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NAG-2, a Novel Transmembrane-4 Superfamily (TM4SF) Protein That Complexes with Integrins and Other TM4SF Proteins

1997; Elsevier BV; Volume: 272; Issue: 46 Linguagem: Inglês

10.1074/jbc.272.46.29181

ISSN

1083-351X

Autores

Isao Tachibana, Jana Bodorova, Fedor Berditchevski, Mary M. Zutter, Martin E. Hemler,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (α6β1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novelantigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (α3β1and α6β1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF·TM4SF and TM4SF-integrin complexes. Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (α6β1) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-2 (novelantigen-2) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets. Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (α3β1and α6β1). Also, two-color immunofluorescence showed that NAG-2 was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-2 in specific TM4SF·TM4SF and TM4SF-integrin complexes. The transmembrane-4 superfamily (TM4SF) 1The abbreviations used are: TM4SF, transmembrane-4 superfamily; mAb, monoclonal antibody; CHO, Chinese hamster ovary cell; MHC, major histocompatibility complex; PAGE, polyacrylamide gel electrophoresis; CHO/P, CHO line stably transfected with the polyoma large T antigen; kb, kilobase pair(s). comprises a group of at least 19 cell-surface proteins (including CD9, CD37, CD53, CD63, CD81, and CD82) each presumed to have four transmembrane domains (1Wright M.D. Tomlinson M.G. Immunol. Today. 1994; 15: 588-594Abstract Full Text PDF PubMed Scopus (334) Google Scholar). Although the precise biological functions of TM4SF proteins remain elusive, multiple experiments using anti-TM4SF mAbs have implicated TM4SF proteins in cell proliferation, activation, adhesion, and motility (1Wright M.D. Tomlinson M.G. Immunol. Today. 1994; 15: 588-594Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 2Hemler M.E. Mannion B.A. Berditchevski F. Biochim. Biophys. Acta. 1996; 1287: 67-71PubMed Google Scholar). In addition, several TM4SF members may regulate signaling events involving tyrosine phosphorylation (3Schick M.R. Nguyen V.Q. Levy S. J. Immunol. 1993; 151: 1918-1925PubMed Google Scholar, 4Shaw A.R.E. Domanska A. Mak A. Gilchrist A. Dobler K. Visser L. Poppema S. Fliegel L. Letarte M. Willett B.J. J. Biol. Chem. 1995; 270: 24092-24099Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and intracellular calcium (5Gil M.L. Vita N. Lebel-Binay S. Miloux B. Chalon P. Kaghad M. Marchiol-Fournigault C. Conjeaud H. Caput D. Ferrara P. Fradelizi D. Quillet-Mary A. J. Immunol. 1992; 148: 2826-2833PubMed Google Scholar). In the plasma membrane, TM4SF proteins interact with each other (2Hemler M.E. Mannion B.A. Berditchevski F. Biochim. Biophys. Acta. 1996; 1287: 67-71PubMed Google Scholar, 6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. 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Engel P. Immunol. Today. 1994; 15: 437-442Abstract Full Text PDF PubMed Scopus (242) Google Scholar), MHC class I (12Szollosi J. Horejsi V. Bene L. Angelisova P. Damjanovich S. J. Immunol. 1996; 157: 2939-2946PubMed Google Scholar), MHC class II (9Angelisova P. Hilgert I. Horejsi V. Immunogenetics. 1994; 39: 249-256Crossref PubMed Scopus (169) Google Scholar, 10Rubinstein E. Le Naour F. Lagaudrière-Gesbert C. Billard M. Conjeaud H. Boucheix C. Eur. J. Immunol. 1996; 26: 2657-2665Crossref PubMed Scopus (330) Google Scholar, 12Szollosi J. Horejsi V. Bene L. Angelisova P. Damjanovich S. J. Immunol. 1996; 157: 2939-2946PubMed Google Scholar), the neural protein L1 (13Schmidt C. Künemund V. Wintergerst E.S. Schmitz B. Schachner M. J. Neurosci. Res. 1996; 43: 12-31Crossref PubMed Scopus (92) Google Scholar), and a discrete subset of integrins (6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. 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Commun. 1996; 222: 13-18Crossref PubMed Scopus (85) Google Scholar, 19Jones P.H. Bishop L.A. Watt F.M. Cell Adh. & Commun. 1996; 4: 297-305Crossref PubMed Scopus (89) Google Scholar). It is hypothesized that within these complexes, a TM4SF protein may function as a new class of membrane adapter that either nucleates assembly and/or regulates signaling activities (20Berditchevski F. Tolias K.F. Wong K. Carpenter C.L. Hemler M.E. J. Biol. Chem. 1997; 272: 2595-2598Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Our recent data have indicated that integrin-TM4SF protein complexes contain additional cell-surface components not yet identified. To characterize these proteins we utilized a systematic approach for generating and selecting mAbs against purified integrin-TM4SF complexes. A panel of mAbs was developed against integrin-TM4SF protein complexes that were purified using anti-CD81 mAb-coated beads. The CD81 protein was chosen for immunization because of its widespread distribution and because it is readily identified as a ∼22-kDa protein following cell-surface labeling. Notably, we found that all selected antibodies recognize either integrins or TM4SF proteins. Moreover, we have identified and characterized a novel TM4SF protein called NAG-2. Human B-cell lines, Raji, JY, and Ramos; T-cell lines, Jurkat and Molt-4; and a promyelocytic cell line, K562 were cultured in RPMI 1640 medium. Chinese hamster ovary (CHO) cell lines were maintained in α-minimum Eagle's medium, and the human breast carcinoma cell line, MDA-MB-435, the fibrosarcoma line HT1080, and all other cell lines were maintained in Dulbecco's modified Eagle's medium. All cell lines were supplemented with 10% fetal calf serum. A CHO line (CHO/P) stably transfected with the polyoma large T antigen was used for expression cloning (21Heffernan M. Dennis J.W. Nucleic Acids Res. 1991; 19: 85-92Crossref PubMed Scopus (94) Google Scholar). K562-α3 and -α6 integrin transfectants (16Berditchevski F. Bazzoni G. Hemler M.E. J. Biol. Chem. 1995; 270: 17784-17790Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) as well as an HT1080-CD9 (6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar) transfectant were previously described. To make CHO-CD63 cells, CHO cells were electroporated with human CD63 cDNA (22Metzelaar M.J. Wijngaard P.L.J. Peters P.J. Sixma J.J. Nieuwenhuis H.K. Clevers H.C. J. Biol. Chem. 1991; 266: 3239-3245Abstract Full Text PDF PubMed Google Scholar) in pCDM8 vector. The CHO-CD63 cells were used for mAb screening 2 days after electroporation. The anti-TM4SF mAbs used were C9-BB, anti-CD9 (6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar); 6H1 (16Berditchevski F. Bazzoni G. Hemler M.E. J. Biol. Chem. 1995; 270: 17784-17790Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and RUU-SP 2.28 (23Toothill V.J. van Mourik J.A. Nieuwenhuis H.K. Metzelaar M.J. Pearson J.D. J. Immunol. 1990; 145: 283-291PubMed Google Scholar), anti-CD63; and M38, anti-CD81 (24Fukudome K. Furuse M. Imai T. Nishimura M. Takagi S. Hinuma Y. Yoshie O. J. Virol. 1992; 66: 1394-1401Crossref PubMed Google Scholar). Anti-integrin mAbs used were A2-2E10, anti-α2 (25Bergelson J.M. St. John N. Kawaguchi S. Pasqualini R. Berditchevski F. Hemler M.E. Finberg R.W. Cell Adh. & Commun. 1994; 2: 455-464Crossref PubMed Scopus (51) Google Scholar); A3-X8, anti-α3 (26Weitzman J.B. Pasqualini R. Takada Y. Hemler M.E. J. Biol. Chem. 1993; 268: 8651-8657Abstract Full Text PDF PubMed Google Scholar); A5-PUJ2, anti-α5 (27Pujades C. Teixidó J. Bazzoni G. Hemler M.E. Biochem. J. 1996; 313: 899-908Crossref PubMed Scopus (39) Google Scholar); A6-ELE, anti-α6 (28Lee R.T. Berditchevski F. Cheng G.C. Hemler M.E. Circ. Res. 1995; 76: 209-214Crossref PubMed Scopus (114) Google Scholar); and TS2/16, anti-β1(29Hemler M.E. Sánchez-Madrid F. Flotte T.J. Krensky A.M. Burakoff S.J. Bhan A.K. Springer T.A. Strominger J.L. J. Immunol. 1984; 132: 3011-3018Crossref PubMed Google Scholar). Other antibodies were 8E11, anti-CD109 (6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar); W6/32, anti-MHC class I (30Barnstable C.J. Bodmer W.F. Brown G. Galfré G. Milstein C. Williams A.F. Zeigler A. Cell. 1978; 14: 9-20Abstract Full Text PDF PubMed Scopus (1600) Google Scholar); and P3, negative control antibody (31Lemke H. Hammerling G.J. Hohmann C. Rajewsky K. Nature. 1978; 271: 249-251Crossref PubMed Scopus (115) Google Scholar). MDA-MB-435 cells were surface-labeled with NHS-LC-biotin (Pierce) or Na125I according to established protocols and lysed in immunoprecipitation buffer (1% Brij 96 or 1% n-octyl glucoside, 25 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) for 1 h at 4 °C. Insoluble materials were pelleted at 12,000 rpm for 10 min, and the cell lysates were precleared by incubation with pansorbin (Calbiochem) for 30 min at 4 °C. Immune complexes were collected onto Sepharose 4B beads (Pharmacia, Uppsala, Sweden) that were pre-bound with mAb, followed by four washes with the immunoprecipitation buffer. For immunoprecipitation under stringent conditions, the Brij 96-immunoprecipitation buffer was supplemented with 0.2% SDS. Immune complexes were eluted from beads with Laemmli sample buffer and resolved by 8–12% SDS-PAGE.125I-Labeled proteins were detected using O-XAR films (Eastman Kodak Co.) for 1–20 days at −70 °C. Biotin-labeled proteins were transferred to nitrocellulose membranes and visualized with peroxidase-conjugated ExtrAvidin (Sigma) using Renaissance Chemiluminescent Reagents (NEN Life Science Products). Re-immunoprecipitation experiments were performed as described earlier (6Berditchevski F. Zutter M.M. Hemler M.E. Mol. Biol. Cell. 1996; 7: 193-207Crossref PubMed Scopus (251) Google Scholar). Briefly, protein complexes were immunopurified using anti-NAG-2 mAb-conjugated Sepharose 4B from nonstringent (without 0.2% SDS) Brij 96 lysates of surface-biotinylated MDA-MB-435 cells. After five washes, the protein complexes were dissociated for 30 min at 4 °C with Brij 96 buffer containing 0.2% SDS. The eluates were subsequently reprecipitated with anti-TM4SF, anti-integrin mAbs, or control mAbs directly coupled to Sepharose 4B. Reciprocal re-immunoprecipitation experiments (in which NAG-2 was reprecipitated) were carried out similarly, except that MDA-MB-435 cells were labeled with125I. CD81-containing protein complexes were purified on mAb M38-coupled Sepharose 4B beads from Brij 96 cellular lysates prepared from MDA-MB-435 cells. After washes, immune complex-coupled Sepharose beads were used for immunization of a RBF/DnJ mouse. After three injections (each time with complexes derived from 1 to 2 × 109 cells), mouse serum was collected and tested by immunoprecipitation to verify antibody production. Four days after the fourth injection, hybridoma clones were produced as described previously (32Pasqualini R. Bodorova J. Ye S. Hemler M.E. J. Cell Sci. 1993; 105: 101-111PubMed Google Scholar). Hybridoma supernatants were first analyzed by flow cytometry followed by immunoprecipitation using surface-biotinylated MDA-MB-435 cell lysates as described above. MDA-MB-435 cells (5 g) were lysed in 500 ml of buffer containing 1% n-octyl glucoside, 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 2 mmphenylmethylsulfonyl fluoride, 15 μg/ml aprotinin, and 60 μg/ml leupeptin. To remove background bead-binding material, the lysate was sequentially preincubated with protein A-Sepharose and Sepharose beads conjugated with irrelevant mouse mAbs. The precleared lysates were incubated with anti-NAG-2 mAb-conjugated Sepharose beads packed in a 2-ml column. After washing the column, the NAG-2 protein was eluted using 50 mm glycine, pH 3.0, and the fractions were immediately neutralized with 0.1 volume of 1 m Tris-HCl, pH 9.0. Eluted fractions were analyzed by SDS-PAGE, and the fraction containing the NAG-2 antigen was determined by silver staining. Larger quantities of this fraction were then subjected to SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride membrane. The 30-kDa band corresponding to the NAG-2 protein was visualized by Ponceau S staining, excised from the membrane, and then amino-terminal sequencing was carried out using an Applied Biosystems 470A gas-phase sequenator equipped with a 120A phenylhydantoin amino acid analyzer (Harvard microsequencing facility, Cambridge, MA). Poly(A)+ RNA was isolated from MDA-MB-435 cells, and double-stranded cDNAs were synthesized using Copy Kit (Invitrogen, San Diego, CA). To facilitate the subcloning procedure,BstXI adapters were placed on the 5′-end of cDNAs. After size fractionation by gel electrophoresis, cDNAs of 0.7–2.0 kb were excised and ligated into pCDM8 expression vectors (Invitrogen). Then MC1061/P3 bacterial cells were transformed with ligated cDNA library and plated to 54 plates at a density of 1,900 clones/plate. Amplified cDNAs (54 separate pools) were collected and purified using QIAwell Plasmid Purification System (Qiagen, Chatsworth, CA). For screening, CHO/P cells (6 × 104/well) were grown on 24-well culture plates and then transiently transfected with each pool of amplified cDNAs using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. After incubation for 18 h, transfected CHO/P cells were stained with anti-NAG-2 mAb, fixed with methanol, and incubated with peroxidase-conjugated goat anti-mouse IgG (Sigma). Positive cells were visualized using ImmunoPure Metal Enhanced DAB Substrate Kit (Pierce) and light microscopy. Pools of cDNA yielding one or more NAG-2-positive CHO/P cells were further subfractionated three more times, until a single clone conferring NAG-2 staining was identified. Both strands of NAG-2 cDNA were sequenced by Sanger's sequencing method using dye-labeled dideoxy nucleotides as terminators. Samples were analyzed on an Applied Biosystem 373A automated DNA sequencer (33Smith L.M. Sanders J.Z. Kaiser R.J. Hughes P. Dodd C. Connell C.R. Heiner C. Kent S.B. Hood L.E. Nature. 1986; 321: 674-679Crossref PubMed Scopus (1288) Google Scholar). Protein data base searches were carried out using BLASTP via the NCBI BLAST network service (34Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71457) Google Scholar). Hydrophobicity plot analysis was performed by the Kyte and Doolittle method (35Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17297) Google Scholar) with a window of seven residues using the computer software DNA Strider. Multiple sequence alignments and similarity calculations were performed with the extended GCG sequence analysis software package (36Devereux J. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11531) Google Scholar). Cells were incubated with saturating concentrations of primary mouse mAbs for 45 min at 4 °C, washed twice, and then labeled with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin. Stained cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA). NAG-2 cDNA insert was excised from pCDM8 vector with XbaI and labeled with [α-32P]dCTP using RadPrime DNA Labeling System (Life Technologies, Inc.). Northern blot filters of multiple human tissues (CLONTECH, Palo Alto, CA) were hybridized with the labeled probe according to the manufacturer's instructions. The probe was stripped off by boiling the filter in sterile H2O containing 0.5% SDS and rehybridized with a 32P-labeled human β-actin cDNA. Radioactive bands were detected by autoradiography. Fresh tissue was obtained from material submitted to the Department of Pathology, Washington University School of Medicine, St. Louis, MO. The tissue was embedded in OCT compound (Miles Laboratory, Elkart, IN), snap-frozen in liquid nitrogen-cooled isopentane, and stored at −70 °C. Frozen sections (6 μm thick) were fixed briefly in acetone and held at −20 °C before staining with anti-NAG-2 mAb and detection with biotinylated anti-mouse IgG and avidin-biotin-peroxidase complex (Vector, Burlingame, CA), as described previously (37Zutter M.M. Blood. 1991; 77: 2231-2236Crossref PubMed Google Scholar). Sections were counterstained with methyl green. To identify new membrane antigens that associate with TM4SF proteins, we have utilized a monoclonal antibody generation and selection strategy, similar to that described in the accompanying paper (38Berditchevski F. Chang S. Bodorova J. Hemler M.E. J. Biol. Chem. 1997; 272: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Beads coated with anti-CD81 mAb were incubated with MDA-MB-435 cell lysate under non-stringent detergent conditions, and then the isolated bead-protein complexes were used to immunize mice. As shown (Fig. 1, lane c) the resulting mouse immune serum immunoprecipitated a protein co-migrating with CD81 (arrow), as well as several additional biotinylated proteins from MDA-MB-435 cells. Most of these proteins were not precipitated by preimmune serum (lane d). The biotinylated proteins directly immunoprecipitated by mouse sera (lane c) using stringent detergent conditions (1% Brij 96, 0.2% SDS) closely resemble those indirectly co-precipitated by an anti-CD81 mAb (lane a) under nonstringent (Brij 96) conditions. Notably, a control experiment (lane b) shows that anti-CD81 mAb precipitated mostly CD81 under stringent conditions. From these results we conclude that the mouse was adequately immunized to make antibodies directly recognizing CD81-associated cell-surface proteins. After four injections with CD81 complexes, 560 hybridomas were prepared, and supernatants were tested by flow cytometry to show that 35 hybridoma clones produced antibodies to cell-surface molecules on MDA-MB-435 cells. Secondary screening by non-stringent immunoprecipitation then was used to confirm whether these 35 mAbs indeed co-precipitated a protein resembling CD81. Immunoprecipitations from four representative antibodies are shown in Fig.2. Under nonstringent conditions, these four antibodies (lanes c, e, g, and i) as well as anti-CD81 (lane a) each precipitated a similar protein pattern that included a ∼22-kDa component co-migrating with CD81. In contrast, under stringent conditions mAb M38 yielded only CD81 (lane b), mAb 4D5 yielded a CD9-like protein (lane f), and mAb 8H6 recognized solely an α6integrin-like protein (lane j). The antigens directly recognized by mAb 1E5 (lane d) and 5C12 (lane h) were not obvious, possibly because these proteins are not very well labeled with biotin. A weak nonspecific 70-kDa band appeared in all lanes upon long exposure. Interestingly, immunoprecipitation data under stringent conditions indicated that none of the 35 selected mAbs recognized CD81 itself. Notably, mAb 1E5, along with 29 other mAb, selectively stained CD63-positive CHO transfectants, thus establishing conclusively that these 30 mAbs recognize CD63. Similarly, mAb 4D5 and another mAb selectively stained CD9-transfected HT1080 cells, and mAb 8H6 selectively stained α6-transfected K562 cells, thus confirming CD9 and α6 assignments from Fig. 2. Two other antibodies (5C12 and 2E12) failed to stain hamster or mouse transfectants expressing human α3, α6, or β1 integrin subunits or either of the TM4SF protein transfectants. Competitive antibody binding assays revealed that the two antibodies of unknown specificity seemed to recognize the same or overlapping antigenic epitopes on the surface of MDA-MB-435 cells (data not shown). This putative novel antigen was named NAG-2. Although not labeled with biotin (Fig. 2, lane h), the NAG-2 protein could be125I-labeled and immunoprecipitated under stringent conditions from MDA-MB-435 cells as a somewhat diffuse protein band of 28–35 kDa (Fig. 3). This band was not seen in CD81 (M38), integrin β1 (TS2/16), or negative control (mAb P3) immunoprecipitations. To characterize further NAG-2, ∼23 pmol of protein was purified from MDA-MB-435 cells using an anti-NAG-2 immunoaffinity column. Amino-terminal analysis of the purified material yielded a "RA-LQAVKY" sequence that was not present in the GenBank data base. A cDNA expression library was prepared from MDA-MB-435 cells and transiently transfected into CHO/P cells for screening, based on cell-surface staining with anti-NAG-2 mAb. One of 54 separate pools of clones yielded a few visibly stained CHO/P cells (out of 6 × 104 cells plated). After three more rounds of subfractionation, a single clone was isolated that was capable of conferring anti-NAG-2 staining upon transfection into CHO/P cells. The entire cDNA sequence of NAG-2 and its corresponding amino acid sequence are shown in Fig. 4 A. It contains a 5′-untranslated sequence of 104 base pairs, a single extended open reading frame of 714 base pairs, followed by 540 base pairs of 3′-untranslated region containing a polyadenylation signal. The first ATG codon of the open reading frame is within a Kozak consensus translation initiation sequence (39Kozak M. Nucleic Acids Res. 1987; 15: 8125-8132Crossref PubMed Scopus (4172) Google Scholar). The deduced 238 amino acids include the partial sequence obtained by amino-terminal analysis (underlined). The predicted protein molecular mass (26,177 Da) is slightly smaller than that observed in Fig. 3. PotentialN-glycosylation sites are located at residues 152 and 161. A hydrophobicity plot revealed four highly hydrophobic domains, each sufficiently long to span cellular membranes (Fig. 4 B). In addition, BLASTP searching revealed sequence similarity between the NAG-2 protein and TM4SF proteins, with CD53 (a TM4SF protein expressed on leukocytes) showing the most similarity (51% similarity, 34% identity). The NAG-2 protein sequence was aligned with known human TM4SF proteins (Fig. 4 C). For NAG-2 and other TM4SF proteins, conserved sequences are mostly within the putative transmembrane domains. In contrast, the extracellular domains are more divergent in terms of length, sequence, and degree of glycosylation. However, NAG-2 does contain four characteristic cysteine residues, within the large extracellular domain between transmembrane domains III and IV, that are highly conserved among nearly all TM4SF proteins. In addition, a search of the dbEST sequence data base revealed five overlapping expressed sequence tags, from which a complete putative murine NAG-2 sequence was constructed (Fig. 4 D). Notably, the level of identity (95%) between the murine and human protein sequences is unusually high. Other TM4SF proteins (e.g.CD53, CD82, CD37, and CD63) typically show 82–83% identity between human and mouse proteins. The TM4SF proteins CD53 and CD63 were previously shown to be associated with phosphatase activity (40Carmo A.M. Wright M.D. Eur. J. Immunol. 1995; 25: 2090-2095Crossref PubMed Scopus (44) Google Scholar). Here we carried out similar phosphatase assays, utilizing immune complexes from MDA 435 cells lysed in 1% Brij 96. Activity was determined by measuring the conversion ofp-nitrophenyl phosphate to the yellow-coloredp-nitrophenol product. As indicated (Fig.5, A and B), phosphatase activity was readily detected in association with CD63 immune complexes in two separate experiments. However, there was minimal phosphatase activity associated with NAG-2 immune complexes (obtained using two different antibodies). Likewise, the activity associated with CD9 or CD81 was not appreciably above the negative control activities seen with mAb P3 or anti-CD109 mAb. Flow cytometric analysis revealed that NAG-2 protein is expressed most strongly on a human melanoma cell line (LOX), a fibrosarcoma line (HT-1080), and the breast carcinoma line (MDA-MB-435) and shows variable levels on other human sarcoma and carcinoma cell lines derived from various tissues. Also, NAG-2 showed little or no expression on several hematopoietic cell lines and was absent from normal peripheral blood T-cells and platelets (Table I). Northern blot analysis showed that a 1.5-kb transcript of NAG-2 is present in all human tissues analyzed with the exception of brain (Fig.6). Expression is especially strong in spleen, colon, and pancreas. Heart and skeletal muscle have an additional 6.5-kb NAG-2 transcript.Table IExpression of NAG-2 protein in cultured human cellsCellsCellsB-cellsFibrosarcoma Raji− HT1080+++ JY− Ramos+Rhabdomyosarcoma RD+T-cellsBreast carcinomas Jurkat+ MDA-MB-435+++ Molt-4− MDA-MB-231− peripheral blood− MCF7−PromyelocyticLung carcinoma K562++ A549+Platelets−Colon carcinomasMelanoma MIP 101− LOX+++ CCL 221+ CCL 227++Neuroblastomas CCL 228+ IMR-32+ SK-N-SH++Vulval carcinoma A431+Cells were stained using anti-NAG-2 mAb and analyzed by FACScan. Staining was scored as −, +, ++, or +++ if mean fluorescence intensity was 10-fold higher than that of P3 control mAb. Open table in a new tab Cells were stained using anti-NAG-2 mAb and analyzed by FACScan. Staining was scored as −, +, ++, or +++ if mean fluorescence intensity was 10-fold higher than that of P3 control mAb. Frozen section immunohistochemistry was carried out to characterize further the distribution of NAG-2 protein. The NAG-2 protein was widely expressed in all tissues evaluated but was present in a restricted repertoire of cell types (Table II). High levels of NAG-2 were observed on vascular endothelial cells and fibroblasts in all tissues. The spleen, composed of a meshwork of branching vascular cords and sinusoids, expressed the highest levels of NAG-2 (Fig. 7 A). In contrast, red pulp cords, thin walled vessels, lymphocytes of the lymphoid follicles, and periarterial lymphoid sheaths were entirely negative. Also, spleen showed greater NAG-2 expression in veins, sinusoids, and small arteries, compared with thick walled arteries (Fig.7 A).Table IIHistologic distribution of the NAG-2 proteinCell typeIntensityFibroblasts3+Endothelial cells3–4+Smooth muscle0–1

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