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

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 ␣, ␤, ␥, ␦, ⑀, and 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 CD3␣␤ chains and formation of CD3-associated, disulfide-linked ␣␤ 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 ␣␤␦⑀␥⑀ complexes.
The antigen receptor expressed on most T lymphocytes is the multisubunit ␣␤ T cell receptor complex (TCR), 1 important for recognition of major histocompatibility complex molecules containing bound peptides (1). The ␣␤TCR is composed of six distinct proteins: clonotypic TCR␣ and -␤ molecules and invariant CD3␥, -␦, -⑀, and -chains (1). TCR assembly is initiated in the endoplasmic reticulum (ER) and occurs via the ordered pairing of: (i) CD3␥, -␦, and -⑀ chains into partial complexes of ␦⑀ and ␥⑀ components; (ii) association of clonotypic proteins with CD3 chains to form ␣␦⑀ and ␤␥⑀ intermediate complexes; (iii) joining of ␣␦⑀ and ␤␥⑀ molecules to create incomplete ␣␤␦⑀␥⑀ complexes, within which disulfide linkage of ␣ and ␤ chains occurs; and finally, (iv) addition of homodimers to form complete ␣␤␦⑀␥⑀ complexes (2,3). 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 (4 -6). Incomplete (␣␤␦⑀␥⑀) and complete (␣␤␦⑀␥⑀) 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 (1).
Four TCR subunits are post-translationally modified by addition of oligosaccharides TCR␣ (3 N-glycans), TCR␤ (4 Nglycans), CD3␦ (3 N-glycans), and CD3␥ (1 N-glycan) (1). N-Glycan chains on newly translated proteins have the structure Glc 3 Man 9 GlcNAc 2 and are sequentially processed by glucosidase I and II ER enzymes to form monoglucosylated Glc 1 Man 9 -GlcNAc 2 species, important for interaction with the endogenous lectins calnexin and calreticulin that function in the quality control system of protein folding (7-10); the final, innermost Glc residue is removed by glucosidase II (gII) before or after chaperone disassembly. Fully trimmed (Glc 0 ) proteins that persist in a malfolded state are modified by UDP-glucose:glycoprotein glucosyltransferase (GT), which transfers a single Glc residue, (re)creating monoglucosylated (Glc 1 ) species that can (re)enter the calnexin, calreticulin assembly pathway (9). GT is proposed to be a major sensor of protein folding in the ER (11)(12)(13) and will only add back Glc residues removed by gII if a glycoprotein has not yet acquired its proper tertiary structure (14). The deglucosylation/reglucosylation cycle continues until correct conformation is achieved (9,14).
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) (15). Both recognition elements must be covalently linked to effectively catalyze Glc transfer (15) 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 (14). The size of the glycan chain, e.g. the oligomannose core, is also important for the efficiency of reglucosylation; Man 8 -9 GlcNAc 2 glycans are reglucosylated much more effectively than shorter Man 5-7 GlcNAc 2 glycans (12,16). As recently demonstrated in mutant BW cell types synthesizing truncated Glc 3 Man 5 GlcNAc 2 N-glycans, TCR␣ molecules having shortened oligosaccharides were reglucosylated much less efficiently than TCR␣ molecules having normal size glycans, which was correlated with TCR␣ instability (16).
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 CD3␣␤ assembly and formation of CD3associated disulfide-linked ␣␤ heterodimers, indicating that TCR folding is finalized convergent with formation of ␣␤␦⑀␥⑀ complexes.
[ 3 H]Galactose Labeling and Biotinylation of Proteins-Metabolic pulse-labeling with [ 3 H]galactose was performed as described previously (16). 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% CO 2 ; cells were centrifuged and resuspended in similar medium containing 0.5 mCi/ml ([6-3 H]galactose) (ICN, Irvine, CA) and labeled for 15-45 min at 37°C in 5% CO 2 . Effectiveness of chx treatment in blocking new protein synthesis was verified by parallel experiments using [ 35 S]methionine (data not shown). In experiments using dmj, cells were cultured overnight in medium containing 75 g/ml dmj at 37°C in 5% CO 2 ; cell viability was identical in medium-and dmj-treated cultures (data not shown). Biotinylation of cell surface proteins was performed as described previously (24).
Cell Lysis, Immunoprecipitation, Gel Electrophoresis, and Immunoblotting-Cells were solubilized in 1% digitonin (Wako, Kyoto, Japan) lysis buffer (20 mM Tris, 150 mM NaCl, plus protease inhibitors) at 1 ϫ 10 8 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 (16). Sequential immunoprecipitation, one-and two-dimensional SDS-PAGE gel electrophoresis, and immunoblotting were performed according to previously published methods (16,25).

RESULTS
Recently we examined the reglucosylation of unassembled TCR␣ and -␤ proteins in BW thymoma cells using [ 3 H]galactose as a radioactive tracer of Glc residues (16). 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, [ 3 H]galactose may be incorporated into N-linked oligosaccharides on glycoproteins via three major pathways: (i) conversion into UDP-[ 3 H]galactose, the sugar donor for galactosyltransferase enzymes that transfer galactose residues to mature, complex-type oligosaccharides in the trans-Golgi; (ii) epimerization of UDP-[ 3 H]galactose to UDP-[ 3 H]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-[ 3 H]glucose into dolichol-phospho[ 3 H]glucose, which is incorporated into nascent Glc 3 Man 9 GlcNAc 2 glycans that are cotranslationally added to newly synthesized polypeptides in the ER ( Fig. 1) (16, 26 -28). In the current study, cycloheximide was included in all experiments to inhibit incorporation of [ 3 H]glucose into newly translated proteins, thereby restricting radiolabeling to galactosylation and reglucosylation routes ( Fig. 1) (16). 2B4 T hybridoma cells were used, which have served as a model cell type for TCR assembly in numerous studies (1). Fig. 2A, multiple TCR subunits were detected in anti-CD3⑀ precipitates of [ 3 H]galactose-labeled 2B4 T cells, including CD3␦ and -␥ glycoproteins and clonotypic TCR␣ and -␤ proteins ( Fig. 2A); as expected, nonglycosylated CD3⑀ and TCR molecules were not visualized ( Fig. 2A). Because anti-CD3⑀ 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) (25). As demonstrated, when supernatants from anti-CD3⑀ precipitates were sequentially precipitated with anti-TCR␤ mAb, radiolabeled TCR␤ proteins were detected ( Fig. 2A), representing unassembled TCR␤ proteins modified via the reglucosylation pathway. Consistent with incorporation of [ 3 H]glucose into glycan chains on ER-localized TCR␤ proteins, the radioactive signal on reglucosylated TCR␤ proteins was sensitive to digestion with endoglycosidase H (EH), specific for immature oligosaccharides (data not shown). The vast majority of radiolabeled CD3␥ glycoproteins associated with CD3⑀ were not simultaneously assembled with TCR␤ but existed in partial complexes of CD3␥⑀ components ( Fig. 2A, anti-TCR␤ 3 anti-CD3⑀ precipitates, respectively), indicating that most radiolabeled CD3␥ chains associated with CD3⑀ were modified by GT. More than half of the CD3⑀-associated CD3␦ chains were assembled with TCR␤ ( Fig. 2A), with remaining CD3␦ proteins existing in partial ␦⑀ complexes containing reglucosylated N-glycans; unassembled CD3␦ glycoproteins were also modified by GT as shown by sequential immunoprecipitation of anti-CD3⑀ precipitates with anti-CD3␦ Ab to capture "free," unassembled CD3␦ chains ( Fig. 2A). Reglucosylation of partially assembled and free CD3␥ and -␦ glycoproteins was verified in 21.2.2 cells (Fig.  2B), a TCR␤-deficient 2B4 variant that cannot assemble CD3 chains into a form capable of ER exit (29). Similar to our results in parental 2B4 cells, significantly more radiolabeled CD3␥ proteins were associated with CD3⑀ than CD3␦ proteins in 21.2.2 cells (Fig. 2B). In agreement with previous studies showing that nascent glycoproteins undergo multiple cycles of deglucosylation and reglucosylation in the ER (9, 16), Glc residues did not persist on CD3⑀-associated ␥ proteins in 21.2.2 cells (Fig. 2C), and CD3␥ proteins were effectively radiolabeled during a secondary pulse period with [ 3 H]galactose (Fig. 2C). Taken together, these results demonstrate that glycosylated TCR subunits were substrates for GT, including invariant CD3␥ and -␦ molecules and clonotypic TCR␤ proteins. In addition, these data show that both "free" and assembled CD3␥ and -␦ proteins contained reglucosylated glycans.

TCR Subunits Bearing N-Glycans Are Substrates for GT-As shown in
Reglucosylation and Disulfide Linkage of Clonotypic TCR␣ and -␤ Proteins-To determine the contribution of reglucosylation in [ 3 H]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 (30); thus, only reglucosylated glycoproteins are visualized in such experiments (Fig. 1). As shown in Fig. 3A, markedly fewer radiolabeled TCR proteins were present in anti-TCR␣ (A2B4) immunoprecipitates of dmj-treated cells relative to media-treated cells with TCR␤ and CD3␥ and -␦ chains being completely absent and only TCR␣ proteins detected (Fig. 3A). Consistent with restriction of radiolabeling to immature N-glycans, TCR␣ proteins in dmj lysates migrated with increased mobility compared with TCR␣ proteins from control lysates (Fig. 3A) and unlike control TCR␣ molecules, disappeared completely following EH digestion (Fig. 3A). Note that increased mobility of EH-digested TCR␣-associated TCR␤ proteins in control lysates results from the fact that several N-glycans on TCR␤ proteins remain in the immature high mannose form, even on surfaceexpressed molecules, which is also true for CD3␦ proteins (31, 32 and see below). Interestingly, increased amounts of reglucosylated (unassembled) TCR␤ proteins existed in dmj lysates relative to control lysates (Fig. 3A), which was accompanied by augmented survival of newly synthesized TCR␤ proteins. 2 These results were specific in that the half-life and reglucosylation of unassembled TCR␣ 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. 3B), demonstrating the effectiveness of dmj in blocking maturation of N-oligosaccharides in these studies and showing that dmj treatment did not perturb TCR assembly. Taken together, these results show that most radiolabeled TCR␤ and CD3␥ and -␦ glycoproteins associated with 2B4 TCR␣ represent galactosylated species and not reglucosylated TCR molecules. We conclude that TCR␤ proteins assembled into TCR␣ and -␤ 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 TCR␤ and CD3␥ and -␦ proteins is terminated following their association with TCR␣ molecules. In addition, these data show that inhibition of mannosidase activity resulted in enhanced reglucosylation of unassembled TCR␤ proteins.
Reglucosylated TCR␣ and -␤ Proteins Are Assembled with 2 K. P. Kearse, unpublished observations. CD3 Subunits-Assembly of TCR␣ and -␤ proteins into disulfide-linked heterodimers is preceded by the association of monomeric TCR␣ and TCR␤ proteins with CD3 components in the form of ␣␦⑀ and ␤␥⑀ intermediates, which join to form ␣␤␦⑀␥⑀ complexes (3). Thus, we next wished to determine whether CD3-associated TCR␣ and -␤ proteins were modified by GT. As shown in Fig. 4, both monomeric and dimeric radiolabeled TCR␣ and -␤ proteins were detected in association with CD3 chains in lysates of [ 3 H]galactose-labeled 2B4 T cells (Fig. 4). Because monomeric TCR␣ and -␤ proteins are restricted to the ER (1, 3), these data indicate that reglucosylated (incompletely folded) TCR␣ and -␤ proteins exist in association with CD3 in 2B4 T cells, most likely as CD3␣ and CD3␤ intermediates because our previous results showed that few, if any, reglucosylated TCR␤ proteins were associated with TCR␣ chains. To determine the contribution of GT modification to [ 3 H]galactose radiolabeling of CD3-associated TCR␣ and -␤ proteins, dmj treatment was utilized to restrict radiolabeling to the reglucosylation pathway as before. In agreement with our previous findings that CD3␥ and -␦ glycoproteins assembled into partial ␦⑀, ␥⑀ complexes were modified by GT, CD3␥ and -␦ chains were effectively labeled in dmj-treated cells (Fig. 5A). As noted earlier, reglucosylation of unassembled TCR␤ chains, captured in sequential precipitates with anti-TCR␤ mAb, was enhanced in dmj-treated cells relative to media-treated cells (Fig. 5A). Importantly, these data show that reduced amounts of TCR␣ and -␤ proteins were associated with CD3 chains in dmj-treated cells compared with media-treated cells (Fig. 5A), which was expected as our previous results showed that a significant portion of radiolabeled TCR␣ and -␤ proteins were assembled into disulfide-linked heterodimers modified by galactosylation. Analysis on two-dimensional nonreducing ϫ reducing (NR ϫ R) gels showed that relatively few radiolabeled TCR␣ and -␤ dimers were detected in anti-CD3 precipitates of dmj-treated cells (Fig. 5B), unlike CD3 chains, which were readily visible (Fig. 5B). Interestingly, radiolabeled TCR␣ proteins existed as both disulfide-linked and monomeric species whereas the vast majority of TCR␤ molecules were present as non-disulfidelinked monomers (Fig. 5B). Formation of TCR␣ and -␤ heterodimers was not precluded in dmj-treated cells as shown by immunoblotting of CD3 precipitates with anti-TCR␣ mAb (Fig.  6); dimeric TCR␣ 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 TCR␣ monomers containing immature (EH-sensitive) glycans (Fig. 6). As demonstrated, only immature TCR␣ proteins were present in lysates of dmj-treated cells (Fig. 6).
These results corroborate our previous findings that TCR␤ subunits associated with TCR␣ proteins were ineffectively modified by GT and that reglucosylated (incompletely folded) CD3-associated monomeric TCR␣ and -␤ proteins exist in normal (untreated) cells. Taken together, these studies demonstrate that reglucosylation of most TCR components is extinguished following the CD3␣ and -␤ assembly and formation of disulfide-linked TCR␣ and -␤ heterodimers, indicating that TCR folding is finalized convergent with formation of ␣␤␦⑀␥⑀ complexes.

DISCUSSION
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 CD3␦ and TCR␣ glycoproteins takes place prior to association with partner TCR chains (33), based on their comigration with calnexin-associated glycoforms following digestion with jack bean ␣-mannosidase. Indeed, the current study utilizes a sensitive radiolabeling method which specifically identifies TCR subunits containing monoglucosylated N-glycans generated via the reglucosylation pathway. The current report establishes that all TCR subunits containing N-glycans are substrates for GT and evaluated reglucosylation as a function of TCR assembly, previously examined only on unassembled TCR␣ and -␤ proteins expressed in BW thymoma cells, which do not efficiently assemble TCR complexes due to deficient CD3␦ synthesis (16).
The results in this study suggest a scheme in which reglucosylation of invariant CD3␥ and -␦ subunits persists until their association with clonotypic TCR␣ and -␤ chains, and GT modification of TCR␤ proteins is terminated following the assembly of ␣␤␦⑀␥⑀ complexes and formation of disulfide-linked TCR␣ and -␤ heterodimers.
Interestingly, unlike CD3-associated TCR␤ molecules, which were ineffectively modified by GT following disulfide linkage with TCR␣ proteins, reglucosylated TCR␣ 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 TCR␣ 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 FIG. 4

. Disulfide linkage status of CD3-associated TCR␣ and -␤ proteins in [ 3 H]galactose-labeled 2B4 T cells.
Digitonin lysates of 2B4 T cells labeled with [ 3 H]galactose for 45 min were sequentially immunoprecipitated with anti-CD3⑀ mAb (145-2C11), followed by anti-TCR␤ mAb (H57-597), and analyzed on 13% SDS-PAGE gels under nonreducing conditions or on two-dimensional nonreducing ϫ reducing (NR ϫ R) gels. The positions of TCR proteins are marked; TCR␣ D ␤ D , dimeric ␣␤ proteins; TCR␣ M and TCR␤ M , monomeric ␣ and ␤ proteins, respectively. Note that the spot below ␣ D in the two-dimensional NR ϫ R samples represents a smudge on the gel that is enhanced by photography and is not a radiolabeled protein.
TCR oligomerization proceeds, similar to ER retention and lysosomal targeting information contained within the polypeptide sequences of certain TCR subunits (1, 34 -36). 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 ␣␤␦⑀␥⑀ 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 (37)(38)(39). 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 (9,16).
Interestingly, we found that reglucosylation of "free" TCR␤ proteins was increased under conditions of mannosidase blockade, which was specific in that GT modification of TCR␣ proteins was relatively unaffected. 2 Because the efficiency of GT modification is inversely correlated with N-glycan chain length (12,16), and dmj inhibits the activity of certain ER mannosidase enzymes (40,41), it is reasonable that increased reglucosylation of unassembled TCR␤ 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 TCR␤ molecules was enhanced under these conditions, similar to what has been described for CD3␦ proteins by Weissman and colleagues (6). 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. (42) 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 (14,43). The data in the current report suggest that determinants that signify malfolded molecules may persist on TCR␣ proteins compared with other TCR subunits, an idea that is consistent with previous findings that TCR␣ survival is uniquely sensitive to perturbations in the ER quality control system (16,27,44). 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.