Calnexin Associates with Monomeric and Oligomeric (Disulfide-linked) CD3δ Proteins in Murine T Lymphocytes
1998; Elsevier BV; Volume: 273; Issue: 23 Linguagem: Inglês
10.1074/jbc.273.23.14152
ISSN1083-351X
Autores Tópico(s)Immunotherapy and Immune Responses
ResumoThe antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic αβ heterodimers noncovalently associated with monomeric CD3γ,δ,ε proteins and disulfide-linked ζζ homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4+CD8+ thymocytes and splenic T cells. These studies demonstrate that CD3δ proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3ε subunits in CD4+CD8+ thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3δ proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem.271, 9660–9665), we found that glucose residues were not invariably removed from CD3δ glycoproteins prior to their assembly with CD3ε subunits in CD4+CD8+thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3δ proteins in murine T cells. The data in the current report demonstrate that CD3δ proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4+CD8+ thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3δ proteins is a function of their oxidation state. The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic αβ heterodimers noncovalently associated with monomeric CD3γ,δ,ε proteins and disulfide-linked ζζ homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4+CD8+ thymocytes and splenic T cells. These studies demonstrate that CD3δ proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3ε subunits in CD4+CD8+ thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3δ proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem.271, 9660–9665), we found that glucose residues were not invariably removed from CD3δ glycoproteins prior to their assembly with CD3ε subunits in CD4+CD8+thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3δ proteins in murine T cells. The data in the current report demonstrate that CD3δ proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4+CD8+ thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3δ proteins is a function of their oxidation state. Most T lymphocytes express on their surfaces a multisubunit complex consisting of clonotypic αβ proteins associated with invariant CD3-γ,δ,ε and ζ chains, designated the T cell antigen receptor (TCR) 1The abbreviations used are: TCR, T cell antigen receptor; ER, endoplasmic reticulum; mAb, monoclonal antibody; EH, endoglycosidase H; JB, jack bean mannosidase; PAGE, polyacrylamide gel electrophoresis; Ab, antibody. complex (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (299) Google Scholar, 2Exley M. Terhorst C. Wileman T. Sem. Immunol. 1991; 3: 283-297PubMed Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Assembly of the TCR occurs in the endoplasmic reticulum (ER) and proceeds in a highly ordered manner involving: (i) formation of noncovalently associated paris of δε and γε proteins, (ii) assembly of α,β proteins with δε,γε pairs to form αδε and βγε intermediate complexes, (iii) joining of αδε and βγε chains followed by disulfide bonding of CD3-associated α,β proteins to yield incomplete αβδεγε complexes, and finally (iv) addition of disulfide-linked ζζ homodimers to form the complete αβδεγεζζ TCR complex (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (299) Google Scholar, 2Exley M. Terhorst C. Wileman T. Sem. Immunol. 1991; 3: 283-297PubMed Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). In general, the surface expression of TCR proteins is tightly associated with their assembly status (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (299) Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Individual, unassembled TCR proteins and partially assembled TCR complexes containing two or three TCR subunits do not effectively exit the ER. Both incomplete αβδεγε and complete αβδεγεζζ TCR egress from the ER to the Golgi complex; however, only complete TCR complexes are efficiently transported to the cell surface (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (299) Google Scholar, 2Exley M. Terhorst C. Wileman T. Sem. Immunol. 1991; 3: 283-297PubMed Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Unlike mature CD4+ and CD8+ (single positive) T cells, which fundamentally express complete αβδεγεζζ TCR (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar), immature CD4+CD8+ (double-positive) thymocytes express both complete TCR and partial complexes of CD3δε, CD3γε proteins associated with calnexin (4Wiest D.L. Kearse K.P. Shores E.W. Singer A. J. Exp. Med. 1994; 180: 1375-1382Crossref PubMed Scopus (54) Google Scholar, 5Wiest D.L. Burgess W.H. McKean D. Kearse K.P. Singer A. EMBO J. 1995; 14: 3425-3434Crossref PubMed Scopus (101) Google Scholar, 6Takase K. Wakizaka K. von Boehmer H. Wada I. Moriya H. Saito T. J. Immunol. 1997; 159: 741-747PubMed Google Scholar), referred to as clonotypic independent complexes (4Wiest D.L. Kearse K.P. Shores E.W. Singer A. J. Exp. Med. 1994; 180: 1375-1382Crossref PubMed Scopus (54) Google Scholar, 5Wiest D.L. Burgess W.H. McKean D. Kearse K.P. Singer A. EMBO J. 1995; 14: 3425-3434Crossref PubMed Scopus (101) Google Scholar). The molecular basis for clonotypic independent complex expression is unknown but is postulated to result from inefficient ER retention mechanisms in CD4+CD8+ thymocytes that do not persist in mature T cells (5Wiest D.L. Burgess W.H. McKean D. Kearse K.P. Singer A. EMBO J. 1995; 14: 3425-3434Crossref PubMed Scopus (101) Google Scholar, 7Wiest D.L. Bhandoola A. Punt J. Kreibich G. McKean D. Singer A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1884-1889Crossref PubMed Scopus (87) Google Scholar). Previous studies by Jin et al. reported that a small subfraction of CD3ε proteins exists as disulfide-linked dimers in human T lymphocytes (8Jin Y.J. Koyasu S. Moingeon P. Steinbrich R. Tarr G.E. Reinherz E.L. J. Biol. Chem. 1990; 265: 15850-15853Abstract Full Text PDF PubMed Google Scholar), which assemble with TCRβ subunits; CD3ε dimers were likewise observed to be present in murine thymocytes, although their assembly status was not evaluated (8Jin Y.J. Koyasu S. Moingeon P. Steinbrich R. Tarr G.E. Reinherz E.L. J. Biol. Chem. 1990; 265: 15850-15853Abstract Full Text PDF PubMed Google Scholar). Disulfide-linked heterodimers of CD3γ-ε proteins have also been described in REX variant human T cell lines, which fail to express TCRα or TCRα,β molecules (9Sancho J. Chatila T. Wong R.C.K. Hall C. Blumberg R. Alarcon B. Geha R.S. Terhorst C. J. Biol. Chem. 1989; 264: 20760-20769Abstract Full Text PDF PubMed Google Scholar). More recently, Huppa and Ploegh demonstrated that human CD3ε molecules translated in vitro in the absence of other TCR proteins have a tendency to form disulfide-linked homooligomers, which assemble with the molecular chaperone calnexin (10Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar). Cotranslation of CD3δ or CD3γ molecules was sufficient to maintain CD3ε proteins in a principally monomeric phase, however, suggesting that CD3δ and CD3γ may guide the folding of CD3ε proteins during the initial stages of their biosynthesis (10Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar). In the current study we evaluated the disulfide linkage status of newly synthesized TCR proteins in murine CD4+CD8+ thymocytes and splenic T cells. These studies show that newly synthesized CD3δ proteins exist as both monomeric and disulfide-linked molecules that differentially assemble with CD3ε molecules in CD4+CD8+ thymocytes and splenic T cells. In addition, these data document that calnexin associates with both monomeric and oligomeric (disulfide-linked) CD3δ proteins in murine T lymphocytes. C57BL/6 (B6) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). CD4+CD8+ thymocytes were isolated by their adherence to plastic plates coated with anti-CD8 mAb (83-12-5) and were typically >95% CD4+CD8+ as described (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Splenic T cells were purified by incubation of single cell suspensions of spleen cells on tissue culture plates coated with rabbit anti-mouse immunoglobulin, (Organo-Technika-Cappel, Malvern, PA) for 60 min at 37 °C, followed by isolation of nonadherent cells. The resultant cell populations were typically 80–85% CD3+ as determined by flow cytometry analysis. The following mAb were used in this study: 145–2C11, specific for CD3ε proteins associated with CD3δ or CD3γ chains (11Leo O. Foo M. Sachs D.H. Samelson L.E. Bluestone J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1374-1378Crossref PubMed Scopus (1705) Google Scholar); HMT 3.1, which recognizes CD3ε proteins irrespective of their assembly state (12Born W. Miles C. White J. O'Brien R. Feed J.H. Marrack P. Kappler J. Kubo R.T. Nature. 1987; 330: 572-575Crossref PubMed Scopus (75) Google Scholar); H28–710, specific for TCRα (13Becker J.L.B. Near R. Mudgett-Hunter M. Margolies M.M. Kubo R.T. Kaye J. Hedrick S.M. Cell. 1989; 58: 911-921Abstract Full Text PDF PubMed Scopus (108) Google Scholar); and H57-597, specific for TCRβ (14Kubo R.T. Born J.W. Kappler J. Marrack P. Pigeon M. J. Immunol. 1989; 142: 2736-2742PubMed Google Scholar), all kindly provided by Dr. Ralph Kubo (Cytel, San Diego, CA). The following antisera were used: R9, specific for CD3δ proteins (15Samelson L.E. Weissman A.M. Robey F.A. Berkower I. Klausner R.D. J. Immunol. 1986; 137: 3254-3258PubMed Google Scholar), kindly provided by Dr. Larry Samelson (National Institutes of Health, Bethesda, MD); 551, specific for TCRζ proteins (16Cenciarelli C. Hou D. Hsu K.C. Rellahan B.L. Weist D.L. Smith H.T. Fried V.A. Weissman A.M. Science. 1992; 257: 795-797Crossref PubMed Scopus (185) Google Scholar), kindly provided by Dr. Allan Weissman (National Institutes of Health, Bethesda, MD); SPA-860 anti-calnexin (Stressgen Biotechnologies, Victoria, BC, Canada) and PA3–900 anti-calreticulin (Affinity BioReagents, Neshanic Station, NJ). Metabolic pulse labeling with [35S]methionine was performed as described previously (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Briefly, cells were pulse-labeled in methionine-deficient medium (Biofluids, Rockville, MD) containing 1 mCi/ml [35S]methionine (Trans 35S-label; ICN, Irvine, CA) for 30 min at 37 °C. Cells were lysed by solubilization in 1% digitonin (Calbiochem, La Jolla, CA) lysis buffer (20 mm Tris, 150 mm NaCl, 10 mmiodoacetamide, plus protease inhibitors) at 1 × 108cells/ml for 20 min at 4 °C; lysates were clarified by centrifugation to remove insoluble material and immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads (Amersham Pharmacia Biotech). Sequential immunoprecipitation and immunoprecipitation/release/recapture procedures were performed as previously detailed (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Digestion with endoglycosidase H (EH) was performed by resuspending precipitates in glycosidase digestion buffer (75 mm sodium phosphate, pH 6.1, 75 mm EDTA, 0.1% Nonidet P-40) containing 10 milliunits of EH (Genzyme, Cambridge, MA) for 16 h at 37 °C; digestion with jack bean mannosidase (JB) (Oxford Glycosystems, Rosedal, NY) was performed according to the manufacturer's instructions. One- and two-dimensional SDS-PAGE was performed as described previously (17Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). As shown in Fig.1 A, analysis of anti-CD3ε immunoprecipitates of radiolabled CD4+CD8+thymocytes on two-dimensional nonreducing × reducing (NR × R) gels shows expected nondisulfide-linked monomeric CD3γ,δ and CD3ε proteins that migrate on and slightly above the diagonal, respectively (Fig. 1 A, top panel), and disulfide-linked TCRαβ heterodimers and ζζ homodimers, which migrate below the diagonal (Fig. 1 A, top panel). Interestingly, a dimeric species of unknown identity was also present in such precipitates, migrating at approximately 26 kDa following reduction of disulfide-linked bonds (Fig. 1 A, top panel, arrow). Disulfide-linked 26-kDa proteins were isolated from CD4+CD8+ thymocyte lysates using two different anti-CD3ε mAbs of distinct specificity, 145-2C11 and HMT3.1 (Fig. 1, A, top panel, and B,top panel, respectively) (see “Experimental Procedures”), and by CD3δ-specific Ab (Figs. 1 B,bottom panel) but not anti-TCRβ mAb (Fig. 1 A,bottom panel) or anti-ζ antiserum (data not shown). Unlike CD4+CD8+ thymocytes, disulfide-linked 26-kDa proteins were not observed in anti-CD3ε precipitates of radiolabeled splenic T cells (Fig. 2, A andB) but were clearly present in anti-CD3δ precipitates of splenic T cells (Fig. 2 B, bottom panel). Disulfide linkage of 26-kDa proteins did not result from artificial formation of disulfide bonds during cell lysis because identical results were observed when cells were solubilized in lysis buffer containing excess (75 mm) iodoacetamide (data not shown). Taken together, these data demonstrate that disulfide-linked 26-kDa proteins differentially associate with CD3ε and TCRβ subunits in CD4+CD8+ thymocytes and splenic T cells.Figure 2Disulfide-linked 26-kDa proteins are not assembled with CD3ε molecules in splenic T cells. A, CD4+CD8+ thymocytes and splenic T cells were radiolabeled with [35S]methionine for 30 min and solubilized in 1% digitonin, and lyates were immunoprecipitated with anti-CD3ε mAb (145–2C11) mAb. Precipitates were analyzed on two-dimensional NR × R SDS-PAGE gels. The positions of TCR proteins and disulfide-linked 26-kDa proteins (arrow) are indicated. B, same as in A, except that splenic T cell lysates were immunoprecipitated with anti-CD3ε mAb (145–2C11) and anti-CD3δ Ab (R9).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because their molecular mass is similar to that of CD3 components, we reasoned that disulfide-linked 26-kDa proteins might represent newly synthesized CD3δ proteins, CD3ε proteins, or both. To determine whether CD3δ,ε proteins were assembled into disulfide-linked dimers in CD4+CD8+ thymocytes, anti-CD3δ precipitates of CD4+CD8+ thymocytes were analyzed on two-dimensional NR × R gels and immunoblotted with antiserum specific for CD3δ and CD3ε molecules. As shown in Fig. 3, CD3δ proteins existed as both monomeric and disulfide-linked molecules in CD4+CD8+ thymocytes (Fig. 3, top panel). In contrast, CD3ε proteins were present exclusively as nondisulfide-linked monomers (Fig. 3, bottom panel). Identical results were obtained in immunoblot experiments of anti-CD3ε precipitates of CD4+CD8+ thymocytes (data not shown). These results show that CD3δ proteins exist as both monomeric and disulfide-linked molecules in CD4+CD8+ thymocytes. CD3δ and CD3ε proteins are easily distinguished from each other in that CD3δ is post-translationally modified by the addition of threeN-linked oligosaccharide chains unlike CD3ε, which does not contain N-glycans (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 6: 403-431Crossref PubMed Scopus (299) Google Scholar, 17Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). To confirm that CD3δ glycoproteins were disulfide-linked in murine T cells, digitonin lysates of radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-CD3δ Ab, CD3δ precipitates were boiled in SDS to release bound material, and CD3δ proteins were recaptured by precipitation with anti-CD3δ Ab. Precipitates were digested with Endo H glycosidase (specific for cleavage of immatureN-linked glycans) and analyzed on one-dimensional SDS-PAGE gels under nonreducing conditions. Most CD3δ proteins radiolabeled during a 30-min pulse period migrated as monomeric 26-kDa proteins (Fig. 4, first lane), which fell to 17 kDa following removal of N-linked glycan chains, as expected (Fig. 4, second lane). Importantly, these data show that remaining CD3δ molecules existed as disulfide-linked proteins that migrated at approximately 52 kDa in mock treated samples (Fig. 4, first lane) and at 34 kDa following glycosidase digestion (Fig. 4, second lane); these results were confirmed by immunoblotting experiments using anti-CD3δ Ab (data not shown). These data are consistent with the assembly of CD3δ glycoproteins into disulfide-linked dimers that are composed of CD3δ proteins linked to itself (CD3δ-δ) or to another molecule of similar size (CD3δ-x), which like CD3δ, must also containN-glycans as the magnitude of decrease in molecular mass following deglycosylation is greater than would be expected for CD3δ associated with a nonglycosylated protein. ImmatureN-glycan chains on newly synthesized glycoproteins having the structure Glc3Man9GlcNAc2 are initially processed by the sequential action of glucosidase I and glucosidase II enzymes in the ER, creating monoglucosylated Glc1Man9GlcNAc2 glycans important for interaction with the lectin-like chaperone calnexin (18Moremen K.W. Trimble R.B. Herscovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (324) Google Scholar, 19Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 20Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 21Spiro R.G. Zhu Q. Bhoyroo V. Soling H.-D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 22Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Calnexin is proposed to function in the quality control of folding and assembly of numerous newly synthesized glycoproteins, including TCRα,β and CD3δ,γ subunits (23Hochstenbach F. David V. Watkins S. Brenner M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4734-4738Crossref PubMed Scopus (217) Google Scholar, 24van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 25van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Previous studies on the processing of TCR glycoproteins in splenic T cells show that Glc residues are removed from newly synthesized CD3δ molecules prior to their assembly with other TCR subunits and that calnexin associates exclusively with unassembled, “free” CD3δ proteins containing incompletely trimmed glycan chains (24van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To determine whether CD3δ proteins were similarly processed in CD4+CD8+thymocytes, cells were pulse-labeled with [35S]methionine for 30 min and solubilized in 1% digitonin, and lysates were immunoprecipitated with anti-CD3δ Ab to purify total CD3δ proteins; alternatively, lysates were sequentially immunoprecipitated with anti-TCRβ mAb to isolate CD3δ proteins assembled into complete αβδεγεζζ and incomplete αβδεγε TCR complexes, followed by precipitation with anti-CD3ε mAb to capture CD3δ chains present in partial complexes of CD3δε components and finally precipitation with anti-CD3δ Ab to purify remaining unassembled, free CD3δ chains. Precipitates were boiled in SDS to release bound material, CD3δ proteins were specifically recaptured with anti-CD3δ Ab, and recapture precipitates were digested with JB and EH glycosidases. JB digestion is useful for evaluating the Glc trimming status of newly synthesized glycoproteins because it removes eight mannoses from fully trimmed N-glycan chains devoid of Glc residues (Man8–9GlcNAc2) but only five mannoses from incompletely trimmed N-glycans containing one to three Glc saccharides (Glc1–3Man8–9GlcNAc2) (19Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (719) Google Scholar, 24van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In contrast, EH removes all but a single GlcNAc fromN-glycan chains irrespective of their Glc content (26Tarentino A.L. Maley F. J. Biol. Chem. 1974; 249: 811-817Abstract Full Text PDF PubMed Google Scholar). Similar to what was previously observed in splenic T cells (24van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), CD3δ proteins synthesized in CD4+CD8+ thymocytes existed in four major glycoforms (A–D), indicative of CD3δ proteins containing three (A), two (B), one (C), and zero (D) incompletely trimmed glycan chains, respectively (Fig.5 A, left-hand side). Interestingly, however, unlike splenic T cells, CD3δ proteins containing incompletely trimmed N-glycans in CD4+CD8+ thymocytes were present as both free, unassembled chains and as assembled molecules associated with CD3ε proteins (Fig. 5, A and B). In contrast, CD3δ proteins associated with TCRβ were totally devoid of Glc residues as shown by their complete sensitivity to JB digestion (Fig. 5,A and B). Taken together, these data show that CD3δ glycoforms are similarly generated in immature CD4+CD8+ thymocytes and splenic T cells and that CD3δ proteins containing incompletely trimmedN-glycans exist as both free and assembled TCR subunits in CD4+CD8+ thymocytes. Next, the assembly of newly synthesized CD3δ proteins with the molecular chaperone calnexin was examined. Metabolically labeled CD3δ proteins coprecipitated with calnexin in CD4+CD8+ thymocytes that, as expected, contained incompletely trimmed glycan chains that were partially resistant to JB digestion (Fig. 5 A, middle lanes). As similarly noted for other T cell types (25van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 25345-25349Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), CD3δ proteins synthesized in CD4+CD8+ thymocytes did not associate with the calnexin-related molecule, calreticulin (data not shown). To determine the disulfide linkage status of CD3δ proteins associated with calnexin, digitonin lysates of radiolabeled CD4+CD8+ thymocytes were immunoprecipitated with anti-calnexin Ab, precipitates were boiled in 1% SDS to release bound material, and CD3δ proteins were specifically recaptured with anti-CD3δ Ab. Analysis of recapture precipitates on two-dimensional NR × R SDS-PAGE gels showed that both monomeric and disulfide-linked CD3δ proteins were assembled with calnexin in CD4+CD8+ thymocytes (Fig.6); similar results were observed in splenic T cells (data not shown). These studies show that both monomeric and disulfide-linked CD3δ proteins associate with calnexin in murine T cells. In the current report we evaluated the disulfide linkage status of newly synthesized TCR proteins in CD4+CD8+thymocytes and splenic T cells. These studies show that: (i) CD3δ proteins exist as both monomeric and oligomeric (disulfide-linked) species in murine T cells; (ii) disulfide-linked CD3δ proteins differentially assemble with CD3ε and TCRβ subunits in CD4+CD8+ thymocytes and splenic T cells; (iii) unlike CD3δ processing in splenic T cells, Glc residues are not invariably removed from CD3δ glycoproteins prior to their assembly with CD3ε chains in CD4+CD8+ thymocytes; and (iv) calnexin associates with both monomeric and disulfide-linked CD3δ proteins in murine T cells. Previous studies by Jin et al. reported that a fraction of CD3ε proteins exists as disulfide-linked dimers in murine T cells, including thymocytes (8Jin Y.J. Koyasu S. Moingeon P. Steinbrich R. Tarr G.E. Reinherz E.L. J. Biol. Chem. 1990; 265: 15850-15853Abstract Full Text PDF PubMed Google Scholar). The data in the current study show that CD3δ proteins were present as disulfide-linked dimers in both CD4+CD8+ thymocytes and splenic T cells, but no evidence was found for disulfide linkage of CD3ε molecules in either cell type. The reason for these apparent discrepancies are unclear but may result from the fact that identification of disulfide-linked CD3ε proteins in previous studies relied on their detection by rabbit antiserum directed against murine CD3ε (8Jin Y.J. Koyasu S. Moingeon P. Steinbrich R. Tarr G.E. Reinherz E.L. J. Biol. Chem. 1990; 265: 15850-15853Abstract Full Text PDF PubMed Google Scholar), which may detect unique epitopes not recognized by the anti-CD3ε mAbs used in our study. Regardless, the current study clearly demonstrates that CD3δ glycoproteins were assembled into disulfide-linked dimers in murine T lymphocytes using several different approaches, including immunoblotting, immunoprecipitation/release/recapture experiments, and glycosidase digestion studies. Our results are consistent with the assembly of CD3δ chains into homodimers containing CD3δ proteins disulfide-linked to itself or heterodimers of CD3δ proteins disulfide-bridged to another molecular of similar molecular mass and carbohydrate content. Huppa and Ploegh have recently shown that human CD3ε molecules translated in vitro in the absence of other TCR proteins have a tendency to form disulfide-linked homooligomers, which assemble with calnexin (10Huppa J.B. Ploegh H.L. J. Exp. Med. 1997; 186: 393-403Crossref PubMed Scopus (82) Google Scholar). The data in the current report importantly extend these studies by showing that murine CD3δ proteins synthesized in intact cells in the presence of other TCR proteins may also exist as disulfide-linked molecules that can assemble with calnexin. Conceivably, disulfide-linked CD3δ proteins in CD4+CD8+ thymocytes and splenic T cells may represent CD3δ molecules that are synthesized in excess of other TCR subunits that rapidly dimerize and bind to calnexin. Curiously, however, disulfide-linked CD3δ proteins were not assembled with other TCR molecules in splenic T cells but were (noncovalently) associated with CD3ε proteins in CD4+CD8+ thymocytes. Although the significance of these findings is currently unclear, it is interesting to note that these results parallel the differential assembly of Glc-containing CD3δ proteins with CD3ε molecules in CD4+CD8+ thymocytes and splenic T cells. The molecular basis for the differential assembly of incompletely trimmed CD3δ chains with CD3ε proteins in CD4+CD8+thymocytes and splenic T cells is unknown but may be influenced by the dissimilar stability of TCRα proteins in these two cell types (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar,17Kearse K.P. Roberts J.L. Munitz T.I. Wiest D.L. Nakayama T. Singer A. EMBO J. 1994; 13: 4504-4514Crossref PubMed Scopus (68) Google Scholar). We have previously noted that CD3δ proteins synthesized in T hybridoma cells under conditions of impaired glucosidase activity (which destabilizes TCRα molecules) show increased assembly with CD3ε subunits, similar to what is naturally observed in CD4+CD8+ thymocytes (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Thus, it is possible that TCRα association with CD3δ or CD3ε proteins retards assembly of disulfide-linked CD3δ proteins with CD3ε molecules, although such αδ,αε intermediates remain to be directly demonstrated in primary murine T cells (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). In both CD4+CD8+thymocytes and splenic T cells, dimeric CD3δ proteins were precluded from incorporation into complete TCR complexes as evidenced by their failure to coprecipitate with TCRβ, ζ, and CD3γ proteins (this study), 2K. P. Kearse, unpublished observations. indicating that quality control mechanisms exist in both cell types that control assembly of dimeric CD3δ proteins into higher ordered TCR complexes. Finally, our results that newly synthesized CD3δ chains bearing incompletely trimmed oligosaccharides were assembled with CD3ε subunits in CD4+CD8+ thymocytes are in agreement with previous reports that Glc-containing CD3δ proteins are expressed on the surfaces of immature thymocytes in association with CD3ε molecules (5Wiest D.L. Burgess W.H. McKean D. Kearse K.P. Singer A. EMBO J. 1995; 14: 3425-3434Crossref PubMed Scopus (101) Google Scholar, 7Wiest D.L. Bhandoola A. Punt J. Kreibich G. McKean D. Singer A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1884-1889Crossref PubMed Scopus (87) Google Scholar). Importantly, however, the data in the current study provide the first assessment of the efficiency of Glc removal from newly synthesized CD3δ proteins in CD4+CD8+ thymocytes and show that CD3δ glycoforms are effectively generated in CD4+CD8+ thymocytes as in splenic T cells (24van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). I thank Drs. Ralph Kubo, Larry Samelson, and Allan Weissman for generosity in providing anti-TCR antibodies and Drs. Mark Mannie and Tom McConnell for critical reading of the manuscript.
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