CD84 leukocyte antigen is a new member of the Ig superfamily

1997 90: 2398-2405 Miguel Angel de la Fuente, Pilar Pizcueta, Marga Nadal, Jaime Bosch and Pablo Engel CD84 Leukocyte Antigen Is a New Member of the Ig Superfamily http://bloodjournal.hematologylibrary.org/content/90/6/2398.full.html Updated information and services can be found at: (5064 articles)Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Copyright 2011 by The American Society of Hematology; all rights reserved. 20036. the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom CD84 Leukocyte Antigen Is a New Member of the Ig Superfamily By Miguel Angel de la Fuente, Pilar Pizcueta, Marga Nadal, Jaime Bosch, and Pablo Engel cDNA isolated from a human B-cell line Raji library was ana- (CD48 and HumLy9) have been mapped. CD84 monoclonal lyzed and shown to encode the full-length cDNA sequence antibodies (MoAbs) were shown to react with cells transof a novel cell-surface glycoprotein, initially termed HLy9b .fected with the cloned cDNA. These MoAbs were further The predicted mature 307-amino acid protein was composed used to show that CD84 is expressed as a single chain cellof two extracellular Ig-like domains, a hydrophobic transsurface glycoprotein of Mr 64,000 to 82,000, which was membrane region, and an 83-amino acid cytoplasmic dohighly glycosylated. CD84 had a unique pattern of expresmain. The extracellular Ig-like domains presented structural sion, being found predominantly on lymphocytes and monoand sequence homology with a group of members of the Ig cytes. Thus, the glycoprotein HLy9b is recognized by MoAbs superfamily that included CD2, CD48, CD58, and Ly9. Northpreviously clustered as CD84 and represents a newly identiern blot analysis showed that the expression of HLy9b was fied member of the Ig superfamily that may play a significant predominantly restricted to hematopoietic tissues. Chromorole in leukocyte activation. some localization studies mapped the HLy9b gene to chroq 1997 by The American Society of Hematology. mosome 1q24, where other members of this Ig superfamily T Ly9, 2B4, and the recently described signaling receptor SLAM. 9-11 HE INTERACTION between leukocytes and other cells regulate normal immune function. Along with the polypeptides forming the T-cell and B-cell receptor comCD84 is a cluster of differentiation that was established during the Fifth International Leukocyte Differentiation An-plexes, a number of surface molecules participate in B-cell and T-cell activation. Some function as cell-cell interaction tigen Workshop (Boston, MA, 1994). Three MoAbs, 2G7, 152-1D5, and 153-4D9, defined CD84 as a new surface anti-molecules or as signal-transducing elements, and some can serve both functions. 1 A significant number of these molegen predominantly expressed by mature B cells and monocytes. 12 More recently, biochemical analysis shows thatcules are members of the Ig superfamily, which are involved in processes such as adhesion, migration, proliferation, difCD84 MoAbs precipitate a single-chain glycoprotein of approximately 72,000 to 86,000 Mr. 13 ferentiation, and effector functions of leukocytes. 2 Extracellular portions of CD2, CD48, and CD58 leukocyte This article describes the molecular cloning, characterization, chromosomal localization, and tissue-specific expres-antigens are predicted to have similar molecular structures, and they form a distinct subgroup within the Ig superfamily, sion of a new gene of the Ig superfamily that encodes for the cell surface leukocyte antigen CD84.named the CD2 family. 3 These proteins have similar patterns of conserved disulfide bonds and comprise N-terminal V-set domains, which lack a disulfide bond, and C-terminal C2 set MATERIALS AND METHODS Ig domains. These transmembrane surface glycoproteins are Isolation of cDNA clones. A cDNA library from the human involved in adhesion interactions between T lymphocytes Burkitt lymphoma cell line Raji in l gt11 (Clontech Laboratories, and accessory cells and generate intracellular activation sigPalo Alto, CA) was used to isolate cDNA clones by cross-species hybridization, with a mixture of different polymerase chain reaction nals. 4-6 In vivo studies have shown that the administration (PCR)-generated probes corresponding to sequences reported for of CD2 and CD48 monoclonal antibodies (MoAbs) can inmurine surface molecule Ly9 9 ; the following primers were used to hibit T-cell responses and prolong allograft survival. 7,8 Other generate the probes: Ly9 #1 (559 bp): sense primer, 5 * -AAGAAGmembers of the growing CD2 family are the surface antigens CTTCCAAGAATGAGTCAGCAGCAG-3 * , and antisense primer, 5 * -CATCGTATGTATTTAAATGGGTGTC-3 * ; Ly9 #2 (470 bp): sense primer, 5 * -GACACCCATTTAAATACATACGATG-3 * , and From the Fundacio ´Clı B nic per a la Recerca Biome `dica, the Uniantisense primer, 5 * -CAGTCTCTTGTAGACAAGCAAGG-3 * ; and versity of Barcelona, Barcelona, Spain; the Departament de Gene `tLy9 #3 (445 bp): sense primer, 5 * -CCTTGCTTGTCTACAAGAica Molecular, the Institut de Recerca Oncolo `gica, Barcelona, GACTG-3 * , and antisense primer, 5 * -CAGAAAATTGTCCATSpain; and the Hepatic Hemodynamic Laboratory, Liver Unit, HosGGCCAGTTG-3 * . pital Clı B nic of Barcelona, Barcelona, Spain. cDNA was obtained by reverse transcription polymerase chain Submitted February 7, 1997; accepted May 19, 1997. reaction (RT-PCR) of total RNA isolated from murine thymus using Supported by Grants No. PB-1562, SAF96-0120, and SAF97-0136 a First Strand cDNA Synthesis Kit for RT-PCR (Avian Myelofrom the Comision Interminsterial de Ciencia y Technologia and blastosis Virus [AMV], Reverse Transcriptase; Boehringer MannGrant No. PIRB-00076 of the Comissio ´Interdepartamental de Reheim, Mannheim, Germany) and the Taq polymerase Expand High cerca Tecnologia 1995. M.A.d.l.F. is a fellow of the Fondo InvestiFidelity (Boehringer Mannheim) following manufacture’s instrucgaciones Sanitarias (97/5246). The GenBank accession number of tions. The PCR products were purified, subcloned into pSP64 vector the cDNA clone is U82988. (Promega, Madison, WI), and random priming labeled using [ g - 32 P] dCTP with the Ready-To-Go DNA labeling kit (Pharmacia Biotech,Address reprint requests to Pablo Engel, MD, Fundacio ´Clı B nic, Villarroel 170, Barcelona 08036, Spain. Uppsala, Sweden). Filter hybridization was performed in ExpressHyb hybridization solution (Clontech Laboratories) at 45 7 C forThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked 2 hours, followed by low stringency washing in 2 1 SSC-0.05% sodium dodecyl sulfate (SDS) at room temperature. Plaques that‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate this fact. were positive on duplicate filters were picked, and phage insert DNA was isolated. cDNA inserts were removed by EcoRI digestion and q 1997 by The American Society of Hematology. 0006-4971/97/9006-0031$3.00/0 subcloned into pSP64 (Promega). Restriction maps were generated 2398 Blood, Vol 90, No 6 (September 15), 1997: pp 2398-2405 AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom CD84 LEUKOCYTE ANTIGEN 2399 and nucleotide sequences were determined by dideoxynucleotide Monocytes were isolated by adherence of blood mononuclear cells to plastic for 60 minutes at 37 7 C. Cell lines were cultured in completechain termination by using the T7 Sequencing Kit (Pharmacia Biotech). 14 A computer search of nucleotide and protein sequences was medium (RPMI 1640 supplemented with 10% fetal calf serum [FCS], antibiotics, and 10 mmol/L glutamine). Cultures of all cellconducted using the Blast GeneSearch (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD). lines were split the day before analysis and were in logarithmic growth.Sequence analysis was performed by using PC/Gene Software (IntelliGenetics Inc, Mountain View, CA). COS cells were transfected with the HLy9b cDNA subcloned into the pMT2 expression vector using the diethylaminoethyl-dextranRNA blot analysis. A positively charged nylon membrane, to which poly A / RNAs from 50 human tissue samples had been method. 16 Cell surface expression was examined after 48 hours by immunohistochemical analysis as described. 17 Stable transfectantsimmobilized in separated dots (The Human RNA Master Blot), was purchased from Clontech Laboratories. Poly A / RNA samples on were produced by cotransfecting the murine pre–B-cell lines 300.19 by using 40 m g of pMT2-HLy9b and 2 m g of pCI-neo vector con-Master Blot have been normalized to the mRNA expression levels of eight different housekeeping genes allowing the determination of taining the neomycin resistance marker (Promega). The 300.19 cells were transfected by electroporation (280 V, 950 m F) with a Genethe relative expression levels of a target mRNA in different tissues. The nylon membrane includes poly A / from the following tissues: Pulser II Apparatus (Bio Rad, Hercules, CA). After electroporation the cells were plated in flat-bottomed 96-well tissue plates by lim-whole brain, amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putaiting dilution, and selection of stable transfectants was performed by using G418 (1 mg/mL; GIBCO/BRL). 18 Twenty clones from themen, substantia nigra, temporal lobe, thalamus, subthalamic nucleus, spinal cord, heart, aorta, skeletal muscle, colon, bladder, uterus, transfection were examined by indirect immunofluorescence screening, and the clone with the highest level of expression was used inprostate, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, mammary gland, kidney, liver, this study and maintained in the continuous presence of the selecting drug.small intestine, spleen, thymus, peripheral leukocytes, lymph node, bone marrow, appendix, lung, trachea, placenta, fetal brain, fetal MoAbs. The CD84 MoAbs 2G7 (IgG1), 152-1D5 (IgG1), and 153-4D9 (IgG1); and the MoAbs of the Unknown Panel were ob-heart, fetal kidney, fetal liver, fetal spleen, fetal thymus, and fetal lung. Northern blot analysis was performed with nylon membranes tained from the Fifth International Workshop on Human Differentiation Antigens, Boston MA, 1994.purchased from Clontech Laboratories (Multiple Tissue Northern blot) to which 2 m g poly A / RNA from different tissues (spleen, Immunofluorescence analysis. Cells were kept at 4 7 C and examined immediately after isolation. Indirect immunofluorescence analy-lymph node, thymus, appendix, lung, and liver) have been blotted after running on a denaturating formaldehyde/1.2% agarose gel. Dot sis of viable cells was performed after washing the cells twice. The cells were then incubated for 20 minutes on ice with each MoAb asblot and Northern blot membranes were hybridized with a 1,040-bp probe that contained the complete reading frame sequence of the ascites fluid diluted to the optimal concentration for immunostaining. Isotype-matched murine MoAbs that were unreactive with humanHLy9b clone and a 2,000-bp human b -actin cDNA as control probe. Hybridization was performed according to the manufacturer’s inleukocytes were used as negative controls. After washing, the cells were treated for 20 minutes at 4 7 C with FITC-conjugated goat anti-structions. Membranes were autoradiographed for 72 hours (HLy9b ) or 4 hours ( b -actin) at 0 80 7 C by using two intensifying screens. mouse Ig antibodies (Caltag, South San Francisco, CA). Singleand double-color immunofluorescence analysis was performed onAutoradiography was scanned with a Bio-Profile densitometer (Vilber Loumart, Marne la Vallee ´, France) and the relative dot intenan FACSCalibur (Becton Dickinson, Immnocytometry Systems, San Jose, CA). At least 5,000 cells were analyzed for each sample.sity was determined with Bio-1D Analysis Software (Vilber Loumart). The relative dot intensity value was calculated as the optical Immunoprecipitation analysis. A total of 40 1 10 6 cells were washed three times in phosphate buffered saline (PBS), resuspendeddensity (OD) of the dot minus the OD of the background. Chromosomal mapping. Fluorescence in situ hybridization in 800 m L saline, and surface labeled with 200 m L of Biotin 1 mg/ mL (Sigma) for 20 minutes at 4 7 C. After washing the cells once in(FISH), using a 1,040-bp cDNA probe that contained the complete reading frame sequence of the HLy9b and a 5.4-kb PCR-generated RPMI media with 10% FCS and four times in PBS, the cells were lysed in 1 mL of buffer containing 0.5% (vol/vol) NP40 and proteaseprobe of genomic DNA of the HLy9b gene (sense oligo: 5 * -TCTGCTTGTTCTCATTCTGTCTTCA-3 * ; antisense oligo: 5 * -CAGinhibitors as described. 19 Immunoprecipitations were carried out using 15 m g of the CD84 MoAbs 2G7 and 152-1D5. Cell lysates wereTAAGAGTTGGGCAGAGAAGAT-3 * ) were performed as previously described. 15 Briefly, 2 m g of each probe was labeled with precleared three times for 2 hours using 50 m L (50% vol/vol) of murine Ig-coated beads at 4 7 C. Cell lysates were precleared againbiotin 16-dUTP (Boehringer Mannheim) by nick translation. A total of 400 ng of labeled probe was precipitated along with 1 m g of Cotovernight. The precleared lysate was then incubated with 50 m Lof CD84 MoAb-coated beads (50% vol/vol) or murine Ig-coated beads1 DNA (GIBCO/BRL, Gaithersberg, MD) and 1 mg of salmon sperm (Sigma, St Louis, MO), and the pellet was resuspended in a hybridwith constant rotation at 4 7 C for 18 hours. Immunoprecipitates were washed and analyzed by sodium dodecyl sulfate-polyacrylamide gelization mix. Ten microliters of the hybridization mix was applied to each slide. After heat denaturation of the probe and the chromosomal electrophoresis (SDS-PAGE) as described. 19 The samples were run in the presence of 5% 2-mercaptoethanol (reducing conditions). Mrspreads, slides were incubated overnight at 37 7 C. After the posthybridization washes the slides were incubated with avidin-fluorescein was determined using prestained standard molecular weight markers (GIBCO/BRL). The proteins were transferred by electroblotting withisothiocyanate (FITC; Vector Laboratories, Burigame, CA). Slides were mounted with 40 m L of antifade solution (Vector Laboratories) glycine transfer buffer (96 mmol/L glycine, 12 mmol/L Tris-base in 10% methanol, pH 8.3) to polyvinylidene difluoride (PVDF) mem-containing 150 ng/mL of 4 * ,6 * -diamidino-2-phenylindole. Slides were studied under an Olympus AH-3 fluorescence microscope branes (Immobilon; Millipore, Boston, MA). After 2 hours of incubation at room temperature with blocking solution (10% nonfat milk(Olympus Optical Co, Ltd, Tokyo, Japan) and images were analyzed with the Cytovision System (Applied Imaging International LTD, in PBS), avidin peroxidase (100 ng/mL; Sigma) was added for 1 hour. The membranes were washed with PBS with 0.1% of Tween-Santa Clara, CA). Cells. Blood was obtained by protocols approved by the Ethics 20, and the blot was developed using Amersham’s Enhancer Chemoluminiscence reagent (Amersham International, Little Chalfont, UK).Committee of the Hospital Clinic of Barcelona. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation. For N-deglycosysation, immunoprecipitates were digested overnight AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom DE LA FUENTE ET AL2400 Fig 1. Nucleotide sequence and predicted amino acid sequence of HLy9b . Potential N-linked glycosylation sites are underlined. Hydrophobic stretches that would serve as a signal peptide region and a transmembrane region are double underlined. * indicates the termination codon. at 37 7 C with 0.4 U of Peptide-N-glycosidase F (PNGase F; Genzyme The 1,040-bp nucleotide sequence of clone HLy9b #1 is Diagnostics, Cambridge, MA) in buffer prepared according to the shown in Fig 1. The open reading frame encodes a 328manufacture’s instructions. amino acid protein, of which the first 21 amino acids comprise a typical hydrophobic signal peptide that is cleaved RESULTS from the immature protein. The remaining 307-amino acid Isolation and characterization of HLy9b cDNA clones protein has a calculated molecular mass of 34,435 Mr before and deduced amino acid sequence. Low stringency screenthe addition of sugars. There are four potential N-linked ing of a human Burkitt lymphoma B-cell line Raji l gt11 glycosylation attachment sites in the mature protein consislibrary using murine Ly9 cDNAs as probes yielded several tent with the observed relative molecular weight on SDSpositive clones. Four independent cDNA clones, named PAGE of 64,000 to 82,000 Mr. The sequence contains 11 HLy9b #1 ( Ç 2.4 kb), HLy9b #2 ( Ç 1.6 kb), HLy9b #3 serine and 18 threonine residues, which could be O-glycosyl- ( Ç 1.3 kb), and HLy9b #4 ( Ç 1.4 kb), were isolated, and ated. There is a single 25-amino acid internal hydrophobic they encoded a cDNA other than that described for HumLy9, segment that represents the transmembrane region and dithe human homologue of murine Ly9 surface molecule. 20 vides the protein in a 189-amino acid predicted external After digestion of these clones with EcoRI two bands were segment and an 83-amino acid intracytoplasmic region. observed. All these clones had an overlapping band of apAnalysis of the sequence suggests that the extracellular porproximately 1,100 bp that showed a single open reading tion of HLy9b is composed of two Ig-like domains. The frame of 987 bp. The other EcoRI bands corresponded to NH 2 -terminal domain consisted of a V-like domain, which lacks the usually conserved disulfide bonds between the b cDNA encoding 3 * untranslated sequences. AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom CD84 LEUKOCYTE ANTIGEN 2401 allowing us to determine the relative expression levels of HLy9b in different tissues and during development. HLy9b expression appeared to be lymphoid tissue specific: the highest mRNA level was detected in lymph node, spleen, and peripheral leukocytes (Fig 2A and B). Although at lower levels, thymus and fetal spleen also expressed significant levels of mRNA (Fig 2B), whereas it was very low in bone marrow. Significant levels of mRNA for HLy9b were detected in kidney and, to a lesser extent, in lung (Fig 2A, C, and data not shown). These two were the only nonlymphoid tissues in which detectable levels of mRNA were observed. Northern blot analysis from different lymphoid tissues and lung showed a band of 4.4 kb (Fig 2C). No smaller RNA species could be detected, even after a prolonged exposure of the membranes, suggesting that the HLy9b mRNA has a long 3 * -untranslated region. HLy9b is located at chromosome 1q24. To determine the chromosomal localization of the HLy9b gene FISH was performed. By using thecDNA probe, pHLy9b , a consistent but not conclusive hybridization signal on chromosome 1 (band q24) was observed. To locate the gene more precisely, the FISH method was performed using a 5.4-kb genomic fragment of the HLy9b gene, obtained by PCR. A total of 85% of the metaphases showed signal on 1q24, confirming the localization obtained with the cDNA probe (Fig 3). Identification of HLy9b as CD84. To check whether preexisting MoAbs were able to recognize the gene product of HLy9b cDNA, the Unknown Panel of MoAbs of the Fifth International Workshop on Differentiation Antigen was screened for MoAbs reactive with pMT2-HLy9b –transfected cells. Three MoAbs, 2G7, 152-1D5, and 153-4D9, reacted with COS cells transfected with the HLy9b cDNA, but did not react with cells transfected with the expression vector alone or with an unrelated cDNA (data not shown). Fig 2. mRNA analysis for HLy9b . (A) Dot blot analysis of poly(A) " These three MoAbs showed an identical and unique pattern RNA from different tissues using labeled HLy9b and b -actin cDNA as probes. Autoradiography was scanned with a Bio-Profile densiof expression and had been clustered as CDw84 during the tometer (Vilber Loumart, Marne la Vallee ´, France). (B) Quantification Fifth International Workshop. 12 These MoAbs also specifiof mRNA in different lymphoid tissues. Dot relative intensity as meacally stained a murine pre–B-cell line (300.19) stably transsured with Bio-1D Analysis Software (Vilber Loumart). (C) Northern fected with the 1,040 bp pHLy9b cDNA (Fig 4). Expression blot analysis of HLy9b . of CD84 on the transfected cells was determined on the basis of anti-CD84 MoAb reactivity, measured by immunofluorescence staining. The CD84 MoAbs did not react with unsheets. The second domain had the structural features of an transfected parent cells nor with cells transfected with vector Ig-truncated C2 set domain with two putative disulfide alone. In all cases the reactivity pattern of the MoAb was bonds. A protein sequence homology search showed that identical. These results show that CD84 MoAbs identify the this protein shared significant sequence identity with a group protein encoded by the HLy9b cDNA. of members of the Ig superfamily, including Ly9, CD48, CD84 is highly glycosylated. The CD84 MoAbs were CD58, and CD2. These data indicate that the HLy9b gene coupled to beads and used to immunoprecipitate the CD84 encodes a protein that belongs to the Ig superfamily. protein from detergent-solubilized extracts of biotinylated The predicted cytoplasmic domain of the mature protein cell lines. Optimum results were obtained using the Raji cell contains five tyrosines (Tyr). Interestingly, four of these Tyr line because the level of CD84 expression was higher than (Tyr 241, Tyr 258, Tyr 278, and Tyr 303) contain motifs in other cell lines (Fig 5). The CD84 antibodies specifically similar to those previously described as potential SH2 doimmunoprecipitated proteins that migrated as a broad single main binding motifs. These are sequence motifs for kinase band in the region from Ç 64,000 to 82,000 Mr in SDSmolecules such as src-kinase type tyrosine kinases. 21 Therepolyacrylamide gels (Fig 5). fore, sequences in the cytoplasmic tail suggest involvement To determine the molecular mass of the protein in the of this molecule in signaling functions. absence of N-linked sugars, surface-labeled CD84 protein Detection of HLy9b mRNA in hematopoietic tissues. was digested with endoglycosydase F and analyzed by SDSmRNA expression was examined by dot blot analysis using poly A / RNA isolated from 50 different human tissues, PAGE. After enzyme digestion, a loss of the Ç 64,000 to AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom DE LA FUENTE ET AL2402 Fig 3. Chromosomal localization of HLy9b gene. Metaphase showing FISH with the HLy9b 5.4-kb genomic probe. Double spots of hybridization signal on chromosome 1 is indicated by an arrow. The HLy9b gene was assigned to 1q24. Fig 4. CD84 MoAbs reactive with the protein product of the pHLy9b cDNA. 300.19 cells transfected with HLy9b cDNA were examined by using the CD84 MoAbs 2G7, 152-1D5, and 153-4D9 in indirect immunofluorescence assays with flow cytometry analysis. Fluorescence histograms generated using 300.19 transfected cells are shown as solid lines. Staining of the 300.19 untransfected cells are shown as dotted lines. A MoAb against CD22 (HB22.7) was unreactive with both transfected and untransfected cell lines. Fluorescence intensity is shown on a threedecade log scale. AID Blood 0021 / 5h3d$$0021 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom CD84 LEUKOCYTE ANTIGEN 2403 Fig 5. Immunoprecipitation of cell surface CD84 protein. Detergent lysates of surface-labeled Raji cell line, 300.19 cells transfected with HLy9b , and 300.19 untransfected cells were immunoprecipitated with the 2G7 plus 152-1D5. The immunoprecipitated materials were treated with or without N-glycosidase (PNGase F) and analyzed under reducing conditions on a 10% SDS-polyacrylamide gel. Molecular weights (kD) were determined by the migration of a known protein standard. 82,000 Mr band and the appearance of a major component protein wassimilar in both cDNA-transfected cells ( Ç 59,000 to 77,000; Fig 5) and Raji cells ( Ç 64,000 to 82,000; Fig 5).at Ç 45,000 and a fainter band of Ç 36,000 Mr was observed (Fig 5). Compatible with this observation CD84 has four Despite this difference in electrophoretic mobility between the immunoprecipitates from the two cells, the bands ob-potential N-linked glycosylation sites (NXT/S) in the extracellular domain. served after treatment with N-glycanase were the same for both cell types (Fig 5). These results show that CD84 under-Immunoprecipitation with CD84 MoAbs of the 300.19 cell stably transfected with 1,040-bp pHLy9b cDNA clone goes extensive post-translational processing as it was expressed as a single-chain molecule, yet the determined Mrshowed a broad band similar to the protein that was immunoprecipitated from the Raji cells, except that the Mr was was more than twice the predicted size of the core protein. In fact, differential use of these N-linked sites may explain Ç 57,000 to 77,000 (Fig 5). After treatment of the immunoprecipitated material with endoglycosydase F, a dramatic the heterogeneity in size of the CD84 protein. reduction in the molecular mass was also observed. A major Comparison of the CD84 amino acid sequences with other band of 45,000 and a lighter one of 36,000 Mr identical to previously identified proteins showed a striking structural those found in immunoprecipitates of the Raji cells line could homology with the extracellular Ig-like domains of a subbe detected. These results showed that the differences in group of the Ig superfamily surface receptors that includes molecular mass between the protein in Raji cells and in the CD48, CD58, Ly9, and CD2, named the CD2 family. CD84 transfected cells were because of differences in the degree is most similar to murine and human Ly9, which are preof glycosylation. dicted to encode a protein with four Ig-like domains in the extracellular region. 9,20 In contrast to Ly9, which has four IgDISCUSSION like domains, CD84 like other members of the CD2 family is composed of only two Ig-like domains. Comparison of IgIn this study, we have isolated cDNA clones encoding for like domains in human Ly9 (HumLy9) and CD84 showed the leukocyte antigen CD84. The structural features of the that the first domain of CD84 had 34.6% amino acid seCD84 protein, predicted from nucleotide sequences derived quence identity with domain 1 and 30.3% with domain 3 of from the cDNA clone HLy9b (Fig 1), showed that it enHumLy9; and the second domain of CD84 had 38.9% idencodes a transmembrane type-I glycoprotein containing two tity with domain 2 and 33.3% with domain 4. Ig-like domains in the extracellular region: a hydrophobic Figure 6 shows sequence homology between CD84, membrane spanning domain, and an 83-amino acid cytoHumLy9, and human CD48. Although the sequences of the plasmic region (Fig 1). It is likely that the entire coding domains share relatively little identity ( Ç 25% to 39%), the region of CD84 was identified as transfection of cell lines core structures of the Ig-like domains are remarkably similar. with the pHLy9b cDNA-generated cell-surface expression of the protein (Fig 4) and the Mr of the immunoprecipitated Domain 1 of CD84 lacks a disulfide bond but is otherwise AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom DE LA FUENTE ET AL2404 Fig 6. Ig-like domains of HLy9b . HLy9b Ig-like domains are compared with HumLy9 and human CD48. Solid lines above the sequence predicted b -sheets. Conserved residues appear in stippled boxes. Gaps are indicated with broken line to allow optimal alignment of the proteins. (A) First Ig-Like domain; (B) Second Ig-like domain. * indicates conserved amino acids of the Ig superfamily. similar to V-like domains found in other proteins of the CD2 chromosome 1. Human CD2 and CD58 genes are located on chromosome 1p13, whereas the CD48 and HumLy9 genesfamily. 2,3 The second domain also shares homology with the truncated C2 domain found in this group of members of the are situated on chromosome 1q21-24. 20,22,23 These genes are tightly linked with the ATP1A genes on chromosome 1,Ig superfamily. Four Cys residues, which are conserved in all members of the CD2 family, were found in the putative which have evolved by gene duplication. 24 By inference, it is likely that duplication of a primordial gene also gave risesecond extracellular domain of CD84 (Fig 6). These Cys are probably involved in the formation of two disulfide bonds. to the structurally related CD2, CD48,CD58, Ly9, and CD84 genes. It has been suggested that Ly9 and mouse CD48 shareOne pair of these residues at positions 134 and172 delineates an Ig-like domain creating the characteristic Ig-like loop a common ancestor that duplicated and diverged, to give rise to a precursor of the Ly9 and CD48 genes, with a secondstructure and this domain contained many of the hallmark amino acids that define the C2 set of Ig-like domains. 2 duplication event producing the four-domain structure observed in Ly9. 9 The sequence similarities between CD84,The 83-amino acid cytoplasmic domain of CD84 contained five tyrosine residues that could serve as potential Ly9, and CD48, together with the mapping of these genes to the same chromosomal region, suggests that these threesites of phosphorylation. Four of these Tyr were contained in two motifs Tyr-X-X-Leu/Ile and Tyr-X-X-Val with a general genes share a common ancestor. CD84 expression appeared predominately to be lymphoid-sequence context that can serve as potential sites for SH2 domain recognition. 21 These types of motifs have been tissue restricted as shown by Dot blot and Northern blot analyses (Fig 2A, B, and C) and immunohistological analysisshown to be sites of interaction with multiple cytosolic proteins, including tyrosine kinases, and are found in the cytoof different tissues (data not shown). The highest levels of CD84 mRNA were detected in lymph node, spleen, andplasmic domain of different signal transduction molecules, indicating that CD84 may be involved in signal transduction. peripheral leukocytes (Fig 2A and B). These results are consistent with the observation that CD84 is expressed by lym-The CD84 gene was mapped to chromosome 1q24 (Fig 3), where other members of this Ig superfamily (CD48 and phocytes and monocytes. 13,25 Staining of lymph node and spleen shows that CD84 is weakly expressed by lymphocytesHumLy9) have also been mapped. The genes of the members of this family are located in two pericentric loci on human but strongly in scattered cells in the interfollicular zone, AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom CD84 LEUKOCYTE ANTIGEN 2405 Stoneman E, Bennett M, Kumar V: Cloning and characterization of which were most likely macrophages. 13 CD84 expression the 2B4 gene encoding a molecule associated with non-MHC-remay be an early event in T-lymphocyte development as thystricted killing mediated by activated natural killer cells and T cells. mocytes showed low but detectable levels of mRNA (Fig 2) J Immunol 151:5328, 1993 and cell surface expression (data not shown). In contrast 11. Cocks BG, Chang CC, Carballido JM, Yssel H, de Vries JE, very low levels of mRNA were detected in bone marrow, Aversa G: A novel receptor involved in T-cell activation. Nature which is consistent with the observation that CD84 is not 376:260, 1995 present in pro-B or pre-B cells. 25,26 In only 2 out of the 39 12. Tedder TF, Wagner N, Engel P: B-cell antigens section report, nonlymphoid tissues analyzed, detectable levels of mRNA in Schlossman S (ed): Leucocyte Typing V. Oxford, UK, Oxford were observed. Significant levels of mRNA for CD84 could University Press, 1995, p 483 13. de la Fuente MA, Pizcueta P, Engel P: CDw84 workshop be detected in the kidney and, to a lesser extent, in the lung report, in Kishimoto T (ed): Leucocyte Typing VI. New York, NY, (Fig 2 and data not shown). The expression of CD84 by Garland Publishing Inc, 1997 (in press) these tissues deserves further investigation. 14. Sanger F, Nicklen S, Coulson AR: DNA sequencing with From the structural information obtained in this study, it chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5413, 1977 appears that CD84 is a newly described cell surface molecule 15. Nadal M, Mila M, Pritchard M, Mur A, Pujals J, Blouin JL, that shares homology and chromosomal localization with a Antonarakis SE, Ballesta F, Estivill X: YAC and cosmid mapping group of Ig superfamily members. Although the function of of an unbalanced chromosomal localization causing partial trisomy CD84 is still unknown, its structural similarity with other 21 and Down syndrome. Hum Genet 98:460, 1996 members of the Ig superfamily, as well as the presence of 16. Engel P, Nojima Y, Rothstein D, Zhou L-J, Wilson GL, Kehrl JH, Tedder TF: The same epitope on CD22 of B lymphocytes medifour potential SH2 domain binding motifs found in the cytoates the adhesion of erythrocytes, T and B lymphocytes, neutrophils plasmic domain, suggest that it may be involved in cellular and monocytes. J Immunol 150:4719, 1993 interactions and signal transduction. 17. Engel P, Wagner N, Miller A, Tedder TF: Identification of the ligand binding domains of CD22, a member of the immunoglobulin ACKNOWLEDGMENT superfamily that uniquely binds a sialic acid-dependent ligand. J Exp Med 181:1581, 1995 We thank Victoria Tovar and Isabel Sa ´nchez for assistance with 18. Chen A, Engel P, Tedder TF: Structural requirements regulate these experiments. endoproteolytic release of the L-selectin (CD62L) adhesion receptor from the cell surface of leukocytes. J Exp Med 182:519, 1995 REFERENCES 19. Tedder TF, Schlossman SF: Phosphorylation of the B1 1. Weiss A, Littman DR: Signal transduction by lymphocyte anti- (CD20) cell surface molecule expressed by normal and malignant gen receptors. Cell 76:263, 1994 human B lymphocytes. J Biol Chem 263:10009, 1988 2. Williams AF, Barclay AN: The immunoglobulin superfamily20. Sandrin MS, Henning MM, Lo MF, Baker E, Sutherland GR, domains for cell surface recognition. 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Oxford, UK, Oxford University Press, 1995, p 62610. Mathew PA, Garni-Wagner BA, Land K, Takashima A, AID Blood 0021 / 5h3d$$$401 08-12-97 13:39:09 blda WBS: Blood For personal use only. at Harvard Libraries on August 27, 2013. bloodjournal.hematologylibrary.orgFrom