Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1)

Gene, 156 (1995) 291-295 Elsevier Science B.V.

291

GENE 08764

Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1) (Neutrophil; inflammation; dog; immunoglobulin supergene family)

Anthony M. Manning a, Hui-Fang Lu b, Gilbert L. Kukielka c, Mary G. Oliver d'* , Theresa Ty d, Carol A. Toman d, Roger F. Drong a, Jerry L. Slightom ~, Christie M. Ballantyne c, Mark L. Entman c, C. Wayne Smith d and Donald C. Anderson a aUpjohn Laboratories, Kalamazoo, MI 49001, USA; and Departments ofblmmunology, CMedicine, and dpediatrics, Baylor College of Medicine, Houston, TX 77030, USA Received by A.M. Chakrabarty: 12 July 1994; Revised/Accepted: 23 August/26 August 1994; Received at publishers: 9 January 1995

SUMMARY

Canine intercellular adhesion molecule-1 (ICAM-1) plays a primary role in the adherence of canine neutrophils to endothelial cells and in the cytotoxicity of canine neutrophils for adult cardiac myocytes. We have cloned the canine I C A M - I gene and have analyzed the conservation of ICAM-1 amino acid (aa) sequences in man, chimpanzee, mouse, rat and dog. Canine ICAM-1 displays 61% identity with human ICAM-1. Cys residues critical to the immunoglobulin (Ig) fold structure and four sites of N-linked glycosylation are absolutely conserved in ICAM-I from all species. Residues in the cytoplasmic tail associated with cytoskeletal a-actinin binding are highly conserved, supporting the hypothesis that intracellular attachment is indeed important for ICAM-1 function. Residues critical for human ICAM-1 binding to the ~2-integrin leukocyte-function-associated antigen 1 (LFA-1) are highly conserved between all species, whereas those residues demonstrated to play an important role in interaction of human ICAM-1 with macrophage activation complex 1 (Mac-l) are not highly conserved. Residues critical for ICAM-1 binding to rhinovirus and malaria-infected red blood cells (IRBC) are not highly conserved.

ICAM-1 (CD54) is a cell adhesion molecule that interacts with the leukocyte 132-integrins, LFA-1 (CD11a/ CD18) and Mac-1 ( C D l l b / C D 1 8 ) . ICAM-1 is a transmembrane protein that has five extracellular Ig-like domains of 76-114 kDa, depending upon tissue-specific

glycosylation (Dustin et al., 1986). ICAM-1 plays an important role in lymphocyte-mediated adhesion, cytotoxic T-cell activity, mixed lymphocyte response and antigen presentation (Springer, 1994). ICAM-1 is also necessary for lymphocyte and granulocyte attachment to endothelium and subsequent transendothelial migration (Smith et al., 1989). While both LFA-1 and Mac-1 bind

Correspondence to: Dr. A.M. Manning, Cell Biology and Inflammation Research, Upjohn Laboratories, Kalamazoo, MI 49001, USA. Tel. (1-616) 385-5450; Fax (1-616) 384-9308; e-mail: [email protected] *Current address: Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Tel. (1-416) 813-1500.

ICAM-1, gene (cDNA) encoding ICAM-1; Ig, immunoglobulin; IRBC, infected red blood cell(s); kb, kilobase(s) or 1000 bp; LFA-1, leukocyte function-associated antigen-l; LPS, lipopolysaccharide; Mac-l, macrophage activation complex-l; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; pfu, plaque-forming units; PCR, polymerase chain reaction; SP, signal peptide; TNFzt, tumor necrosis factor ~.

INTRODUCTION

Abbreviations: aa, amino acid(s); bp, base pair(s); CJVEC, canine jugular vein endothelial cells; ICAM-I, intercellular adhesion molecule 1;

SSD1 0378-1119(95)00045-3

292

(b) Sequence analysis of canine ICAM-1 The ICAM-1 cDNA sequence of 2895 bp encoded a polypeptide of 528 aa (Fig. 2) comprising an N-terminal signal peptide (SP), five Ig-like domains, a hydrophobic transmembrane domain and a short cytoplasmic tail. Clone pLdi encoded the first Ig-like domain of canine ICAM-1 preceded by a 24-aa SP but did not encode a start codon. Addition of sequences encoding the first 4 aa of human ICAM-1 SP to the existing canine ICAM-1 resulted in efficient cell-surface expression of canine ICAM-1 following transfection in COS-7 cells (H.-F.L., K. Youker, C.M.B., M.L.E. and C.W.S., data not shown), suggesting that the canine ICAM-1 SP is of similar length to human. Canine ICAM-1 was 61% identical to human ICAM-1. Ig2 and 4, and the cytoplasmic tail were most identical to human ICAM-1. Cys residues identified by secondary structure prediction to form disulfide bridges as part of the Ig fold are absolutely conserved (Fig. 3). Four additional Cys residues are conserved in all species, suggesting they may be important for ICAM-1 function. The sequence and charge characteristics of a 9-aa stretch in the cytoplasmic domain of human ICAM-1 (478-486) critical for cytosketal association (Carpen et al., 1992; Fig. 4B) are highly conserved, suggesting that cytosketal association is indeed critical for ICAM-1 function.

to ICAM-1 their sites of interaction appear distinct (Diamond et al., 1991). ICAM-1 also serves as the receptor for a major group of rhinoviruses and malariainfected erythrocytes, and these binding sites appear distinct from those for LFA-1 and Mac-1 (Ockenhouse et al., 1992). Canine ICAM-1 plays an important role in adherence of canine neutrophils to canine endothelial cells and in cytotoxicity of canine neutrophils for adult canine cardiac myocytes (Smith et al., 1991; Entman et al., 1992). ICAM-1 mRNA and protein levels are elevated in ischemic and reperfused canine myocardium, and in cultured canine endothelial cells and isolated cardiac myocytes incubated with pro-inflammatory cytokines or postischemic cardiac lymph (Kukielka et al., 1993; Youker et al., 1992). The aim of this study was the cloning and sequence analysis of the canine cDNA for ICAM-1.

EXPERIMENTAL AND DISCUSSION

(a) Isolation of cDNA clones encoding canine ICAM-1 A lipopolysaccharide (LPS)-stimulated canine jugular vein endothelial cell (CJVEC) oligo(dT)-primed eDNA library was screened with a canine ICAM-1 probe (PCR 740) encoding the equivalent of nt 575-1315 of human I C A M - I (Smith et al., 1991). 27 positive ~ eDNA clones were purified. The largest of these, CI26, contained an insert of 2.3 kb (Fig. 1). A random-primed cDNA library prepared from TNF~-stimulated CJVEC was screened with a 5' probe from CI26 and clone 3A was isolated. Clone pLdi was isolated by screening with a 5' probe from 3A. DNA sequence and restriction mapping established the relationship of each of the cDNA clones.

0 1

(c) Conservation of ICAM-1 binding sites for LFA-1 and Mac-1 Residues in human ICAM-1 critical for LFA-1 binding (Staunton et al., 1990; Fig. 3) are conserved in all species. Arg13Gly is conserved in all but murine ICAM-1 (Arg~Glu). Gln 34 and Asp6°Ser are conserved in all species, and Glu 73 is found in all species, except canine (Glu~Gln). Asn 1°3 is present in human, rat and canine,

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Fig. 1. Structure of canine ICAM-1 and clones isolated in this study. The ICAM-1 coding region is depicted as an open box and the 3' untranslated region as a solid line. A, ApaI; H, HindlII; Hi, HinclI; K, KpnI; S, SalI. PCR 740 is depicted as a solid bar. Methods: An LPS-stimulated CJVEC cDNA library in the ~,ZAPII vector (Stratagene, La Jolla, CA, USA) was screened by plaque filter hybridization with a canine ICAM-1 probe (PCR 740) labelled with 32p by random hexanucleotide priming (Sambrook et al., 1989). Plaque-purified clones were rescued in pBluescript SK(-) by in vivo excision. A random hexamer-primed cDNA library prepared from TNF~t-stimulated CJVEC RNA in the ~.ZAPII vector was screened with a 200-bp probe from the 5' end of CI26 and clone 3A isolated. This library was also screened with a probe derived from the 5' end of 3A and clone pLdi was isolated.

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Fig. 2. Nucleotide sequence of the available canine I C A M - I and the deduced aa sequence. Underlined is the putative transmembrane region. Arrows indicate the beginning of each Ig domain, numbered 1 through 5. The nt sequence reported in this paper has been deposited with the GenEMBL Database (accession No. L31625). Methods: The nt sequence was determined for both strands of clones CI26, 3A and pLdi by the dideoxy chaintermination method of Sanger et al. (1977). A nested set of deletion clones for both CI26 and 3A were generated by exonuclease lII digestion a n d the sequence of pLdi determined by walking with oligo primers. Sequence reactions were performed using the Sequenase Version 2.0 kit (US Biochemical, Cleveland, OH, USA). Sequences were compiled and analyzed using the Genetics Computing Group (Madison, WI, USA) suite of DNA sequence manipulation programs. [-)i H

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Fig. 3. Comparison of the aa sequences of human, chimpanzee, murine, rat and canine ICAM-1. The N-terminal residue of each of five Ig-like extracellular domains is marked by an arrow and numbered. Residues comprising a transmembrane region are noted by a solid line overhead. Dashed lines represent identity of aa, dots signify no aa at that position for that sequence. Conserved Cys residues are marked with a star (*). Conserved N-linked glycosylation sites are marked with a plus sign (+). Those residues discussed in the text as important for human ICAM-I binding to LFA-1, Mac-1 and rhinovirus are bolded. _HH,human (Staunton et al., 1988); C, chimpanzee (GenBank/EMBL accession No. M86848); M, mouse (Ballantyne et al., 1989); R, rat (Kita et al., 1992); D, canine. but

Asn--+Asp).

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conclude that the LFA-1 highly conserved.

in i n c r e a s e d I C A M - 1 b i n d i n g to M a c - 1 ( D i a m o n d et al.,

b i n d i n g site o n I C A M - 1

is

1991), is c o n s e r v e d in c h i m p a n z e e b u t n o t in m u r i n e , rat

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Fig. 4. Comparison of conservation of aa sequences comprising the malaria-IRBC site (A) and ¢t-actinin binding site (B) of human ICAM-1. (A) Residue 13-20 of the mature human ICAM-1 (_H_)polypeptide are aligned with those of chimpanzee (_C_),routine (M_), rat ~ and canine (._D_). Dashed lines mark identical residues. (B) Residues 478-486 of human ICAM-1 are aligned with those other species as in A.

or canine, suggesting that N-linked glycosylation at this site is not a common mode of regulating ICAM-1 binding to Mac-1. Those aa in Ig3 important for Mac-1 binding (Diamond et al., 1991) also are not highly conserved (Fig. 3). Aspn9GluArg are conserved in chimp, but differ in mouse, rat and canine. Gln254AspGln are conserved in chimp and canine, but differ in mouse and rat. Why there should be such strong conservation of residues involved in LFA-1 binding but not Mac-1 binding is unclear. It is possibile that ICAM-1 binding to Mac-1 is more dependent upon secondary than primary sequence constraints. With the cloning of canine 1CAM-l, studies to define the binding properties of CL18/6, an anti-canine ICAM-1 monoclonal antibody which blocked Mac-l-dependent neutrophil adhesion to canine cardiac myocytes, and a non-blocking antibody CL18/I (Smith et al., 1991) are now possible. These studies may clarify the role of distinct Ig-like domains of canine ICAM-1 in the binding of LFA-1 and Mac-1.

(d) Conservation of ICAM-1 binding sites for rhinovirus and malaria-IRBC Human ICAM-1 is a receptor for the major group of rhinoviruses, with Igl containing the primary site of contact (Staunton et al., 1990). Those aa playing a role in this binding are conserved in chimpanzee, but not in mouse, rat and canine. Several aa important for both LFA-1 and rhinovirus binding (Arg13Gly), however, are conserved. Human ICAM-1 has also been implicated as a cytoadhesi0n receptor for Plasmodium falciparuminfected red blood cells (IRBC; Ockenhouse et al., 1992). The residues critical for IRBC binding (Fig. 4A) are conserved in all species, except for Leu 18, which in human ICAM-1 is pivotal for IRBC binding. This suggests that only human ICAM-1 can effectively bind malaria-IRBC. (e) Conclusions (1) Canine ICAM-1 is highly homologous to human, chimpanzee, mouse and rat ICAM-1. (2) Cys residues defining the Ig fold are absolutely con-

served in all species. Four additional Cys residues are also conserved and may contribute to ICAM-1 function. (3) Sequences defining the LFA-1 binding site are highly conserved in all species whereas those for Mac-l, rhinovirus and malaria-IRBC are not. These data form the basis for further studies investigating ICAM-1 structure and function.

ACKNOWLEDGEMENTS

The authors would like to thank Robert L. Heinrikson for critical review of this manuscript. This work was supported in part by NIH grant HL42550.

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