Modification of the T Cell Antigen Receptor (TCR) Complex by UDP-glucose:Glycoprotein Glucosyltransferase
TCR FOLDING IS FINALIZED CONVERGENT WITH FORMATION OF alpha beta delta epsilon gamma epsilon COMPLEXES*

Terrence G. Gardner and Kelly P. KearseDagger

From the Department of Microbiology and Immunology, East Carolina University, School of Medicine, Greenville, North Carolina 27858-4354

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha , beta , gamma , delta , epsilon , and zeta  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 CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta 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 alpha beta delta epsilon gamma epsilon complexes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The antigen receptor expressed on most T lymphocytes is the multisubunit alpha beta T cell receptor complex (TCR),1 important for recognition of major histocompatibility complex molecules containing bound peptides (1). The alpha beta TCR is composed of six distinct proteins: clonotypic TCRalpha and -beta molecules and invariant CD3gamma , -delta , -epsilon , and -zeta chains (1). TCR assembly is initiated in the endoplasmic reticulum (ER) and occurs via the ordered pairing of: (i) CD3gamma , -delta , and -epsilon chains into partial complexes of delta epsilon and gamma epsilon components; (ii) association of clonotypic proteins with CD3 chains to form alpha delta epsilon and beta gamma epsilon intermediate complexes; (iii) joining of alpha delta epsilon and beta gamma epsilon molecules to create incomplete alpha beta delta epsilon gamma epsilon complexes, within which disulfide linkage of alpha  and beta  chains occurs; and finally, (iv) addition of zeta zeta homodimers to form complete alpha beta delta epsilon gamma epsilon zeta zeta 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 (alpha beta delta epsilon gamma epsilon ) and complete (alpha beta delta epsilon gamma epsilon zeta zeta ) 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 TCRalpha (3 N-glycans), TCRbeta (4 N-glycans), CD3delta (3 N-glycans), and CD3gamma (1 N-glycan) (1). 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 (7-10); 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 (9). GT is proposed to be a major sensor of protein folding in the ER (11-13) 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; Man8-9GlcNAc2 glycans are reglucosylated much more effectively than shorter Man5-7GlcNAc2 glycans (12, 16). As recently demonstrated in mutant BW cell types synthesizing truncated Glc3Man5GlcNAc2 N-glycans, TCRalpha molecules having shortened oligosaccharides were reglucosylated much less efficiently than TCRalpha molecules having normal size glycans, which was correlated with TCRalpha 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 CD3alpha beta assembly and formation of CD3-associated disulfide-linked alpha beta heterodimers, indicating that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Reagents-- 2B4 T hybridoma cells and the TCRbeta -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 (17-19). The following monoclonal antibodies (mAb) were used in this study: H57-597, specific for TCRbeta proteins (20); A2B4 specific for 2B4 TCRalpha proteins (18); 145-2C11 specific for CD3gamma epsilon , delta epsilon molecules (21); HMT3.2, which recognizes both murine CD3gamma and CD3delta proteins (22); the following antiserum was used: R9, specific for CD3delta molecules (23). Deoxymannojirimycin (dmj) was purchased from Roche Molecular Biochemicals and was used at a final concentration of 75 µg/ml.

[3H]Galactose Labeling and Biotinylation of Proteins-- Metabolic pulse-labeling with [3H]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% 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 (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 × 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 (16). Sequential immunoprecipitation, one- and two-dimensional SDS-PAGE gel electrophoresis, and immunoblotting were performed according to previously published methods (16, 25).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recently we examined the reglucosylation of unassembled TCRalpha and -beta proteins in BW thymoma cells using [3H]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, [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) (16, 26-28). 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) (16). 2B4 T hybridoma cells were used, which have served as a model cell type for TCR assembly in numerous studies (1).


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Fig. 1.   Radiolabeling of glycoproteins with [3H]galactose. Diagram illustrating the routes of incorporation of [3H]galactose into glycoproteins containing N-linked oligosaccharides and sensitivity of labeling to chx and dmj. +, incorporation via this route; -, no incorporation via this route. See text for details.

TCR Subunits Bearing N-Glycans Are Substrates for GT-- As shown in Fig. 2A, multiple TCR subunits were detected in anti-CD3epsilon precipitates of [3H]galactose-labeled 2B4 T cells, including CD3delta and -gamma glycoproteins and clonotypic TCRalpha and -beta proteins (Fig. 2A); as expected, nonglycosylated CD3epsilon and TCRzeta molecules were not visualized (Fig. 2A). Because anti-CD3epsilon 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-CD3epsilon precipitates were sequentially precipitated with anti-TCRbeta mAb, radiolabeled TCRbeta proteins were detected (Fig. 2A), representing unassembled TCRbeta proteins modified via the reglucosylation pathway. Consistent with incorporation of [3H]glucose into glycan chains on ER-localized TCRbeta proteins, the radioactive signal on reglucosylated TCRbeta proteins was sensitive to digestion with endoglycosidase H (EH), specific for immature oligosaccharides (data not shown). The vast majority of radiolabeled CD3gamma glycoproteins associated with CD3epsilon were not simultaneously assembled with TCRbeta but existed in partial complexes of CD3gamma epsilon components (Fig. 2A, anti-TCRbeta right-arrow anti-CD3epsilon precipitates, respectively), indicating that most radiolabeled CD3gamma chains associated with CD3epsilon were modified by GT. More than half of the CD3epsilon -associated CD3delta chains were assembled with TCRbeta (Fig. 2A), with remaining CD3delta proteins existing in partial delta epsilon complexes containing reglucosylated N-glycans; unassembled CD3delta glycoproteins were also modified by GT as shown by sequential immunoprecipitation of anti-CD3epsilon precipitates with anti-CD3delta Ab to capture "free," unassembled CD3delta chains (Fig. 2A). Reglucosylation of partially assembled and free CD3gamma and -delta glycoproteins was verified in 21.2.2 cells (Fig. 2B), a TCRbeta -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 CD3gamma proteins were associated with CD3epsilon than CD3delta 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 CD3epsilon -associated gamma  proteins in 21.2.2 cells (Fig. 2C), and CD3gamma proteins were effectively radiolabeled during a secondary pulse period with [3H]galactose (Fig. 2C). Taken together, these results demonstrate that glycosylated TCR subunits were substrates for GT, including invariant CD3gamma and -delta molecules and clonotypic TCRbeta proteins. In addition, these data show that both "free" and assembled CD3gamma and -delta proteins contained reglucosylated glycans.


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Fig. 2.   Reglucosylation of TCR glycoproteins. A, digitonin lysates of 2B4 T cells labeled with [3H]galactose for 30 min were sequentially immunoprecipitated with anti-CD3epsilon mAb (145-2C11), followed by anti-TCRbeta mAb (H57-597); alternatively, lysates were sequentially precipitated with anti-TCRbeta mAb (H57-597), followed by anti-CD3epsilon mAb (145-2C11); or with anti-CD3epsilon mAb (145-2C11), followed by anti-CD3delta Ab (R9). B, 21.2.2 cells were radiolabeled with [3H]galactose for 30 min and solubilized in 1% Nonidet P-40, and lysates were sequentially immunoprecipitated with 145-2C11 anti-CD3epsilon mAb, followed by HMT3.2 anti-CD3gamma mAb, which also recognizes murine CD3delta molecules. C, 21.2.2 cells were radiolabeled with [3H]galactose for 15 min in the presence of chx, chased in medium without radiolabel in the presence of chx for 30 min, and then radiolabeled a second time with [3H]galactose for 15 min in the presence of cycloheximide. Nonidet P-40 lysates were precipitated with 145-2C11 mAb and analyzed as in B.

Reglucosylation and Disulfide Linkage of Clonotypic TCRalpha and -beta Proteins-- 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 (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-TCRalpha (A2B4) immunoprecipitates of dmj-treated cells relative to media-treated cells with TCRbeta and CD3gamma and -delta chains being completely absent and only TCRalpha proteins detected (Fig. 3A). Consistent with restriction of radiolabeling to immature N-glycans, TCRalpha proteins in dmj lysates migrated with increased mobility compared with TCRalpha proteins from control lysates (Fig. 3A) and unlike control TCRalpha molecules, disappeared completely following EH digestion (Fig. 3A). Note that increased mobility of EH-digested TCRalpha -associated TCRbeta proteins in control lysates results from the fact that several N-glycans on TCRbeta proteins remain in the immature high mannose form, even on surface-expressed molecules, which is also true for CD3delta proteins (31, 32 and see below). Interestingly, increased amounts of reglucosylated (unassembled) TCRbeta proteins existed in dmj lysates relative to control lysates (Fig. 3A), which was accompanied by augmented survival of newly synthesized TCRbeta proteins.2 These results were specific in that the half-life and reglucosylation of unassembled TCRalpha 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 TCRbeta and CD3gamma and -delta glycoproteins associated with 2B4 TCRalpha represent galactosylated species and not reglucosylated TCR molecules. We conclude that TCRbeta proteins assembled into TCRalpha and -beta 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 TCRbeta and CD3gamma and -delta proteins is terminated following their association with TCRalpha molecules. In addition, these data show that inhibition of mannosidase activity resulted in enhanced reglucosylation of unassembled TCRbeta proteins.


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Fig. 3.   GT modification of TCR glycoproteins associated with TCRalpha . A, 2B4 T cells were cultured in medium or deoxymannojirimycin (dmj) as indicated and labeled with [3H]galactose in the presence of cycloheximide for 45 min. The presence of dmj was maintained in appropriate groups throughout the entire experiment. Digitonin lysates of equivalent numbers of cells were sequentially immunoprecipitated with A2B4 anti-TCRalpha mAb, followed by H57-597 anti-TCRbeta mAb, and precipitates were treated -/+ EH glycosidase as indicated. The positions of TCR proteins are marked. B, media- and dmj-treated 2B4 T cells were surface-labeled by biotinylation and solubilized in 1% digitonin; digitonin lysates of equivalent numbers of cells were precipitated with 145-2C11 anti-CD3epsilon mAb and digested with EH as indicated. Precipitates were analyzed on one-dimensional 13% SDS-PAGE gels under reducing conditions, and labeled proteins were visualized by chemiluminescence. The positions of TCR proteins are marked. R subscript denotes Endo H-resistant proteins; S subscript denotes Endo H-sensitive proteins.

Reglucosylated TCRalpha and -beta Proteins Are Assembled with CD3 Subunits-- Assembly of TCRalpha and -beta proteins into disulfide-linked heterodimers is preceded by the association of monomeric TCRalpha and TCRbeta proteins with CD3 components in the form of alpha delta epsilon and beta gamma epsilon intermediates, which join to form alpha beta delta epsilon gamma epsilon complexes (3). Thus, we next wished to determine whether CD3-associated TCRalpha and -beta proteins were modified by GT. As shown in Fig. 4, both monomeric and dimeric radiolabeled TCRalpha and -beta proteins were detected in association with CD3 chains in lysates of [3H]galactose-labeled 2B4 T cells (Fig. 4). Because monomeric TCRalpha and -beta proteins are restricted to the ER (1, 3), these data indicate that reglucosylated (incompletely folded) TCRalpha and -beta proteins exist in association with CD3 in 2B4 T cells, most likely as CD3alpha and CD3beta intermediates because our previous results showed that few, if any, reglucosylated TCRbeta proteins were associated with TCRalpha chains. To determine the contribution of GT modification to [3H]galactose radiolabeling of CD3-associated TCRalpha and -beta proteins, dmj treatment was utilized to restrict radiolabeling to the reglucosylation pathway as before. In agreement with our previous findings that CD3gamma and -delta glycoproteins assembled into partial delta epsilon , gamma epsilon complexes were modified by GT, CD3gamma and -delta chains were effectively labeled in dmj-treated cells (Fig. 5A). As noted earlier, reglucosylation of unassembled TCRbeta chains, captured in sequential precipitates with anti-TCRbeta mAb, was enhanced in dmj-treated cells relative to media-treated cells (Fig. 5A). Importantly, these data show that reduced amounts of TCRalpha and -beta 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 TCRalpha and -beta proteins were assembled into disulfide-linked heterodimers modified by galactosylation. Analysis on two-dimensional nonreducing × reducing (NR × R) gels showed that relatively few radiolabeled TCRalpha and -beta dimers were detected in anti-CD3 precipitates of dmj-treated cells (Fig. 5B), unlike CD3 chains, which were readily visible (Fig. 5B). Interestingly, radiolabeled TCRalpha proteins existed as both disulfide-linked and monomeric species whereas the vast majority of TCRbeta molecules were present as non-disulfide-linked monomers (Fig. 5B). Formation of TCRalpha and -beta heterodimers was not precluded in dmj-treated cells as shown by immunoblotting of CD3 precipitates with anti-TCRalpha mAb (Fig. 6); dimeric TCRalpha 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 TCRalpha monomers containing immature (EH-sensitive) glycans (Fig. 6). As demonstrated, only immature TCRalpha proteins were present in lysates of dmj-treated cells (Fig. 6). These results corroborate our previous findings that TCRbeta subunits associated with TCRalpha proteins were ineffectively modified by GT and that reglucosylated (incompletely folded) CD3-associated monomeric TCRalpha and -beta proteins exist in normal (untreated) cells. Taken together, these studies demonstrate that reglucosylation of most TCR components is extinguished following the CD3alpha and -beta assembly and formation of disulfide-linked TCRalpha and -beta heterodimers, indicating that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.


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Fig. 4.   Disulfide linkage status of CD3-associated TCRalpha and -beta proteins in [3H]galactose-labeled 2B4 T cells. Digitonin lysates of 2B4 T cells labeled with [3H]galactose for 45 min were sequentially immunoprecipitated with anti-CD3epsilon mAb (145-2C11), followed by anti-TCRbeta mAb (H57-597), and analyzed on 13% SDS-PAGE gels under nonreducing conditions or on two-dimensional nonreducing × reducing (NR × R) gels. The positions of TCR proteins are marked; TCRalpha Dbeta D, dimeric alpha beta proteins; TCRalpha M and TCRbeta M, monomeric alpha  and beta  proteins, respectively. Note that the spot below alpha D in the two-dimensional NR × R samples represents a smudge on the gel that is enhanced by photography and is not a radiolabeled protein.


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Fig. 5.   Reglucosylated TCRalpha and -beta 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-CD3epsilon mAb, followed by H57-597 anti-TCRbeta mAb. The positions of TCR proteins are marked. B, anti-CD3epsilon precipitates of dmj-treated [3H]galactose-labeled 2B4 cells shown in A were analyzed on two-dimensional nonreducing x reducing (NR × R) gels. The positions of TCR proteins are marked.


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Fig. 6.   Dimerization of TCRalpha proteins in media- and dmj-treated 2B4 T cells. Aliquots of the same cells used for [3H]galactose labeling in Fig. 5 were lysed in digitonin and immunoprecipitated with 145-2C11 anti-CD3epsilon 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-TCRalpha mAb. The positions of immature (EH-sensitive) and mature (EH-resistant) TCRalpha proteins are indicated; alpha D, dimeric alpha  proteins; alpha M, monomeric alpha  proteins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 CD3delta and TCRalpha glycoproteins takes place prior to association with partner TCR chains (33), based on their comigration with calnexin-associated glycoforms following digestion with jack bean alpha -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 TCRalpha and -beta proteins expressed in BW thymoma cells, which do not efficiently assemble TCR complexes due to deficient CD3delta synthesis (16).

The results in this study suggest a scheme in which reglucosylation of invariant CD3gamma and -delta subunits persists until their association with clonotypic TCRalpha and -beta chains, and GT modification of TCRbeta proteins is terminated following the assembly of alpha beta delta epsilon gamma epsilon complexes and formation of disulfide-linked TCRalpha and -beta heterodimers.

Interestingly, unlike CD3-associated TCRbeta molecules, which were ineffectively modified by GT following disulfide linkage with TCRalpha proteins, reglucosylated TCRalpha 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 TCRalpha 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 (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 alpha beta delta epsilon gamma epsilon 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 (37-39). 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" TCRbeta proteins was increased under conditions of mannosidase blockade, which was specific in that GT modification of TCRalpha 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 TCRbeta 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 TCRbeta molecules was enhanced under these conditions, similar to what has been described for CD3delta 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 TCRalpha proteins compared with other TCR subunits, an idea that is consistent with previous findings that TCRalpha 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R29 AI42104 (to K. P. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 252-816-2703; Fax: 252-816-3104; E-mail: kearse{at}brody.med.ecu.edu.

2 K. P. Kearse, unpublished observations.

    ABBREVIATIONS

The 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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