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Aug 31, 1998 - cells and T cells. J Immunol 151:5328–5337. Melton E, Sarner N, Torkar M, van der Merwe PA, Russell JQ,. Budd RC, Mamalaki C, Tolaini M, ...
Immunogenetics (1999) 49 : 249–255

Q Springer-Verlag 1999

ORIGINAL PAPER

Miguel Angel de la Fuente 7 Victoria Tovar Pilar Pizcueta 7 Marga Nadal 7 Jaime Bosch Pablo Engel

Molecular cloning, characterization, and chromosomal localization of the mouse homologue of CD84, a member of the CD2 family of cell surface molecules Received: 1 July 1998 / Revised: 31 August 1998

Abstract CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative 329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%, considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of the CD84 glycoprotein may be similar in humans and mice.

M.A. de la Fuente 7 V. Tovar 7 P. Engel (Y) Immunology Unit, Department of Cellular Biology and Pathology, IDIBAPS, Medical School, University of Barcelona, C/Casanovas 143, Barcelona, Spain, E-mail: engel6medicina.ub.es, Tel.: c34-93-4035267, Fax: c34-93-4515272 P. Pizcueta 7 J. Bosch Hepatic Hemodynamic Laboratory, Liver Unit, IDIBAPS, Hospital Clínic, Barcelona, Spain M. Nadal Departament de Genètica Molecular, Institut de Recerca Oncològica, Barcelona, Spain

Key words Surface molecule 7 Leukocyte 7 Immunoglobulin superfamily 7 CD84 7 Chromosome 1

Introduction Interactions between leukocyte cell surface proteins and their counter-receptors regulate immune responses. Cellular immune responses are modulated by cross-talk between stimulatory and inhibitory signalling pathways triggered by cell surface receptors. Particular interest has been generated by the finding that members of the CD2 family regulate lymphocyte development, activation, and adhesion (Bierer et al. 1989; Cocks et al. 1995; Mathew et al. 1993; Melton et al. 1996; Moingeon et al. 1989; Qin et al. 1994; Teh et al. 1997). The CD2 family includes a number of cell-surface receptors (CD2, CD48, CD58, Ly-9, 2B4, and SLAM) which belong to the immunoglobulin superfamily (IgSF) and which are characterized by similar patterns of conserved disulfide bonds. They comprise N-terminal V-set domains that lack a disulfide bond, and C-terminal C2 set Ig domains (Davis and van der Merwe 1996). The CD2 family member genes are located in close proximity in mouse and human genomes, and it has been proposed that all these molecules arose from a series of gene duplication events (Kingsmore et al. 1995; Wong et al. 1990). The human CD84 (hCD84) cDNA has recently been isolated and shown to encode a new member of the IgSF with one variable and one constant Ig-like domain (de la Fuente et al. 1997). Comparison of the CD84 amino acid sequence with other previously identified proteins reveals striking structural similarity to the extracellular Ig-like domains of other CD2 family members, especially mouse and human Ly-9 and CD48 (de la Fuente et al. 1997). The hCD84 gene has been mapped to chromosome 1q24, close to human Ly-9 and CD48 (de la Fuente et al. 1997). Human CD84 is a singlechain cell surface glycoprotein of Mr 72 000–86 000,

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which is highly glycosylated. Human CD84 expression is restricted to hematopoietic tissues, predominantly on the cell surface of mature B lymphocytes and monocytes (de la Fuente et al. 1998). Although the function of CD84 is unknown, the presence of potential SH2 domain binding motifs in its cytoplasmic tail similar to those found in several receptors involved in leukocyte activation suggests that it is involved in signal transduction, like all the other molecules of the CD2 family. This report describes the molecular cloning, characterization, chromosomal localization, and tissue-specific expression of the mouse homologue of the cell surface leukocyte antigen CD84.

Materials and methods

Chromosome spreads from mouse B6SJL peripheral blood were obtained according to the protocol described by Davisson and Akeson (1987). Slides were kept at -20 7C until use. Prior to hybridization, slides were baked at 55 7C for 30 min. Fluorescence in situ hybridization (FISH) was performed as previously described (Nadal et al. 1996). Briefly, 2 mg aliquots of each genomic DNA were used as probes. Ly-9 clone 10 [insert F14 kilobases (kb)] and mCD84 clone 15 (insert F12 kb) were labeled with digoxigenin 11-dUTP and biotin 16-dUTP, respectively, by standard nick translation. For detection of the signals, slides were incubated with sheep anti-digoxigenin-TRITC and avidin (Vector Laboratories, San Francisco, Calif.). The signals were amplified once by incubating the slides with biotinylated anti-avidin (Vector Laboratories) and avidin-FITC (Vector Laboratories). Slides were mounted in an anti-fade solution containing 150 ng/ml of DAPI and were viewed under an Olympus Vanox fluorescence microscope equipped with the appropriate filter set. Images were analyzed and recorded with the Cytovision system (Applied Imaging Ltd., UK).

Isolation of cDNA clones

Cells

A cDNA library from mouse peritoneal macrophages in lgt11 (Stratagene, La Jolla, Calif.) was used to isolate cDNA clones, with a mouse polymerase chain reaction (PCR) probe generated using human primers (sense oligonucleotide 5b-ACCTGGCCGGAAGCAGCTGGAAA-3b, antisense oligonucleotide, 5bTGTTGCTGACAGGGTTCTGGGCTGT-3b) corresponding to the first and second Ig-like domains (de la Fuente et al. 1997). PCR probes were obtained from cDNA produced by reverse transcription (RT)-PCR of total RNA isolated from mouse peripheral lymph nodes using a first-strand cDNA Synthesis Kit for RT-PCR (AMV) and the Taq polymerase Expand High Fidelity (Boehringer Mannheim, Mannheim, Germany) following the manufacturer’s instructions. The PCR products were purified, subcloned into pSP64 vector (Promega, Madison, Wis.), sequenced, and random priming labeled with [a-32P] dCTP using the Ready-To-Go DNA labeling kit (Pharmacia Biotech, Uppsala, Sweden). Filter hybridization was performed in ExpressHyb hybridization solution (Clontech, Palo Alto, Calif.), according to the manufacturer’s instructions. Plaques that were positive on duplicate filters were picked up, and phage insert DNA was isolated and excised as pBluescript II (SK-) plasmid by the addition of helper phage (Stratagen).

Cell lines were cultured in RPMI 1640 medium (Gibco-BRL, Gaithersburg, Md.) containing 10% heat-inactivated fetal calf serum, 10 mM glutamine and penicillin/streptomycin. Cultures of all cell lines were split the day before analysis and were in logarithmic growth. The following mouse cell lines were used: 300.19 (preB-cell line), WEHI 231 (immature B-cell line), NS1 (myeloma cell line), CTLL-2 (IL-2 dependent T-cell line), and RAW 264-7 (monocytic cell line). Cell lines were obtained from the American Tissue Collection (Rockville, Md.), except 300.19 which was provided by M. Streuli (Dana Farber Cancer Institute, Boston, Mass.).

RNA blot analysis A positively charged nylon membrane with poly A c RNAs from mouse tissue samples was purchased from Clontech. Total cellular RNA was isolated from Balb/c mouse spleen, peripheral lymph nodes, bone marrow, thymus, kidney, and the 300.19, WEHI 231, NS1, RAW 264-7, and CTLL-2 cell lines. Tissues and cell lines were homogenized in RNAzol (Gibco/BRL, Gaithersberg, M.D.). Ten micrograms of total RNA/lane was size fractionated by electrophoresis in 1% agarose gel and transferred by capillary blotting. These membranes were hybridized with a 256 base pair (bp) (Ssp I/Sal I) probe that contained a sequence corresponding to the first Ig domain of mouse CD84 cDNA. Hybridization was performed according to the manufacturer’s instructions (Clontech). Autoradiography was scanned with a BioProfile densitometer (Vilber Loumart, Marne la Valleé, France). Chromosomal mapping Genomic DNA clones of the CD84 and Ly-9 genes were isolated from a l FIXII genomic DNA library (Stratagene) generated from 129/SV mice, using two probes of cDNA encoding the first domain of CD84 and the third domain of Ly-9. Screening was performed as described for the isolation of the cDNA clones.

Sequence analysis Nucleotide sequences were determined by dideoxynucleotide chain termination using the T7 Sequencing Kit (Pharmacia Biotech). Sequence comparisons with other molecules and alignments between human and mouse CD84 were performed by using BCM Search Launcher analysis software (Human Genome Center, Baylor College of Medicine, Houston, Tex.). Identification of putative leader peptides and transmembrane regions were analyzed using the PSORT program (Nenta Nakai, Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan). The numbering system used throughout this paper refers to the amino acid sequence of the mature protein, following cleavage of the leader peptide.

Results Isolation of the mouse homologue of CD84 cDNA clones PCR primers according to the human CD84 sequence (de la Fuente et al. 1997) allowed the amplification of a 553 bp fragment from cDNA of mouse peripheral lymph node cells, which showed 76.9% similarity at the nucleotide level to the corresponding region of human CD84 cDNA. Twelve different cDNA clones were isolated from a lgt11 mouse macrophage library using this PCR product as probe. The sequence of clone mCD84 no. 7 (F1500 bp) revealed an open reading frame encoding a 329 amino acid protein that displayed an overall amino acid sequence identity of 57.3% with hCD84

251 Fig. 1 Nucleotide sequence and predicted amino acid sequence of mouse CD84. Nucleotide sequence (1132 bp) of clone mCD84 no.7. Potential N-linked glycosylation sites are underlined. Hydrophobic stretches that would serve as a signal peptide region and a transmembrane region are double underlined. An asterisk indicates the termination codon. cDNA sequence was deposited in GenBank with the accession number AF043445

(88.7% if conserved amino acid substitutions are considered) (Figs. 1, 2), indicating that it represents the mouse homologue for this molecule. Analysis of structural features of predicted protein

Fig. 2 Amino acid comparison of mouse and human CD84. The upper sequence corresponds to mouse CD84 (mCD84) and the lower to human CD84 (hCD84). Identical amino acids are indicated by two dots; conserved substitutions are indicated by one dot; gaps are indicated by a broken line to allow optimal alignment of the proteins

Similarly to hCD84, the sequence begins with a typical hydrophobic signal peptide of 21 amino acids followed by an extracellular region composed of two IgSF domains, a transmembrane region, and an 87 amino acid cytoplasmic tail. Mouse CD84 mature protein is composed of 308 amino acids and has a calculated relative molecular mass (Mr) of 37 300 before the addition of sugars. There are three potential N-linked glycosylation attachment sites, which are also conserved in the human protein, at the same positions. These sites are located in the second IgSF domain. The NH2-terminal domain consisted of a V-like domain that lacks the usually conserved disulfide bonds between the b sheets. Four cysteines residues (Cys125, Cys131, Cys169, and Cys188), which are conserved in all members of the CD2 family, were found in the putative second extracellular domain of mCD84 (Fig. 3). The second domain had the structural features of an IgSF truncated C2 set domain with two putative disulfide bonds. A protein sequence similarity search showed that this protein shared significant sequence identity with a group of members of the Ig superfamily, including Ly-9, CD48, 2B4, and CD2 (Fig. 3)

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Fig. 3 Mouse CD84 Ig-like domains compared with Ly-9, CD48, 2B4, and CD2. Solid lines above the sequence, predicted b-sheets. Conserved residues appear in stippled boxes. Gaps are indicated by a broken line to allow optimal alignment of the proteins. An asterisk indicates conserved amino acids of the Ig superfamily. A cross-hatch indicates conserved cysteine residues

species observed in lymphoid tissues, whereas no significant levels of CD84 mRNA could be detected in myeloma cell line NS1, T-cell line CTLL-2, or monocytic cell line RAW 264-7 (Fig. 5). Chromosomal localization

CD84 mRNA is primarily expressed in hematopoietic tissues Expression of mouse CD84 mRNA from several tissue sources and cell lines was analyzed by northern blot. A major mRNA species of F3.6 kb and a minor species of F1.5 kb, were detected in all lymphoid tissues tested, which included lymph node, spleen, thymus, and bone marrow (Fig. 4 A,B). High levels of mRNA for mCD84 were detected in lung and very low levels in heart. These two were the only nonlymphoid tissues in which significant levels of mRNA for CD84 were observed. The pre-B-cell line 300.19 and the immature Bcell line WEHI 231 expressed the same CD84 mRNA

To determine the chromosomal localization of the mouse CD84 gene, FISH was performed. The FISH method was carried out on metaphase spreads using genomic clones of mouse CD84 and of mouse Ly-9 genes as probes. Eighty percent of the metaphases resulted in specific labeling of chromosome 1 for both genes (Fig. 6). Note that both signals appear at the distal third of chromosome 1 and that mCD84 is located telomeric to Ly-9, lying close together but at a different locus.

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Fig. 5 Northern blot analysis of mouse CD84 with total RNA from different cell lines. B-cell lines 300.19, WEHI 231, and NS1. T-cell line CTLL-2, and monocytic cell line RAW 264–7

Discussion

Fig. 4A,B Northern blot analysis of mouse CD84. A Northern blot analysis of poly(A) c RNA from different tissues. B Northern blot analysis of total RNA in different lymphoid tissues and kidney. Autoradiography was scanned with a Bio-Profile densitometer

Fig. 6A,B Chromosomal localization of mouse CD84 and Ly-9 genes. Chromosome spread of B6SJL mouse counterstained with DAPI and showing the hybridization signals obtained with probes Ly9 (red) and mCD84 (green) to chromosome 1. Boxes A and B show in detail the signals on both homologues 1 of probes Ly-9 (A) and mCD84 (B). Note that both signals appear in the distal third of chromosome 1 and that mCD84 is located more distally to Ly-9

The structural features of the mouse CD84 protein showed that it is a transmembrane type-I glycoprotein containing two Ig-like domains in the extracellular region, a hydrophobic membrane spanning domain, and a 87-amino acid cytoplasmic region (Fig. 1). Comparison of the IgSF domains between human and mouse showed 53.7% amino acid sequence identity in domain 1, and 67% identity in domain 2 (79.6% and 94.6%, respectively, if conserved amino acid substitutions are considered) (Fig. 2). Alignment of the transmembrane region showed 47% amino acid identity. The mCD84 similarity to the human sequence extends into the cytoplasmic tail (48.3% identity). In this domain two threonines, five serines, and three tyrosines were con-

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served between human and mouse, and these residues could serve as potential sites of phosphorylation. These conserved tyrosines residues (Tyr228, Tyr243 and Tyr263) are present in three Tyr-containing motifs (Yx-x-I/V) suggestive of SH2-binding sites (Songyang et al. 1994). The conserved tyrosine based motifs found in the cytoplasmic tail of human and mouse CD84 implicate this cell-surface molecule in signaling functions. The cytoplasmic tail of hCD84 has a deletion of 7 amino acids as compared with mCD84, which suggests that these amino acids may be encoded by a single exon that has been deleted in humans, or alternatively this represents a splice variant of hCD84. Mouse CD84 shared structural similarity with the extracellular Ig-like domains of members of the CD2 family, especially mouse and human Ly-9 (Sandrin et al. 1992, 1996) (Fig. 3). In contrast to Ly-9, which has four Ig-like domains, CD84 like all the other members of the CD2 family is composed of only two Ig-like domains. Comparison of Ig-like domains in mCD84 and Ly-9 showed that the two extracellular Ig-like domains of mCD84 had 36.3% amino acid sequence identity with Ig-like domain 1 and 2 of Ly-9; and 31.4% identity with domain 3 and 4 of Ly-9. Interestingly, Ly-9 is more similar to CD84 than to CD48 (25% identity with domains 1 and 2; and 22.3% with domains 3 and 4) (Fig. 3). The tissue distribution of mCD84 is similar to that observed for the human counterpart, as Northern blot and flow cytometric analysis show that hCD84 expression is also lymphoid tissue specific (de la Fuente et al. 1997). Mouse CD84 expression appears to be an early event in T lymphocyte development, as thymocytes expressed high levels of mRNA. Strikingly, prominent expression of mCD84 was detected in mouse bone marrow, in contrast to human, in which mRNA is barely detectable and no staining occurs with monoclonal antibodies (de la Fuente et al. 1997). This is consistent with the expression of mCD84 mRNA in the pre-B cell line 300.19 and in the immature B cell line WEHI 231. Mouse CD84 probably disappears with differentiation to plasma cells, since no significant levels of mCD84 mRNA could be detected in the myeloma cell line NS1 (Fig. 5). The strong mCD84 signal observed in lung (Fig. 3A) was unexpected. However, staining of human lung sections with hCD84 MoAbs shows CD84 c alveolar macrophages (unpublished data). Low levels of mRNA for mCD84 were detected in heart. Since no CD84 transcript has been detected in human heart, its expression in mice deserves further investigation. Together all these data indicate that CD84 expression in the mouse, like that in humans, is largely confined to the hematopoietic system. We cannot discern whether the two different sized bands of F3.6 kb and of F1.5 kb represent an alternatively spliced mRNA or arise from the use of differential polyadenylation signals. Cross-hybridization of the mCD84 probe to related protein mRNA is unlikely, since the filters were washed in high stringency conditions.

The mouse CD84 gene was mapped to distal chromosome 1 adjacent to Ly-9 gene. Ly-9 gene had already been mapped on mouse distal chromosome 1 within 1100 kb of CD48 (Kingsmore et al 1995). These results are consistent with the observation that the human CD84 gene maps to chromosome 1q24, whereas human Ly-9 and CD48 have been mapped to 1q21–23 (Kingsmore et al. 1995). It has been suggested that Ly-9 and mouse CD48 share a common ancestor that duplicated and diverged, to give rise to a precursor of the Ly-9 and CD48 genes, with a second duplication event producing the four-domain structure observed in Ly-9 (Sandrin et al. 1992). The sequence similarities between CD84, Ly9 and CD48, together with the mapping of these genes to the same chromosomal regions, both in human and mouse genomes, suggests that these three genes share a common ancestor, and reinforces the hypothesis that the CD2 family genes have evolved from a primordial gene in a series of duplication events. The ligand of CD84 is unknown. Assignment of CD84 to the CD2 gene family suggests that CD84 may be a ligand for CD48, Ly-9 or another member of the CD2 family, in a manner analogous to CD2 with CD48 and CD58 (Arulanandam et al. 1993; Davis and van der Merwe 1996). The possibility that a member of the CD2 family could be a natural ligand of CD84 is currently under investigation using CD84 fusion proteins. Characterization of mouse CD84 may allow questions about the developmental and signaling functions of CD84 to be approached by gene targeting techniques. Acknowledgments We thank Isabel Sánchez for assistance with these experiments and Josep M. Estanyol for help with the preparation of the photographs. This study was supported by grants from the Comisión Interministerial de Ciencia y Tecnología: SAF97–0136 and SAF96–0120; and Promoción General del Conocimiento: PB94–1562. M.A. de la F. is a fellow of the Fondo de Investigaciones Sanitarias (97/5246). V.T. is a fellow of the Programa Nacional de Formación de Personal Investigador. All the experiments presented in this paper comply with the current laws of the country in which the experiments were performed.

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