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Modification of the T Cell Antigen Receptor (TCR) Complex by UDP-glucose:Glycoprotein Glucosyltransferase

1999; Elsevier BV; Volume: 274; Issue: 20 Linguagem: Inglês

10.1074/jbc.274.20.14094

ISSN

1083-351X

Autores

Terrence G. Gardner, Kelly P. Kearse,

Tópico(s)

Immune Cell Function and Interaction

Resumo

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing α, β, γ, δ, ε, and ζ molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3αβ chains and formation of CD3-associated, disulfide-linked αβ heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of αβδεγε complexes. Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing α, β, γ, δ, ε, and ζ molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3αβ chains and formation of CD3-associated, disulfide-linked αβ heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of αβδεγε complexes. The antigen receptor expressed on most T lymphocytes is the multisubunit αβ T cell receptor complex (TCR), 1The abbreviations used are: TCR, T cell antigen receptor; ER, endoplasmic reticulum; GT, UDP-glucose:glycoprotein glucosyltransferase; mAb, monoclonal antibody; dmj, deoxymannojirimycin; chx, cycloheximide; PAGE, polyacrylamide gel electrophoresis; EH, endoglycosidase H important for recognition of major histocompatibility complex molecules containing bound peptides (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). The αβTCR is composed of six distinct proteins: clonotypic TCRα and -β molecules and invariant CD3γ, -δ, -ε, and -ζ chains (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). TCR assembly is initiated in the endoplasmic reticulum (ER) and occurs via the ordered pairing of: (i) CD3γ, -δ, and -ε chains into partial complexes of δε and γε components; (ii) association of clonotypic proteins with CD3 chains to form αδε and βγε intermediate complexes; (iii) joining of αδε and βγε molecules to create incomplete αβδεγε complexes, within which disulfide linkage of α and β chains occurs; and finally, (iv) addition of ζζ homodimers to form complete αβδεγεζζ complexes (2Exley M Terhorst C. Wileman T. Semin. 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 most T cell types, intracellular transport and expression of TCR proteins is tightly regulated by their assembly status. Unassembled and partially assembled TCR proteins are retained within the ER and disposed of by poorly understood mechanisms involving retrograde transport to the cytosol and degradation by proteasomes (4Huppa J.B. Ploegh H.L. Immunity. 1997; 7: 113-122Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 5Yu H. Kaung G. Kobayashi S. Kopito R.R. J. Biol. Chem. 1997; 272: 20800-20804Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 6Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Incomplete (αβδεγε) and complete (αβδεγεζζ) TCR complexes egress from the ER to the Golgi; however, incomplete TCR complexes are sorted to lysosomes where they are degraded. Only complete TCR complexes efficiently traffic to the cell surface (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). Four TCR subunits are post-translationally modified by addition of oligosaccharides TCRα (3 N-glycans), TCRβ (4N-glycans), CD3δ (3 N-glycans), and CD3γ (1N-glycan) (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). N-Glycan chains on newly translated proteins have the structure Glc3Man9GlcNAc2 and are sequentially processed by glucosidase I and II ER enzymes to form monoglucosylated Glc1Man9GlcNAc2 species, important for interaction with the endogenous lectins calnexin and calreticulin that function in the quality control system of protein folding (7Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-634Crossref PubMed Scopus (3779) Google Scholar, 8Ware F.E. Vassilakos A. Peterson P.A. Jackson M.A. Lehrman M.A. Williams D.B. J. Biol. Chem. 1995; 270: 4697-4704Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 10Spiro R.G. Zhu Q. Bhoyroo V. Soling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar); the final, innermost Glc residue is removed by glucosidase II (gII) before or after chaperone disassembly. Fully trimmed (Glc0) proteins that persist in a malfolded state are modified by UDP-glucose:glycoprotein glucosyltransferase (GT), which transfers a single Glc residue, (re)creating monoglucosylated (Glc1) species that can (re)enter the calnexin, calreticulin assembly pathway (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). GT is proposed to be a major sensor of protein folding in the ER (11Gannan S. Cazzulo J.J. Parodi A.J. Biochemistry. 1991; 30: 3098-3104Crossref PubMed Scopus (52) Google Scholar, 12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 13Fernandez F.S. Trombetta S.E. Hellman U. Parodi A.J. J. Biol. Chem. 1994; 269: 30701-30706Abstract Full Text PDF PubMed Google Scholar) and will only add back Glc residues removed by gII if a glycoprotein has not yet acquired its proper tertiary structure (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). The deglucosylation/reglucosylation cycle continues until correct conformation is achieved (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). GT modification of incompletely folded proteins involves interaction with both polypeptide and glycan determinants, including recognition of hydrophobic amino acids and interestingly, the innermost GlcNAc residue of the glycan chain (the site of attachment of oligosaccharide to protein) (15Sousa M.C. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (242) Google Scholar). Both recognition elements must be covalently linked to effectively catalyze Glc transfer (15Sousa M.C. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (242) Google Scholar) and accessible to GT modification, which for certain glycoproteins may be concealed by molecular chaperone association in vivo, particularly under conditions of extreme ER stress (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). The size of the glycan chain,e.g. the oligomannose core, is also important for the efficiency of reglucosylation; Man8–9GlcNAc2glycans are reglucosylated much more effectively than shorter Man5–7GlcNAc2 glycans (12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). As recently demonstrated in mutant BW cell types synthesizing truncated Glc3Man5GlcNAc2 N-glycans, TCRα molecules having shortened oligosaccharides were reglucosylated much less efficiently than TCRα molecules having normal size glycans, which was correlated with TCRα instability (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To evaluate the folding status of TCR glycoproteins as a function of their assembly into multisubunit complexes in the ER, we studied the GT modification of TCR proteins in 2B4 T hybridoma cells. These studies show that all TCR subunits bearing N-glycan chains were modified by GT and that TCR proteins were differentially reglucosylated during their assembly into multisubunit complexes. Furthermore, these data demonstrate that reglucosylation of most TCR subunits was extinguished following CD3αβ assembly and formation of CD3-associated disulfide-linked αβ heterodimers, indicating that TCR folding is finalized convergent with formation of αβδεγε complexes. 2B4 T hybridoma cells and the TCRβ-deficient 2B4 variant 21.2.2 were maintained by weekly passage in RPMI 1640 medium containing 5% fetal calf serum at 37 °C in 5% CO2 (17Hedrick S.M. Matis L.A. Hecht T.T. Samelson L.E. Longo D.L. Heber-Katz E. Schwartz R.H. Cell. 1982; 30: 141-152Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 18Samelson L.E. Germain R.M. Schwartz R.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6972-6976Crossref PubMed Scopus (230) Google Scholar, 19Sussman J.J. Saito T. Shevach E.M. Germain R.N. Ashwell J.D. J. Immunol. 1988; 140: 2520-2526PubMed Google Scholar). The following monoclonal antibodies (mAb) were used in this study: H57-597, specific for TCRβ proteins (20Kubo R.T. Born W. Kappler J.W. Marrack P. Pigeon M. J. Immunol. 1989; 142: 2736-2743PubMed Google Scholar); A2B4 specific for 2B4 TCRα proteins (18Samelson L.E. Germain R.M. Schwartz R.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6972-6976Crossref PubMed Scopus (230) Google Scholar); 145-2C11 specific for CD3γε, δε molecules (21Leo O. Foo M. Sachs D.J. Samelson L.E. Bluestone J.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1374-1378Crossref PubMed Scopus (1704) Google Scholar); HMT3.2, which recognizes both murine CD3γ and CD3δ proteins (22Dietrich J. Neisig A. Hou X. Wegener A.M.K. Gajhede M. Geisler C. J. Cell Biol. 1996; 132: 299-310Crossref PubMed Scopus (66) Google Scholar); the following antiserum was used: R9, specific for CD3δ molecules (23Samelson L.E. Weissman A.M. Robey F.A. Berkower I. Klausner R.D. J. Immunol. 1986; 137: 3254-3258PubMed Google Scholar). Deoxymannojirimycin (dmj) was purchased from Roche Molecular Biochemicals and was used at a final concentration of 75 μg/ml. Metabolic pulse-labeling with [3H]galactose was performed as described previously (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Briefly, cells were incubated in glucose-free RPMI 1640 medium (Life Technologies, Inc.) containing 10% dialyzed fetal calf serum, 5 mm sodium pyruvate (Life Technologies, Inc.), and 1 mm cycloheximide (chx) for 3 min at 37 °C in 5% CO2; cells were centrifuged and resuspended in similar medium containing 0.5 mCi/ml ([6-3H]galactose) (ICN, Irvine, CA) and labeled for 15–45 min at 37 °C in 5% CO2. Effectiveness of chx treatment in blocking new protein synthesis was verified by parallel experiments using [35S]methionine (data not shown). In experiments using dmj, cells were cultured overnight in medium containing 75 μg/ml dmj at 37 °C in 5% CO2; cell viability was identical in medium- and dmj-treated cultures (data not shown). Biotinylation of cell surface proteins was performed as described previously (24Wu W. Harley P.H. Punt J. Sharrow S.O. Kearse K.P. J. Exp. Med. 1996; 184: 759-794Crossref PubMed Scopus (51) Google Scholar). Cells were solubilized in 1% digitonin (Wako, Kyoto, Japan) lysis buffer (20 mm Tris, 150 mmNaCl, plus protease inhibitors) at 1 × 108 cells/ml for 20 min at 4 °C. Cell lysates were clarified by centrifugation to remove insoluble material and immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads as described previously (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Sequential immunoprecipitation, one- and two-dimensional SDS-PAGE gel electrophoresis, and immunoblotting were performed according to previously published methods (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Kearse 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). Recently we examined the reglucosylation of unassembled TCRα and -β proteins in BW thymoma cells using [3H]galactose as a radioactive tracer of Glc residues (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here we extended these studies in 2B4 T hybridoma cells to approximate at which stage(s) of TCR complex formation folding of individual TCR glycoproteins is completed, with the rationale that GT modification (reglucosylation) will cease upon attainment of proper conformation. As shown in Fig.1, [3H]galactose may be incorporated into N-linked oligosaccharides on glycoproteins via three major pathways: (i) conversion into UDP-[3H]galactose, the sugar donor for galactosyltransferase enzymes that transfer galactose residues to mature, complex-type oligosaccharides in the trans-Golgi; (ii) epimerization of UDP-[3H]galactose to UDP-[3H]glucose, the sugar donor for GT that transfers Glc residues to high mannose glycans on incompletely folded glycoproteins in the ER; and (iii) conversion of UDP-[3H]glucose into dolichol-phospho[3H]glucose, which is incorporated into nascent Glc3Man9GlcNAc2 glycans that are cotranslationally added to newly synthesized polypeptides in the ER (Fig. 1) (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hubbard S.C. Robbins P.W. J. Biol. Chem. 1979; 254: 4568-4576Abstract Full Text PDF PubMed Google Scholar, 27Suh K. Bergmann J.E. Gabel C.A. J. Cell Biol. 1989; 108: 811-819Crossref PubMed Scopus (57) Google Scholar, 28Varki A. Methods Enzymol. 1994; 230: 16-32Crossref PubMed Scopus (56) Google Scholar). In the current study, cycloheximide was included in all experiments to inhibit incorporation of [3H]glucose into newly translated proteins, thereby restricting radiolabeling to galactosylation and reglucosylation routes (Fig. 1) (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 2B4 T hybridoma cells were used, which have served as a model cell type for TCR assembly in numerous studies (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar). As shown in Fig. 2 A, multiple TCR subunits were detected in anti-CD3ε precipitates of [3H]galactose-labeled 2B4 T cells, including CD3δ and -γ glycoproteins and clonotypic TCRα and -β proteins (Fig.2 A); as expected, nonglycosylated CD3ε and TCRζ molecules were not visualized (Fig. 2 A). Because anti-CD3ε precipitates contain a mixture of TCR components at various stages of their assembly superimposed upon one another, sequential precipitation techniques were used to separate more completely assembled TCR proteins (capable of becoming galactosylated in the Golgi) from partially assembled and unassembled TCR subunits (retained in the ER) (25Kearse 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 demonstrated, when supernatants from anti-CD3ε precipitates were sequentially precipitated with anti-TCRβ mAb, radiolabeled TCRβ proteins were detected (Fig. 2 A), representing unassembled TCRβ proteins modified via the reglucosylation pathway. Consistent with incorporation of [3H]glucose into glycan chains on ER-localized TCRβ proteins, the radioactive signal on reglucosylated TCRβ proteins was sensitive to digestion with endoglycosidase H (EH), specific for immature oligosaccharides (data not shown). The vast majority of radiolabeled CD3γ glycoproteins associated with CD3ε were not simultaneously assembled with TCRβ but existed in partial complexes of CD3γε components (Fig. 2 A, anti-TCRβ → anti-CD3ε precipitates, respectively), indicating that most radiolabeled CD3γ chains associated with CD3ε were modified by GT. More than half of the CD3ε-associated CD3δ chains were assembled with TCRβ (Fig. 2 A), with remaining CD3δ proteins existing in partial δε complexes containing reglucosylatedN-glycans; unassembled CD3δ glycoproteins were also modified by GT as shown by sequential immunoprecipitation of anti-CD3ε precipitates with anti-CD3δ Ab to capture “free,” unassembled CD3δ chains (Fig. 2 A). Reglucosylation of partially assembled and free CD3γ and -δ glycoproteins was verified in 21.2.2 cells (Fig. 2 B), a TCRβ-deficient 2B4 variant that cannot assemble CD3 chains into a form capable of ER exit (29Bonifacino J.S. Chen C. Lippincott-Schwartz J. Ashwell J.D. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 35: 6929-6933Crossref Scopus (62) Google Scholar). Similar to our results in parental 2B4 cells, significantly more radiolabeled CD3γ proteins were associated with CD3ε than CD3δ proteins in 21.2.2 cells (Fig. 2 B). In agreement with previous studies showing that nascent glycoproteins undergo multiple cycles of deglucosylation and reglucosylation in the ER (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), Glc residues did not persist on CD3ε-associated γ proteins in 21.2.2 cells (Fig. 2 C), and CD3γ proteins were effectively radiolabeled during a secondary pulse period with [3H]galactose (Fig. 2 C). Taken together, these results demonstrate that glycosylated TCR subunits were substrates for GT, including invariant CD3γ and -δ molecules and clonotypic TCRβ proteins. In addition, these data show that both “free” and assembled CD3γ and -δ proteins contained reglucosylated glycans. To determine the contribution of reglucosylation in [3H]galactose radiolabeling of TCR proteins, studies were performed using the mannosidase inhibitor dmj, which precludes conversion of immature, high mannose glycans to mature (galactosylated) glycans in the Golgi (30Elbein A.D. Annu. Rev. Biochem. 1987; 56: 497-534Crossref PubMed Google Scholar); thus, only reglucosylated glycoproteins are visualized in such experiments (Fig. 1). As shown in Fig.3 A, markedly fewer radiolabeled TCR proteins were present in anti-TCRα (A2B4) immunoprecipitates of dmj-treated cells relative to media-treated cells with TCRβ and CD3γ and -δ chains being completely absent and only TCRα proteins detected (Fig. 3 A). Consistent with restriction of radiolabeling to immature N-glycans, TCRα proteins in dmj lysates migrated with increased mobility compared with TCRα proteins from control lysates (Fig. 3 A) and unlike control TCRα molecules, disappeared completely following EH digestion (Fig. 3 A). Note that increased mobility of EH-digested TCRα-associated TCRβ proteins in control lysates results from the fact that several N-glycans on TCRβ proteins remain in the immature high mannose form, even on surface-expressed molecules, which is also true for CD3δ proteins (31, 32 and see below). Interestingly, increased amounts of reglucosylated (unassembled) TCRβ proteins existed in dmj lysates relative to control lysates (Fig.3 A), which was accompanied by augmented survival of newly synthesized TCRβ proteins. 2K. P. Kearse, unpublished observations. These results were specific in that the half-life and reglucosylation of unassembled TCRα molecules were relatively unaffected; similar results were observed in BW thymoma cells (data not shown). Biochemical analysis of surface-labeled molecules showed that the vast majority of TCR glycoproteins expressed on dmj-treated cells contained immature, EH-sensitive oligosaccharides (Fig. 3 B), demonstrating the effectiveness of dmj in blocking maturation ofN-oligosaccharides in these studies and showing that dmj treatment did not perturb TCR assembly. Taken together, these results show that most radiolabeled TCRβ and CD3γ and -δ glycoproteins associated with 2B4 TCRα represent galactosylated species and not reglucosylated TCR molecules. We conclude that TCRβ proteins assembled into TCRα and -β heterodimers are ineffectively modified by GT (and thus no longer perceived by the ER quality control system as incompletely folded) and relatedly, that reglucosylation of TCRβ and CD3γ and -δ proteins is terminated following their association with TCRα molecules. In addition, these data show that inhibition of mannosidase activity resulted in enhanced reglucosylation of unassembled TCRβ proteins. Assembly of TCRα and -β proteins into disulfide-linked heterodimers is preceded by the association of monomeric TCRα and TCRβ proteins with CD3 components in the form of αδε and βγε intermediates, which join to form αβδεγε complexes (3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar). Thus, we next wished to determine whether CD3-associated TCRα and -β proteins were modified by GT. As shown in Fig. 4, both monomeric and dimeric radiolabeled TCRα and -β proteins were detected in association with CD3 chains in lysates of [3H]galactose-labeled 2B4 T cells (Fig. 4). Because monomeric TCRα and -β proteins are restricted to the ER (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar, 3Kearse K.P. Roberts J.L. Singer A. Immunity. 1995; 2: 391-399Abstract Full Text PDF PubMed Scopus (69) Google Scholar), these data indicate that reglucosylated (incompletely folded) TCRα and -β proteins exist in association with CD3 in 2B4 T cells, most likely as CD3α and CD3β intermediates because our previous results showed that few, if any, reglucosylated TCRβ proteins were associated with TCRα chains. To determine the contribution of GT modification to [3H]galactose radiolabeling of CD3-associated TCRα and -β proteins, dmj treatment was utilized to restrict radiolabeling to the reglucosylation pathway as before. In agreement with our previous findings that CD3γ and -δ glycoproteins assembled into partial δε, γε complexes were modified by GT, CD3γ and -δ chains were effectively labeled in dmj-treated cells (Fig.5 A). As noted earlier, reglucosylation of unassembled TCRβ chains, captured in sequential precipitates with anti-TCRβ mAb, was enhanced in dmj-treated cells relative to media-treated cells (Fig. 5 A). Importantly, these data show that reduced amounts of TCRα and -β proteins were associated with CD3 chains in dmj-treated cells compared with media-treated cells (Fig. 5 A), which was expected as our previous results showed that a significant portion of radiolabeled TCRα and -β proteins were assembled into disulfide-linked heterodimers modified by galactosylation. Analysis on two-dimensional nonreducing × reducing (NR × R) gels showed that relatively few radiolabeled TCRα and -β dimers were detected in anti-CD3 precipitates of dmj-treated cells (Fig. 5 B), unlike CD3 chains, which were readily visible (Fig. 5 B). Interestingly, radiolabeled TCRα proteins existed as both disulfide-linked and monomeric species whereas the vast majority of TCRβ molecules were present as non-disulfide-linked monomers (Fig. 5 B). Formation of TCRα and -β heterodimers was not precluded in dmj-treated cells as shown by immunoblotting of CD3 precipitates with anti-TCRα mAb (Fig. 6); dimeric TCRα proteins in media-treated groups existed as two species: an upper band representing mature (EH-resistant) proteins and a lower band migrating parallel with non-disulfide-linked TCRα monomers containing immature (EH-sensitive) glycans (Fig. 6). As demonstrated, only immature TCRα proteins were present in lysates of dmj-treated cells (Fig. 6). These results corroborate our previous findings that TCRβ subunits associated with TCRα proteins were ineffectively modified by GT and that reglucosylated (incompletely folded) CD3-associated monomeric TCRα and -β proteins exist in normal (untreated) cells. Taken together, these studies demonstrate that reglucosylation of most TCR components is extinguished following the CD3α and -β assembly and formation of disulfide-linked TCRα and -β heterodimers, indicating that TCR folding is finalized convergent with formation of αβδεγε complexes.Figure 5Reglucosylated TCR α and -β proteins are assembled with CD3 components in 2B4 T cells. A, 2B4 T cells were cultured in medium or dmj as indicated and labeled with [3H]galactose for 45 min. Digitonin lysates of equivalent numbers of cells were sequentially immunoprecipitated with 145-2C11 anti-CD3ε mAb, followed by H57-597 anti-TCRβ mAb. The positions of TCR proteins are marked.B, anti-CD3ε precipitates of dmj-treated [3H]galactose-labeled 2B4 cells shown in Awere analyzed on two-dimensional nonreducing x reducing (NR × R) gels. The positions of TCR proteins are marked.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Dimerization of TCR α proteins in media- and dmj-treated 2B4 T cells. Aliquots of the same cells used for [3H]galactose labeling in Fig. 5were lysed in digitonin and immunoprecipitated with 145-2C11 anti-CD3ε mAb. Lysates of equivalent numbers of cells were analyzed in media and dmj groups. Precipitates were analyzed on two-dimensional NR × R gels or digested with Endo H and analyzed on one-dimensional gels under reducing conditions and immunoblotted with anti-TCRα mAb. The positions of immature (EH-sensitive) and mature (EH-resistant) TCRα proteins are indicated; αD, dimeric α proteins; αM, monomeric α proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The current report has examined the modification of TCR glycoproteins by the ER folding sensor enzyme GT and provides the first example where GT modification of a multisubunit protein complex has been studied. The data in the current report significantly extend previous studies on TCR processing in splenic T lymphocytes, which showed that significant Glc trimming of newly synthesized CD3δ and TCRα glycoproteins takes place prior to association with partner TCR chains (33Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1996; 271: 9660-9665Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), based on their comigration with calnexin-associated glycoforms following digestion with jack bean α-mannosidase. Indeed, the current study utilizes a sensitive radiolabeling method which specifically identifies TCR subunits containing monoglucosylatedN-glycans generated via the reglucosylation pathway. The current report establishes that all TCR subunits containingN-glycans are substrates for GT and evaluated reglucosylation as a function of TCR assembly, previously examined only on unassembled TCRα and -β proteins expressed in BW thymoma cells, which do not efficiently assemble TCR complexes due to deficient CD3δ synthesis (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The results in this study suggest a scheme in which reglucosylation of invariant CD3γ and -δ subunits persists until their association with clonotypic TCRα and -β chains, and GT modification of TCRβ proteins is terminated following the assembly of αβδεγε complexes and formation of disulfide-linked TCRα and -β heterodimers. Interestingly, unlike CD3-associated TCRβ molecules, which were ineffectively modified by GT following disulfide linkage with TCRα proteins, reglucosylated TCRα molecules existed as both CD3-associated monomers and dimers. Although the exact significance of these findings remain to be determined, these data suggest that folding of TCRα may be one of the final steps of ER quality control that precedes TCR egress to the Golgi. It is conceivable that folding of the TCR complex occurs concomitant with the ordered assembly of TCR subunits and that GT recognition motifs become progressively “masked” as TCR oligomerization proceeds, similar to ER retention and lysosomal targeting information contained within the polypeptide sequences of certain TCR subunits (1Klausner R.D. Lippincott-Schwartz J. Bonifacino J.S. Annu. Rev. Cell Biol. 1990; 54: 403-431Crossref Scopus (300) Google Scholar, 34Bonifacino J.S. Cosson P. Klausner R.D. Cell. 1990; 63: 503-513Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 35Letourner F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 36Dietrich J. Geisler C. J. Biol. Chem. 1998; 273: 26281-26284Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Consistent with this idea, the results in the current study provide evidence that most TCR glycoproteins are no longer perceived by the ER quality control system as incompletely folded following the assembly of αβδεγε TCR complexes, which, interestingly, is the stage at which TCR complexes become competent for ER exit. It is possible that reglucosylation of higher ordered TCR complexes ceases due to relocalization from the ER to the Golgi complex; however, we favor the idea that assembly, folding, and intracellular transport of TCR proteins are closely coupled events, similar to what has been observed for other multimeric immune protein complexes, i.e. major histocompatibility complex molecules (37Cresswell P. Cell. 1996; 84: 505-507Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 38Solheim J. Carreno B.M. Hansen T. J. Immunol. 1997; 158: 541-543PubMed Google Scholar, 39Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (874) Google Scholar). Indeed, previous studies have demonstrated that protein reglucosylation is not static but proceeds in a rapid, cyclic fashion in concert with Glc removal by glucosidase II enzymes (9Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar,16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Interestingly, we found that reglucosylation of “free” TCRβ proteins was increased under conditions of mannosidase blockade, which was specific in that GT modification of TCRα proteins was relatively unaffected.2 Because the efficiency of GT modification is inversely correlated with N-glycan chain length (12Sousa M.C. Ferrero-Garcia M.A. Parodi A.J. Biochemistry. 1992; 31: 97-105Crossref PubMed Scopus (269) Google Scholar, 16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and dmj inhibits the activity of certain ER mannosidase enzymes (40Weng S. Spiro R.G. J. Biol. Chem. 1993; 268: 25656-25663Abstract Full Text PDF PubMed Google Scholar,41Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar), it is reasonable that increased reglucosylation of unassembled TCRβ proteins in dmj-treated cells results from persistence of Man residues on N-glycan chains. However, it was also noted that the stability of newly synthesized TCRβ molecules was enhanced under these conditions, similar to what has been described for CD3δ proteins by Weissman and colleagues (6Yang M. Omura S. Bonifacino J.S. Weissman A. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Thus, the relationship between increased reglucosylation and increased survival of TCR proteins under conditions of mannosidase blockade remains to be determined. Moreover, despite the fact that GT modification of certain TCR subunits was enhanced by prevention of Man removal, relatively few reglucosylated TCR proteins assembled into higher ordered TCR complexes were detected under these conditions, indicating that GT modification (folding) of TCR subunits is tightly regulated. Finally, it is unknown to what extent specific N-glycans on TCR proteins containing multiple oligosaccharides may be differentially modified by GT enzymes. Recent studies by Dessen et al. (42Dessen A. Gupta D. Sabewsan S. Brewer C.F. Sacchettini J.C. Biochemistry. 1995; 34: 4933-4942Crossref PubMed Scopus (152) Google Scholar) demonstrate that N-acetylglucosamine residues interact with neighboring amino acids of proteins in native conformations, which may be one of the major mechanisms by which GT modification of newly synthesized proteins is regulated (14Fernandez F. Alessio C.D. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar, 43Fanchiotti S. Fernandez F. Alessio C.D. Parodi A.J. J. Cell Biol. 1998; 143: 625-631Crossref PubMed Scopus (72) Google Scholar). The data in the current report suggest that determinants that signify malfolded molecules may persist on TCRα proteins compared with other TCR subunits, an idea that is consistent with previous findings that TCRα survival is uniquely sensitive to perturbations in the ER quality control system (16Van Leeuwen J.E.M. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Suh K. Bergmann J.E. Gabel C.A. J. Cell Biol. 1989; 108: 811-819Crossref PubMed Scopus (57) Google Scholar, 44Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar). Identification of polypeptide and N-glycan domains important for GT recognition of TCR glycoproteins should provide valuable information regarding the molecular basis of GT modification and the regulation of quality control mechanisms that monitor the presence of unassembled and incompletely folded TCR proteins in the ER. We thank Drs. Velislava Karaivanova and Tom McConnell for critical reading of the manuscript. We are also grateful to Dr. Ralph Kubo for the gift of HMT3.2 Ab and Dr. Larry Samelson for the gift of R9 Ab.

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