Calnexin Associates Exclusively with Individual CD3 (cid:100) and T Cell Antigen Receptor (TCR) (cid:97) Proteins Containing Incompletely Trimmed Glycans That Are Not Assembled into Multisubunit TCR Complexes*

Most T lymphocytes express on their surfaces an oli- gomeric protein complex consisting of clonotypic (cid:97)(cid:98) polypeptides associated with invariant CD3- (cid:103)(cid:100)(cid:101) and (cid:122) chains, designated the T cell antigen receptor (TCR) complex. Assembly and intracellular transport of nascent TCR proteins is believed to be assisted by their interaction with the molecular chaperone calnexin, which for certain molecules functions as a lectin for monoglucosylated glycans. However, as most of our knowledge about calnexin-TCR protein associations has been obtained under conditions of limited TCR assembly, the role of calnexin in the formation of nascent TCR complexes is unclear. Here, we studied the role of glucose (Glc) trimming and calnexin association in the oli- gomerization of TCR (cid:97) and CD3 (cid:100) glycoproteins in murine splenic T lymphocytes, a model cell type for efficient assembly of complete TCR complexes. We show that removal of Glc residues from both CD3 (cid:100) proteins and TCR (cid:97) proteins occurred prior to their association with any other TCR components and that calnexin spe- cifically interacted with unassembled TCR (cid:97) and CD3 (cid:100) proteins containing incompletely trimmed oligosaccha- rides. Interestingly, we found that removal of Glc residues from glycan chains was necessary for efficient as- sociation of calnexin with TCR (cid:97) glycoproteins but not with CD3 (cid:100) glycoproteins. These studies define Glc trimming and calnexin association as initial molecular events in the translation of CD3 (cid:100) and TCR (cid:97) proteins, occurring coincident with or immediately after their translocation into the endoplasmic reticulum and pre-ceding the ordered pairing of TCR chains. In addition, these data document that calnexin assembly with CD3 (cid:100) and TCR (cid:97) glycoproteins involves both glycan-depend-ent and glycan-independent mechanisms. in the of TCR subunits within the we studied the role of Glc trimming and calnexin association in the assembly of TCR (cid:97) and CD3 (cid:100) glycoproteins into TCR complexes in murine splenic T Our studies show that removal of Glc residues from nascent glycan chains on both CD3 (cid:100) and TCR (cid:97) proteins occurs to their association with other TCR proteins and that associates with unassembled CD3 (cid:100) and TCR proteins containing incompletely trimmed glycans.

The molecular chaperone calnexin is a nonglycosylated resident ER transmembrane protein that associates with numerous oligomeric protein complexes within the ER, including ␤ 1 and ␣ 6 integrins (5), major histocompatibility class I (6, 7) and class II molecules (8), and the antigen receptors expressed on T and B lymphocytes (7,9,10). Regarding the TCR complex, four individual TCR proteins have been shown to associate with calnexin, including clonotypic TCR␣ and TCR␤ polypeptides (7,10) and CD3␦ and CD3⑀ chains (7,9,11). Association between calnexin and proteins has never been observed (12). While convincing evidence exists for association of calnexin with individual, unassembled TCR proteins, much less is known about the assembly of calnexin with incompletely assembled TCR complexes containing multiple TCR subunits. Indeed, the assembly status of most nascent TCR proteins associated with calnexin has not been rigorously evaluated (7,9). Recently, Wiest and co-workers reported that calnexin is expressed on the surfaces of immature thymocytes in association with CD3␥⑀ and CD3␦⑀ pairs (13,14). Expression of such clonotype-independent complexes appears to be developmentally regulated as they were not detected on the surfaces of mature T cells (14). Unlike mature T cells, immature thymocytes do not efficiently assemble CD3 components into complete ␣␤␦⑀␥⑀ TCR complexes (15), leading to the suggestion that calnexin association with partial TCR complexes is exaggerated in cell types that are deficient in formation of complete TCR complexes (14).
A growing body of evidence signifies that removal of glucose (Glc) residues from nascent oligosaccharide chains is important for initial association of glycoproteins with calnexin (10,16,17). Removal of Glc residues from immature Glc 3 Man 9 GlcNAc 2 species (Glc, glucose; Man, mannose; GlcNAc, N-acetyl glucosamine) is accomplished by the sequential action of ER glucosidase I and glucosidase II enzymes, which remove the outermost and two innermost Glc residues, respectively (18), a process referred to as Glc trimming. Recent studies by several laboratories indicate that calnexin recognizes glycan chains on nascent glycoproteins bearing monoglucosylated (Glc 1 Man 9 GlcNAc 2 ) saccharides (10, 16 -19), indicating that both glucosidase I and glucosidase II activities are necessary for creation of glycan substrates for calnexin binding (20). Oligo-saccharide chains are not strictly required for calnexin association, however, as several nonglycosylated molecules interact stably with calnexin, including recombinant multidrug resistance P glycoprotein lacking N-linked addition sites (21) and the CD3⑀ subunit of the TCR complex (11).
The role of Glc trimming and calnexin association in the oligomerization of TCR proteins within the ER is poorly understood. Indeed, it is unknown at which stage(s) of TCR assembly Glc residues are removed from oligosaccharide chains on nascent TCR glycoproteins in any cell type. Regarding the role of calnexin in TCR assembly, it has been suggested that calnexin interacts with individual newly synthesized TCR proteins to facilitate their folding within the ER and to prevent their escape to the Golgi compartment (7,9,10,12). Moreover, since calnexin has been shown to associate with all TCR components except , it has also been proposed that calnexin functions in the oligomerization of TCR proteins into incomplete ␣␤␦⑀␥⑀ TCR complexes (9,12). To determine at which stage(s) of TCR assembly Glc trimming and calnexin association occurs and to see if calnexin does, in fact, participate in the oligomerization of TCR subunits within the ER, we studied the role of Glc trimming and calnexin association in the assembly of TCR␣ and CD3␦ glycoproteins into TCR complexes in murine splenic T cells. Our studies show that removal of Glc residues from nascent glycan chains on both CD3␦ and TCR␣ proteins occurs prior to their association with other TCR proteins and that calnexin associates exclusively with unassembled CD3␦ and TCR␣ proteins containing incompletely trimmed glycans. Moreover, we demonstrate that Glc trimming is required for effective interaction of calnexin with TCR␣ proteins, but not with CD3␦ proteins, and report that removal of oligosaccharides does not affect the stability of existing protein complexes of calnexin and TCR␣, CD3␦ proteins. The implications of these findings on our current knowledge of TCR assembly are discussed.

EXPERIMENTAL PROCEDURES
Animals, Cell Preparation, and Reagents-C57BL/6 (B6) mice 6 -8 weeks of age were obtained from the Jackson Laboratory (Bar Harbor, ME). T cell receptor transgenic V␣11 ϩ mice (22) were generously provided by Dr. Stephen Hedrick. Splenic T cells were obtained by incubation of single cell suspensions of spleen cells on tissue culture plates coated with rabbit anti-mouse immunoglobulin (Ig) (Organon-Technika-Cappel, Malvern, PA) for 60 min at 37°C, followed by isolation of non-adherent cells. The resulting cell populations were typically 85-90% CD3 ϩ , as determined by flow cytometry analysis. Castanospermine (Cas) was obtained from Calbiochem (La Jolla, CA) and was used at a final concentration of 100 g/ml.
Metabolic Labeling, Cell Lysis, and Immunoprecipitation-Metabolic pulse-labeling of splenic T cells was performed as described (4). Briefly, cells were incubated in methionine-free RPMI 1640 medium (Biofluids, Rockville, MD) containing 10% fetal calf serum and 1 mCi/ml [ 35 S]methionine (Tran 35 S-label) (ICN, Irvine, CA) for 30 min at 37°C in 5% CO 2 . In experiments using Cas, cells were preincubated with medium or Cas for 30 min at 37°C prior to metabolic labeling. The presence of Cas was maintained throughout the pulse and chase periods. Cells were lysed by solubilization 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 25 min at 4°C. Cell lysates were clarified by centrifugation to remove insoluble material. Sequential immunoprecipitation and release/recapture procedures were performed as described previously (4).
Glycosidase Digestions and Gel Electrophoresis-Digestion with Endoglycosidase H (Endo H) was performed by resuspending precipitates in glycosidase digestion buffer (75 mM sodium phosphate, pH 6.1, 75 mM EDTA, 0.1% Nonidet P-40) containing 10 milliunits of Endo H (Genzyme, Cambridge, MA); digestion with jack bean mannosidase (JB) (Oxford Glycosystems, Rosedale, NY) was performed according to the manufacturer's instructions. Samples were placed at 37°C for 12-16 h and digestions stopped by addition of 3 ϫ electrophoresis sample buffer. For experiments examining the effects of oligosaccharide release on association of TCR glycoproteins with calnexin, lysates were precipitated with anti-calnexin Abs; precipitates were either mock-treated or digested with Endo H, washed, boiled in 1% SDS, and TCR proteins recaptured with anti-TCR Abs as described above. SDS-PAGE gel electrophoresis was performed as described previously (15).

Differential Glucose Trimming of Individual Glycan Chains on CD3␦ Proteins Results in the Generation of Glycoforms-In
our initial studies, the Glc trimming status of nascent CD3␦ glycoproteins synthesized in splenic T cells was examined. For these experiments, splenic T cells were metabolically pulselabeled for 30 min with [ 35 S]methionine, solubilized in 1% digitonin, and lysates immunoprecipitated with anti-CD3␦ Ab. Radiolabeled TCR␣, CD3⑀, CD3␥, and chains coprecipitated with CD3␦ chains in splenic T cell lysates ( Fig. 1, left-hand side), reflecting their assembly into TCR complexes. For specific isolation of CD3␦ proteins, precipitates were boiled in 1% SDS, Nonidet P-40 detergent added, and released material immunoprecipitated with anti-CD3␦ Ab to recapture CD3␦ chains (4). The Glc trimming status of glycan chains on CD3␦ proteins was evaluated by digestion with jack bean mannosidase (JB), which removes eight mannose residues from fully trimmed (Man 9 GlcNAc 2 ) oligosaccharides, but only five mannoses from incompletely trimmed (Glc 1-3 Man 9 GlcNAc 2 ) glycan chains (17). Because CD3␦ proteins are modified by addition of 3 N-linked sugar chains, 4 glycoforms of CD3␦ can exist, reflecting the presence of 0 -3 incompletely trimmed saccharide chains on CD3␦ proteins. Indeed, as shown in Fig. 1, JB digestion of CD3␦ proteins revealed the existence of one minor (A) and three major (B-D) CD3␦ glycoforms in splenic T cell lysates ( Fig. 1, middle), denoting CD3␦ proteins with 3 (A), 2 (B), 1 (C), and 0 (D) incompletely trimmed glycan chains, respectively. In contrast, digestion with Endo H, which removes all but a single GlcNAc residue from both incompletely and fully trimmed glycan chains, generated a single CD3␦ species, which migrated just below the D glycoform (Fig. 1, CD3␦ EH S ). Existence of CD3␦ glycoforms in splenic T cells was dependent on glucosidase activity, as they were not present in lysates of splenic T cells treated with the glucosidase inhibitor castanospermine (18) (Fig. 1, right-hand side). Taken together, these results show that distinct glycoforms of CD3␦ proteins are generated in splenic T cells that reflect the differential removal of Glc residues from individual glycan chains.
Calnexin Associates Specifically with Unassembled CD3␦ Proteins Containing Incompletely Trimmed Glycan Chains-To determine the Glc trimming status of CD3␦ proteins associated with calnexin, lysates of radiolabeled splenic T cells were precipitated with anti-calnexin Ab, precipitated material was released by boiling, and CD3␦ proteins recaptured by precipitation with anti-CD3␦ Ab; recaptured CD3␦ proteins were digested with JB enzymes. As shown in Fig. 2A, CD3␦ proteins associated with calnexin in splenic T cell lysates contained incompletely trimmed glycan chains, with most existing in the B glycoform, and to a lesser extent, the C glycoform ( Fig. 2A, right-hand side).
To determine if calnexin remained associated with CD3␦ proteins following their assembly with other TCR proteins and to ascertain at which stage(s) of TCR assembly Glc residues are completely removed from CD3␦ proteins, a series of sequential immunoprecipitation studies was performed to examine the Glc trimming status of CD3␦ proteins containing incompletely trimmed and fully trimmed glycan chains. For these experiments, digitonin lysates of radiolabeled splenic T cells were precipitated with anti-CD3␦ antiserum to capture total CD3␦ proteins, or were sequentially immunoprecipitated with: (i) anti-TCR␤ mAb to isolate CD3␦ proteins existing in complete ␣␤␦⑀␥⑀ TCR and incomplete ␣␤␦⑀␥⑀ TCR complexes, followed by (ii) anti-CD3⑀ Ab to capture CD3␦ proteins existing in intermediate ␣␦⑀ and partial ␦⑀ protein complexes, and finally (iii) anti-CD3␦ Ab to isolate free, unassembled CD3␦ proteins. CD3␦ glycoproteins were recaptured from precipitates and their Glc trimming status evaluated by JB digestion. As previously established, four distinct glycoforms of CD3␦ chains existed in splenic T cell lysates (Fig. 2B, left-hand side, A-D). Clearly, both incompletely trimmed and fully trimmed CD3␦ glycoforms were present in the pool of unassembled CD3␦ proteins (Fig. 2B, right-hand side, A-D). In contrast, CD3␦ proteins associated with CD3⑀ and TCR␤ molecules contained glycan chains that were devoid of Glc residues as they were completely susceptible to JB digestion (Fig. 2B, middle, D). Chase studies showed that Glc residues were completely removed from CD3␦ glycoproteins within several hours of their synthesis in splenic T cells, which correlated with their assembly into TCR complexes and transport to the Golgi system (data not shown).
Taken together, these results demonstrate that calnexin associates with unassembled CD3␦ proteins containing incompletely trimmed oligosaccharide chains. Moreover, since CD3␦ proteins assembled with CD3⑀ containing complexes contained fully trimmed glycan chains, these studies indicate that Glc trimming of CD3␦ glycoproteins occurs prior to their assembly with other TCR proteins.
To confirm these results, we wished to assess the Glc trimming status of CD3␦ proteins at an earlier time point prior to their association with CD3⑀ and TCR␤ proteins. For these studies, splenic T cells were pulse-labeled for only 5 min and chased for 10 and 20 min. Sequential precipitations of assembled and unassembled CD3␦ chains and release/recapture procedures were performed as described in Fig. 2B. As expected, the vast majority of CD3␦ proteins existing at the end of the short pulse period were not associated with CD3⑀ chains, but existed as free, unassembled CD3␦ chains (Fig. 3, top). During the chase period, nascent CD3␦ proteins assembled with CD3⑀ chains (Fig. 3, middle), and successively with TCR␤ molecules (Fig. 3, bottom). These results show that the first detectable CD3␦ proteins assembled with other TCR proteins in our studies contained fully trimmed glycan chains. Note that most nascent CD3␦ proteins synthesized during a 5 min pulse period were susceptible to JB digestion (Fig. 3, top, A-D), indicating that processing of nascent CD3␦ proteins by ER glucosidase enzymes occurs coincident with or immediately after their translation and insertion into the ER lumen (28,29).
Calnexin Associates with Unassembled TCR␣ Proteins Containing Incompletely Trimmed Oligosaccharides-Next, we examined the Glc trimming status of TCR␣ proteins associated with calnexin and CD3 proteins in splenic T cells. Since the TCR␣ protein is clonotypic and thus quite heterogeneous, transgenic mice expressing a single rearranged TCR␣ gene product, the V␣11 protein, were used for these experiments (22). Splenic T cells of V␣11 ϩ transgenic mice were pulselabeled for 30 min, solubilized in 1% digitonin, and lysates immunoprecipitated with anti-calnexin Ab; alternatively, lysates were sequentially precipitated with anti-CD3⑀ mAb to isolate CD3-associated TCR␣ proteins, followed by anti-V␣11 mAb to capture unassembled TCR␣ proteins. Precipitated material was released by boiling in SDS and recaptured TCR␣ proteins were digested with JB or Endo H glycosidases. Unassembled TCR␣ proteins existed in two major glycoforms in splenic T cell lysates, marked A and B, which represent "free" TCR␣ proteins containing incompletely trimmed and fully trimmed glycan chains, respectively (Fig. 4, right-hand side). As demonstrated, TCR␣ proteins associated with calnexin contained incompletely trimmed glycans (Fig. 4, left-hand side, A), whereas TCR␣ proteins assembled with CD3⑀ chains possessed fully trimmed oligosaccharide chains (Fig. 4, middle, B). Thus, similar to what was observed for CD3␦ proteins, these results show that calnexin associated specifically with unassembled TCR␣ proteins containing incompletely trimmed N-linked sugar chains, and that Glc residues were removed from glycan chains on nascent TCR␣ proteins prior to their assembly with CD3⑀ proteins. Although transgenic mice were used in these experiments, it should be realized that similar conclusions were reached in studies using normal, nontransgenic mice (data not shown). It should also be appreciated that radiolabeled TCR␤ proteins are not visible in these studies as formation of CD3-associated disulfide-linked ␣␤ heterodimers involves pairing of newly synthesized TCR␣ proteins with preexisting nonradiolabeled TCR␤ proteins in murine T cells (15).

Requirement for Oligosaccharide Chains for Association of
Calnexin with TCR␣ and CD3␦ Glycoproteins-To assess the importance of Glc trimming in the initial association of nascent TCR␣ and CD3␦ proteins with calnexin, studies were performed on splenic T cells treated with the glucosidase inhibitor Cas. As shown in Fig. 5A, Glc trimming was required for efficient association of nascent TCR␣ proteins with calnexin as only trace amounts (approximately 10%) of TCR␣ proteins were recaptured from anti-calnexin precipitates of Cas-treated splenic T cells relative to untreated splenic T cells (Fig. 5A, top). Surprisingly, we found that association of calnexin with CD3␦ chains was only modestly affected by blockade of glucosidase activity, as significant amounts of CD3␦ proteins were associated with calnexin in Cas-treated splenic T cells (Fig. 5A,  bottom). These data indicate that Glc trimming is required for effective interaction of TCR␣ proteins, but not CD3␦ proteins, with calnexin in splenic T cells. We reasoned that CD3␦ proteins might associate directly with calnexin molecules in Cas-treated splenic T cells, or indirectly, via pairing with CD3⑀ proteins (11). To examine this issue, we evaluated the ability of various anti-CD3⑀ mAbs to preclear calnexin-associated CD3␦ proteins from splenic T cell lysates. As demonstrated in Fig. 5B, precipitation of Castreated splenic T cell lysates with anti-CD3␦ Abs effectively removed calnexin-associated CD3␦ chains, as expected (Fig.  5B, compare lanes 1 and 4). In contrast, precipitation with two different anti-CD3⑀ mAbs failed to preclear calnexin-associated CD3␦ proteins from lysates of Cas-treated splenic T cells (Fig.  5B, compare lanes 1, 2, and 3). Thus, we conclude that CD3␦ proteins associate directly with calnexin molecules in Cas- FIG. 4. Calnexin associates with unassembled TCR␣ proteins containing incompletely trimmed oligosaccharides. Digitonin lysates of radiolabeled splenic T cells were immunoprecipitated with anti-calnexin Ab, or sequentially precipitated with anti-CD3⑀ mAb, followed by anti-V␣11 mAb. Precipitated material was released by boiling in SDS, TCR␣ proteins recaptured by precipitation with anti-TCR␣ mAb, and samples digested with glycosidases as indicated. Precipitates were analyzed on 13% SDS-PAGE gels under reducing conditions. The positions of TCR␣ glycoforms (A and B) and Endo H-sensitive TCR␣ glycoproteins (TCR␣ EH S ) are marked. Note that higher percentage acrylamide gels reveal additional TCR␣ glycoforms that are compressed between the A and B glycoforms displayed here (data not shown); because resolution of other TCR␣ glycoforms was not optimal on such gels, however, material was analyzed on 13% gels for these studies.
FIG. 5. Glc trimming is required for effective association of TCR␣ proteins, but not CD3␦ proteins, with calnexin. A, splenic T cells were treated with medium (MED) or castanospermine (CAS), metabolically pulse-labeled, lysed in digitonin, and precipitated with anti-calnexin Ab. The presence of Cas was maintained throughout the pulse period. Calnexin precipitates were boiled in SDS, TCR proteins recaptured, and precipitates were digested with Endo H. The relative amount of TCR proteins present in Endo H-digested samples was quantitated by densitometry scanning, with the amount of calnexin-associated TCR proteins in medium groups set at 1.0. Multiple exposures of autoradiographs were scanned to ensure linearity. B, digitonin lysates of Cas-treated splenic T cells were incubated with three rounds each of protein A-Sepharose (control), 145-2C11 anti-CD3⑀ mAb, HMT3.1 anti-CD3⑀ mAb, or anti-CD3␦ Ab. Material was then sequentially precipitated with anti-calnexin Ab, precipitates boiled in SDS, and CD3␦ proteins recaptured with anti-CD3␦ Ab. For quantitative purposes, precipitates were digested with Endo H. The position of digested CD3␦ proteins (CD3␦ EH S ) is indicated. treated splenic T cells. While these experiments do not formally exclude the possibility that anti-CD3⑀ mAbs do not effectively recognize calnexin-associated ␦⑀ protein complexes in Castreated splenic T cells, it should be noted that these mAbs are capable of precipitating calnexin-associated CD3␦⑀ proteins formed in immature T cells (14).
Finally, we wished to evaluate whether N-linked glycan chains on TCR␣ and CD3␦ glycoproteins were required for maintaining stable interaction with calnexin. Calnexin precipitates of radiolabeled splenic T cell lysates were either mocktreated or digested with Endo H, and association of TCR glycoproteins with calnexin before and after deglycosylation was compared. As demonstrated, equivalent amounts of TCR␣ and CD3␦ proteins were recaptured from mock-treated and Endo H-digested anti-calnexin precipitates (Fig. 6), showing that once formed, complexes of calnexin and individual CD3␦ and TCR␣ proteins do not require oligosaccharide chains to maintain their association. DISCUSSION The quality control system of the ER ensures that properly folded, fully assembled protein complexes are expressed on the cell surface. The molecular chaperone calnexin is believed to assist in the oligomerization of nascent TCR proteins within the ER and to play a role in regulating the transport of assembled TCR complexes from the ER to the Golgi compartment. In the current study, we evaluated the role of Glc trimming and calnexin association in the oligomerization of CD3␦ and TCR␣ proteins in splenic T lymphocytes. Our results show that: (i) removal of Glc residues from nascent CD3␦ proteins and TCR␣ proteins occurs prior to their association with partner TCR chains within the ER; (ii) calnexin associates specifically with unassembled CD3␦ proteins and unassembled TCR␣ proteins containing incompletely trimmed glycan chains; (iii) Glc trimming is required for effective association of TCR␣ proteins, but not CD3␦ proteins, with calnexin; and (iv) oligosaccharide chains are important in the initial assembly of TCR␣-calnexin protein complexes, but that once formed, are not required to maintain their association. Taken together, these data effectively rule out the postulate that calnexin functions as a scaffold for assembly of nascent TCR complexes and show that calnexin assembly with TCR glycoproteins involves both glycan-dependent and glycan-independent mechanisms.
Recent studies suggest that association of nascent glycoproteins with calnexin proceeds in a two-step fashion involving initial binding of monoglucosylated glycans by calnexin, followed by protein-protein interactions which stabilize these associations (19,30). In agreement with this model, we found that inhibition of glucose trimming markedly impaired association of nascent TCR␣ proteins with calnexin in splenic T cells and, similar to what has been reported for major histocompatibility class I proteins (19) and major histocompatibility class II proteins (31), removal of oligosaccharide chains from existing calnexin-TCR␣ complexes did not affect the stability of their interaction. Interestingly, the transmembrane domain of all of these proteins has been implicated in maintaining their association with calnexin (31,32), suggesting that calnexin interactions may be stabilized within the lipid bilayer of the ER. The finding that blockade of Glc trimming severely limits the assembly of nascent TCR␣ proteins with calnexin in splenic T cells is in agreement with previous studies showing that association of nascent TCR␣ proteins with calnexin is impaired in glucosidase II-deficient BW PHAR2.7 thymoma cells and in Cas-treated wild type BW thymoma cells (10). Importantly, the current study extends these findings by showing that, unlike calnexin association with TCR␣ proteins, assembly of calnexin with CD3␦ proteins was only modestly affected by inhibition of Glc trimming. Thus, CD3␦ represents the second TCR component that has been described to interact with calnexin in a glycan-independent manner, with CD3⑀ being the first (11). Because CD3␦ and CD3⑀ proteins are structurally homologous (1), it is reasonable to speculate that CD3␦ and CD3⑀ molecules share a common region that mediates their association with calnexin. It remains to be determined why some CD3␦ chains synthesized in Cas-treated splenic T cells failed to associate with calnexin in our studies. The presence of monoglucosylated glycans on CD3␦ proteins, although not absolutely required, may increase the efficiency with which CD3␦ proteins interact with calnexin. Alternatively, as persistence of Glc residues on oligosaccharide chains has been observed to result in increased association of CD3␦ chains with CD3⑀ molecules (4), it is conceivable that enhanced assembly of CD3␦ chains into ␦⑀ pairs in Cas-treated splenic T cells precludes their association with calnexin.
The role of calnexin in the oligomerization of TCR proteins has been controversial as most studies on calnexin association with TCR proteins have been performed in cell types that are markedly impaired or completely deficient in the assembly of complete ␣␤␦⑀␥⑀ TCR complexes (10,14). Studies on calnexin interaction with TCR proteins are further complicated by the fact that multiple TCR proteins coprecipitate with calnexin, which may or may not be assembled with each other into oligomeric TCR complexes (7,9). To overcome these problems, we performed studies in murine splenic T lymphocytes, which efficiently assemble complete ␣␤␦⑀␥⑀ TCR complexes (15) and, most importantly, implemented a precipitation/recapture/ glycosidase digestion protocol that allowed us to rigorously identify the Glc trimming status of TCR␣ and CD3␦ proteins associated with calnexin and partner TCR chains. Indeed, our results demonstrate that calnexin associates exclusively with unassembled CD3␦ and TCR␣ proteins containing incompletely trimmed oligosaccharide chains in splenic T cells and show that processing of oligosaccharides on CD3␦ and TCR␣ glycoproteins by ER glucosidase enzymes precedes their assembly into intermediate ␣␦⑀ and incomplete ␣␤␦⑀␥⑀ TCR complexes (4). Thus, we believe that calnexin assists in the folding of newly synthesized, individual TCR proteins within the ER but do not think that calnexin plays a significant role in the oligomerization of multiple TCR protein subunits. It remains possible that calnexin release occurs coincident with assembly of individual TCR proteins with partner chains and that such assembly FIG. 6. Oligosaccharide chains are not necessary to maintain stable association of calnexin with TCR␣ and CD3␦ glycoproteins. Digitonin lysates of radiolabeled splenic T cells were immunoprecipitated with anti-calnexin Ab; precipitates were resuspended in Endo H digestion buffer in the presence or absence of Endo H and incubated at 37°C for 14 h. Following digestion, precipitates were washed in PBS; supernatants containing material released by Endo H digestion were removed, pellets (precipitates) were boiled in SDS, and TCR proteins recaptured by precipitation with anti-TCR specific Abs. The positions of CD3␦ and TCR␣ proteins are indicated. Note that negligible amounts of TCR␣ and CD3␦ proteins were recovered from supernatants of mock-treated and Endo H-digested calnexin precipitates (data not shown). facilitates displacement of calnexin; importantly, however, our data show that calnexin does not remain associated with TCR␣ and CD3␦ proteins assembled into multisubunit TCR complexes in splenic T cells. Finally, it is noteworthy to mention that our results on calnexin association with unassembled TCR components in splenic T cells parallel recent findings on the assembly of IgM complexes in B cells, which showed that calnexin associated with free IgM heavy chains but not with IgM chains assembled with Ig␣ proteins (33).
In summary, the current report has evaluated the role of Glc trimming and calnexin association in the assembly of CD3␦ and TCR␣ proteins into TCR complexes in splenic T cells. These studies define Glc trimming and calnexin association as initial molecular events in the assembly cascade of TCR glycoproteins that precede the ordered pairing of TCR chains within the ER.