCTC GCTGGCCACG TTTCTACTCG GCCGTTCACG CCCGGGAGAG CTCCTGCCGC CGGCCAGGTC TCCCAAACTC GGACTTTGTG TCTCAAGATT CAGGATTGAA CCACACAGGC GCCACCTAAT CATCTTTTGC TGCAGTTTGT CTGTGGCCCT GACCACCTAC GAATGATGGT ATCACTCCCA ATATGGAGGG AACCACAGCA CTATGAGCAA ATCACCTCAT TGATGCTGGC TAGCAACATC TGACATTGGA AATGTATGTC ACCCTATCCA CAAGGACAGC GAGGTGGTTT GTATGGAGTC TTTTTAACTT TTGGCAGCGA CACCCAAAAA CCAGCCTTAC ACCCAGTGTA CATACTGGCT TCTTATTGAA

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460

The methionine initiation codon (ATG), the termination codon (TAAJ and the polyadenylation signal (CATAAA) are double underlined.

Accession numbers Human2

D

Y09926

Deficiency No deficiency has been identified.

D

Polymorphic variants No polymorphic variants have been identified.

References 1 Ji, Y.H. et al. (1988) J. Immunol. 141, 4271-4275. 2 Thiel, S. et al. (1997) Nature 386, 506-510. 5 Stover, CM. et al. (1999) J. Immunol. 162, 3481-3490.

Factor D

C3 convertase activator, adipsin, EC 3.4.21.46

Jurg Schifferli and Sylvie Miot, Department of Immunonephrology, Kantonsspital Basel, Basel, Switzerland

Physicochemical properties Factor D is synthesized as a precursor single-chain molecule of 246 amino acids including a 13 amino acids partial signal peptide and a five amino acid potential activation peptide. Mature protein: pP'2 74 Mr(Kp predicted 24.4 observed 25 N-linked glycosylation sites'^ none In rodents, adipsin is heavily glycosylated.

Structure Factor D is the first serine protease whose three-dimensional structure has been determined. The structure was solved at 0.2 n m resolution by a combination of multiple isomorphous and molecular replacement methods^. The structure shows the characteristic chymotrypsin structural fold, but presents certain key differences from other serine proteases in the conformation of residues of the catalytic triad and the substrate-binding region^. Regulation of factor D activity depends on reversible conformational changes for expression and control of catalytic activity. These changes are believed to be induced by the single natural substrate, C3bB, and to result in alignment of the catalytic triad, the specificity pocket and the non-specific substrate-binding site, all of which have atypical conformations^. Mutational studies identified structural elements responsible for these structural features of factor D*'^.

Function Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor activation by a serpin for its controP. Factor D is a protein of the alternative complement pathway. It is a highly specific serine protease and cleaves factor B bound to C3b, generating the C3bBb enzyme which is the alternative pathway C3 convertase. It cleaves R259-K260 of substrate factor B only when B is complexed with activated C3. It is the rate-limiting enzyme of the alternative pathway. Factor D may play some particular role in fatty tissue distinct from its role as a complement protein.

Tissue distribution Serum protein: 2 /xg/ml in plasma. Primary site of synthesis: adipose tissue^*^. Secondary sites: monocytic macrophage lineage^^.

Factor D

Regulation of expression In astroglioma cell line^^, IFN7 exerts no effect on the level of factor D mRNA. In mouse, adipsin is deficient in several genetically and acquired models of obesity^2,i3 insulin reduces adipsin in vitro and is negatively correlated with adipsin expression in vivo^"^.

Protein sequence AVLVLLGAAA CAARPRGRIL GGREAEAHAR

PYMASVQLNG AHLCGGVLVA

50

EQWVLSAAHC

LEDAADGKVQ VLLGAHSLSQ

PEPSKRLYDV LRAVPHPDSQ

100

PDTIDHDLLL

LQLSEKATLG

PAVRPLPWQR VDRDVAPGTL CDVAGWGIVN

HAGRRPDSLQ HVLLPVLDRA TCNRRTHHDG AITERLMCAE

SNRRDSCKGD

SGGPLVCGGV L E G W T S G S R

IDSVLA

VCGNRKKPGI YTRVASYAAW

150 2 00

The leader sequence is underlined and the potential activation peptide is double-underlined (RPRGR). Conflicting sequence data (above sequence stated first): I19M^^ HISW, M33V^^ H42E^6,i7 G45A^«, Q 5 2 R ^ S56T^^ D66G^^ HSLS76-79 -> THLP«, HS76-77 ^ ST^^ deletion LY87-88^^ D89E^^ Q129G^ TCNRRTHHDGAITE171-184^KCRLYDVL^^ S236T^«, S243H^«, deletion 5243^^

Protein modules 1-13 Leader sequence 14-18 Potential activation peptide 19-246 Serine protease domain Catalytic triad: H59, D105, S201.

Chromosomal location Exact location and structure of the human factor D gene not reported. May lie on the X chromosome.

cDNA sequence GCAGTTCTGG GGCGGCAGAG GCGCACCTGT CTGGAGGACG CCGGAGCCCT CCCGACACCA CCTGCTGTGC TGCGACGTGG CACGTGCTCT GCCATCACCG TCCGGGGGCC GTTTGCGGCA ATCGACAGCG AGTCCCGAGC ATATGCAGAA ATGGGCCACG GGGCATGGAG TTGAACGCAG

TCCTCCTAGG AGGCCGAGGC GCGCAGGCGT CGGCCGACGG CCAAGCGCCT TCGACCACGA GCCCCCTGCC CCGGCTGGGG TGCCAGTGCT AGCGCTTGAT CGCTGGTGTG ACCGCAAGAA TCCTGGCCTA AATGAAGTCA GGGGAGGCCG TAGCGCGACT GTGGGTGCTT GAGGCTGAGG

AGCGGCCGCC GCACGCGCGG CCTGGTGGCG GAAGGTGCAG GTACGACGTG CCTCCTGCTG CTGGCAGCGC CATAGTCAAC GGACCGCGCC GTGCGCGGAG CGGGGGCGTG GCCCGGGATC GGGTGCCGGG TCCACTCCTG AGGTGGGAGG CCATCTCTAC GTAGTTCCAG CTGCAGTGAG

TGCGCGGCGC CCCTACATGG GAGCGGTGGG GTTCTCCTGG CTCCGCGCAG CTACAGCTGT GTGGACCGCG CACGCGGGCC ACCTGCAACC AGCAATCGCC CTCGAGGGCG TACACCCGCG GCCTGAAGGT CATCTGGTTG ATCATTGGAT AAATAAATAA CTACTCAGGA TTGTGATTGC

GGCCCCGTGG TCGGATGCTG 60 CGTCGGTGCA GCTGAACGGC 12 0 TGCTGAGCGC GGCGCACTGC 180 GCGCGCACTC CCTGTCGCAG 2 40 TGCCCCACCC GGACAGCCAG 3 00 CGGAGAAGGC CACACTGGGC 3 60 ACGTGGCACC GGGAACTCTC 42 0 GCCGCCCGGA CAGCCTGCAG 4 80 GGCGCACGCA CCACGACGGC 540 GGGACAGCTG CAAGGGTGAC 600 TGGTCACCTC GGGCTCGCGC 660 TGGCGAGCTA TGCGGCCTGG 72 0 CAGGGTCACC CAAGCAACAA 780 GTCTTTATTG AGCACCTACT 84 0 CTCAGGAGTT GGAGATCAGC 900 AAATTAGCTG GGCAATTGGC 9 60 GGCTGAGGTG GGAGGATGAC 102 0 ACCACTGCCCT

Factor D

The methionine initiation codon (ATG), the termination codon (TAG) and the probable polyadenylation signal (AATAAA) are indicated.

D

Genomic structure^^ The human gene structure is unknown.

Accession numbers Human5^6,io,i5-i9 Mouse2o-22

Rat23-24 p^g25-26

M84526 Gl78626 Ml 1768 G202167 X04673 G581866 M13386 G387105 S73894 G693722 U29948 G915533 Z49058 G773265

Deficiency^^ X-linked mode of inheritance is suggested. One case of patient with a complete deficiency in factor D has been reported. The patient suffers from recurrent Neisseria meningitidis and Neisseria gonorrhoeae infections. No activity of the alternative pathway is reported (reduced capacity of the patient to promote phagocytosis of Escherichia coh). Decreased haemolytic complement activity.

Polymorphic variants Factor D is unusual among serum protein in having a single-banded pattern in isoelectric focusing. One variant was found in three people of West African parentage among 120 tested^^. Presence of the variant band was associated with weakening of the normal band. Both bands are of equal strength^^, indicating that variants are inherited in the usual autosomal codominant way. References ' Lesavre, P.H. et al. (1979) J. Immunol. 123, 529-534. 2 Gotze, O. et al. (1976) Adv. Immunol. 24, 1-35. ^ Volanakis, J.E et al. (1998) In The Human Complement System in Health and Disease, chapter 4 (Volanakis, J.E. and Frank, M.M. eds). Marcel Dekker, New York, pp. 48-82. ^ Tomana, M. et al. (1985) Mol. Immunol. 22, 107-111.

5 Narayana, S.V.L. et al. (1994) J. Mol. Biol. 235, 695-708. 6 Davis, A.E. (1980) Proc. Natl Acad. Sci. USA 11, 4938-4942. 7 Volanakis, J.E. et al. (1996) Protein Sci. 5, 553-564. « Kim, S. et al. (1995) Biochemistry^ 33, 14393-14399. ^ Kim, S. et al. (1995) J. Immunol. 154, 6073-6079. ^« White, R.T. et al. (1992) J. Biol. Chem. 267, 9210-9213. ^^ Barnum, S.R. et al. (1992) Biochem J. 287, 595-601. ^2 Rosen, B.S. et al. (1989) Science 244, 1483-1487. ^^ Flier, J.S. et al. (1987) Science 237, 405-408. ^^ Miner, J.L. et al. (1993) Physiol. Behav. 54, 207-212. ^5 Volanakis, J.E. et al. (1980) Proc. Natl Acad. Sci. USA 11, 1116-1119. ^6 Johnson, D.M.A. et al. (1984) FEBS Lett. 166, 347-351. ^7 Johnson, D.M.A. et al. (1980) Biochem. J. 187, 863-874. ^s Niemann, M.A. et al. (1984) Biochemistry 23, 2482-2486. ^^ Kim, S. et al. (1995) J. Biol. Chem. 270, 24399-24405. 20 Phillips, M. et al. (1986) J. Biol. Chem. 261, 10821-10827. 2^ Min, H.Y. et al. (1986) Nucleic Acids Res. 14, 8879-8892. 22 Cook, K.S. et al. (1985) Proc. Natl Acad. Sci. USA 82, 6480-6484. 23 Zhu, L. et al. ( 1994) J. Clin. Invest. 94, 1163-1171. 24 Baker, B.C. et al. (1991) Biochem. J. 279, 115-119. 25 Miner, J.L. et al. (1995) Submitted to EMBL/GenBank. 26 Nicolas, N. (1995) Submitted to EMBL/GenBank. 27 Hiemstra, P.S. et al. (1989) J. Clin. Invest. 84, 1957-1961. 28 Hobart, M.J. et al. (1976) Transplant. Rev. 32, 26-42. 29 Martin, A. et al. (1976) Immunochemistry 13, 317-324.

EC 3.4.21.43

Yuanyuan Xu, University of Alabama at Birmingham, AL, USA and John E. Volanakis, BSCR "A. Fleming'', Vari, Greece

Physicochemical properties C2 is a single polypeptide chain glycoprotein synthesized as a pre molecule of 752 amino acid residues including a 20 amino acid leader peptide. Mature protein contains 15.9% carbohydrate^. Mature protein: pF M,(K) predicted observed N-linked glycosylation sites^ (probably all occupied)

-6.0-6.3 (depending on isoform) 81 100 8 (29, 112, 290, 333, 467, 471, 621, 651)

Structure C2 is structurally similar to factor B, exhibiting 39% amino acid residue identity with it and a similar trilobed structure demonstrated by electron microscopy^. The N-terminal lobe of C2 is formed by the C2b region, which is cleaved off during cleavage/activation by Cls. The middle lobe is formed by the von Willebrand factor type A module and the C-terminal one by the serine protease domain.

Function C2 provides the catalytic subunit for the C3 and C5 convertases of the classical and lectin pathways of complement activation. Formation of the C3 convertase requires the Mg^'-'dependent binding of C2 to activatorattached C4b and the subsequent cleavage of C2 by Cls or MASP. The C2a fragment remains bound to C4b and expresses proteolytic activity against C3 only in the context of the C4b2a complex. Attachment of a C3b fragment to the C3 convertase generates the trimolecular complex C4b2a3b, which is the C5 convertase. Both enzymes share the same active centre provided by the serine protease domain of C2a. The proteolytic activity of the convertases is restricted to single peptide bonds on C3 and C5, respectively.

Tissue distribution Serum protein: 11-35/ig/ml in serum^. Primary site of synthesis: liver^. Secondary sites: monocytes/macrophages^'^, fibroblasts^, type II alveolar epithelial cells^^ and astroglioma cells^^

Regulation of expression C2 synthesis by macrophages, fibroblasts, alveolar type II epithelial cells, astroglioma cells and hepatoma cells is induced mainly in response to IFN^io,i2,/3 jn most cells, IFN7 enhances the transcriptional rate of the gene so that mRNA increases even though its half-life may be shortened^^. Steroids upregulate C2 synthesis by human monocytes by stabilizing the C2 message^^.

Protein sequence^-'^ MGPLMVLFCL CPQGLYPSPA TPRLGSYPVG CPNPGISLGA EPICRQPYSY GHLNLYLLLD EPKVLMSVLN NNQMRLLGME KRNDYLDIYA HMLDVSKLTD WVLTAAHCFR NQGILEFYGD RDHENELLNK NLTDVREWT LYNPCLGSAD PL

LFLYPGLADS SRLCKSSGQW GNVSFECEDG VRTGFRFGHG DFPEDVAPAL CSQSVSENDF DNSRDMTEVI TMAWQEIRHA IGVGKLDVDW TICGVGNMSA DGNDHSLWRV DIALLKLAQK QSVPAHFVAL DQFLCSGTQE KNSRKRAPRS

APSCPQNVNI QTPGATRSLS FILRGSPVRQ DKVRYRCSSN GTSFSHMLGA LIFKESASLM SSLENANYKD IILLTDGKSN RELNELGSKK NASDQERTPW NVGDPKSQWG VKMSTHARPI NGSKLNINLK DESPCKGESG KVPPPRDFHI

SGGTFTLSHG KAVCKPVRCP CRPNGMWDGE LVLTGSSERE TNPTQKTKES VDRIFSFEIN HENGTGTNTY MGGSPKTAVD DGERHAFILQ HVTIKPKSQE KEFLIEKAVI CLPCTMEANL MGVEWTSCAE GAVFLERRFR NLFRMQPWLR

WAPGSLLTYS APVSFENGIY TAVCDNGAGH CQGNGVWSGT LGRKIQIQRS VSVAIITFAS AALNSVYLMM HIREILNINQ DTKALHQVFE TCRGALISDQ SPGFDVFAKK ALRRPQGSTC WSQEKTMFP FFQVGLVSWG QHLGDVLNFL

50 10 0 15 0 2 00 2 50 3 00 3 50 400 450 500 550 60 0 65 0 7 00 7 50

The leader sequence and the cleavage site (RK) between C2b and C2a are underlined. Potential N-linked glycosylation sites (probably all occupied) are indicated (N).

Protein modules 1-20 Leader peptide 21-85 CCPl CCP2 86-147 148-206 CCP3 Linker 207-236 VWFA 237-452 Linker 453-466 Serine protease domain 467-752 Catalytic triad: H507, D561, S679.

exon 1 exon 2 exon 3 exon 4 exon 5 exon 6-10 exon 11 exon 11-1

Chromosomal location Human^^'^^: 6p21.3 (MHC class III region). Telomere ... HSP70 ... C2 ... Bf ... Centromere Mouse^*: chromosome 17. Telomere ... HSP70 ... C2 ... Bf ... Centromere

cDNA sequence3,15 GGGGGCAGTA GGGAGACAGG CTAACAGAAG AGCTGCTGAC CTACCTCTCG CTGTTCCTGT TCGGGTGGCA TGCCCCCAGG CAGACCCCAG GCCCCTGTCT GGCAATGTGA TGTCGCCCCA TGCCCCAACC GACAAGGTCC TGCCAGGGCA GACTTCCCTG ACCAATCCCA GGTCATCTGA CTCATCTTCA GTGAGCGTTG GACAACTCCC CATGAAAATG AACAACCAAA ATCATCCTTC CATATCAGAG ATCGGGGTGG GATGGTGAGA CATATGCTGG AACGCCTCTG ACCTGCCGGG GATGGCAACG AAAGAATTGC AACCAGGGAA GTAAAGATGT GCTCTGCGGA CAGAGTGTTC ATGGGAGTGG AACTTGACAG GATGAGAGTC TTTTTTCAGG AAAAACTCCC AATCTCTTCC CCCCTCTAGC CTTCTACCTC AATCCGGGTC TACCAGCAGG CCCTGGTTGA TAATAAAAAT

CACAAAGCCT GCAAAGGTTT ACCATCCCCC CCAGCTCTAG CCGCCCCTAG ACCCAGGTCT CCTTCACCCT GCCTGTACCC GAGCCACCCG CCTTTGAGAA GCTTCGAGTG ACGGCATGTG CAGGCATTTC GCTATCGCTG ACGGGGTCTG AGGACGTGGC CCCAGAAGAC ACCTCTACCT AGGAGAGCGC CCATTATCAC GGGATATGAC GAACTGGGAC TGCGACTCCT TGACAGATGG AGATCCTGAA GCAAGCTGGA GGCATGCCTT ATGTCTCCAA ACCAGGAGAG GGGCCCTCAT ACCACTCCCT TTATTGAGAA TCCTGGAGTT CCACCCATGC GACCTCAAGG CTGCTCATTT AGTGGACAAG ATGTCAGGGA CCTGCAAGGG TGGGTCTGGT GCAAAAGGGC GCATGCAGCC CATGGCCACT TGAATGGCCA TCTAGGATGC ACTGCCTCGC CTTGACTCAT CAATGGTTTC

GTGGGGGAGA CACCCTTCAG TTGCCACTCC TTTTCGGGAA GGAGGACACC GGCAGACTCG CAGCCATGGC ATCCCCAGCA GTCTCTGTCT TGGCATTTAT TGAGGATGGC GGATGGAGAA ACTGGGCGCA CTCCTCGAAT GAGTGGAACG CCCTGCCCTG AAAGGAAAGC GCTCCTGGAC CTCCCTCATG CTTTGCCTCA TGAGGTGATC TAACACCTAT CGGCATGGAA AAAGTCCAAT CATCAACCAG TGTGGACTGG CATTCTGCAG GCTCACAGAC GACACCCTGG CTCCGACCAA GTGGAGGGTC GGCGGTGATC CTATGGTGAT CAGGCCCATC CAGCACCTGT TGTCGCCTTG CTGTGCCGAG GGTGGTGACA AGAATCTGGG GAGCTGGGGT CCCTCGTAGC CTGGCTGAGG GAGCCCTCTG CCCTTAGACC CAGAGGCAGC TGCCCCACCT GCTTGTTTCA GAG

TCTATTGACC TTCAGTCCCC CTGGTTTTTC GTCAGATGAC ATGGGCCCAC GCTCCCTCCT TGGGCTCCTG TCACGGCTGT AAGGCGGTCT ACCCCACGGC TTCATATTGC ACAGCTGTGT GTGCGGACAG CTTGTGCTCA GAGCCCATCT GGCACTTCCT CTGGGCCGTA TGTTCGCAGA GTGGACAGGA GAGCCCAAAG AGCAGCCTGG GCGGCCTTAA ACGATGGCCT ATGGGTGGCT AAGAGGAATG AGAGAACTGA GACACAAAGG ACCATCTGCG CATGTCACTA TGGGTCCTGA AATGTGGGAG TCCCCAGGGT GACATAGCTC TGCCTTCCCT AGGGACCATG AATGGGAGCA GTTGTCTCCC GACCAGTTCC GGAGCAGTTT CTTTACAACC AAGGTCCCGC CAGCACCTGG CTGCCCTGCC CTGTGATCCA GCACACAAGC CCCGCTCCTT CTTTCACATG

CTATAGATAT AATCCCTGCT TTCTCTGGCA CTTTTCCCTC TGATGGTTCT GCCCTCAGAA GGAGCCTTCT GCAAGAGCAG GCAAACCTGT TGGGGTCCTA GGGGCTCGCC GTGATAATGG GCTTCCGCTT CGGGGTCTTC GCCGCCAACC TCTCCCACAT AAATCCAAAT GTGTGTCGGA TCTTCAGCTT TCCTCATGTC AAAATGCCAA ACAGTGTCTA GGCAGGAAAT CTCCCAAGAC ACTATCTGGA ATGAGCTAGG CTCTGCACCA GGGTGGGGAA TTAAGCCCAA CAGCAGCTCA ACCCCAAATC TTGATGTCTT TGCTGAAGCT GCACGATGGA AGAATGAACT AACTGAACAT AAGAAAAAAC TATGCAGTGG TCCTTGAGCG CCTGCCTTGG CGCCACGAGA GGGATGTCCT AGAATCTGGG TCCTCTCTCC TGGGAAATCC GGCCTGTCCC GAATTTCCCA

ATTAGCATCA TATTATTTCC GCAATGAAGC CCGCGGCTCT TTTTTGCCTG CGTGAATATC CACCTACTCC CGGACAGTGG GCGCTGTCCA TCCCGTGGGT TGTGCGTCAG GGCTGGCCAC TGGTCATGGG GGAGCGGGAG CTACTCTTAT GCTTGGGGCC CCAGCGCTCT AAATGACTTT TGAGATCAAT TGTCCTGAAC CTATAAAGAT TCTCATGATG CCGACATGCC AGCTGTTGAC CATCTATGCC GTCCAAGAAG GGTCTTTGAA CATGTCAGCA GAGCCAAGAG TTGCTTCCGC CCAGTGGGGC TGCCAAAAAG GGCCCAGAAA GGCCAATCTG GCTGAACAAA TAACCTTAAG CATGTTCCCC GACCCAGGAG GAGATTCAGG CTCTGCTGAC CTTTCACATG GAATTTTTTA GCCCCTCCAT TAGCTGAGTA TCAGGGCTCC CAGATTCCTT GTTATGAAAT

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820

The first five nucleotides in each exon are underUned to indicate intron-exon boundaries. The methionine initiation codon (ATG), the termination codon (TAG) and the polyadenylation signal (AATAAA) are indicated.

Genomic structure^^ The gene spans 18kb of DNA and contains 18 exons. The poly(A) signal is located only 421 bp upstream of the cap site of the factor B gene^^. The largest intron (4.5 kb) follows exon 3 and contains a human-specific^^ SINE-type retroposon, derived from the human endogenous retrovirus HERV-KIO^^ This retroposon, termed SINE-R.C2, is associated with a variable number tandem repeats (VNTR) locus, which gives rise to a multiallelic RFLP. 1kb 18

lf+

III

I

I—h

411

SINE.R-C2

Accession numbers Human^'^5,19 Mouse^^

cDNA X04481 K1236 M26301 M57891 J05661

Genomic L09706 L09707 L09708 M60563-M60579 J05661

Deficiency^"^'^^ Autosomal recessive. This is the most common complement component genetic deficiency among individuals of European descent with an estimated frequency of the null allele of about 1%. Deficient individuals have an increased incidence of SLE and SLE-like syndromes, glomerulonephritis, vasculitis and pyogenic infections. Two molecular mechanisms can cause C2 deficiency: In type I deficiency no protein is detected either in the blood or intracellularly. In type II deficiency a mutant C2 polypeptide is retained intracellularly. Mutations identified: Type I: 28 bp deletion removes 9bp of the 3' end of exon 6 and 19bp of the 5' end of the adjoining intron. This causes skipping of exon 6 during RNA splicing, resulting in a shift of the reading frame and a premature termination codon^^. In linkage disequilibrium with HLA haplotype, HLAA25, B18, Drw227. Many cases described. Type IF8^29.

G662 to A, C131 to Y; one patient C896 to T, S209 to F; one patient G1660 to A, G464 to R; one patient

Polymorphic variants A common (C) and two relatively rare allotypes (A, B) have been identified by isoelectric focusing. In addition, a number of acidic (A) and basic (B)

variants have been described using polyacrylamide gel isoelectric focusing followed by western blotting and immunofixation^^. At the gene level a multiallelic and several dimorphic RFLPs have been described^^.

References ^ 2 ^ ^ 5 6 7 « 9 0

Tomana, M. et al. (1985) Mol. Immunol. 22, 107-111. Alper, C.A. (1976) J. Exp. Med. 144, 1111-1115. Bentley, D.R. (1986) Biochem. J. 239, 339-345. Smith, C.A. et al. (1984) J. Exp. Med. 159, 324-329. Oglesby, T.J. et al. (1988) J. Immunol. Methods 110, 55-62. Perlmutter, D.H. et al. (1984) J. Biol. Chem. 259, 10380-10385. Whaley, K. (1980) J. Exp. Med. 151, 501-516. Cole, F.S. et al. (1983) Clin. Immunol. Immunopathol. 27, 153-159. Katz. Y. et al. (1988) J. Exp. Med. 167, 1-14. Strunk, R.C. et al. (1988) J. Clin. Invest. 81, 1419-1426. Barnum, S.R. et al. (1992) Biochem. J. 287, 595-601. Strunk, R.C. et al. (1985) J. Biol. Chem. 260, 15280-15285. Lappin, D.F. et al. (1992) Biochem. J. 281, 437-442. Lappin, D.F. et al. (1991) Biochem. J. 280, 117-123. Horiuchi, T. et al. (1989) J. Immunol. 142, 2105-2111. Carroll, M.C. et al. (1984) Nature 307, 237-241. Carroll, M.C. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8535-8539. Steinmetz, M. et al. (1986) Cell 44, 895-904. Ishii, Y. et al. (1993) J. Immunol. 151,170-174. 20 Wu, L.-C. et al. (1987) Cell 48, 331-342. 2^ Zhu, Z.-B. et al. (1994) Hum. Genet. 93, 545-551. 22 Zhu, Z.-B. et al. (1992) J. Exp. Med. 175, 1783-1787. 23 Ishikawa, N. et al. (1990) J. Biol. Chem. 265, 19040-19046. 2^ Glass, D. et al. (1976) J. Clin. Invest. 58, 853-861. 25 Johnson, C.A. et al. (1992) New Engl. J. Med. 326, 871-874. 26 Johnson, C.A. et al. (1992) J. Biol. Chem. 267, 9347-9353. 2^ Awdeh, Z.L. et al. (1981) J. Clin. Invest. 67, 581-583. 2s Wetsel, R.A. (1996) J. Biol. Chem. 271, 5824-5831. 29 Zhu, Z.-B. et al. (1998) J. Immunol. 161, 578-584. ^0 Hauptmann, G. et al. (1990) Complement Inflamm. 7, 252-254. 3^ Cross, S.J. et al. (1985) Immunogenetics 21, 39-48.

Factor B Antonella Circolo, University of Alabama Medical School, Birmingham, AL, USA and Harvey R. Colten, Northwestern University School of Medicine, Chicago, IL, USA

D

Other names Complement component C3/C5 convertase (alternative), properdin factor B, C3 proactivator, C3 convertase, C5 convertase, EC 3.4.21.47.

Physicochemical properties Factor B is synthesized as a single-chain molecule of 764 amino acids, including a leader peptide of 25 amino acids^'^. Mature protein: pP -5.6-6.1 Mr(K) predicted 83 observed 90 N-linked glycosylation sites (all occupied) 4 (122, 142, 285, 378)

Structure^ Electron microscopy analysis of the native molecule reveals a tri-lobed structure. In the assembled bimolecular complex C3 convertase, Bb appears as a bi-lobed molecule with only one lobe bound to the C3b, indicating that the N-terminal lobe is lost after cleavage by factor D. This structure is similar to that observed for C2 and the C4b-C2a complex. Amino acid sequence alignment of the Bf serine protease domain reveals high homology Mrith the serine protease domain of chymotrypsinogen, trypsin, human factor D, Clr, Cls, factor I and MASPs, and with murine Bf and C2.

Function Bf provides the catalytic subunit of the C3/C5 convertases of the alternative complement pathway. Assembly of the C3 convertase (C3bBb) requires binding of Bf to C3b (C3.H2O) and factor D-mediated cleavage of bound Bf resulting in the release of Ba (M, (K) -33). The C3 convertase is stabilized by the binding of properdin. This provides a positive amplification loop for the classical and alternative complement pathways. After cleavage of C3, the C5 convertase ((C3b)2Bb) is formed. The initial binding of Bf to C3b is mediated by two binding sites, one on Ba (mapped on the second and third CCP domain)^, and the other (Mg^^ or Ni^+ ion dependent) on the VWFA domain of Bb and similar to the metal ions-binding domain of C2^. In addition to complement activation, Bf proteolytic fragments participate in other immunological functions (macrophage spreading, monocyte-mediated cytotoxicity and B lymphocyte proliferation). The C3 site cleaved by the active C3bBb: L744-G-L-A-R-S-N-L-D752.

Tissue distribution Plasma concentration: 180jag/ml (range 74-286)^. (Serum concentration significantly higher in homozygous BF*F, lower in homozygous BF*S and intermediate in heterozygous individuals*.) Primary site of synthesis: liver. Secondary sites: mononuclear phagocytes, fibroblasts, endothelial cells, alveolar type II epithelial cells^.

Regulation of expression Bf is an acute-phase protein. Serum Bf levels increase during inflammation. Bf expression is increased in a time- and concentration-dependent fashion by LPS and cytokines (particularly IL-la and f^, TNFcc and, to a lesser extent, IL-6 and IFN/and E). Polypeptide growth factors (PDGF, EGF and FGF)^^ and IL-4^^ counter-regulate the stimulation induced by cytokines (IL-1, lL-6 and TNFa). The effects of the cytokines and growth factors are exerted primarily at pretranslational levels and are gene- and cellspecific^'^2 Cis-acting elements for the IL-1 and IFN7 responsiveness and for constitutive expression have been localized in the 5'-flanking region of the Bf gene; constitutive expression is controlled by the nuclear factor HNF-4.

Protein sequence^^ MGSNLSPOLC LLQEGQALEY IHCPRPHDFE WSGQTAICDN QRRTCQEGGS HGPGEQQKRK ASYGVKPRYG SGTNTKKALQ VIDEIRDLLY HVFKVKDMEN VIRPSKGHES EWLFHPNYN EGTTRALRLP DKKGSCERDA GPLIVHKRSR WLKEKLQDED

LMPFILGLLS VCPSGFYPYP NGEYWPRSPY GAGYCSNPGI WSGTEPSCQD IVLDPSGSMN LVTYATYPKI AVYSMMSWPD IGKDRKNPRE LEDVFYQMID CMGAWSEYF INGKKEAGIP PTTTCQQQKE QYAPGYDKVK FIQVGVISWG LGFL

GGVTTTPWSL VQTRTCRSTG YNVSDEISFH PIGTRKVGSQ SFMYDTPQEV lYLVLDGSDS WVKVSEADSS DVPPEGWNRT DYLDVYVFGV ESQSLSLCGM VLTAAHCFTV EFYDYDVALI ELLPAQDIKA DISEWTPRF WDVCKNQKR

AQPQGSCSLE SWSTLKTQDQ CYDGYTLRGS YRLEDSVTYH AEAFLSSLTE IGASNFTGAK NADWVTKQLN RHVIILMTDG GPLVNQVNIN VWEHRKGTDY DDKEHSIKVS KLKNKLKYGQ LFVSEEEKKL LCTGGVSPYA QKQVPAHARD

GVEIKGGSFR KTVRKAECRA ANRTCQVNGR CSRGLTLRGS TIEGVDAEDG KCLVNLIEKV EINYEDHKLK LHNMGGDPIT ALASKKDNEQ HKQPWQAKIS VGGEKRDLEI TIRPICLPCT TRKEVYIKNG DPNTCRGDSG FHINLFQVLP

50 10 0 150 2 00 2 50 3 00 3 50 400 45 0 500 550 600 65 0 700 750

Amino acid sequence of allelic variant BF*F, characterized by a Q at position 32 (BF*S: R32). The leader peptide and the factor D cleavage site (RK) are underlined. The four N-linked glycosylation sites are indicated (N).

Protein modules^^ 1-25 Leader peptide 34-100 CCP 101-161 CCP 163-220 CCP 221-252 Connecting segment 253-468 VWFA 469-481 Connecting segment 482-764 Serine protease domain Catalytic triad: H526, D576, S699.

exon 1/2 exon 2 exon 3 exon 4 exon 5 exon 5-10 exon 11 exon 11-18

Chromosomal location^^-^^ Human: 6p21.1-6p21.3 within MHC. Mouse: chromosome 17 within H-2. Major histocompatibility complex gene cluster (MHC class III), immediately 3' to the C2 gene (Bf mRNA start site: 421 bp downstream of the C2 polyadenylation site).

cDNA sequence^^'^^-2^ AGGGGAAGGG AGACAAGCAA TCCAACGCCA CTCTTGTCTG TCTCTGGAGG CTGGAGTACG TCTACGGGGT TGCAGAGCAA TCTCCCTACT CGGGGCTCTG TGTGACAACG GGCAGCCAGT CGTGGCTCCC TGCCAAGACT CTGACAGAGA AAGCGGAAGA TCAGACAGCA GAGAAGGTGG CCCAAAATTT CAGCTCAATG GCCCTCCAGG AACCGCACCC CCAATTACTG CCAAGGGAGG AACATCAATG ATGGAAAACC TGTGGCATGG AAGATCTCAG GAGTACTTTG AAGGTCAGCG AACTACAACA

AATGTGACCA AGCAAGCCAG TGGGGAGCAA GAGGTGTGAC GGGTAGAGAT TGTGTCCTTC CCTGGAGCAC TCCACTGTCC ACAATGTGAG CCAATCGCAC GAGCGGGGTA ACCGCCTTGA AGCGGCGAAC CCTTCATGTA CCATAGAAGG TCGTCCTGGA TTGGGGCCAG CAAGTTATGG GGGTCAAAGT AAATCAATTA CAGTGTACAG GCCATGTCAT TCATTGATGA ATTATCTGGA CTTTGGCTTC TGGAAGATGT TTTGGGAACA TCATTCGCCC TGCTGACAGC TAGGAGGGGA TTAATGGGAA

GGTCTAGGTC GACACACCAT TCTCAGCCCC CACCACTCCA CAAAGGCGGC TGGCTTCTAC CCTGAAGACT AAGACCACAC TGATGAGATC CTGCCAAGTG CTGCTCCAAC AGACAGCGTC GTGTCAGGAA CGACACCCCT AGTCGATGCT CCCTTCAGGC CAACTTCACA TGTGAAGCCA GTCTGAAGCA TGAAGACCAC CATGATGAGC CATCCTCATG GATCCGGGAC TGTCTATGTG CAAGAAAGAC TTTCTACCAA CAGGAAGGGT TTCAAAGGGA AGCACATTGT GAAGCGGGAC AAAAGAAGCA

TGGAGTTTCA CCTGCCCCAG CAACTCTGCC TGGTCTTTGG TCCTTCCGAC CCGTACCCTG CAAGACCAAA GACTTCGAGA TCTTTCCACT AATGGCCGGT CCGGGCATCC ACCTACCACT GGTGGCTCTT CAAGAGGTGG GAGGATGGGC TCCATGAACA GGAGCCAAAA AGATATGGTC GACAGCAGTA AAGTTGAAGT TGGCCAGATG ACTGATGGAT TTGCTATACA TTTGGGGTCG AATGAGCAAC ATGATCGATG ACCGATTACC CACGAGAGCT TTCACTGTGG CTGGAGATAG GGAATTCCTG

GCTTGGACAC GCCCAGCTTC TGATGCCCTT CCCAGCCCCA TTCTCCAAGA TGCAGACACG AGACTGTCAG ACGGGGAATA GCTATGACGG GGAGTGGGCA CCATTGGCAC GCAGCCGGGG GGAGCGGGAC CCGAAGCTTT ACGGCCCAGG TCTACCTGGT AGTGTCTAGT TAGTGACATA ATGCAGACTG CAGGGACTAA ACGTCCCTCC TGCACAACAT TTGGCAAGGA GGCCTTTGGT ATGTGTTCAA AAAGCCAGTC ACAAGCAACC GTATGGGGGC ATGACAAGGA AAGTAGTCCT AATTTTATGA

TGAGCCAAGC TCTCCTGCCT TATCTTGGGC GGGATCCTGC GGGCCAGGCA TACCTGCAGA GAAGGCAGAG CTGGCCCCGG TTACACTCTC GACAGCGATC AAGGAAGGTG GCTTACCCTG GGAGCCTTCC CCTGTCTTCC GGAACAACAG GCTAGATGGA CAACTTAATT TGCCACATAC GGTCACGAAG CACCAAGAAG TGAAGGCTGG GGGCGGGGAC TCGCAAAAAC GAACCAAGTG AGTCAAGGAT TCTGAGTCTC ATGGCAGGCC TGTGGTGTCT ACACTCAATC ATTTCACCCC CTATGACGTT

60 12 0 18 0 240 3 00 3 60 42 0 4 80 540 600 660 72 0 7 80 84 0 900 9 60 102 0 108 0 1140 12 00 12 60 132 0 13 80 1440 15 00 15 60 162 0 16 80 174 0 18 00 18 6 0

Factor B

cDNA sequence

continued

GCCCTGATCA CCCTGCACCG CAAAAGGAAG AAAAAGCTGA AGAGATGCTC CCTCGGTTCC GATTCTGGCG AGCTGGGGAG GCCCGAGACT GATGAGGATT AAAACAGCTG

TAAGCTGAAA TCGAGCTTTG TGCACAGGAT GGTCTACATC AGGCTATGAC AGGAGTGAGT AGTTCACAAG CTGCAAAAAC CCTCTTTCAA ATAAGGGGTT

AGCTCAAGAA AGGGAACAAC AGCTGCTCCC CTCGGAAGGA AATATGCCCC TTTGTACTGG GCCCCTTGAT TAGTGGATGT TTCACATCAA TGGGTTTTCT CGACAAC

TATGGCCAGA AGGCTTCCTC ATCAAAGCTC AAGAATGGGG AAAGTCAAGG CCCTATGCTG AGAAGTCGTT CAGAAGCGGC GTGCTGCCCT TCCTGCTGGA

CTATCAGGCC CAACTACCAC TGTTTGTGTC ATAAGAAAGG ACATCTCAGA ACCCCAATAC TCATTCAAGT AAAAGCAGGT GGCTGAAGGA CAGGGGCGTG

CATTTGTCTC TTGCCAGCAA TGAGGAGGAG CAGCTGTGAG GGTGGTCACC TTGCAGAGGT TGGTGTAATC ACCTGCTCAC GAAACTCCAA GGATTGAATT

192 0 19 80 2 04 0 210 0 2160 222 0 22 8 0 23 40 2 40 0 2 4 60

Complete cDNA sequence of the factor BF*F allele. The first five nucleotides in each exon are underlined to indicate intron-exon boundaries. The methionine initiation codon (ATG), the termination codon (TAAj and the polyadenylation signal (ATTAAA) are indicated.

Genomic structure^^'^^ The gene consists of 18 exons spanning 6kb of genomic DNA and has an exon-intron organization similar to the murine Bf and to human and murine C2. In the mouse tissue-specific utilization of an upstream initiation site leads to the expression of an additional, 302 bp longer transcript. Similar transcripts have not been identified in humans. 0.5kb I 1

•• n i l I I II IIII III! Accession numbers Human Mouse

cDNA L15702 (BF*F) X72875 (BF*S) M57890 J05660

Genomic AF019413

Deficiency With the exception of a preliminary report^^, no ''natural'' Bf deficiencies have been described in humans or animals. Recently, Bf genetically deficient mice have been generated by targeted disruption of the Bf gene. In pathogen-free conditions, Bf null mice exhibit no overt phenotype23'24

Polymorphic variants Four allelic variants BF*S (slow), BF*F (fast), BF*S0.7 and BF*F1 (allele frequency in Caucasian population 0.731, 0.239, 0.025 and 0.005,

Factor B

respectively) were first identified by differences of electrophoretic mobility in agarose at pH 8.6^^. Each allelic variant consists of five electrophoretic isoforms resulting from variable sialic acid content^^. Additional studies have identified approximately 11 allelic variants. No significant differences in haemolytic activity were found among Bf allelic variants. A224G; Q32R; F/S

References 1 2 3 ^ 5 6 7 « 9 ^« " ^2

^^ ^^ ^5 ^6 ^^ ^s ^^ 20 2^ 22 23 2^ 25 26

Lesavre, P.H. et al. (1979) J. Immunol. 123, 529-534. Niemann, M.A. et al. (1980) Biochemistry 19, 1576-1583. Curman, B. et al. (1977) Biochemistry 16, 5368-5375. Smith, C.A. et al. (1984) J. Exp. Med. 159, 324-329. Hourcade, D.E. et al. (1995) J. Biol. Chem. 270, 19716-19722. Tuckwell, D.S. et al. (1997) Biochemistry 36, 6605-6613. Oglesby, T.J. et al. (1988) J. Immunol. Meth. 110, 55-62. Mortensen, J.P. and Lamm, L.U. (1981) Immunology 42, 505-511. Colten, H.R. and Strunk, R.C. (1993) In Complement in Health and Disease (Whaley, K., Loos, M. and Weiler, J.M. eds). Academic Publisher, Dordrecht, pp. 127-158. Circolo, A. et al. (1990) J. Biol. Chem. 265, 5066-5071. Katz, Y. and Strunk, R.C. (1990) J. Immunol. 144, 4675-4680. Colten, H.R. and Gamier, G. (1998) In The Human Complement System in Health and Disease (Volanakis, J.E. and Frank, M.M. eds). Marcel Dekker, New York, pp. 217-240. Mole, J.E. et al. (1984) J. Biol. Chem. 259, 3407-3412. Volanakis, J.E. and Arlaud, G.J. (1998) In The Human Complement System in Health and Disease (Volanakis, J.E. and Frank, M.M. eds). Marcel Dekker, New York, pp. 49-81. Carroll, M.C. et al. (1984) Nature 307, 237-241. Dunham, I. et al. (1987) Proc. Natl Acad. Sci. USA 84, 7237-7241. Chaplin, D.D. et al. (1983) Proc. Natl Acad. Sci. USA 80, 6947-6951. Wu, L.C. et al. (1987) Cell 48, 331-342. Campbell, R.D. et al. (1984) Phil. Trans. R. Soc. Lond. B306, 367-378. Woods, D.E. et al. (1982) Proc. Natl Acad. Sci. USA 79, 5661-5665. Ishikawa, N. et al. (1990) J. Biol. Chem. 265, 19040-19046. Densen, P. et al. (1996) Mol. Immunol. 33, 68 (Abstract). Matsumoto, M. et al. (1997) Proc. Natl Acad. Sci. USA 94, 8720-8725. Taylor, P.R. et al. (1998) J. Biol. Chem. 273, 1699-1704. Alper, C.A. et al. (1972) J. Exp. Med. 135, 66-80. Gamier, G. et al. (1988) Complement 5, 77-88.

Factor I

C3b inactivator, C 3 b / C 4 b inactivator, EC 3.4.21.45

Bernard J. Morley, Imperial College School of Medicine, Hammersmith Campus, London, UK

Physicochemical properties Factor I is synthesized as a prepro single-chain molecule of 583 amino acids including an 18 amino acid leader sequence and a four amino acid cleavage site. Mature protein: pP -4.5-6 (depending on isoform) -7.5 (after neuraminidase treatment)

Amino acids M, (K) predicted observed N-linked glycosylation sites^ (all occupied) Interchain disulfide bonds 1 heavy-light

Heavy chain 19-335 35.4 50

Light chain 340-583 27.6 38

3(70, 103, 177)

3 (474, 494, 536)

? (33, 284, 327)

453

Structure Factor I has a bilobed structure, each lobe formed by one chain shown to be 13 ± 1.4 n m by electron microscopy^, confirmed by neutron scattering^. The serine protease domain is very similar to that of ^-trypsin as determined by neutron and X-ray scattering^.

Function Factor I functions as a control protein for the activation of the complement pathways. It is a highly specific serine protease and cleaves the a' chains of C3b and C4b in the presence of cofactors (factor H for C3b, C4BP for C4 and CRl and MCP for both), inactivating these proteins and preventing the formation of the C3/C5 convertases. In C3, it cleaves at two positions in the a' chain to yield iC3b and C3f, while in C4b, it cleaves on either side of the thioester to yield C4c and C4d.

Tissue distribution Serum protein: 35 jiig/ml in plasma. Primary site of synthesis: liver"^. Secondary sites: monocytes, fibroblasts, endothelial cells, myoblasts, glial cells^-*.

Regulation of expression Factor I is an acute-phase protein and is upregulated by LPS and by IFNy in endothelial cells, fibroblasts and hepatocytes^'^. In myoblasts, IFN7 has no effect*.

Protein sequence 9,10 MKLLHVFLLF WQRCIEGTCV LNNGTCTAEG NVACLDLGFQ RRTMGYQDFA SDELCCKACQ QEETEILTAD LPWQVAIKDA HPDLKRIVIE PACVPWSPYL NRFYEKEMEC GKPEFPGFYT

LCFHLRFCKV CKLPYQCPKN KFSVSLKHGN QGADTQRRFK DWCYTQKAD GKGFHCKSGV MDAERRRIKS SGITCGGIYI YVDRIIFHEN FQPNDTCIVS AGTYDGSIDA KVANYFDWIS

TYTSQEDLVE GTAVCATNRR TDSEGIVEVK LSDLSINSTE SPMDDFFQCV CIPSQYQCNG LLPKLSCGVK GGCWILTAAH YNAGTYQNDI GWGREKDNER CKGDSGGPLV YHVGRPFISQ

KKCLAKKYTH SFPTYCQQKS LVDQDKTMFI CLHVHCRGLE NGKYISQMKA EVDCITGEDE NRMHIRRKRI CLRASKTHRY ALIEMKKDGN VFSLQWGEVK CMDANNVTYV YNV

LSCDKVFCQP LECLHPGTKF CKSSWSMREA TSLAECTFTK CDGINDCGDQ VGCAGFASVA VGGKRAQLGD QIWTTWDWI KKDCELPRSI LISNCSKFYG WGWSWGENC

50 10 0 15 0 2 00 2 50 3 00 3 50 40 0 450 500 550 583

The leader sequence and the cleavage site (RRKRI between the heavy (Nterminal) and light (C-terminal) chains, which is lost from the mature protein, are underlined. N-linked glycosylation sites (all occupied) are indicated (N).

Protein modules Leader peptide 1-18 23-89 FIMAC 95-200 C D 5 domain LDLRA 202-239 241-275 LDLRA 300-583 Serine protease domain Catalytic triad: H362, D411, S507.

exon 1 exon2 exon 3/4 exon 5 exon 6 exon 9-13

Cliromosomal location Human^O'^^: 4q25. Telomere ... IL-2 ... IF ... EGF ... Centromere Mouse^^: chromosome 3, 66.6 cM. Telomere ... Adh3 ... Cfi ... Egf ... Centromere

cDNA sequence 9,10,13 GAGAGACAAA TGTGCTTCCA AAAAGTGCTT GGCAGAGATG GCACTGCAGT TGGAATGTCT AGTTTAGTGT TTGTGGACCA ACGTGGCCTG TGTCTGATCT CCAGTTTGGC ATGTGGTTTG

GACCCCGAAC CTTAAGGTTT AGCAAAAAAA CATTGAGGGC GTGTGCAACT TCATCCAGGG TTCCTTGAAG AGATAAGACA CCTTGACCTT CTCTATAAAT TGAATGTACT TTATACACAG

ACCTCCAACA TGCAAGGTCA TATACTCACC ACCTGTGTTT AACAGGAGAA ACAAAGTTTT CATGGAAATA ATGTTCATAT GGGTTTCAAC TCCACTGAAT TTTACTAAGA AAAGCAGATT

TGAAGCTTCT CTTATACATC TCTCCTGCGA GTAAACTACC GCTTCCCAAC TAAATAACGG CAGATTCAGA GCAAAAGCAG AAGGTGCTGA GTCTACATGT GAAGAACTAT CTCCAATGGA

TCATGTTTTC TCAAGAGGAT TAAAGTCTTC GTATCAGTGC ATACTGTCAA AACATGCACA GGGAATAGTT CTGGAGCATG TACTCAAAGA GCATTGCCGA GGGTTACCAG TGACTTCTTT

CTGTTATTTC CTGGTGGAGA TGCCAGCCAT CCAAAGAATG CAAAAGAGTT GCCGAAGGAA GAAGTAAAAC AGGGAAGCCA AGGTTTAAGT GGATTAGAGA GATTTCGCTG CAGTGTGTGA

60 120 180 240 300 360 420 480 540 600 660 720

Factor I

• | cDNA sequence continued ^ H ^ H ^^1

^H ^^m ^ H ^ H ^^1 ^^1 ^^1 ^^1 ^ H ^ H ^^1 ^^m ^^1 ^ H ^ H ^^1 ^ H ^^1

ATGGGAAATA CATTTCTCAG ATGAAAGCCT GTGATGGTAT GTGATGAACT GTGTTGTAAA GCATGCCAAG GCAAAGGCTT GCATTCCAAG CCAGTATCAA TGCAATGGTG AGGTGGACTG AGGCTTTGCA TCTGTGGCTC AAGAAGAAAC TGGATGCAGA AAGAAGACGG ATAAAATCAT TATTACCTAA ACAGAATGCA CATTCGAAGG AAACGAATTG TGGGAGGAAA TCCCATGGCA GGTGGCAATT AAGGATGCCA GTGGAATCAC GTGGCTGTTG GATTCTGACT GCTGCACATT GTCTCAGAGC AAATATGGAC AACAGTAGTA GACTGGATAC ACCCCGACCT ACGTGGATAG AATTATTTTC CATGAAAACT ACAATGCAGG CTTTGATTGA AATGAAAAAA GACGGAAACA AAAAAGATTG CTGCCTGTGT CCCCTGGTCT CCTTACCTAT TCCAACCTAA GCTGGGGACG AGAAAAAGAT AACGAAAGAG TCTTTTCACT TAATAAGCAA CTGCTCTAAG TTTTACGGAA ATCGTTTCTA CAGGTACATA TGATGGTTCC ATCGATGCCT GTAAAGGGGA GTATGGATGC CAACAATGTG ACTTATGTCT GGGGTGTTGT GAAAACCAGA GTTCCCAGGT TTTTACACCA AAGTGGCCAA ACCATGTAGG AAGGCCTTTT ATTTCTCAGT ACAATGTATA ATTCTATTCT TTTTCTCTCA AGAGTTCCAT TTAATGGAAA TTCTCTAGGG GGGAAAAATG AAGCAAATCT CATTGGATAT TTTATGCCAT ATTGGAATTT TGTTGTATAA TTCTCAAATA

CAATGATTGT CCATTGCAAA CATTACAGGG AGAAATTTTG ACTATCTTGT GCGAGCACAA CTGTGGGGGA CAGTAAAACT TAAACGTATA CACTTACCAA TGAGCTGCCT TGATACATGC TCAGTGGGGT TGAAAAAGAA CTCTGGAGGC GAGTTGGGGG TTATTTTGAC AAATTGTGAT TAAAACGGTA TTTTAAAGGT AATATTTTGG

GGAGACCAAA TCGGGTGTTT GAAGATGAAG ACTGCTGACA GGAGTTAAAA CTGGGAGACC ATTTATATTG CATCGTTACC GTAATTGAAT AATGACATCG CGTTCCATCC ATCGTTTCTG GAAGTTAAAC ATGGAATGTG CCCTTAGTCT GAAAACTGTG TGGATTAGCT CTCTCTCTTC TAATTAATAA CTCCACAGAG TGAAGCAT

780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920

The first five nucleotides in each exon are underlined to indicate intron-exon boundaries. The methionine initiation codon (ATG), the termination codon (TAA) and the probable polyadenylation signal (AATAAA) are indicated.

Genomic structure^^ The gene spans 63 kb and is encoded by 13 exons illustrated below. The first intron is 36 kb in size while the others vary from 0.19 to 5.9 kb. 4kb

4-h-h

-h-H 13

1

Accession numbers Human^'^O'^^ Mouse (BALB/c)^2

Xenopus^"^

cDNA Y00318 J02770 U47810 X59958

Genomic X78594

Deficiency^-'^ Autosomal recessive. Uncontrolled activation of the amplification loop of alternative pathway results in consumptive loss of C3. Patients show an

increased incidence of recurrent pyogenic infection, glomerulonephritis and SLE-like illness. Mutations identified: A1282 to T, H418 to L; three chromosomes G801 to A; splicing defect, loss of exon 5; one chromosome

Polymorphic variants G833A; silent^. Two common (A and B)^^ ^^d four rare (Al, A2, B2 and C)^^'^* variants have been identified by isoelectric focusing of neuraminidase-treated plasma followed by western blotting.

References ^ Sim, R.B. et al. (1993) Int. Rev. Immunol. 10, 65-86. 2 Perkins, S.J. et al. (1993) Biochem. J. 295, 101-108. 3 DiScipio, R.G. (1992)}. Immunol. 149, 2592-2599. ^ Morris, K.M. et al. (1982) J. Clin. Invest. 70, 906-913. 5 Whaley, K. (1980) J. Exp. Med. 151, 501-516. 6 Vyse, T.J. et al. (1996) J. Clin. Invest. 97, 925-933. ^ Dauchel, H. et al. (1990) Eur. J. Immunol. 20, 1669-1675. « Legoedec, J. et al. (1995) Eur. J. Immunol. 25, 3460-3466. ^ Catterall, C.F. et al. (1987) Biochem. J. 242, 849-856. '0 Goldberger, G. et al. (1987) J. Biol. Chem. 262,10065-10071. ^^ Shiang, R. et al. (1989) Genomics 4, 82-86. ^2 Minta, J.O. et al. (1996) Mol. Immunol. 33, 101-112. ^3 Vyse, T.J. et al. (1994) Genomics 24, 90-98. ^^ Kunnath-Muglia, L.M. et al. (1993) Mol. Immunol. 30, 1249-1256. ^5 Vyse, T.J. et al. (1994) Q. J. Med. 87, 385-401. ^6 Nakamura, S. and Abe, K. (1985) Hum. Genet. 71, 45-48. ^7 Nakamura, S. et al. (1991) Hum. Hered. 41, 403-408. ^8 Umetsu, K. et al. (1994) Hum. Biol. 66, 339-348.

Parts C3 Family

Marina Botto, Imperial College School of Medicine, Hammersmith Campus, London, UK

Physicochemical properties C3 is synthesized as a prepro single-chain molecule of 1663 amino acids including a 22 amino acid leader peptide and a four amino acid cleavage site^ Mature protein^: pl M, (K) predicted Amino acids M, (K) predicted N-linked glycosylation sites Interchain disulfide bonds I p-a

-5.9 -190 P chain 23-667 -75 (645 aa) 1(85)

a chain 672-1663 -115 (992 aa) 2(939, 1617-potential)

559

816

Structure C3 has a two-domain shape, consisting of a flat ellipsoid about 18nm long, 2nm thick and 8-10 nm wide, with a smaller flat domain of 2 X 4 X 9nm, confirmed by scattering solution analysis^. The larger domain represents the C3c and the smaller is the C3d region. On proteolytic activation and removal of C3a there is a large conformational change with the two domains moving closer together. Recently, X-ray crystallographic analysis has shown C3d to be folded into a highly helical structure known as an a-a barrel with a convex surface at one end which contains the thioester-dependent covalent attachment site (see under Function below) and a concave depression at the opposite end of the barreP.

Function C3 precursor is first processed by the removal of four Arg residues, forming two chains, /3 and a, linked by a disulfide bond. C3 convertase activates C3 by cleaving the a chain, releasing C3a anaphylotoxin and generating C3b {p chain and a' chain). C3b can bind to, via its reactive thioester, cell surface carbohydrates or immune aggregates and have opsonic and immunoregulatory functions. C3b is rapidly split in two positions by factor I and a cofactor to form iC3b (inactivated C3b) and C3f which is released. iC3b is slowly cleaved (possibly by factor I) to form C3c and C3dg. Other proteases produce other fragments such as C3d or C3g. C3a anaphylatoxin is a mediator of local inflammatory processes. It induces the contraction of smooth muscles, increases vascular permeability and causes histamine release from mast cells and basophilic leukocytes.

Thioester site _^4 a chain C3 p chain

-N

I

C3 convertase

a' chain

C3a

C3b p chain

I 1 s-s

• a'-67

1 1 C3f II

a-40

1

1

Factor I with cofactor ( H , MCP, CR1)

1

iC3b

chain

I C3dg

Factor I with CR1 as cofactor

a-40

C3c p chain

C3 Degradation Pathway

Tissue distribution Serum protein: 1-1.5 mg/ml^. Primary site of synthesis: liver^. Secondary sites: monocytes/macrophages^, neutrophils^, fibroblasts^, capillary^'^ and umbilical vein endothelial cells^^, myoblasts^^, synovium^^, T cells^'*, endometrium^^, astrocytes'^, glomerular mesangial cells'^, proximal tubular epithelium'*, glomerular epithelium'^, pulmonary alveolar type II epithelium^o, adipocytes^', osteoblasts^^, intestinal epithelial cells^-^.

Regulation of expression C3 is the most prominent of the acute-phase complement components. C3 synthesis is tissue-specific and is modulated in response to a variety of stimulatory agents. It is upregulated in most cell types by IL-1, IL-6, TNFa and LPS, the exception being neutrophils, where IL-1 downregulates C3. IFN7 also generally upregulates expression, except in neutrophils, pneumocytes and intestinal epithelial cells. In the endometrium, oestrogens lower C3 expression, while progesterone increases synthesis. Expression levels are controlled both at the level of transcription^^ and mRNA stability25.

I

Protein sequence^ MGPTSGPSLL GDVPVTVTVH GRNKFVTVQA TVNHKLLPVG MGQWKIRAYY LEVTITARFL VLSRKVLLDG SPYQIHFTKT TQGDGVAKLS NSNNYLHLSV LKAGRQVREP VWVDVKDSCV GVFVLNKKNK GQQTAQRAEL NPMRFSCQRR LDEDIIAEEN TWEILAVSMS LYNYRQNQEL VPLKTGLQEV RLGREGVQKE KHLIVTPSG^ KGYTQQLAFR CGAVKWLILE LQEAKDICEE RLKGPLLNKF PWRWLNEQR PSRSSKITHR KAKDQLTCNK SILDISMMTG VSHSEDDCLA KLNKLCRDEL KVQLSNDFDE HYLMWGLSSD AFTESMWFG

LLLLTHLPLA DFPGKKLVLS TFGTQWEKV RTVMVNIENP ENSPQQVFST YGKKVEGTAF VQNLRAEDLV PKYFKPGMPF INTHPSQKPL LRTELRPGET GQDLWLPLS GSLWKSGQS LTQSKIWDW QCPQPAARRR TRFISLGEAC IVSRSEFPES DKKGICVADP KVRVELLHNP EVKAAVYHHF DIPPADLSDQ GEQNMIGMTP QPSSAFAAFV KQKPDGVFQE QVNSLPGSIT LTTAKDKNRW YYGGGYGSTQ IHWESASLLR FDLKVTIKPA FAPDTDDLKQ FKVHQYFNVE CRCAEENCFI YIMAIEQTIK FWGEKPNLSY CPN

LGSPMYSIIT SEKTVLTPAT VLVSLQSGYL EGIPVKQDSL EFEVKEYVLP VIFGIQDGEQ GKSLYVSATV DLMVFVTNPD SITVRTKKQE LNVNFLLRMD ITTDFIPSFR EDRQPVPGQQ EKADIGCTPG RSVQLTEKRM KKVFLDCCNY WLWNVEDLKE FEVTVMQDFF AFCSLATTKR ISDGVRKSLK VPDTESETRI TVIAVHYLDE KRAPSTWLTA DAPVIHQEMI KAGDFLEANY EDPGKQLYNV ATFMVFQALA SEETKENEGF PETEKRPQDA LANGVDRYIS LIQPGAVKVY QKSDDKVTLE SGSDEVQVGQ IIGKDTWVEH

PNILRLESEE NHMGNVTFTI FIQTDKTIYT SSQNQLGVLP SFEVIVEPTE RISLPESLKR ILHSGSDMVQ GSPAYRVPVA LSEAEQATRT RAHEAKIRYY LVAYYTLIGA MTLKIEGDHG SGKDYAGVFS DKVGKYPKEL ITELRRQHAR PPKNGISTKL IDLRLPYSW RHQQTVTIPP WPEGIRMNK LLQGTPVAQM TEQWEKFGLE YWKVFSLAV GGLRNNNEKD MNLQRSYTVA EATSYALLAL QYQKDAPDHQ TVTAEGKGQG KNTMILEICT KYELDKAFSD AYYNLEESCT ERLDKACEPG QRTFISPIKC WPEEDECQDE

TMVLEAHDAQ PANREFKSEK PGSTVLYRIF LSWDIPELVN KFYYIYNEKG IPIEDGSGEV AERSGIPIVT VQGEDTVQSL MQALPYSTVG TYLIMNKGRL SGQREWADS ARWLVAVDK DAGLTFTSSS RKCCEDGMRE ASHLGLARSN MNIFLKDSIT RNEQVEIRAV KSSLSVPYVI TVAVRTLDPE TEDAVDAERL KRQGALELIK NLIAIDSQVL MALTAFVLIS lAGYALAQMG LQLKDFDFVP ELNLDVSLQL TLSWTMYHA RYRGDQDATM RNTLIIYLDK RFYHPEKEDG VDYVYKTRLV REALYKTRLV ENQKQCQDLG

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650

The leader sequence and the cleavage site (RRRRI between the a and j8 chains are underlined. CIO 10 and Q1013, whose side-chains form the thioester bond, are double underlined and N-linked glycosylation sites are indicated (N).

Protein modules 1-22 23-667 672-1663

Leader peptide /3 chain a chain

672-748 955-1303 955-1001

C3a anaphylatoxin C3dg fragment C3g fragment

1002-1303 1303-1321

C3d fragment C3f fragment

1010-1013

Thioester domain (C-Q)

1424-1456 1209-1271 749-790 1227-1236 1383-1403

Properdin-binding site Factor H-binding site^^ Factor H, factor B^^ and CRPs binding sites CR2-binding site^^ CR3-binding site^^

Chromosomal location Human^^: Mouse: Rat:

19pl3.3-pl3.2. chromosome 17, 34.3 cM. chromosome 9.

cDNA sequence^ CTCCTCCCCA ATGGGACCCA CTGGGGAGTC ACCATGGTGC GACTTCCCAG AACCACATGG GGGCGCAACA GTGCTGGTCA CCTGGCTCCA CGGACGGTCA TCTTCTCAGA ATGGGCCAGT GAGTTTGAGG AAATTCTACT TACGGGAAGA AGGATTTCCC GTGCTGAGCC GGGAAGTCTT GCAGAGCGCA CCCAAGTACT GGCTCTCCAG ACCCAGGGAG AGCATCACGG ATGCAGGCTC CTACGTACAG CGCGCCCACG TTGAAGGCGG ATCACCACCG AGCGGCCAGA GGCTCGCTGG ATGACCCTGA GGCGTGTTCG GAGAAGGCAG GACGCAGGGC CAGTGCCCGC GACAAAGTCG

TCCTCTCCCT CCTCAGGTCC CCATGTACTC TGGAGGCCCA GCAAAAAACT GCAACGTCAC AGTTCGTGAC GCCTGCAGAG CAGTTCTCTA TGGTCAACAT ACCAGCTTGG GGAAGATCCG TGAAGGAGTA ACATCTATAA AAGTGGAGGG TGCCTGAATC GGAAGGTACT TGTACGTGTC GCGGGATCCC TCAAACCAGG CCTACCGAGT ATGGCGTGGC TGCGCACGAA TGCCCTACAG AGCTCAGACC AGGCCAAGAT GACGCCAGGT ACTTCATCCC GGGAGGTGGT TGGTAAAAAG AGATAGAGGG TGCTGAATAA ACATCGGCTG TGACCTTCAC AGCCAGCCGC GCAAGTACCC

CTGTCCCTCT CAGCCTGCTG TATCATCACC CGACGCGCAA AGTGCTGTCC CTTCACGATC CGTGCAGGCC CGGGTACCTC TCGGATCTTC TGAGAACCCG CGTCTTGCCC AGCCTACTAT CGTGCTGCCC CGAGAAGGGC AACTGCCTTT CCTCAAGCGC GCTGGACGGG TGCCACCGTC CATCGTGACC AATGCCCTTT CCCCGTGGCA CAAACTCAGC GAAGCAGGAG CACCGTGGGC CGGGGAGACC CCGCTACTAC GCGAGAGCCC TTCCTTCCGC GGCCGACTCC CGGCCAGTCA TGACCACGGG GAAGAACAAA CACCCCGGGC GAGCAGCAGT CCGCCGACGC CAAGGAGCTG

GTCCCTCTGA CTCCTGCTAC CCCAACATCT GGGGATGTTC AGTGAGAAGA CCAGCCAACA ACCTTCGGGA TTCATCCAGA ACCGTCAACC GAAGGCATCC TTGTCTTGGG GAAAACTCAC AGTTTCGAGG CTGGAGGTCA GTCATCTTCG ATTCCGATTG GTGCAGAACC ATCTTGCACT TCTCCCTACC GACCTCATGG GTCCAGGGCG ATCAACACAC CTCTCGGAGG AACTCCAACA CTCAACGTCA ACCTACCTGA GGCCAGGACC CTGGTGGCGT GTGTGGGTGG GAAGACCGGC GCCCGGGTGG CTGACGCAGA AGTGGGAAGG GGCCAGCAGA CGTTCCGTGC CGCAAGTGCT

CCCTGCACTG TAACCCACCT TGCGGCTGGA CAGTCACTGT CTGTGCTGAC GGGAGTTCAA CCCAAGTGGT CAGACAAGAC ACAAGCTGCT CGGTCAAGCA ACATTCCGGA CACAGCAGGT TCATAGTGGA CCATCACCGC GGATCCAGGA AGGATGGCTC TCCGAGCAGA CAGGCAGTGA AGATCCACTT TGTTCGTGAC AGGACACTGT ACCCCAGCCA CAGAGCAGGC ATTACCTGCA ACTTCCTCCT TCATGAACAA TGGTGGTGCT ACTACACGCT ACGTCAAGGA AGCCTGTACC TACTGGTGGC GTAAGATCTG ATTACGCCGG CCGCCCAGAG AGCTCACGGA GCGAGGACGG

TCCCAGCACC CCCCCTGGCT GAGCGAGGAG TACTGTCCAC CCCTGCCACC GTCAGAAAAG GGAGAAGGTG CATCTACACC ACCCGTGGGC GGACTCCTTG ACTCGTCAAC CTTCTCCACT GCCTACAGAG CAGGTTCCTC TGGCGAACAG GGGGGAGGTT AGACCTGGTG CATGGTGCAG CACCAAGACA GAACCCTGAT GCAGTCTCTA GAAGCCCTTG TACCAGGACC TCTCTCAGTG GCGAATGGAC GGGCAGGCTG GCCCCTGTCC GATCGGTGCC CTCCTGCGTG TGGGCAGCAG CGTGGACAAG GGACGTGGTG TGTCTTCTCC GGCAGAACTT GAAGCGAATG CATGCGGGAG

60 12 0 180 240 3 00 3 60 42 0 480 54 0 6 00 66 0 72 0 7 80 84 0 900 9 60 102 0 108 0 114 0 12 0 0 12 6 0 132 0 13 8 0 144 0 15 0 0 15 60 162 0 1680 174 0 180 0 18 6 0 192 0 19 8 0 2 04 0 210 0 2160

1

1

cDNA sequence AACCCCATGA AAGAAGGTCT GCCAGCCACC ATCGTTTCCC CCACCGAAAA ACGTGGGAGA TTCGAGGTCA CGAAACGAGC AAGGTGAGGG CGTCACCAGC GTGCCGCTAA ATCAGTGACG ACTGTGGCTG GACATCCCAC CTCCTGCAAG AAGCACCTCA ACGGTCATCG AAGCGGCAGG CAACCCAGCT TACGTGGTCA TGCGGGGCTG GATGCGCCCG ATGGCCCTCA CAGGTCAACA ATGAACCTAC AGGCTGAAGG GAGGACCCTG CTGCAGCTAA TACTACGGTG CAATACCAAA CCCAGCCGCA TCAGAAGAGA ACCTTGTCGG TTCGAGGTCA AAGAACACTA TCTATATTGG CTGGCCAATG AGGAACACCC TTCAAAGTTC GCCTATTACA AAGCTGAACA CAAAAGTCGG GTGGACTATG TACATCATGG CAGCGCACGT CACTACCTCA ATCATCGGGA GAGAACCAGA TGCCCCAACT

GGTTCTCGTG TCCTGGACTG TGGGCCTGGC GAAGTGAGTT ATGGAATCTC TTCTGGCTGT CAGTAATGCA AGGTGGAAAT TGGAACTACT AGACCGTAAC AGACCGGCCT GTGTCAGGAA TTCGCACCCT CTGCAGACCT GGACCCCAGT TTGTGACCCC CTGTGCATTA GGGCCTTGGA CTGCCTTTGC AGGTCTTCTC TTAAATGGCT TGATACACCA CGGCCTTTGT GCCTGCCAGG AGAGATCCTA GGCCTCTTCT GTAAGCAGCT AAGACTTTGA GTGGCTATGG AGGACGCCCC GCTCCAAGAT CCAAGGAAAA TGGTGACAAT AGGTCACCAT TGATCCTTGA ACATATCCAT GTGTTGACAG TCATCATCTA ACCAATACTT ACCTGGAGGA AGCTCTGCCG ATGACAAGGT TGTACAAGAC CCATTGAGCA TCATCAGCCC TGTGGGGTCT AGGACACTTG AACAATGCCA GACCACACCC

continued CCAGCGCCGG CTGCAACTAC CAGGAGTAAC CCCAGAGAGC TACGAAGCTC CAGCATGTCG GGACTTCTTC CCGAGCCGTT CCACAATCCA CATCCCCCCC GCAGGAAGTG GTCCCTGAAG GGATCCAGAA CAGTGACCAA GGCCCAGATG CTCGGGCTGC CCTGGATGAA GCTCATCAAG GGCCTTCGTG TCTGGCTGTC GATCCTGGAG AGAAATGATT TCTCATCTCG CAGCATCACT CACTGTGGCC TAACAAATTT CTACAACGTG CTTTGTGCCT CTCTACCCAG TGACCACCAG CACCCACCGT TGAGGGTTTC GTACCATGCT AAAACCAGCA GATCTGTACC GATGACTGGC ATACATCTCC CCTGGACAAG TAATGTAGAG AAGCTGTACC TGATGAACTG CACCCTGGAA CCGACTGGTC GACCATCAAG CATCAAGTGC CTCCTCCGAT GGTGGAGCAC GGACCTCGGC CCATTCCCCC

ACCCGTTTCA ATCACAGAGC CTGGATGAGG TGGCTGTGGA ATGAATATAT GACAAGAAAG ATCGACCTGC CTCTACAATT GCCTTCTGCA AAGTCCTCGT GAAGTCAAGG GTCGTGCCGG CGCCTGGGCC GTCCCGGACA ACAGAGGATG GGGGAACAGA ACGGAGCAGT AAGGGGTACA AAACGGGCAC AACCTCATCG AAGCAGAAGC GGTGGATTAC CTGCAGGAGG AAAGCAGGAG ATTGCTGGCT CTGACCACAG GAGGCCACAT CCCGTCGTGC GCCACCTTCA GAACTGAACC ATCCACTGGG ACAGTCACAG AAGGCCAAAG CCGGAAACAG AGGTACCGGG TTTGCTCCAG AAGTATGAGC GTCTCACACT CTTATCCAGC CGGTTCTACC TGCCGCTGTG GAACGGCTGG AAGGTTCAGC TCAGGCTCGG AGAGAAGCCC TTCTGGGGAG TGGCCTGAGG GCCTTCACCG ACTCCAGATA

TCTCCCTGGG TGCGGCGGCA ACATCATTGC ACGTTGAGGA TTTTGAAAGA GGATCTGTGT GGCTACCCTA ACCGGCAGAA GCCTGGCCAC TGTCCGTTCC CTGCCGTCTA AAGGAATCAG GTGAAGGAGT CCGAGTCTGA CCGTCGACGC ACATGATCGG GGGAGAAGTT CCCAGCAGCT CCAGCACCTG CCATCGACTC CCGACGGGGT GGAACAACAA CTAAAGATAT ACTTCCTTGA ATGCTCTGGC CCAAAGATAA CCTATGCCCT GTTGGCTCAA TGGTGTTCCA TTGATGTGTC AATCTGCCAG CTGAAGGAAA ATCAACTCAC AAAAGAGGCC GAGACCAGGA ACACAGATGA TGGACAAAGC CTGAGGATGA CTGGAGCAGT ATCCGGAAAA CTGAGGAGAA ACAAGGCCTG TGTCCAATGA ATGAGGTGCA TGAAGCTGGA AGAAGCCCAA AGGACGAATG AGAGCATGGT AAGCTTCAGT

CGAGGCGTGC GCACGCGCGG AGAAGAGAAC CTTGAAAGAG CTCCATCACC GGCAGACCCC CTCTGTTGTT CCAAGAGCTC CACCAAGAGG ATATGTCATC CCATCATTTC AATGAACAAA GCAGAAAGAG GACCAGAATT GGAACGGCTG CATGACGCCC CGGCCTAGAG GGCCTTCAGA GCTGACCGCC CCAAGTCCTC CTTCCAGGAG CGAGAAAGAC TTGCGAGGAG AGCCAACTAC CCAGATGGGC GAACGGCTGG CTTGGCCCTA TGAACAGAGA AGCCTTGGCT CCTCCAACTG CCTCCTGCGA AGGCCAAGGC CTGTAATAAA TCAGGATGCC TGCCACTATG CCTGAAGCAG CTTCTCCGAT CTGTCTAGCT CAAGGTCTAC GGAGGATGGA TTGCTTCATA TGAGCCAGGA CTTTGACGAG GGTTGGACAG GGAGAAGAAA CCTCAGCTAC CCAAGACGAA TGTCTTTGGG TATATCTC

2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040

The first five nucleotides in each exon are underlined to indicate the intron-exon boundaries. The methionine initiation codon (ATGj, the termination codon (TGA) and the polyadenylation signal (GATAAA) are indicated^^'^^.

Genomic structure^^^^^ The gene spans 41 kb and is encoded by 41 exons illustrated below.

Mil m m III—^—H+-HH—II nil iiiiiiii Accession numbers Human: K02765 Mouse: K02782

Deficiency Autosomal recessive. Patients show an increased susceptibility to pyogenic infections, membranoproliferative glomerulonephritis and SLE-like illness. Mutations identified: G to A, intron 18; splicing defect, loss of 61 bp of exon 18 one patient^^ G to T intron 10; splicing defect, partial deletion of exon 10 one patient-^^ Gl 705 to A; D549 to N one patient^^ Deletion of exons 22 and 23 one patient^^

Polymorphic variants Two common variants (CSS and C3F)^^ and more than 20 rare variants have been identified on the basis of their relative electrophoretic mobilities. C364G; R102G; C3S/C3F^9 A4915C; S992R; C3S/C3*S025^o CI00IT; P314L, -ve/+ve for monoclonal antibody HAV4-P^ C4956T; silent^o

References ' 2 ^ ^ 5 6 7 « 9 ^0 " ^2 ^3 ^^

De Bmijn, M.H.L. et al. (1985) Proc. Natl Acad. Sci. USA 82, 708-712. Tack, B.F. et al. (1976) Biochemistry 15, 4513-4521. Perkins, S.J. et al. (1986) Eur. J. Biochem. 157, 155-168. Nagar, B. et al. (1998) Science 280, 1277-1281. Kohler, P.F. et al. (1967) J. Immunol. 99, 1211-1216. Alper, C.A. et al. (1969) Science 163, 286-288. Eistein, L.P. et al. (1977) J. Clin. Invest. 60, 963-969. Botto, M. et al. (1992) J. Immunol. 149, 1348-1355. Katz, Y. et al. (1989) Eur.}. Immunol. 19, 983-988. Ueki, A. et al. (1987) Immunology 61, 11-14. Warren, H.B. et al. (1987) Am. J. Pathol. 129, 9-13. Legoedec, J. et al. (1995) Eur. J. Immunol. 25, 3460-3466. Ruddy, S. et al. (1974) N. Engl. J. Med. 290, 1284-1288. Pantazis, P. et al. (1990) Mol. Immunol. 27, 283-289.

^5 Sundstrom, S.A. et al. (1989) J. Biol. Chem. 264, 16941-16947. ^6 Barnum, S.R. et al. (1992) J. Neuroimmunol. 38, 275-282. ^7 Sacks, S.H. et al. (1993) Clin. Exp. Immunol. 93, 411-417. ^s Brooimans, R.A. et al. (1991) J. Clin. Invest. 88, 379-384. ^9 Sacks, S.H. et al. (1993) Immunology 19, 348-354. 20 Strunk, R. et al. (1988) J. Clin. Invest. 81, 1419-1426. 2^ Choy, L.N. et al. (1992) J. Biol. Chem. 267, 12736-12741. 22 Hong, M.H. et al. (1991) Endocrinology 129, 2774-2779. 23 Molmenti, E.P. et al. (1993) J. Biol. Chem. 268, 14116-14124. 24 Wilson, D.R. et al. (1990) Mol. Cell. Biol. 10, 6181-6191. 25 Mitchell, T.J. (1996) J. Immunol. 156, 4429-4434. 26 Lambris, J.D. et al. (1988) J. Biol. Chem. 263, 12147-12150. 27 Lambris, J.D. et al. (1996) J. Immunol. 156, 4821-4832. 2s Becherer J.D. et al. (1988) J. Biol. Chem. 263, 14586-14591. 29 Lambris, J.D. et al. (1985) Proc. Natl Acad. Sci. USA 82, 4235-4239. 30 Wright, S.D. et al. (1987) Proc. Natl Acad. Sci. USA 84, 1965-1968. 3^ Whitehead, A.S. et al. (1982). Proc. Natl Acad. Sci. USA 19, 5021- 5025. 32 Fong, K.Y.et aL (1990) Genomics 7, 579-586. 33 Vik, D.P. et aL (1991) Biochemistry 30,1080-1085. 3^* Botto, M. et al. (1990) J. Clin. Invest. 86, 1158-1163. 35 Huang, J.L. et al. (1994) Clin. Immunol. Immunopathol. 73, 267-273. 36 Singer, L. et al. (1994) J. Biol. Chem. 269, 28494-28499. 37 Botto, M. et al. (1992) Proc. Natl Acad. Sci. USA 89, 4957-4961. 3« Alper, C.A. et al. (1968) J. Clin. Invest. 47, 2181-2192. 39 Botto, M. et aL (1990) J. Exp. Med. 172, 1011-1017. ^» Hohler, T. et al. (1995) Hum. Genet. 96, 539-541.

D

David E. Isenman, Department of Biochemistry, University of Toronto, Toronto, Canada Other names None in human; in mouse formerly also known as Ss protein.

Physicochemical properties Human C4 is synthesized as a prepro single chain of 1744 amino acids including a 19 amino acid leader sequence and two tetrabasic cleavage sites for a furin-like enzyme. The three chains of mature C4 are linked by disulfide bonds and these chains have the order p-a-y in the precursor molecule. In primates, sheep and cattle there are two isotypes of C4, C4A and C4B^, the A/B designations reflecting their more Acidic or 5asic electrophoretic mobilities at alkaline pH. Mature protein: pi ==7.9 (calculated for C4A, however is allotype dependent)

P chain Amino acids

M, (K)

20-675

predicted 71.8 observed 75 N-linked glycosylation sites 1 (226) (all occupied^ Interchain disulfide bonds-^'"^ /3567 1 p-a, 2 a-y (Parings inferred from C3 data^J

a chain 680-1449 (secreted) 680-1427 (plasma) 84.6 93

/chain 1454-1744

33.1 33

3(862,1328,1391)

a820 aS76 al394

7(1588, 1590, 1595)? 71566

Structure A high-resolution structure of C4 is not available. Electron microscopic^ as well as X-ray and neutron scattering studies suggest that C4 is a multidomain molecule^. The shape is that of an oblate ellipsoid of approximate dimension 2 x 8 X 18 nm. The scattering data further suggest that C4c and C4d form independent domains^. Recently, X-ray crystallographic analysis has shown C3d to be folded into a highly helical structure known as an a-a barrel with a convex surface at one end which contains the thioester-dependent covalent attachment site (see under Function below) and a concave depression at the opposite end of the barreP. Given the - 4 0 % identity between C4d and C3d and the fact that the models derived from the X-ray and neutron-scattering studies of C3 are similar to those for C4^, it seems highly likely that the C4d domain will adopt a similar structure.

Function Following secretion into the plasma, an elastase-like metalloprotease removes a 22 residue peptide from the C-terminus of the a chain^^. Other post-translational modifications include formation of the intramolecular thioester bond, glycosylation and tyrosine sulfation. The chain structure of the mature plasma form of C4 and its degradation products are depicted below. C4 is proteolytically activated through both the classical pathway by Cls and the lectin pathway by MASP-2. The resulting C4b fragment is a modulatory subunit of both the classical pathway C3 convertase (C4b2a) and classical pathway C5 convertase (C3b4b2a). Only when bound to C4b can C2a cleave C3 and similarly, only when bound to the C4b3b heterodimer can C5 be efficiently cleaved by C4b-associated C2a^^. Thus in terms of propagating the pathway, C4b has stable binding sites for C2, C5 and nascently activated C3b^2 j ^ also has binding sites for the CCP domain-containing complement regulatory molecules C4BP, CRl, MCP and DAF and the regulatory serine protease factor I. The C4a activation peptide has weak anaphylatoxin activity (depending on the experimental system -100-1000 less potent than C3a)^^ and although it was originally thought to bind to the same receptor as did C3a, more recent work suggests that C4a binds to a distinct receptor^^'^^. The thioester formed between the side chains of CyslOlO and Gin 1013 within the C4d region of the a chain mediates covalent attachment to the target surface bearing activated forms of Cls or MASP. The human C4A and C4B isotypes not only differ in their preference of acceptor nucleophiles, amino groups for C4A and hydroxyl groups for C4B, but also in the mechanism through which transacylation is accomplished^^. In nascently activated C4b of the A isotype, amino group nucleophiles are believed to directly attack the thioester carbonyl. However, due to the presence in C4B of a histidine at residue 1125 in place of the non-nucleophilic residue aspartic acid in C4A, activation first leads to the formation of an intramolecular acyl-imidazole intermediate involving the transacylation of the thioester carbonyl to the ring nitrogen of H1125. The released thiolate anion can then act as a general base to catalyse the attack of a hydroxyl-bearing nucleophile on the very reactive acyl-imidazole intermediate. Species which have only one functional isotype of C4 have the C4B isotype equivalent and can thus undergo the catalysed form of the transacylation reaction. Target-bound C4b is a ligand of CRl, also known as the immune adherence receptor. There is evidence that C4b of the A isotype is a somewhat better ligand for this receptor than is C4b of the B isotype^''. All functional interaction sites of C4b are lost when a C4b-cofactor complex (i.e. C4b-C4BP, C4b-MCP or C4b-CR1) is cleaved by factor I C-terminal to residues 956 and 1336 to yield fragments C4c and C4d.

cC

i;

NC

:^

C4

Thioester site

1-c

iXs-L^ C-j

Y chain

C1S/MASP2

o£

|3 chain

s-s-l

C4i

e

l-C

N{

C^

±D_ V chain

C4b

|-N

Factor I + Cofactor (C4BP, CR1, MCP)

c£

e

N-l

l-N

(3 chain

s-s-l ^^4

a3

C-|

±ZL_n

y chain

C4c

^-C

NC

C4cl

C4d

C4 Degradation Pathway

Tissue distribution Serum protein: ~0.6nig/ml. Primary site of synthesis: liver^*. Secondary sites: monocytes, macrophages, mammary gland, lung, spleen, kidney, brain, testis, and intestinal epithelial cells^*-^^.

Regulation of expression C4 is an acute-phase protein which is upregulated by INF/in hepatocytes, monocytes and intestinal epithelial cells^^^'^i. INF7 increases the stability of the C4 mRNA22. Upregulation of C4 synthesis in intestinal epithelial cells is also mediated by 11-6^^. No other cytokines have been reported to affect C4 expression. LPS downregulates the synthesis of C4 and counteracts the upregulation of synthesis produced by INF/^^

I

Protein sequence23 MRLLWGLIWA WKGSVFLRN QLLRGPEVQL NPGQRVRYRV DFVIPDISEP YILTVPGHLD SQTKLVNGQS EMEEAELTSW IPVKVSATVS GSPHPAIARL TFSHYYYMIL DHPVANSLRV LGALDTALYA GLAFSDGDQW RCCQDGVTRL QAGLQRALEI DSLTTWEIHG LRPVLYNYLD WPTAAAAVS LDHRGRTLEI ASLLRLPRGC KGYMRIQQFR QETSNWLLSQ HGLAVFQDEG TLTKAPVDLL SDPMPQAPAL STQDTVIALD IRGLEEELQF VTVKGHVEYT RRREAPKWE LEKLTSLSDR PASATLYDYY ALERGLQDED LHFTKDVKAA YLLDSNSWIE

SSFFTLSLQK PSRNNVPCSP VAHSPWLKDS FALDQKMRPS GTWKISARFS EMQLDIQARY HISLSKAEFQ YFVSSPFSLD SPGSVPEVQD TVAAPPSGGP SRGQIVFMNR DVQAGACEGK AGSKSHKPLN TLSRKRLSCP PMMRSCEQRA LQEEDLIDED LSLSKTKGLC KNLTVSVHVS LKWARGSFE PGNSDPNMIP GEQTMIYLAP KADGSYAAWL QQADGSFQDP AEPLKQRVEA GVAHNNLMAM WIETTAYALL ALSAYWIASH SLGSKINVKV MEANEDYEDY EQESRVHYTV YVSHFETEGP NPERRCSVFY GYRMKFACYY ANQMRNFLVR EMPSERLCRS

PRLLLFSPSV KVDFTLSSER LSRTTNIQGI TDTITVMVEN DGLESNSSTQ lYGKPVQGVA DALEKLNMGI LSKTKRHLVP IQQNTDGSGQ GFLSIERPDS EPKRTLTSVS LELSVDGAKQ MGKVFEAMNS KEKTTRKKRN ARVQQLDCRE DIPVRSFFPE VATPVQLRVF PVEGLCLAGG FPVGDAVSKV DGDFNSYVRV TLAASRYLDK SRDSSTWLTA CPVLDRSMQG SISKANSFLG AQETGDNLYW HLLLHEGKAE TTEERGLNVT GGNSKGTLKV EYDELPAKDD CIWRNGKVGL HVLLYFDSVP GAPSKSRLLA PRVEYGFQVK ASCRLRLEPG TRQRAACAQL

VHLGVPLSVG DFALLSLQVP NLLFSSRRGH SHGLRVRKKE FEVKKYVLPN YVRFGLLDED TDLQGLRLYV GAPFLLQALV VSIPIIIPQT RPPRVGDTLN VFVDHHLAPS YRNGESVKLH YDLGCGPGGG VNFQKAINEK PFLSCCQFAE NWLWRVETVD REFHLHLRLP GGLAQQVLVP LQIEKEGAIH TASDPLDTLG TEQWSTLPPE FVLKVLSLAQ GLVGNDETVA EKASAGLLGA GSVTGSQSNA MADQAAAWLT LSSTGRNGFK LRTYNVLDMK PDAPLQPVTP SGMAIADVTL TSRECVGFEA TLCSAEVCQC VLREDSRAAF KEYLIMGLDG NDFLQEYGTQ

VQLQDVPRGQ LKDAKSCGLH LFLQTDQPIY VYMPSSIFQD FEVKITPGKP GKKTFFRGLE AAAIIEYPGG REMSGSPASG ISELQLSVSA LNLRAVGSGA FYFVAFYYHG LETDSLALVA DSALQVFQAA LGQYASPTAK SLRKKSRDKG RFQILTLWLP MSVRRFEQLE AGSARPVAFS REELVYELNP SEGALSPGGV TKDHAVDLIQ EQVGGSPEKL LTAFVTIALH HAAAITAYAL VSPTPAPRNP RQGSFQGGFR SHALQLNNRQ NTTCQDLQIE LQLFEGRRNR LSGFHALRAD VQEVPVGLVQ AEGKCPRQRR RLFETKITQV ATYDLEGHPQ GCQV

50 100 150 200 2 50 3 00 350 400 450 500 550 600 650 7 00 7 50 800 85 0 900 950 1000 1050 1100 115 0 12 0 0 12 50 13 00 1350 14 00 145 0 1500 1550 1600 1650 17 00

The leader sequence and the cleavage sites (RKKR and RRRR) between the P-a and a-y chains, respectively, are underlined. iV-linked glycosylation sites (all utilized) are indicated (N) and residues CIOIO and Q1013, whose side-chains form the thioester bond, are double underlined.

Protein modules 1-19 20-675 680-1449 1454-1744

Leader peptide P chain a chain /chain

680-756 7S7-9S6 957-1336 1337-1427

C4a anaphylatoxin o& fragment of C4c C4d fragment a^ fragment of C4c

1010-1013

CG/AEQ thioester motif common to C3, C4 and ci^-macroglobulin of all species Human isotype-specific sequence, C4A = PCPVLD, C4B = LSPVIH

1120-1125 474-488

Putative location within ^ chain of a site of interaction with 052^^ Putative location within the o& fragment of a site of interaction with C4BP25

757-845

The isotype-specific residues, in addition to four other residues that generally, although not absolutely, segregate as a set with either C4A or C4B produce the serological antigenic determinants of h u m a n C4 known as Chido (Chi,2,3,4,5,6) and Rodgers (Rgl,2). Shown below are the residues that have been deduced to form the various Ch and Rg antigenic determinants^^. Rgl, Rg2, C h i , Ch4, Ch5, and Ch6 are linear sequence determinants, whereas Ch2 and Ch3 are conformational determinants. 2

C4A

° pS-C-,

PC pv LD

D

^

C4B

,

CO

o

A F

_J

——• O

[

LO

II

^

1

1

1

2

o

5

(

1

O

1—1 V

LS PV IH

G j

Rg

M

tt

I II

r^ o o ^

IV

Ch residue number C4d polymorphic cluster no.

Chromosomal location^^ Human: 6p21.3 within the class III region of the MHC. Telomere ... HLA-B ... C2 ... Bf ... C4A ... CYP21A ... C4B ... CYP21B ... HLA-DR Mouse: chromosome 17, locus position 18.8 within the class III region of the MHC. Telomere ... H2-D ... C2 ... Bf ... Sip ... CYP21A ... C4 ... CYP21B ... H2-Ea Sip is 94% identical to mouse C4, but because it is not readily cleaved by C l s , its function in complement is questionable.

cDNA sequence23,28 AGAAGGTAGC CTCTGGGCAT CCTTCTGTGG CGAGGACAGG TGCTCCCCAA CAGGTGCCCT GTCCAGCTGG CAGGGTATCA CCCATTTACA CGCCCGAGCA AAGAAGGAGG TCAGAGCCAG AGCACCCAGT GGAAAGCCCT GCCAGGTACA GATGAGGATG GGACAGAGCC ATGGGCATTA CCAGGTGGGG TCCTTGGATC GCCTTGGTCC ACGGTGTCTT AGCGGCCAAG GTATCTGCAG GGAGGCCCCG ACTCTGAACC ATGATCCTAT TCGGTCTCGG TACCATGGAG GAGGGCAAGC AAGCTCCACT CTGTATGCTG ATGAACAGCT CAGGCAGCGG AGCTGTCCCA AATGAGAAAT ACACGTCTGC TGCCGGGAGC GACAAGGGCC GATGAGGATG ACAGTGGACC ATCCATGGCC CGGGTGTTCC CAGCTGGAGC CACGTGTCCC CTGGTGCCTG GCTGTGTCTC TCCAAGGTTC CTCAACCCCT ATGATCCCTG ACTTTAGGCT CGAGGCTGTG CTGGACAAGA CTGATCCAGA GCTTGGTTGT

AGACAGACAG CCAGCTTCTT TTCATCTGGG TAGTGAAAGG AGGTGGACTT TGAAAGATGC TGGCCCATTC ACCTGCTCTT ACCCTGGCCA CTGACACCAT TGTACATGCC GGACCTGGAA TTGAGGTGAA ACATCCTGAC TCTATGGGAA GTAAGAAGAC ACATTTCCCT CTGACCTCCA AGATGGAGGA TTAGCAAGAC GTGAGATGTC CTCCTGGGTC TCAGCATTCC GCTCCCCACA GGTTTCTGTC TGAACTTGCG CCCGAGGGCA TGTTTGTGGA AGCACCCAGT TGGAGCTCAG TAGAAAGCGA CAGGCAGCAA ATGACCTCGG GCCTGGCCTT AGGAGAAGAC TGGGTCAGTA CCATGATGCG CCTTCCTGTC AGGCGGGCCT ACATTCCCGT GCTTTCAAAT TGAGCCTGTC GCGAGTTCCA TGCGGCCTGT CAGTGGAGGG GGGGCTGTGC TGAAGGTGGT TGCAGATTGA TGGACCACCG ATGGGGACTT GTGAGGGGGC GGGAGCAAAC CAGAGCAGTG AAGGCTACAT CACGGGACAG

ACGGATCTAA CACCTTATCT GGTCCCCCTA ATCAGTGTTC CACCCTTAGC GAAGAGCTGT GCCATGGCTA CTCCTCTCGC GCGGGTTCGG CACAGTCATG CTCGTCCATC GATCTCAGCC GAAATATGTC GGTGCCAGGC GCCAGTGCAG TTTCTTTCGG CTCAAAGGCA GGGGCTGCGC GGCAGAGCTC CAAGCGACAC AGGCTCCCCA TGTTCCTGAA AATAATTATC TCCAGCGATA TATTGAGCGG AGCCGTGGGC GATCGTGTTC CCATCACCTG GGCCAACTCC CGTGGACGGT CTCCCTAGCC GTCCCACAAG CTGTGGTCCT TTCTGATGGA AACCCGGAAA TGCTTCCCCG TTCCTGCGAG CTGCTGCCAA CCAACGAGCC GCGCAGCTTC ATTGACACTG CAAAACCAAA CCTGCACCTC CCTCTATAAC GCTGTGCCTG CCGGCCTGTT GGCTCGAGGG GAAGGAAGGG AGGCCGGACC TAACAGCTAC CTTGTCACCA CATGATCTAC GAGCACACTG GCGGATCCAG CAGCACCTGG

CCTCTCTTGG CTGCAGAAGC TCGGTGGGGG CTGAGAAACC TCAGAAAGAG GGCCTCCATC AAGGACTCTC CGGGGGCACC TACCGGGTCT GTGGAGAACT TTCCAGGATG CGATTCTCAG CTTCCCAACT CATCTTGATG GGGGTGGCAT GGGCTGGAGA GAGTTCCAGG CTCTACGTTG ACATCCTGGT CTTGTGCCTG GCTTCTGGCA GCCCAGGACA CCTCAGACCA GCCAGGCTCA CCGGATTCTC AGTGGGGCCA ATGAATCGAG GCACCCTCCT CTGCGAGTGG GCCAAGCAGT CTGGTGGCGC CCCCTCAAGA GGGGGTGGGG GACCAGTGGA AAGAGAAACG ACAGCCAAGC CAGCGGGCAG TTTGCTGAGA CTGGAGATCC TTCCCAGAGA TGGCTCCCCG GGCCTATGTG CGCCTGCCCA TACCTGGATA GCTGGGGGCG GCCTTCTCTG TCCTTCGAAT GCCATCCATA TTGGAAATAC GTCAGGGTTA GGAGGCGTGG TTGGCTCCGA CCTCCCGAGA CAGTTTCGGA CTCACAGCCT

ATCCTCCAGC CCAGGTTGCT TGCAGCTCCA CATCTCGTAA ACTTCGCACT AACTCCTCAG TGTCCAGAAC TCTTTTTGCA TTGCTCTGGA CTCACGGCCT ACTTTGTGAT ATGGCCTGGA TTGAGGTGAA AAATGCAGTT ATGTGCGCTT GTCAGAGCAA ACGCCCTGGA CTGCAGCCAT ATTTTGTGTC GGGCCCCCTT TTCCTGTCAA TTCAGCAAAA TCTCAGAGCT CTGTGGCAGC GACCTCCTCG CCTTTTCTCA AGCCCAAGAG TCTACTTTGT ATGTCCAGGC ACCGGAACGG TGGGAGCCTT TGGGCAAGGT ACAGTGCCCT CCTTATCCAG TGAACTTCCA GCTGCTGCCA CCCGCGTGCA GTCTGCGCAA TGCAGGAGGA ACTGGCTCTG ACTCTCTGAC TGGCCACCCC TGTCTGTCCG AAAACCTGAC GAGGGCTGGC TGGTGCCCAC TCCCTGTGGG GAGAGGAGCT CTGGCAACTC CAGCCTCAGA CCTCCCTCTT CACTGGCTGC CCAAGGACCA AGGCGGATGG TTGTGTTGAA

CATGAGGCTG CTTGTTCTCT GGATGTGCCC TAATGTCCCC CCTCAGTCTC AGGCCCTGAG GACAAACATC GACGGACCAG TCAGAAGATG GGGGGTGGGG CCCAGACATC ATCCAACAGC GATCACCCCT AGACATCCAG TGGGCTCCTA GCTGGTGAAT GAAGCTGAAT CATTGAGTCT ATCTCCCTTC CCTGCTGCAG AGTTTCTGCC CACAGACGGG GCAGCTGTCA CCCACCTTCA TGTTGGGGAC TTACTACTAC GACCCTGACC GGCCTTCTAC TGGGGCCTGC GGAGTCCGTG GGACACAGCT CTTTGAAGCT TCAGGTGTTC AAAGAGACTA AAAGGCGATT GGATGGGGTG GCAGCCGGAC GAAGAGCAGG GGACCTGATT GAGAGTGGAA CACGTGGGAG AGTCCAGCTC CCGCTTTGAG TGTGAGCGTC CCAGCAGGTG GGCAGCCGCC AGATGCGGTG GGTCTATGAA TGATCCCAAT TCCATTGGAC GAGGCTTCCT TTCCCGGTAC CGCCGTGGAT TTCCTATGCG GGTCCTGAGT

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300

cDNA sequence TTGGCCCAGG CTGTCCCAGC ATGCAGGGGG GCCCTTCATC GTGGAAGCCT CTGGGTGCCC GACCTGCTCG CTGTACTGGG CGCAACCCAT GCCCTGCTGC TGGCTCACCC GCCCTGGATG AATGTGACTC AACCGCCAGA GTGAAGGTGG GACATGAAGA GAGTACACGA AAGGATGACC AGGAACCGCC TACACCGTGT GTCACCCTCC TCTGACCGTT TCGGTCCCCA CTGGTGCAGC GTGTTTTACG TGCCAGTGTG GACGAGGATG CAGGTTAAGG ACCCAAGTCC CTGGTTCGAG CTGGATGGGG TGGATCGAGG GCCCAGCTCA CCCTCCCACC GTGTCCGCTT GTGTCAGTGT

AGCAGGTAGG AGCAGGCTGA GTTTGGTGGG ATGGGCTGGC CCATCTCAAA ACGCAGCTGC GTGTTGCCCA GCTCAGTCAC CCGACCCCAT ACCTCCTGCT GTCAGGGCAG CCCTGTCTGC TCAGCTCCAC TTCGCGGCCT GAGGAAACAG ACACGACCTG TGGAAGCAAA CAGATGCCCC GCAGGAGGGA GCATCTGGCG TGAGTGGATT ACGTGAGTCA CCTCCCGGGA CGGCCAGCGC GGGCACCAAG CTGAGGGGAA GCTACAGGAT TTCTCCGAGA TGCACTTCAC CCTCCTGCCG CCACCTATGA AGATGCCCTC ACGACTTCCT TCCGCTGGGA TCATGAACAC TGGC

continued AGGCTCGCCT CGGCTCGTTC CAATGATGAG CGTCTTCCAG GGCAAACTCA CATCACGGCC CAACAACCTC TGGTTCTCAG GCCCCAGGCC TCACGAGGGC CTTCCAAGGG CTACTGGATT AGGCCGGAAT GGAGGAGGAG CAAAGGAACC CCAGGACCTA CGAGGACTAT TCTGCAGCCC GGCGCCCAAG GAACGGCAAG CCACGCCCTG CTTTGAGACC GTGCGTGGGC AACCCTGTAC TAAGAGCAGA GTGCCCTCGC GAAGTTTGCC AGACAGCAGA CAAGGATGTC CCTTCGCTTG CCTCGAGGGA TGAACGCCTG CCAGGAGTAT GGAACCTGAA AGCCTGGGAC

GAGAAACTGC CAGGACCCCT ACTGTGGCAC GATGAGGGTG TTTTTGGGGG TATGCCCTGT ATGGCAATGG AGCAATGCCG CCAGCCCTGT AAAGCAGAGA GGATTCCGCA GCCTCCCACA GGGTTCAAGT CTGCAGTTTT CTGAAGGTCC CAGATAGAAG GAGGACTATG GTGACACCCC GTGGTGGAGG GTGGGGCTGT CGTGCTGACC GAGGGGCCCC TTTGAGGCTG GACTACTACA CTCTTGGCCA CAGCGTCGCG TGCTACTACC GCTGCTTTCC AAGGCCGCTG GAACCTGGGA CACCCCCAGT TGCCGGAGCA GGCACTCAGG CCTGGGAACC CAGGGCATAT

AGGAGACATC GTCCAGTGTT TCACAGCCTT CAGAGCCATT AGAAAGCAAG CACTGACCAA CCCAGGAGAC TGTCGCCCAC GGATTGAAAC TGGCAGACCA GTACCCAAGA CCACTGAGGA CCCACGCGCT CCTTGGGCAG TTCGTACCTA TGACAGTCAA AGTACGATGA TGCAGCTGTT AGCAGGAGTC CTGGCATGGC TGGAGAAGCT ACGTCCTGCT TGCAGGAAGT ACCCCGAGCG CCTTGTGTTC CCCTGGAGCG CCCGTGTGGA GCCTCTTTGA CTAATCAGAT AAGAATATTT ACCTGCTGGA CCCGCCAGCG GGTGCCAGGT ATGAAGCTGG TAAAGGCTTT

TAACTGGCTT AGACAGGAGC TGTGACCATC GAAGCAGAGA TGCTGGGCTC GGCGCCTGTG TGGAGATAAC CCCGGCTCCT CACAGCCTAC GGCTTCGGCC CACGGTGATT GAGGGGTCTC GCAGCTGAAC CAAGATCAAT CAATGTCCTG AGGCCACGTC GCTTCCAGCC TGAGGGTCGG CAGGGTGCAC CATCGCGGAC GACCTCCCTC GTATTTTGAC GCCGGTGGGG CAGATGTTCT TGCTGAAGTC GGGTCTGCAG GTACGGCTTC GACCAAGATC GCGCAACTTC GATCATGGGT CTCGAATAGC GGCAGCCTGT GTGAGGGCTG AAGCACTGCT TGGCAGCAAA

33 60 3 42 0 3480 3 540 3 600 3 660 3 72 0 3 780 3 84 0 3 9 00 3 9 60 4 02 0 40 80 414 0 42 00 42 6 0 43 2 0 43 8 0 4440 45 00 4 5 60 4 62 0 4 6 80 474 0 4800 4860 492 0 49 8 0 5 04 0 5100 5160 52 2 0 52 80 53 4 0 54 0 0

The first five nucleotides in each exon are underHned to indicate the intron-exon boundaries. The methionine initiation codon (ATGJ, the termination codon (TGA) and the polyadenylation signal (ATTAAA) are indicated.

Genomic structure^^^^^ Human C4A and C4B are normally tandem loci that are separated by -10 kb. Whereas C4A is -21 kb in length, C4B can be either 14.6 kb or 21 kb in length. The size difference is solely due to a larger intron 9 in the long C4B gene, which in turn is due to the insertion of an -6.4 kb retroposon element within this intron^^. Both human genes, as well as the mouse C4 gene consist of 41 exons.

1

6.4 kb Intron in C4A andC4B longl

. ,. , ^'^^ , 20

UtiHWiimillllllllilllllHI

41

llll

HHH

Accession numbers Human

C4A23,29

cDNA K02403*

C4B30

Mouse Xenopus Chicken

C 4 (B10.WRp2,33 Q^34

C4

Ml 1729 D78003

Genomic M59815 M59816 AF019413 U24578 J05095 AL023516

* K02403 provides the cDNA for human precursor C4A as it was originally published^^. This sequence is not fully correct in that it is missing part of the leader peptide and 9 nucleotides (GACTATGAG) coding for a repeating DYE peptide sequence near the C-terminus of a chain, a sequence that has been found in other C4A and C4B clones and at the protein leveP*. Both of these segments have been corrected in the cDNA of human C4A presented in the section above. The protein sequence presented above, as well as the protein sequence links available from the C4A gene accession numbers, have been corrected for these problems. The protein sequence link for human C4B from accession number AF019413 is full length, whereas that from U24578 is not and contains sequence ambiguities.

Deficiency Given the tandem nature of the C4 locus, in most individuals there will be expression from two C4A genes and two C4B genes. The population frequency for non-expression of 1, 2 or 3 of these alleles is respectively 3 5 % , 8-10% and 1%, regardless of racial backgrounds^. Non-expression of all four alleles is extremely rare, but most individuals who are totally deficient in C4 have systemic or discoid lupus erythematosus, i m m u n e complex disease of the kidney and increased susceptibility to infections'^^. Even partial deficiencies, especially at the C4A locus, predispose individuals to i m m u n e complex disease^^. Null alleles for C4 have three origins: (1) about 40% are due to deletion of the entire gene, (2) some C4A null alleles are due to the presence of pseudogenes at this locus, and (3) non-deleted C4B nulls have C4A isotype-specific sequences and thus likely represent gene conversions^*^.

Polymorphic variants In addition to the non-expressed alleles at both C4 loci in humans, there are at least 13 allelic polymorphisms of C4A and 16 of C4Bs^. Within an isotype, allotypes are given numbers in increasing order of their anodal migration at alkaline pH. The two most common allotypes in all ethnic groups are C4A3 and C4BP''. The isotype-specific polymorphic residues have been identified within the C4d region as have some allotype-specific residues in all three chains^^. In most cases, allotypic amino acid interchanges are without functional consequence, but an exception is C4A6 in which p chain residue R477 is replaced by W (as a result of a C to T transition in the first nucleotide of the codon), which results in the molecule losing the ability to bind C5^*'^^.

References ' 2 3 ^ 5 6 ^ « ^

20 2^ 22 23 2^ 25 26 27 28 29 30 3^ 32 33 3^ 35 36 37 38 39

Dodds, A.W. and Law, S.K.A. (1990) Biochem. J. 265, 495-502. Chan A.C. and Atkinson, J.P. (1985) J. Immunol. 134, 1790-1798. Janatova, J. (1986) Biochem. J. 233, 819-825. Seya, T. et al. (1986) J. Immunol. 136, 4152-4156. Dolmer, K. and Sottrup-Jensen, L. (1993) FEBS Lett. 315, 85-90. Smith, C.A. et al. (1984) J. Exp. Med. 159, 324-329. Perkins, S.J. et al. (1990) Biochemistry 29, 1167-1175. Nagar, B. et al. (1998) Science 280, 1277-1281. Perkins, S.J. et al. (1990) Biochemistry 29, 1175-1180. Hortin, G. et al. (1986) J. Biol. Chem. 261, 9065-9069. Takata, Y. et al. (1987) J. Exp. Med. 165, 1494-1507. Kim, U.K. et al. (1992) J. Biol. Chem. 267, 4171-4176. Hugh, T.E. et al. (1983) Mol. Immunol. 20, 637-645. Murakami, Y. et al. (1993) Immunol. Lett. 36, 301-304. Tornetta, M.A. et al. (1997) J. Immunol. 158, 5277-5282. Law, S.K.A. and Dodds, A.W. (1997) Protein Sci. 6, 263-274. Reilly, B.D. and Mold, C. (1997) Clin. Exp. I m m u n o l 110, 310-316. Cox, B.J. and Robins, D.M. (1988) Nucleic Acids Res. 16, 6857-6870. Whaley, K. (1980) J. Exp. Med. 151, 501-516. Andoh, A. et al. (1993) J. Immunol. 151, 4239-4247. Kulics, J. et al. (1990) J. Clin. Invest. 85, 943-949. Mitchell, T.J. (1996) J. Immunol. 156, 4429-4434. Belt, K.T. et al. (1984) Cell 36, 907-914. Ebanks, R.O. et al. (1995) J. Immunol. 154, 2808-2820. Hessing, M. et al. (1990) FEBS Lett. 271, 131-136. Yu, C.Y. et al. (1988) Immunogenetics 27, 399-405. Campbell, R.D. et al. (1988) Annu. Rev. Immunol. 6, 161-195. Belt, K.T. et al. (1985) Immunogenetics 21, 173-180. Yu, C.Y. (1991) J. Immunol. 146, 1057-1066. Ulgiati, D. et al. (1996) Immunogenetics 43, 250-252. Dangel, A.W. et al. (1994) Immunogenetics 40, 425-436. Ogata, R.T. et al. (1989) J. Biol. Chem. 264, 16565-16572. Sepich, D.S. et al. (1985) Proc. Natl Acad. Sci. USA 82, 5895-5899. Mo, R. et al. (1996) Immunogenetics 43, 360-369. Hauptmann, G. et al. (1988) Immunodeficiency Rev. 1, 3-22. Braun, L. et al. (1990) J. Exp. Med. 171, 129-140. Mauff, G. et al. (1990) Complement Inflamm. 7, 261-268. Anderson, M.J. et al. (1992) J. Immunol. 148, 2795-2802. Ebanks, R.O. et al. (1992) J. Immunol. 148, 2803-2811.

Rick A. Wetsel, University of Texas-Houston Institute of Molecular Medicine, Houston, TX, USA

Physicochemical properties C5 is synthesized as an intracellular single-chain precursor, pro-C5^, of 1676 amino acids including an 18 amino acid leader peptide, and an arginine-rich linker sequence (RPRR) located between the N-terminal /3 chain and the C-terminal a chain. The signal and linker peptides are processed from the promolecule during secretion, yielding the mature, native two-chain structure that is held together by a disulfide bond(s) and non-covalent forces. Mature protein: pF 4.7-5.5 6.0 (calculated) Amino acids^ Mr (K) predicted observed^ N-linked glycosylation site^ Interchain disulfide bonds 1 ^a (putative)^

j8 chain 19-673 73.3 75.0

a chain 678-1676 112.5 115.0 1 (741)

567

810

Structure Electron microscopy^, neutron^ and X-ray scattering, and physical chemical studies have indicated that C5 is an asymmetrical two-domain molecule with a multilobal, irregular ultrastructure of dimensions 10.4x14x16.8 nm. NMR modelling of C5a peptide suggests a drumstick shape with high helical content through residue 70. The C-terminal segment containing the activation domain is seen as a dynamic random coiP-^^.

Function C5 is cleaved during complement activation by both C5 convertase enzymes at a specific site within the a chain R751-L752, into C5a and C5b fragments". After activation, the Mr(K) 11.2 C5a peptide of 74 amino acids that is derived from the N-terminus of the a chain (residues 678-751) is released from the parent molecule. C5a acts as a potent anaphylatoxin causing smooth muscle contraction, increased vascular permeability, basophil and mast cell degranulation and lysosomal enzyme release*. In addition, C5a stimulates the directed migration of neutrophils, eosinophils, basophils and monocytes. Recent studies indicate that C5a also modulates the hepatic acute-phase gene response" and augments the overall immune response by increasing protein synthesis of TNFa, IL-1/3, IL-6, and IL-8 from certain cell types. Plasma enzyme carboxypeptidase N (EC 3.4.12.7) removes the C-terminal arginine from the C5a peptide forming the C5 desArg derivative. On a molar basis, human C5a des-Arg expresses only 1 % as much anaphylatoxic activity and 1 % as PMN chemotactic activity as does

undigested C5a. The C5b fragment initiates the assembly of the membrane attack complex (MAC) that mediates cytolysis of viral and bacterial pathogens. C5a a-chain (3-chain

400 aa

Tissue distribution Serum protein: IS A^g/ml in plasma^^. Primary site of synthesis: liver (hepatocytes). Secondary sites: lung, spleen, fetal intestine, monocytes, macrophages, and type II alveolar cells.

Regulation of expression Regulated expression of C5 has not been explored in depth. In studies that have been performed, C5 expression was not regulated by inflammatory stimuli to any great degree. Cytokines such as IL-6 and IL-ljS that regulate the hepatic expression of numerous acute-phase proteins do not affect C5 expression.

Protein sequence3,13 MGLLGILCFL DATISIKSYP YVYLEWSKH DLKPAKRETV IKAKYKEDFS KARYFYNKW FDSETAVKEL PYKLNLVATP TSDLDPSKSV GYRAIAYSSL YLILSKGKII TAELVSDSVW ALAAVDSAVY LTFLTNANAD DGACVNNDET RLHMKTLLPV GIGISNTGIC

IFLGKTWGQE DKKFSYSSGH FSKSKRMPIT LTFIDPEGSE TTGTAYFEVK TEADVYITFG SYYSLEDLNN LFLKPGIPYP TRVDDGVASF SQSYLYIDWT HFGTREKFSD LNIEEKCGNQ GVQRGAKKPL DSQENDEPCK CEQRAARISL SKPEIRSYFP VADTVKAKVF

QTYVISAPKI VHLSSENKFQ YDNGFLFIHT VDMVEEIDHI EYVLPHFSVS IREDLKDDQK KYLYIAVTVI IKVQVKDSLD VLNLPSGVTV DNHKALLVGE ASYQSINIPV LQVHLSPDAD ERVFQFLEKS EILRPRRTLQ GPRCIKAFTE ESWLWEVHLV KDVFLEMNIP

FRVGASENIV NSAILTIQPK DKPVYTPDQS GIISFPDFKI lEPEYNFIGY EMMQTAMQNT ESTGGFSEEA QLVGGVPVIL LEFNVKTDAP HLNIIVTPKS TQNMVPSSRL AYSPGQTVSL DLGCGAGGGL KKIEEIAAKY CCWASQLRA PRRKQLQFAL YSWRGEQIQ

IQVYGYTEAF QLPGGQNPVS VKVRVYSLND PSNPRYGMWT KNFKNFEITI MLINGIAQVT EIPGIKYVLS NAQTIDVNQE DLPEENQARE PYIDKITHYN LVYYIVTGEQ NMATGMDSWV NNANVFHLAG KHSWKKCCY NISHKDMQLG PDSLTTWEIQ LKGTVYNYRT

50 100 150 200 2 50 3 00 3 50 400 450 500 550 600 650 7 00 750 800 850

1

1

Protein sequence SGMQFCVKMS LPLEIGLHNI GTISRRKEFP THLPKGSAEA EGMLSIMSYR CNSLLWLVEN GIRKAFDIGP THPQFRSIVS TAYALLTSLN SLLVKQLRLS GFGSGLATVH RIVAGASYKP TDYQIKDGHV PDKQCTMFYS TAGKPEIAYA IKKVTCTNAE DTTCSSCQAF

continued

AVEGICTSES NFSLETWFGK YRIPLDLVPK ELMSWPVFY NADYSYSVWK YQLDNGSFKE LVKIDTALIK ALKREALVKG LKDINYVNPV MDIDVSYKHK VTTWHKTST SREESSSGSS ILQLNSIPSS TSNIKIQKVC YKVSITSITV LVKGRQYLIM LANLDEFAED

PVIDHQGTKS EILVKTLRW TEIKRILSVK VFHYLETGNH GGSASTWLTA NSQYQPIKLQ ADNFLLENTL NPPIYRFWKD IKWLSEEQRY GALHNYKMTD SEEVCSFYLK HAVMDISLPT DFLCVRFRIF EGAACKCVEA ENVFVKYKAT GKEALQIKYN IFLNGC

SKCVRQKVEG PEGVKRESYS GLLVGEILSA WNIFHSDPLI FALRVLGQVN GTLPVEAREN PAQSTFTLAI NLQHKDSSVP GGGFYSTQDT KNFLGRPVEV IDTQDIEASH GISANEEDLK ELFEVGFLSP DCGQMQEELD LLDIYKTGEA FSFRYIYPLD

SSSHLVTFTV GVTLDPRGIY VLSQEGINIL EKQKLKKKLK KYVEQNQNSI SLYLTAFTVI SAYALSLGDK NTGTARMVET INAIEGLTEY LLNDDLIVST YRGYGNSDYK ALVEGVDQLF ATFTVYEYHR LTISAETRKQ VAEKDSEITF SLTWIEYWPR

900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650

The signal peptide sequenc(I and cleavaee site, (RPRRl. between the Band a chains are underUned. The known N-Hnked glycosylation site in the C5a peptide is indicated (N). The linker peptide is lost from the mature protein.

Protein modules 1-18 19-673 678-1676 678-751 752-1676

Leader peptide P chain a chain C5a peptide CSa' chain

Chromosomal location Human^^: 9q33. Telomere ... C8G ... ABL ... C5 ... PAPPA ... Centromere Mouse^^: chromosome 2. Telomere ... C8g ... Abl ... He ... Pappa ... Centromere

cDNA sequence3,13 CTACCTCCAA TGGGGACAGG GAAAATATTG AAAAGTTATC AATAAATTCC AACCCAGTTT ATGCCAATAA CCAGACCAGT AGAGAAACTG ATTGATCATA

CCATGGGCCT AGCAAACATA TGATTCAAGT CTGATAAAAA AAAACTCTGC CTTATGTGTA CCTATGACAA CAGTAAAAGT TCTTAACCTT TTGGAATTAT

TTTGGGAATA TGTCATTTCA TTATGGATAC ATTTAGTTAC AATCTTAACA TTTGGAAGTT TGGATTTCTC TAGAGTTTAT CATAGATCCT CTCTTTTCCT

CTTTGTTTTT GCACCAAAAA ACTGAAGCAT TCCTCAGGCC ATACAACCAA GTATCAAAGC TTCATTCATA TCGTTGAATG GAAGGATCAG GACTTCAAGA

TAATCTTCCT TATTCCGTGT TTGATGCAAC ATGTTCATTT AACAATTGCC ATTTTTCAAA CAGACAAACC ACGACTTGAA AAGTTGACAT TTCCGTCTAA

GGGGAAAACC 60 TGGAGCATCT 12 0 AATCTCTATT 18 0 ATCCTCAGAG 240 TGGAGGACAA 3 00 ATCAAAAAGA 3 60 TGTTTATACT 42 0 GCCAGCCAAA 4 80 GGTAGAAGAA 54 0 TCCTAGATAT 600

cDNA sequence GGTATGTGGA TTTGAAGTTA TTCATTGGTT AATAAAGTAG GATGATCAAA GCTCAAGTCA GATTTAAACA TCTGAAGAGG GTTGCTACTC GATTCGCTTG GTAAACCAAG GTAGCTTCCT ACTGATGCTC TACTCATCTC CTAGTGGGAG ACTCACTATA AAATTTTCAG TCATCCCGAC GATTCAGTCT CCTGATGCAG GATTCCTGGG AAAAAGCCCT GGTGGTGGCC GCAAATGCAG AGAACGCTGC AAATGTTGTT CGGATTAGTT CAGCTCCGTG CTGTTACCAG GTTCATCTTG TGGGAAATTC GCAAAGGTGT GAACAGATCC GTTAAAATGT GGCACAAAGT ACATTCACTG TGGTTTGGAA GAAAGCTATT AAGGAGTTCC TTGAGTGTAA ATCAATATCC CCAGTATTCT GACCCATTAA ATGTCCTACA TGGTTAACAG CAAAATTCAA TCTTTCAAGG GCCCGAGAGA GATATATGCC GAAAATACAC CTGGGAGATA TTGGTTAAAG AGCTCTGTAC ACCAGTCTGA

CGATCAAGGC AAGAATATGT ACAAGAACTT TCACTGAGGC AAGAAATGAT CATTTGATTC ACAAGTACCT CAGAAATACC CTCTTTTCCT ACCAGTTGGT AGACATCTGA TTGTGCTTAA CAGATCTTCC TCAGCCAAAG AACATCTGAA ATTACTTGAT ATGCATCTTA TTCTGGTCTA GGTTAAATAT ATGCATATTC TGGCATTAGC TGGAAAGAGT TCAACAATGC ATGACTCCCA AAAAGAAGAT ACGATGGAGC TAGGGCCAAG CTAATATCTC TAAGCAAGCC TTCCCAGAAG AAGGCATTGG TCAAAGATGT AATTGAAAGG CTGCTGTGGA CCTCCAAATG TGCTTCCTCT AAGAAATCTT CTGGTGTTAC CATACAGGAT AAGGACTGCT TAACCCACCT ATGTTTTTCA TTGAAAAGCA GAAATGCTGA CTTTTGCTTT TTTGTAATTC AAAATTCACA ACAGCTTATA CCCTGGTGAA TGCCAGCCCA AAACTCACCC GTAATCCACC CTAACACTGG ACTTGAAAGA

continued TAAATATAAA CTTGCCACAT TAAGAATTTT TGACGTTTAT GCAAACAGCA TGAAACAGCA TTATATTGCT TGGCATCAAA GAAGCCTGGG AGGAGGAGTC CTTGGATCCA TCTCCCATCT AGAAGAAAAT TTACCTTTAT TATTATTGTT TTTATCCAAG TCAAAGTATA TTATATCGTC TGAAGAAAAA TCCAGGCCAA AGCAGTGGAC ATTTCAATTC CAATGTGTTC AGAAAATGAT AGAAGAAATA CTGCGTTAAT ATGCATCAAA TCATAAAGAC AGAAATTCGG AAAACAGTTG CATTTCAAAC CTTCCTGGAA AACTGTTTAC GGGAATCTGC TGTGCGCCAG GGAAATTGGC AGTAAAAACA TTTGGATCCT ACCCTTAGAT TGTAGGTGAG CCCCAAAGGG CTAGCTGGAA GAAACTGAAG CTACTCTTAC AAGAGTACTT TTTATTGTGG GTATCAACCA TCTTACAGCC AATCGACACA GAGCACCTTT ACAGTTTCGT CATTTATCGT TACGGCACGT TATAAATTAT

GAGGACTTTT TTTTCTGTCT GAAATTACTA ATCACATTTG ATGCAAAACA GTCAAAGAAC GTAACAGTCA TATGTCCTCT ATTCCATATC CCAGTAATAC AGCAAAAGTG GGAGTGACGG CAGGCCAGGG ATTGATTGGA ACCCCCAAAA GGCAAAATTA AACATTCCAG ACAGGAGAAC TGTGGCAACC ACTGTGTCTC AGTGCTGTGT TTAGAGAAGA CACCTAGCTG GAACCTTGTA GCTGCTAAAT AATGATGAAA GCTTTCACTG ATGCAATTGG AGTTATTTTC CAGTTTGCCC

CAACAACTGG CAATCGAGCC TAAAAGCAAG GAATAAGAGA CAATGTTGAT TGTCATACTA TAGAGTCTAC CTCCCTACAA CCATCAAGGT TGAATGCACA TAACACGTGT TGCTGGAGTT AAGGTTACCG CTGATAACCA GCCCATATAT TCCATTTTGG TAACACAGAA AGACAGCAGA AGCTCCAGGT TTAATATGGC ATGGAGTCCA GTGATCTGGG GACTTACCTT AAGAAATTCT ATAAACATTC CCTGTGAGCA AATGTTGTGT GAAGGCTACA CAGAAAGCTG TACCTGATTC ACTGGTATAT' GTGTTGCTGA ATGAATATAC CATATTCTGT AACTATAGGA CTTCTGGGAT ACTTCGGAAA GCCCAGTCAT AAAGTAGAGG GCTCCTCCAG CTTCACAACA TCAATTTTTC TTACGAGTGG TGCCAGAAGG AGGGGTATTT ATGGTACCAT TTGGTCCCCA AAACAGAAAT ATCTTGTCTG CAGTTCTAAG AGTGCAGAGG GGGAGCTGAT ACAGGAAATC ATTGGAACAT AAAAAATTAA AAGAAGGGAT AGTGTGTGGA AGGGTGGAAG GGACAAGTAA ATAAATACGT CTAGTTGAGA ATTATCAATT ATAAAATTAC AGGGTACCTT TTTACTGTGA TTGGAATTAG GCTCTAATTA AAGCTGACAA ACATTGGCCA TTTCTGCGTA TCAATTGTTT CAGCTTTGAA TTTTGGAAAG ACAATCTTCA ATGGTAGAAA CAACTGCCTA GTTAACCCAG TCATCAAATG

AACCGCATAT AGAATATAAT ATATTTTTAT AGACTTAAAA AAATGGAATT CAGTTTAGAA AGGTGGATTT ACTGAATTTG GCAGGTTAAA AACAATTGAT TGATGATGGA TAATGTCAAA AGCAATAGCA TAAGGCTTTG TGACAAAATA CACGAGGGAG CATGGTTCCT ATTAGTGTCT TCATCTGTCT AACTGGAATG AAGAGGAGCC CTGTGGGGCA CCTCACTAAT CAGGCCAAGA AGTAGTGAAG GCGAGCTGCA CGTCGCAAGC CATGAAGACC GTTGTGGGAA TCTAACCACC TACTGTCAAG TGTACGAGGA GCAGTTCTGT TGATCATCAG TCACTTGGTG ACTGGAGACT TGTCAAAAGG TAGCAGACGA CAAAAGGATT TCAGGAAGGC GAGCGTTGTC TTTTCATTCT GTTGAGCATT TGCTAGCACT AGAGCAGAAC AGATAATGGA GCCTGTTGAA AAAGGCTTTC CTTTCTGCTT TGCTCTTTCC GAGAGAAGCT GCATAAAGAC TGCTTTACTC GCTATCAGAA

660 720 780 840 900 960 1020 108 0 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 210 0 2160 222 0 22.8 0 2340

2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840

1 cDNA sequence GAGCAGAGGT CTGACGGAAT TACAAGCATA CCAGTAGAGG GCTACAGTAC TTTTATTTGA TCTGATTACA TCTGGATCCT GAAGACTTAA GATGGACATG TTCCGGATAT GAATACCACA CAGAAAGTCT GAAGAATTGG ATTGCATATG TACAAGGCAA GAGATTACCT TACTTAATTA TACCCTTTAG TGTCAAGCAT TAAAATTCCT CGTTTTTTTG TTTACTTAGA CTGAAATAAC CGGAAACAAT GAAAGAACAG ACCAAGGAAC

continued

ATGGAGGTGG ATTCACTCCT AAGGTGCCTT TGCTTCTCAA ATGTAACAAC AAATCGATAC AACGCATAGT CTCATGCGGT AAGCCCTTGT TTATTCTGCA TTGAACTCTT GACCAGATAA GTGAAGGAGC ATCTGACAAT CTTATAAAGT CCCTTCTGGA TCATTAAAAA TGGGTAAAGA ATTCCTTGAC TTTTAGCTAA GAAGTTCAGC TTTTCTTCTT ATTAGTGGCA ATGGCCTTGG AAATTGGAAC TCCATTGAAA AGGAAACTGA

CTTTTATTCA GGTTAAACAA ACATAATTAT TGATGACCTC TGTAGTTCAC TCAGGATATT AGCATGTGCC GATGGACATG GGAAGGGGTG ACTGAATTCG TGAAGTTGGG ACAGTGTACC CGCGTGCAAG CTCTGCAGAG TAGCATCACA TATCTACAAA GGTAACCTGT AGCCCTCCAG CTGGATTGAA TTTAGATGAA TGCATACAGT TTTTTAAACA CTTGCTTTTA AGGGCATGAA ACCTCCTCAA GGGAGTATTA TCATTAAAGC

ACCCAGGACA CTCCGCTTGA AAAATGACAG ATTGTCAGTA AAAACCAGTA GAAGCATCCC AGCTACAAGC TCCTTGCCTA GATCAACTAT ATTCCCTCCA TTTCTCAGTC ATGTTTTATA TGTGTAGAAG ACAAGAAAAC TCCATCACTG ACTGGGGAAG ACTAACGCTG ATAAAATACA TACTGGCCTA TTTGCCGAAG TTGCACTTAT TTCATAGCTG TTAGAGAATG GACAGATACT ACCTACCACT CAAAAACATG CTGAGTTTGC

CCATCAATGC GTATGGACAT ACAAGAATTT CAGGATTTGG CCTCTGAGGA ACTACAGAGG CCAGCAGGGA CTGGAATCAG TCACTGATTA GTGATTTCCT CTGCCACTTT GCACTTCCAA CTGATTGTGG AAACAGCATG TAGAAAATGT CTGTTGCTGA AGCTGGTAAA ATTTCAGTTT GAGACACAAC ATATCTTTTT GGACTCCTGT GTCTTATTTG ATTTCAAATG CCTCCAAGGT CAGGAATGTT GCCTTTGCTT TTTC

CATTGAGGGC CGATGTTTCT CCTTGGGAGG CAGTGGCTTG AGTTTGCAGC CTACGGAAAC AGAATCATCA TGCAAATGAA CCAAATCAAA TTGTGTACGA CACAGTTTAC TATCAAAATT GCAAATGCAG TAAACCAGAG TTTTGTCAAG GAAAGACTCT AGGAAGACAG CAGGTACATC ATGTTCATCG AAATGGATGC TGTTGAAGTT TAAAGCTCAC CTGTAACTTT TATTGGACAC TGCTGGGGCC GAAAGAAAAT

3 9 00 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400

The first five nucleotides in each exon are underlined to indicate the intron-exon boundaries. The methionine initiation codon (ATG), the termination codon (TAAl and the polyadenylation signal (ATTAAAl are indicated.

Genomic structure^^' 17 The human C5 structural gene is 79 kb in length and is highly interrupted by 41 intron-exon boundaries. Although C5 is much larger than the C3 (41 kb) and C4 (20 kb) genes, the intron-exon organization of all three family members is very similar. Two alternatively processed human C5 polyadenylated transcripts have been cloned and sequenced. No in vivo function has been attributed to them, however. 5kb, 21

I I II

I I I iiiimii

Accession numbers Human: Mouse:

cDNA M57729 J05234

41

III—H+fl—III II iiMiiii I II

Genomic M72430 M64852

Deficiency Autosomal recessive. Several C5-deficient families have been reported from different ethnic backgrounds and from different geographic regions. Sera from homozygous deficient individuals lack bactericidal activity and have a severely impaired ability to induce chemotaxis. Consequently, all C5deficient individuals display a propensity for severe recurrent infections, particularly to neisserial species, including meningitis and extragenital gonorrhoea. In addition, one individual with complete C5 protein deficiency has SLE, but it is not clear if this is directly related to her C5 deficiency. Human mutations identified^*: African-American families. Two nonsense mutations: C67toT;Q19tostop C4438 to T; R1476 to stop Mouse mutations identified^^: 2 bp gene deletion TA768-769 in exon 7

Polymorphic variants Polymorphisms of C5 have been examined only cursorily. A protein polymorphism of C5 has been reported in populations of the South Pacific region^^ and from cDNA sequence studies^. T1565C;F518S

References ' Ooi, Y.M. and Colten, H.R. (1979) J. Immunol. 123, 2494-2498. 2 DiScipio, R.G. et al. (1983) J. Biol. Chem. 258, 10629-10636. 3 Haviland, D.L. et al. (1991) J. Immunol. 146, 362-368. ^ Tack, B.F. et al. (1979) Biochemistry 18, 1490-1497. 5 Fernandez, H.N. and Hugh, T.E. (1978) J. Biol. Chem. 253, 6955-6962. 6 Dolmer, K. and Sottrup-Jensen, L. (1993) FEBS Lett. 315, 85-90. 7 Perkins, S.J. et al. (1990) Biochemistry 29, 1175-1180. « Gerard, C. and Gerard, N.P. (1994) Annu. Rev. Immunol. 12, 775-808. 9 Greer, J. (1985) Science 228, 1055-1060. ^0 Zuiderweg, R.P. et al. (1988) Proteins Struct., Funct. Genet. 3, 139-145. ^^ Lambris, J.D. et al. (1998) In The Human Complement System in Health and Disease (Volanakis, J.E. and Frank, M.F. eds). Marcel Dekker, New York, pp. 83-118. ^2 Kohler, P.F. and Muller-Eberhard, H.J. (1967) J. Immunol. 99, 1211-1216. ^3 Wetsel, R.A. et al. (1990) J. Biol. Chem. 265, 2435-2440. ^^ Wetsel, R.A. et al. (1988) Biochemistry 27, 1226-1232. ^5 DTustachio, P. et al. (1986) J. Immunol. 137, 3990-3995. ^6 Haviland, D.L. et al. (1991) J. Biol. Chem. 266, 11818-11825. 1^ Carney, D.F. et al. (1991) J. Biol. Chem. 266, 18786-18791. ^« Wang, X. et al. (1995) J. Immunol. 154, 5464-5471. ^^ Hobart, M. et al. (1981) Ann. Hum. Genet. 45, 1-4.

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Part 4 Terminal Pathway Components

Michael Hobart, Department of Biological Sciences, De Montfort University, Leicester, UK

Physicochemical properties C6 is synthesized as a single chain precursor of 934 amino acids including a leader peptide of 21 amino acids^-^. pi -6-6.5 (multiple bands and polymorphisms'^) -6.5-7 (after neuraminidase treatment) M,(K) -100 N-linked glycosylation sites 2 (324, 855)

D

Structure Not known.

Function C6 connects the components of the terminal complement complex to C5b, the last cleavage product of the complement enzyme cascades. The C5b6 complex avidly binds C7, but in its absence, the bimolecular complex has a prolonged half-life. On binding of C5b6 to C7 a site is revealed by which the complex binds to local substrates. These would normally be antigen on which the complement system was activated, including the membrane of cells or bacteria, but also carbohydrates (e.g. agarose)^. It is assumed that the binding site resides in C7. The bound complex attracts C8 and subsequently C9 to form the terminal complement complex which damages the osmotic integrity of cells.

Tissue distribution I

Serum protein: 45 mg/mP. Primary site of synthesis: liver^. ! Secondary site: macrophage.

D

Regulation of expression C6 is an acute-phase reactant^. CCAAT enhancer binding site is essential*.

Protein sequence2,3 MARRSVLYFI WDKYYQENF SKVRSVLRPS CIARKLECNG AGEPRGEVLD KTDFYKDLTS QAIQASHKKD

LLNALINKGQ CEQICSKQET QFGGQPCTEP ENDCGDNSDE NSFTGGICKT LGHNENQQGS SSFIRIHKVM

ACFCDHYAWT RECNWQRCPI LVAFQPCIPS RDCGRTKAVC VKSSRTSNPY FSSQGGSSFS KVLNFTTKAK

QWTSCSKTCN NCLLGDFGPW KLCKIEEADC TRKYNPIPSV RVPANLENVG VPIFYSSKRS DLHLSDVFLK

SGTQSRHRQI SDCDPCIEKQ KNKFRCDSGR QLMGNGFHFL FEVQTAEDDL ENINHNSAFK ALNHLPLEYN

50 100 150 2 00 2 50 3 00 3 50

Protein sequence SALYSRIFDD RIETKKRVLF YGAALAWEKG VTKRNNLRKA NCEKQSPDYK CEGEKRQEED ENGFIRNEKQ RTECIKPWQ WTPPISNSLT CVFDTDSNDY RLSSNSTKKE GSSTSEKTLN

continued

FGTHYFTSGS AKKTKVEHRC SSGLEEKTFS LQEYAAKFDP SNAVDGQWGC CTFSIMENNG LYLVGEDVEI EVLTITPFQR CEKDTLTKLK FTSPACKFLA SCGYDTCYDW ICEVGTIRCA

LGGVYDLLYQ TTNKLSEKHE EWLESVKENP CQCAPCPNNG WSSWSTCDAT QPCINDDEEM SCLTGFETVG LYRIGESIEL GHCQLGQKQS EKCLNNQQLH EKCSASTSKC NRKMEILHPG

FSSEELKNSG GSFIQGAEKS AVIDFELAPI RPTLSGTECL YKRSRTRECN KEVDLPEIEA YQYFRCLPDG TCPKGFWAG GSECICMSPE FLHIGSCQDG VCLLPPQCFK KCLA

LTEEEAKHCV ISLIRGGRSE VDLVRNIPCA CVCQSGTYGE NPAPQRGGKR DSGCPQPVPP TWRQGDVECQ PSRYTCQGNS EDCSHHSEDL RQLEWGLERT GGNQLYCVKM

40 0 450 500 550 60 0 650 7 00 750 800 85 0 9 00

The leader sequence is underlined and the TV-linked glycosylation sites are indicated (N).

Protein modules 1-21 22-80 81-137 138-175 176-516 517-553 563-611 643-700 703-763 766-840 858-934

Leader peptide TSPl TSPl LDLRA MACPF

EGF TSPl

CCP CCP FIMAC FIMAC

exon 1 exon 1/2 exon 2/3 exon 3/4 exon 4-10 exon 10 exon 11 exon 12/13 exon 14 exon 15/16 exon 16/17

Chromosomal location Human^-^^: 5 p l 2 - 1 4 . 5' C6 3 ' - 3 ' C7 5^^2,13 Orientation on chromosome unknown, near C9. Mouse^'*: chromosome 15. Two loci in some strains.

cDNA sequence^^ AGCTTAGGTC GCCAGACGCT TGCTTCTGTG GGAACCCAGA GAACAGATTT TGCCTCCTGG AAAGTTAGAT GTAGCCTTTC AATAAATTTC AATGACTGTG CGGAAGTATA

CGAGGACACC CTGTCTTGTA ATCACTATGC GCAGACACAG GCAGCAAGCA GAGATTTTGG CTGTCTTGCG AACCATGCAT GCTGTGACAG GAGACAATTC ATCCCATCCC

ACAAACTCTG CTTCATCCTG ATGGACTCAG ACAAATAGTA GGAGACTAGA ACCATGGTCA TCCCAGTCAG TCCATCTAAG TGGCCGCTGC AGATGAAAGG TAGTGTACAG

CTTAAAGGGC CTGAATGCTC TGGACCAGCT GTAGATAAGT GAATGTAACT GACTGTGACC TTTGGGGGAC CTCTGCAAAA ATTGCCAGAA GACTGTGGGA TTGATGGGCA

CTGGAGGCTC TGATCAACAA GCTCAAAAAC ACTACCAGGA GGCAAAGATG CTTGTATTGA AGCCATGCAC TTGAAGAGGC AGTTAGAATG GGACAAAGGC ATGGGTTTCA

TCAAGGCATG GGGCCAAGCC TTGCAATTCT AAACTTTTGT CCCCATCAAC AAAACAGTCT TGAGCCTCTG TGACTGCAAG CAATGGAGAA AGTATGCACA TTTTCTGGCA

60 120 180 240 300 360 420 480 540 600 660

cDNA sequence GGAGAGCCCA AAAAGCAGTA GAGGTACAAA GGACACAATG CCAATTTTTT GCCATTCAAG GTCTTAAACT CTTAACCATC GGGACTCATT AGCAGTGAGG ATTGAAACAA ACCAACAAGC TCCCTGATTC TCTGGTCTGG GTGATTGACT ACAAAACGGA CAGTGTGCTC GTGTGTCAGA AATGCAGTAG AAGAGATCGA GAGGGGGAGA CCATGTATCA TCCGGGTGTC TACTTGGTTG ^CAGTACTTCA ACGGAGTGCA TATAGAATTG TCAAGGTACA GAAAAAGATA TCTGAATGCA GTGTTTGACA AAATGTTTAA CAGTTAGAAT TGTGGCTATG TGCCTATTGC TCATCAACAA AGGAAGATGG CAGCACAATG ACAAACAGCA ATCATTATTC GCCCTGTAGC CATTGTACAA TCACAATTAG CTCCAGAACC ATTTCTGAGT AATAAAACAG CAAAAAATGA

GAGGAGAAGT GGACAAGTAA CTGCAGAAGA AAAATCAACA ATTCCTCAAA CCTCTCACAA TCACAACGAA TGCCTCTAGA ACTTCACCTC AACTAAAGAA AGAAACGCGT TGTCAGAGAA GAGGTGGAAG AGGAGAAGAC TTGAGCTTGC ACAACCTCAG CATGCCCTAA GTGGCACCTA ACGGACAGTG GAACCCGAGA AGCGACAAGA ATGATGATGA CTCAGCCAGT GAGAAGATGT GATGCTTACC TCAAGCCAGT GTGAATCCAT CATGCCAGGG CTCTAACAAA TTTGTATGTC CAGACTCCAA ATAATCAGCA GGGGTCTTGA ACACCTGCTA CCCCACAGTG GTGAGAAAAC AAATACTGCA AACAGATTTA GACTGGCATG TCCCCTGACT ATACCCCTAG AAATAATGTG AAATAAGAAT TCTGAAACAC GTGTATACAG ACAAAAGCCT AATAAACAGG

continued CCTTGATAAC TCCATACCGT TGACTTGAAA AGGCTCATTC GAGAAGTGAA AAAGGATTCT AGCTAAAGAT ATACAACTCT TGGCTCCCTG CTCAGGTTTA TTTATTTGCT ACATGAAGGT GAGTGAATAT ATTTTCTGAG CCCCATCGTG GAAAGCTTTG TAATGGCCGA TGGTGAGAAC GGGTTGTTGG ATGCAATAAT GGAAGACTGC AGAAATGAAA TCCTCCAGAA TGAAATTTCA AGACGGGACC TGTGCAGGAA TGAGCTAACT GAATTCCTGG ATTAAAAGGC TCCAGAAGAA CGATTACTTT ACTCCATTTT AAGGACAAGA TGACTGGGAA CTTCAAGGGT ATTGAACATC TCCTGGAAAG CCATCCCGAA CTCAAAGTTA CTCCTGTTTG GTACCAACTT ACTTCTGAGG AAAACCCATA ATTCTTGAAG GATGTCAAGT TTGCCTTCAT TAAAATATGT

TCTTTCACTG GTTCCGGCCA ACAGATTTCT TCAAGTCAGG AATATCAACC AGTTTTATTA CTGCACCTTT GCTTTGTACA GGAGGCGTGT ACCGAGGAAG AAGAAAACAA TCATTTATAC GGAGCAGCTT TGGTTAGAAT GACTTGGTAA CAAGAGTATG CCCACCCTCT TGTGAGAAAC TCTTCCTGGA CCTGCCCCCC ACATTTTCAA GAGGTCGATC AATGGATTTA TGCCTTACTG TGGAGACAAG GTCCTGACAA TGCCCCAAAG ACACCACCCA CATTGTCAGC GACTGTAGCC ACTTCACCCG CTACATATTG CTTTCATCCA AAATGTTCAG GGAAACCAAC TGTGAAGTGG TGTTTGGCCT GAACCAACTC CTGACAAAAA GGCATGTCTT CCACAGCAGT CCCTTATGTA ATTTTCTTCA CCCAGCTTTC ACTGACCAAA GAAGCATACA AGC

GAGGAATATG ATCTGGAAAA ACAAGGATTT GGGGGAGCTC ATAATTCTGC GGATCCATAA CTGATGTCTT GCCGAATATT ATGACCTTCT AAGCCAAACA AAGTGGAACA AGGGAGCAGA TGGCATGGGA CAGTGAAGGA GAAACATCCC CAGCCAAGTT CAGGGACTGA AGTCTCCAGA GTACCTGTGA AACGAGGAGG TCATGGAAAA TTCCTGAGAT TCCGGAATGA GCTTTGAAAC GGGATGTGGA TTACACCATT GCTTTGTTGT TTTCAAACTC TGGGACAGAA ATCATTCAGA CTTGTAAGTT GTTCCTGCCA ACAGCACAAA CCTCCACTTC TCTACTGTGT GAACTATAAG AGCACAATTA CTACAAATGA TTATTTTCTG ATTCAGTTCC CTCGTAAATT GCCTGTGACA ATGAGTTAAT ATATCTTCAT GTCCTGAGAA TTCATTCAGG

TAAAACTGTC TGTCGGCTTT AACTTCTCTT TTTCAGTGTA CTTCAAACAA AGTGATGAAA TTTGAAAGCA CGATGACTTT CTATCAGTTT CTGTGTCAGG TAGGTGCACC GAAATCCATA GAAAGGGAGC AAATCCTGCT CTGTGCAGTG CGATCCTTGC ATGTCTGTGT TTATAAATCC TGCTACTTAT GAAACGCTGT CAATGGACAA AGAAGCAGAT AAAGCAACTA TGTTGGATAC ATGCCAACGG TCAGAGATTG TGCTGGGCCA TCTCACCTGT ACAATCAGGA AGATCTCTGT TTTGGCTGAG AGACGGCCGC GAAAGAATCC CAAATGTGTC CAAAATGGGA ATGTGCAAAC CTGCTAGGCC GAATTCTTGC TTAGTTTGAG AGCTCATGAC CTCCTGTTCA TTAAGCATTC AAACAGAAAT TCAACAAATA CTCGGCAGAT GGTAGACACA

720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420

The first five nucleotides in each, exon are underiined to indicate the intron-exon boundaries. The methionine initiation codon (ATGJ, the termination codon (TAG) and the polyadenylation signal (AATAAA) are indicated.

Genomic structure^^ The gene spans approximately 80kbp and is encoded in 18 exons (starting with exon 0, to align exon numbering with C9, the first member of the gene family investigated). The introns vary from 450bp to 12 kb. Note that the majority of the exons are asymmetric with respect to boundary phase type and that there is a strong counter-correspondence of protein modules (domains) and exons.

-+H—\

H-H

INI II

II—I

Accession numbers Human:

cDNA HSC6A

Genomic HSC6X1-HSC6X17

Deficiency Complete Autosomal recessive^^. Loss of lytic function of complement. Patients show increased susceptibility to recurrent meningococcal meningitis and systemic N. gonorrhoeae infection, though many, including siblings sharing the deficiency may be healthy^^-^^. Mutations identified: 291 delC^^ One Japanese patient 879 delG^^ 30 Cape Coloured South African chromosomes,- three Dutch chromosomes 1195 delC^^'^^ Seven African chromosomes 1396 delG^^^^ Four African chromosomes Subtotap3.24 Autosomal recessive. Very substantial but incomplete loss of lytic function of complement (approximately 2% normal haemolytic activity and protein). Patients show no clinical effects. Rare (6 patients), but often associated with subtotal C7 deficiency. Mutation identified: Splice defect at 5' donor site of intron 15 (GT to GC)^^; 8 patients

Polymorphic variants Two common A (0.65) and B (0.35) and at least 10 rare phenotypic variants have been identified by isoelectric focusing^'^^ and detection by functional overlay, immunoblotting or immunofixation and are found at approximately these frequencies in all h u m a n populations. In addition, there are two known RFLPs and an unusual combined insertion/deletion polymorphism in intron 10. The D N A polymorphisms are tabulated below, including the D N A origin of the A/B phenotype.

Locus 1^'27,28 A413C; El 19A; A/B frequency 0.35 Locus 229-31 CCGG/CTGG in intron 3 frequency 0.35 Locus 3^2 Combined insertion/deletion in intron 10 frequency 0.05 Locus 4^^'^"^ T(3gl polymorphism in intron 16? frequency 0.4 Markers at loci 1 and 2 show strong allelic association in North Europeans^^, but not in other populations. Other markers do not show associations.

References ' 2 3 ^

DiScipio, R.G. et al. (1982) Mol. Immunol. 19, 1425-1431. DiScipio, R.G. and Hugli, T.E. (1989) J. BioL Chem. 264,16197-16206. Haefliger, J.-A. et al. (1989) J. BioL Chem. 264,18041-18051. Hobart, M.J. et al. (1975) In Protides of the Biological Fluids, 22nd Colloquium (Peeters, H. ed.), Pergamon Press, Oxford, pp. 575-580. 5 Wurzner, R. et al. (1991) Clin. Exp. Immunol. 83, 430-437. 6 Hobart, M.J. et al. (1977) J. Exp. Med. 146, 629. 7 Thompson, R.A. and Lachmann, P.J. (1970) J. Exp. Med. 131, 629-641. s Gonzalez, S. and Lupez-Larrea, C. (1996) J. Immunol. 157, 2282-2290. ^ Abbott, C. et aL (1989) Genomics 4, 606-609. ^0 Jeremiah, S.J. et al. (1990) Ann. Hum. Genet. 54, 141-147. '' Rogde, S. et al. (1991) J. Med. Genet. 28, 587-590. ^2 Hobart, M.J. et al. (1993) Hum. Mol. Genet. 2, 1035-1036. " Setien, F. et al. (1993) Immunogenetics 38, 341-344. ^"^ Hayakawa, J. et al. (1985) Immunogenetics 22, 637-642. ^5 Hobart, M.J. et aL (1993) Biochemistry 32, 6198-6205. ^6 Leddy, J.P. et aL (1974) J. Clin. Invest. 53, 544-553. ^^ Wurzner, R. et al. (1992) Immunodeficiency Rev. 3, 123-147. ^s Ross, S.C. and Denson, P. (1984) Medicine 63, 243-273. 29 Orren, A. et al. (1987) Immunology 62, 249-253. 20 Nishizaka, H. et aL (1996) J. Immunol. 156, 2309-2315. 2i Hobart, M.J. et al. (1998) Hum. Genet. 103, 506-512. 22 Zhu, Z.B. et al. (1998) Clin. Exp. Immunol. I l l , 91-96. 23 Lachmann, P.J. et al. (1978) Clin. Exp. Immunol. 33, 193-803. 2-^ Morgan, B.P. et al. (1989) Clin. Exp. Immunol. 75, 396-401. 25 Fernie, B.A. et aL (1996) J. ImmunoL 157, 3648-3657. 26 Wiirzner, R. et al. (1999) Exp. Clin. Immunogenet. 15, 268-285. 27 Fernie, B.A. et al. (1993) Hum. Mol. Genet. 2, 591-592. 2« Dewald, G. et al. (1993) Biochem. Biophys. Res. Commun. 194, 458-464. 39 Goto, E. et al. (1991) Nucleic Acids Res. 19, 194. 30 Potter, P.C. et al. (1993) Exp. Clin. Immunogenet. 10, 38-44. 3^ Fernie, B.A. et al. (1996) Exp. Clin. Immunogenet. 13, 92-103. ^^ Fernie, B.A. and Hobart, M.J. (1997) Hum. Genet. 100, 104-108. ^^ Goto, E. et al. (1991) Immunogenetics 33, 184-187. 3^ Fernie, B.A. et al. (1995) Ann. Hum. Genet. 59, 163-181.

Michael Hobart, Department of Biological Sciences, De Montfort University, Leicester, UK

Physicochemical properties C7 is synthesized as a single-chain precursor of 843 amino acids including a leader peptide of 22 amino acids^. pi -6-6.5 (multiple bands and polymorphisms^) -6.3-7 (after neuraminidase treatment-^) Mr (K) -95 N-linked glycosylation site 1 (754)^

D

Structure Unknown.

Function The C5b6 complex avidly binds C7 and a site is revealed by which the complex binds to local substrates. These would normally be antigen on which the complement system was activated, including the membrane of cells or bacteria, but also carbohydrates (e.g. agarose)^. It is assumed that the binding site resides in C7. The bound complex attracts C8 and subsequently C9 to form the terminal complement complex which damages the osmotic integrity of cells.

Tissue distribution Serum protein: 90 mg/mP. Primary site of synthesis: granulocytes^.

Regulation of expression C7 is only a weak acute-phase reactant. This leads to a molar deficit compared with C5 and C6 during acute inflammation^.

Protein sequence^ MKVISLFILV SVAVYGQYGG LVCNGDSDCD FRNRVINTKS YNSTWSYVKH LWENTVEVA THYLQSGSLG HGCKELENAL RRYSAWAESV

GFIGEFQSFS QPCVGNAFET EDSADEDRCE FGGQCRKVFS TSTEHTSSSR QFINNNPEFL GEYRVLFYVD KAASGTQNNV TNLPQVIKQK

SASSPVNCQW QSCEPTRGCP DSERRPSCDI GDGKDFYRLS KRSFFRSSSS QLAEPFWKEL SEKLKQNDFN LRGEPFIRGG LTPLYELVKE

DFYAPWSECN TEEGCGERFR DKPPPNIELT GNVLSYTFQV SSRSYTSHTN SHLPSLYDYS SVEEKKCKSS GAGFISGLSY VPCASVKKLY

GCTKTQTRRR CFSGQCISKS GNGYNELTGQ KINNDFNYEF EIHKGKSYQL AYRRLIDQYG GWHFWKFSS LELDNPAGNK LKWALEEYLD

50 100 15 0 2 00 2 50 3 00 3 50 400 4 50

Protein sequence EFDPCHCRPC GGWSCWSSWS EHLRLLEPHC EGYSLIGNPV VGEKVTVSCS KCQRWEKLQN QGRNYTLTGR GFSICVEVNG

continued

QNGGLATVEG PCVQGKKTRS FPLSLVPTEF ARCGEDLRWL GGMSLEGPSA SRCVCKMPYE DSCTLPASAE KEQTMSECEA

THCLCHCKPY RECNNPPPSG CPSPPALKDG VGEMHCQKIA FLCGSSLKWS CGPSLDVCAQ KACGACPLWG GALRCRGQSI

TFGAACEQGV GGRSCVGETT FVQDEGPMFP CVLPVLMDGI PEMKNARCVQ DERSKRILPL KCDAESSKCV SVTSIRPCAA

LVGNQAGGVD ESTQCEDEEL VGKNWYTCN QSHPQKPFYT KENPLTQAVP TVCKMHVLHC CREASECEEE ETQ

500 550 600 650 7 00 750 800

The leader sequence is underlined and the N-linked glycosylation site is indicated (N).

Protein modules 1-22 24-83 84-121 122-450 451-487 497-545 570-627 630-689 694-770 771-843

Leader peptide TSPl LDLRA MACPF

EGF TSPl

CCP CCP FIMAC FIMAC

exon 0/1 exon 2/3 exon 3/4 exon 4-10 exon 10 exon 11 exon 12/13 exon 14 exon 15/16 exon 16/17

Chromosomal location Human^-^'^: 5pl2-14. 5' C6 3'-3' C7 5'^^'^2 Orientation on chromosome unknown, near C9. Mouse^^'^'^: chromosome 15.

cDNA sequence^ ATGAAGGTGA AGTGCCTCCT GGCTGTACCA CAGCCTTGTG ACAGAGGAGG TTGGTTTGCA GAGTCAGAAA GGAAATGGTT TTTGGTGGTC GGAAATGTCC TACAATAGTA AAGCGCTCCT GAAATCCATA CAGTTCATTA TCCCACCTCC ACACATTATC

TAAGCTTATT CTCCAGTCAA AGACTCAGAC TTGGAAATGC GATGTGGAGA ATGGGGATTC GGAGACCTTC ACAATGAACT AATGTAGAAA TGTCCTATAC CTTGGTCTTA TTTTTAGATC AAGGAAAGAG ATAACAATCC CCTCTCTGTA TGCAATCTGG

CATTTTGGTG CTGCCAGTGG TCGCAGGCGG TTTTGAAACA GCGTTTCAGG TGACTGTGAT CTGTGATATC CACTGGCCAG GGTGTTTAGT ATTCCAGGTG TGTAAAACAT TTCATCATCT TTACCAACTG AGAATTTTTA TGACTACAGT GTCGTTAGGA

GGATTTATAG GACTTCTATG TCAGTTGCTG CAGTCCTGTG TGCTTTTCAG GAAGACAGTG GATAAACCTC TTTAGGAACA GGGGATGGAA AAAATAAATA ACGTCGACAG TCTTCACGCA CTGGTTGTTG CAACTTGCTG GCCTACCGAA GGAGAATACA

GAGAGTTCCA CCCCTTGGTC TGTATGGGCA AACCTACAAG GTCAGTGCAT CTGATGAAGA CTCCTAACAT GAGTCATCAA AAGATTTCTA ATGATTTTAA AACACACATC GTTATACTTC AGAACACTGT AGCCATTCTG GATTAATCGA GAGTTCTATT

AAGTTTTTCA AGAATGCAAT GTATGGAGGC AGGATGTCCA CAGCAAATCA CAGATGTGAG AGAACTTACT TACCAAAAGT CAGGCTGAGT TTATGAATTT ATCTAGTCGG ACATACCAAT TGAAGTGGCT GAAG