CKB8-1

CK 8-1 Byung-S. Youn1 and Byoung S. Kwon2,3,* 1

Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, Indiapolis, IN 46202, USA 2 The Immunomodulation Research Center, University of Ulsan, Ulsan, Korea 3 Department of Ophthalmology, LSUMC, 2020 Gravier Street Suite B, New Orleans, LA 70112, USA * corresponding author tel: 504-412-1200 ex 1379, fax: 504-412-1315, e-mail: [email protected] DOI: 10.1006/rwcy.2000.11013.

SUMMARY

Structure

cDNA for two new members of the human CC chemokine family was isolated based on structural and functional similarities to leukotactin 1 (Lkn-1). One of these cDNA clones was identical to the previously isolated CC chemokine, CK 8 (MPIF1), whereas the other is a major splicing variant, therefore named CK 8-1. The mature proteins of CK 8-1 and CK 8 consisted of 116 and 99 amino acids with calculated molecular weights of 12,500 and 10,950, respectively. Both CK 8-1 and CK 8 were potent agonists for CCR1. These chemokines induced migration of neutrophils, monocytes, and lymphocytes, which is similar to Lkn-1. They also significantly suppressed colony formation by human bone marrow, granulocyte-macrophage, erythroid, and multipotential progenitor cells stimulated by combinations of growth factors. Lkn-1 and CK 8-1 gene were mapped to the same bacterial artificial chromosome. To our knowledge, this is the first example showing that an alternative splicing produces two active CC chemokines from a single gene.

CK 8-1 has the basic frame of a CC chemokine (Figure 1). In addition to this, two structural features of its structure are prominent: it has a fairly long Nterminus to the first two cysteines (49 amino acids) and six conserved cysteines. The additional two cysteines besides the four cysteines conserved in other CC chemokine family are also conserved in Lkn-1 (MIP-5/HCC-2), MPIF1, MIP-1 /MRP-2, and Mu C10. Therefore, we have referred to this subgroup of CC chemokines as C6 CC chemokines.

BACKGROUND

Discovery The cDNA encoding CK 8-1 was isolated from the THP1 cDNA library with the use of Lkn-1 cDNA as a probe, meaning that CK 8-1 and Lkn-1 have a significant degree of the nucleotide identity (over 80% identity) (Youn et al., 1998a). As a matter of fact, the nucleotide sequence of the CK 8-1 signal peptide is 95% identical to that of Lkn-1.

Main activities and pathophysiological roles The recombinant CK 8-1 chemoattracts lymphocytes, monocytes, and, to a lesser degree, neutrophils.

GENE AND GENE REGULATION

Accession numbers cDNA: 058913

Sequence The cDNA encoding CK 8-1 is 642 bp, which includes a 50 UTR (71 bp), an open reading frame (45 bp), and a 30 UTR (156 bp). The 30 UTR contains a typical polyadenylation signal (AATAAA) (Figure 1).

1260 Byung-S. Youn and Byoung S. Kwon Figure 1 Nucleotide sequence of cDNA encoding CK 8-1 and the deduced amino acid sequence. The nucleotide sequence of the message strand is numbered in the 50 to 30 direction. The 50 untranslated sequence is indicated by negative numbers. The predicted amino acid sequence is shown below the nucleotide sequence. The stop codon is indicated by asterisks. The six cysteine residues are depicted in boxes. CKβ8 -1 -71

C TGGCATCCCG

-60

AGAAGCCAGG AAGCAGTGAG CCCAGGAGTC CTCGGCCAGC CCTGCCTGCC CACCAGGAGG

1 1

ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTG ATG CTT GTT ACT GCC CTT M K V S V A A L S C L M L V T A L

52 18

GGA TCC CAG GCC CGG GTC ACA AAA GAT GCA GAG ACA GAG TTC ATG ATG TCA G S Q A R V T K D A E T E F M M S

103 35

AAG CTT CCA TTG GAA AAT CCA GTA GTT CTG GAC ATG CTC TGG AGG AGA AAG K L P L E N P V L L D M L W R R K

154 52

ATT GCT CCT CAG ATG ACC CTT TCT CAT GCT GCA GGA TTC CAT GCT ACT AGT I G P Q M T L S H A A G F H A T S

205 69

GCT GAC TGC TGC ATC TCC TAC ACC CCA CGA AGC ATC CCG TGC TCA CTC CTG L D C C I S Y T P R S I P C S L L

256 86

GAG AGT TAC TTT GAA ACG AAC AGC GAG TGC TCC AAG CCG GGT GTC ATC TTC E S Y F E T N S E C S K P G V I F

307 103

CTC ACC AAG AAG GGG CGA CGT TTC TGT GCC AAC CCC AGT GAT AAG CAA GTT L T K K G R R F C A N P S D K Q V

358 120

CAG GTT TGC ATG AGA ATG CTG AAG CTG GAC ACA CGG ATC AAG ACC AGG AAG Q V C M R M L K L D T R I K T R K

409 137

AAT TGA ACTTGTCAAG GTGAAGGGAC ACAAGTTGCC AGCCACCAAC TTTCTTGCCT CAAC N ***

469

TACCT CCTGAATTAT TTTTTTAAGA AGCATTTATT CTTGTGTTCT GGATTTAGAG CAATT

530

CATCT AATAAACAGT TTCTCACTTA AAAAAAAAAA AAAAA

Chromosome location Using bacterial artificial clones (BAC), the gene for CK 8-1 has been mapped to human chromosome 17 (Youn et al., 1998a). Interestingly, these BAC clones also carry the genes for Lkn-1 and LMC (HCC-4/ LEC), thus the gene for CK 8-1 is located at a cluster of CC chemokine genes as previously described (Youn et al., 1998b).

Cells and tissues that express the gene To investigate the tissue distribution of CK 8-1 mRNA, CK 8-1-specific oligonucleotide was labeled

and hybridized to the human multiple tissue blot and the human master blot containing poly(A) RNAs derived from a variety of tissues. CK 8-1 mRNA was only detected in the pancreas and skeletal muscle. THP1 cells (a human monocytic leukemic cell) express the CK 8-1 as mRNA, suggesting that human monocytes may express the CK 8-1 mRNA.

PROTEIN

Sequence The mature CK 8-1 protein consists of 116 amino acids with a calculated molecular weight of 12 kDa. The mature CK 8-1 is preceded by a 21 amino acid

CK 8-1 1261 signal peptide. CK 8-1 has the longest N-terminus (49 amino acids) ever found in CC chemokines. This notable feature may imply a novel function of CK 8-1, because the N-terminus of CC chemokine is thought to be important for receptor activation. When the THP1 cDNA library was screened with the Lkn-1 cDNA probe, a structurally related cDNA to CK 8-1 was isolated. The subsequent cDNA library screening and RT-PCR analysis revealed that the cDNA encoded MPIF (CK 8), which is an alternative splicing product transcribed from the CK 8-1 gene (Patel et al., 1997; Forssmann et al., 1997). Several BAC clones representing the CK 8-1 gene were isolated, and exon±intron organization of the gene was determined. As seen in Figure 2, the CK 8-1 gene is comprised of four exons, whereas most CC chemokine genes

contain three exons. Alternative splicing occurred at a region of exon 3 before the CC motif in such a way that MPIF1 is shortened by 17 amino acids at the N-terminus. Among these 17 amino acids was one amino acid created as a unique residue (arginine), which is not found in CK 8-1.

Important homologies The CK 8-1 signal peptide has a 86% amino acid identity to that of Lkn-1. The N-terminal region has a 50% and 60% amino acid identity to those of Lkn-1 and MPIF1, respectively. More importantly, six conserved cysteines are found in CK 8-1, along with Lkn-1, MIP-1 /MRP-2, and Mu C10 (Figure 3).

Figure 2 MPIF1 is an alternative splicing form of CK 8-1. (a) Two bacterial artificial clones (BAC) (110-F-6 and 125-F-14) representing the CK 8-1/MPIF1 gene were isolated and further analyzed for determination of exon and intron organization. A 3.0 kb HindIII fragment was isolated from 110-F-6 and the entire nucleotide sequence covering a portion of exons 2, 3, and 4 and introns 2 and 3 was determined. A 1.3 kb HindIII fragment was also isolated from 125-F14 and the entire nucleotide sequence was determined. This sequence included portions of exon 2 and intron 1. Two independent cDNA clones representing CK 8-1 and MPIF1 were isolated from a THP1 cDNA library. Their sequences were determined. Exons are shown in boxes. The putative splicing donor (gt) or acceptor (cag or tag) consensus sequences are also shown. The Cys±Cys motifs are represented in circles in exon 3,whereas the extra two cysteines are denoted by asterisks in cDNAs. (b) Alternative splicing deleted 57 bp from exon 3 in CK 8-1 in such a way that MPIF1 was formed. A 17 amino acid deletion point is indicated in MPIF1 cDNA. Genomic DNA 5'

CC

> 5 kb

ATG

G

423 bp

cag AT A gt

Exon 1 (76 bp)

STOP

434 bp

cag TG

Exon 2 (80 bp)

AT gt

tag C

Exon 3 (166 bp)

TGA

3'

Exon 4 (112 bp)

CKβ8-1 cDNA N

* C

C C

C

C

C

* C

116AA

Genomic DNA 5'

G

474 bp

cag AT A gt

Exon 1 (76 bp)

MPIF1

CC

> 5 kb

ATG

434 bp

cag GA AT gt

Exon 2 (60 bp)

Exon 3 (115 bp)

C C

* C

C

99AA

C

* C

STOP

TGA Exon 4 (112 bp)

cDNA 17 AA N

tag C

C

3'

Figure 3 Alignment of CK 8-1 and MPIF1 with Lkn-1, Mu C10, MIP-1 /MRP-2, MIP-1, and MIP-1 . The putative signal peptide sequence is not shown. Shown in boxes are four conserved cysteine residues, whereas two extra cysteines conserved in C6 CC chemokine family members are indicated by filled circles. Gaps were introduced for optimum alignment. CKb8-1 Lkn-1 MPIF1 Mu C10 MIP-1r/MRP-2 hMIP-1a mMIP-1a hMIP-1b

RVTKDAETEF QFTNDAETEL RVTKDAETEF G–––––LIQE QITHATETKE A––––––––– A––––––––– A–––––––––

MMSKLPLENP MMSKLPLENP MMSKLPLENP ME–––KEDRR VQSSLKAQQG –––––––––– –––––––––– ––––––––––

VLLDMLWRRK VVL––––––– VLL––––––– Y––––––––– L––––––––– –––––––––– –––––––––– ––––––––––

IGPQMTLSHA –––––––––– –– ––––––D –––NPPIIH– –––EIEMFH– ––––––––SL ––––––––PY ––––––––PM

AGFHATSADC NSFHF–AADC R–FHATSADC QGFQDTSSDC MGFQD-SSDC A-A-DTPTAC G-A-DTPTAC G-S-DPPTAC

Consensus

CISYTPRSIP CTSYISQSIP CISYTPRSIP CFSYATQ–IP CLSYNSR-IQ CFSYTSRQIP CFSYSRK-IP CFSYTARKLP

C

CKb8-1 Lkn-1 MPIF1 Mu C10 MIP-1r/MRP-2 hMIP-1a mMIP-1a hMIP-1b

·CSLLESYFET CSLMKSYFET CSLLESYFET CKRFIYYFPT CSRFIGYFPT QNFIADYFET RQFIVEVFET RNFVVDYYET

NSECSKPGVI SSECSKPGVI NSECSKPGVI SGGCIKPGII SGGCTRPGII SSQCSKPGVI SSLCSQPGVI SSLCSQPAVV

FLTKKGRRFC FLTKKGRQVC FLTKKGRRFC FISRRGTQVC FISKRGFQVC FLTKRSRQVC FLTKRNRQIC FQTKRSKQVC

ANPSDKQVQV AKPSGPGVQD ANPSDKQVQV ADPSDRRVQR ANPSDRRVQR ADPSEEWVQK ADSKETWVQE ADPSESWVQE

YFET

SS CS PGVI

F TK

A PS

Consensus

Q C

C SY

· CMRMLKLDTR

VQ

P

I-KTRKN –––PYSI I-KTRKN SGNKVIA P-RTYYK

CMKKLK–––– CMRMLKLDTR CLSTLKQGPR CIERLEQNSQ YVSDLE–––– YITDLE–––– VYYDLE––––

50 42 43 37 45 21 20 21

106 92 99 95 101 70 69 69

L

Figure 4 (a) Chemotactic activities of the recombinant CK 8-1 (rCK 8-1) and MPIF1 (rMPIF1). Lymphocytes, monocytes, and neutrophils were isolated from human peripheral blood buffy coat. Chemotaxis assays were performed using a Boyden chamber kit with varying concentrations of rCK 8-1 and rMPIF1. Microscopic observation at 100 was used to count each cell type. Three independent regions were selected and cell numbers were counted. The chemotactic index was driven by the following formula: (the number of cells migrated in response to rCK 8-1 or rMPIF1)/(the number of cells migrated in response to medium alone). (b) Each leukocyte population was isolated and examined for its responsiveness to rCK 8-1 or rMPIF1, along with RANTES and MIP-1. Two million cells were loaded with Fura-2/AM, stimulated with 25 nM of the chemokine indicated, and used for calcium flux assay. Lymphocytes

(a)

Monocytes Chemotactic index

Chemotactic index

CKβ8-1

8

MPIF1 RANTES

6 4 2 0

Neutrophils 10

10

0

0.1

1

10

100

1000 10000

CK β 8-1

8

MPIF1 MIP-1α

6 4 2 0

0.1

1

10

100

1000

CK β 8-1 MPIF1 IL-8

8 6 4 2 0

0

0

0.1

Chemokine (ng/ml)

Chemokine (ng/ml)

1

10

100 5000 1000 10000

Chemokine (ng/ml)

Lymphocytes

Lymphocytes

Monocytes

Monocytes

RANTES CKβ8-1

CKβ 8-1 RANTES

MIP-1α CK β8-1

CKβ 8-1 MIP-1α

Neutrophils IL-8

CKβ 8-1

Neutrophils CK β 8-1

IL-8

Relative fluorescence

(b)

Chemotactic index

10

50 100 150 200 250 Lymphocytes

Lymphocytes MPIF1 RANTES

Monocytes MIP-1α

MPIF1

50 100 150 200 250 50 100 150 200 250 50 100 150 200 250 Monocytes MPIF1 MIP-1α

Neutrophils IL-8

MPIF1

Neutrophils MPIF1

IL-8

Relative fluorescence

RANTES MPIF1

50 100 150 200 250 50 100 150 200 250

50 100 150 200 250

50 100 150 200 250 50 100 150 200 250

50 100 150 200 250 50 100 150 200 250 50 100 150 200 250

Time (seconds)

CK 8-1 1263

Posttranslational modifications CK 8-1 does not appear to have putative N- or O-glycosylation sites.

RECEPTOR UTILIZATION The capacity of CK 8-1 to induce rapid calcium flux was assessed on lymphocytes, monocytes, and neutrophils along with MPIF1. CK 8-1 and MPIF1 induced a robust calcium flux at 25 nM in these cells (Figure 4). CK 8-1 and MPIF1 were able to desensitize the response of lymphocytes and monocytes to a subsequent stimulation with RANTES and MIP-1, respectively. RANTES and MIP-1 were also able to desensitize the responsiveness of these

cells to subsequent stimuli with CK 8-1 and MPIF1. These data indicate that CK 8-1 and MPIF1 share a receptor(s) with RANTES and MIP-1. To identify receptor(s) for CK 8-1 along with MPIF1, a transient calcium flux assay was performed, using human osteogenic sarcoma cell lines (HOS cells) expressing CCR1, CCR2B, CCR3, CCR4, CCR5, or CXCR4. A robust calcium flux was only detected in the CCR1 transfectants at 25 nM CK 8-1, or in response to the same dose of MPIF1 (Figure 5). CK 8-1 and MPIF1 completely desensitized the responsiveness of the CCR1-transfected cells primarily stimulated with MIP-1, suggesting that MIP-1, CK 8-1, and MPIF1 all use CCR1 as a common receptor. To compare agonistic potential among CK 8-1, MPIF1, and MIP-1, dose-response experiments were conducted. These data suggest that CK 8-1 and MPIF1 exhibited comparable agonistic potential to that of MIP-1.

Figure 5 (a) CCR1 is a receptor for CK 8-1 and MPIF. HOS cells expressing CCR1 were loaded with Fura-2/AM for 30 min and washed. Two million cells in 2 mL were used for calcium flux assay. 25 nM chemokine was used for the assay. CCR1 HOS cells were sequentially stimulated with IL-8, MIP-1, and MIP-1. Their calcium response was measured. For desensitization experiments, the HOS cells were stimulated with 25 nM MIP-1 and CK 8-1 or CK 8-1 and MIP-1, or were stimulated with 25 nM MIP-1 and MPIF1 or MPIF1 and MIP-1. (b) Comparison of agonistic potential. Fura-2/AM-loaded CCR1 HOS cells were stimulated with the indicated concentrations of chemokines, and relative fluorescence was measured. The peak amplitude of calcium response versus chemokine concentration was plotted on a semilog scale. MIP-1α CKβ 8-1

CKβ 8-1 MIP-1α

MIP-1α MPIF1

MPIF1 MIP-1α

Relative fluorescence

(a) IL-8 MIP-1α MIP-1α

100

200

300

50 100 150 200 250

50 100 150 200 250

50 100 150 200 250

50 100 150 200 250

Time (seconds)

Peak amplitude

4

3

Peak amplitude

4

(b)

CKβ 8-1

2 MIP-1α

1 0

0

1

10

Concentration (nM)

100

3

MPIF1 MIP-1α

2

1 0

0

1

10

Concentration (nM)

100

1264 Byung-S. Youn and Byoung S. Kwon

IN VITRO ACTIVITIES

In vitro findings Based on chemotaxis assay, CK 8-1 exhibited a potent chemotactic effect on lymphocytes, monoFigure 6 Effect of CK 8-1 and MPIF1 on colony formation by low-density human marrow cells. Lowdensity human bone marrow cells were plated at 5  104 cells/mL with 10±30% FBS, growth factors, and chemokines (50 ng/mL) in a 0.3% agar or 1% methylcellulose culture medium. Colony formation was scored 14 days after incubation in 5% CO2 and lowered (5%) O2. Results are representative of three separate samples.

Colony formation

70 60 50

Colony formation

Control medium

Preimmune serum

Anti-CKβ8-1

Preimmune serum

Anti-CKβ8-1

(b) BFU-E

Control medium

Control medium Control diluent

Colony formation

The CK 8-1 message can be upregulated in THP1 cells by IL-4 and lipopolysaccharide (Youn et al., 1998a).

References

20 10 0

20 18 16 14 12 10 8 6 4 2 0

Regulatory molecules: Inhibitors and enhancers

(a) CFU-GM

40 30

100 90 80 70 60 50 40 30 20 10 0

cytes, and neutrophils. Except for its inability to chemoattract eosinophils, CK 8-1 appears to be indistinguishable from Lkn-1 (MIP-5/HCC-2). As has been shown for Lkn-1 (MIP-5/HCC-2) and MIP-1, CK 8-1 showed a potent suppressive effect on colony formation by different lineages of hematopoietic progenitor cells (Figure 6).

CKβ8-1 MPIF1

(c) CFU-GEMM

Forssmann, U., Delgado, M. B., Uguccioni, M., Loetscher, P., Garotta, G., and Baggiolini, M. (1997). CK 8, a novel CC chemokine that predominantly acts on monocytes. FEBS Lett. 408, 211±216. Patel, V. P., Kreider, B. L., Li, Y., Li, H., Leung, K., Salcedo, T., Nardelli, B., Pippalla, V., Gentz, S., Thotakura, R., Parmelee, R., Gentz, R., and Garotta, G. (1997). Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors. J. Exp. Med. 185, 1163±1172. Youn, B.-S., Zhang, S., Broxmeyer, H. E., Cooper, S., Antol, K., Fraser Jr, M. J., and Kwon, B. S. (1998a). Characterization of CK 8 and CK 8-1: two alternatively spliced forms of human -chemokine, chemoattractants for neutrophils, monocytes, and lymphocytes, and potent agonists at CC chemokine receptor 1. Blood 91, 3118±3128. Youn, B.-S., Zhang, S., Broxmeyer, H. E., Antol, K., Fraser Jr, M. J., Hangoc, G., and Kwon, B. S. (1998b). Isolation and characterization of LMC, a novel lymphocyte and monocyte chemoattractant human CC chemokine, with myelosuppressive activity. Biochem. Biophys. Res. Commun. 247, 217±222.

MIP-1α

ACKNOWLEDGEMENTS Control medium

Preimmune serum

Anti-CKβ8-1

SRC funds to IRC from the Korean Ministry of Science and Technology are greatly appreciated.