The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum.

Assembly of the multisubunit T cell antigen receptor (TCR) complex is an intricate process requiring coordinated regulation of at least six different gene products (α, β, γ, δ, ε, and ζ) and the ordered pairing of partner chains within the endoplasmic reticulum (ER). To date, two proteins have been implicated as functioning as molecular chaperones in the assembly of nascent TCR proteins: calnexin, a resident ER transmembrane protein, which associates with all TCR components except ζ, and T cell receptor-associated protein, which selectively associates with CD3γε pairs. In this study, we examined the association of calreticulin, a soluble protein with significant sequence homology to calnexin, with newly synthesized TCR proteins. Analogous to calnexin, processing of glycan chains by glucosidase enzymes was required for initial association of TCRα and -β proteins with calreticulin; however, several major differences were noted regarding interaction of calnexin and calreticulin chaperones with TCR proteins. First, TCRα and -β proteins showed prolonged association with calnexin molecules compared with calreticulin; interaction of TCRα proteins with calreticulin was particularly transient, with most calreticulin-TCRα protein complexes dissociating within 15 min of their initial assembly. Second, we found that, unlike calnexin, which associated with clonotypic TCRα and -β proteins and invariant CD3δ and -ε polypeptides, calreticulin associated specifically with clonotypic TCRα and -β proteins. These studies identify calreticulin as a molecular chaperone for nascent clonotypic TCRα and -β proteins and demonstrate that calreticulin and calnexin differentially associate with newly synthesized TCR proteins within the ER.

Assembly of complete ␣␤␦⑀␥⑀ T cell antigen receptor (TCR) 1 complexes occurs within the endoplasmic reticulum (ER) and proceeds in a precisely ordered fashion (1,2). Oligomerization of TCR proteins into TCR complexes is believed to be facilitated by their association with the molecular chaperone calnexin (3)(4)(5), a resident ER transmembrane protein that participates in the assembly of other multisubunit immune recep-tor complexes, including major histocompatibility class I molecules (4,6,7), class II molecules (8), and the B cell antigen receptor (4,9). Efficient association of TCR glycoproteins with calnexin requires removal of two Glc residues from nascent immature Glc 3 Man 9 (GlcNAc) 2 glycan chains (5), a process referred to as Glc trimming (5,10,11). Glc trimming occurs in the ER by the sequential action of glucosidase I and II enzymes, which remove the outermost and two innermost Glc residues, respectively (12,13). Because calnexin specifically recognizes monoglucosylated (Glc 1 Man 9 (GlcNAc) 2 ) oligosaccharide species, both glucosidase I and II activities are necessary for creation of glycan substrates for calnexin binding (10,14). Indeed, association of nascent glycoproteins with calnexin is significantly impaired by drug-induced blockade of glucosidase activity and in mutant cell lines that are deficient in expression of glucosidase enzymes (5,12). Oligosaccharide chains are not strictly required for calnexin association, however, as several nonglycosylated molecules associate stably with calnexin, including recombinant multidrug resistance P glycoprotein (15) and CD3⑀ proteins (16).
Four individual TCR proteins have been demonstrated to interact with calnexin: clonotypic TCR␣ and TCR␤ polypeptides (3,5) and invariant CD3␦ and CD3⑀ chains (3,16,17). Association of calnexin with CD3␥⑀ and CD3␦⑀ pairs has also been observed, which, interestingly, are localized on the cell surfaces of immature T cells (18,19). Assembly of calnexin with proteins has never been reported (3). In the current study we examined the association of calreticulin with newly synthesized TCR proteins (20,21). We found that the interaction of TCR␣ and -␤ proteins with calnexin and calreticulin chaperones was quite distinct in that TCR␣ and -␤ proteins associated much more transiently with calreticulin molecules than with calnexin. Moreover, in contrast to calnexin, which associated with both clonotypic TCR␣ and -␤ proteins and invariant CD3␦ and -⑀ chains, we found that calreticulin assembly was restricted to clonotypic, antigen-reactive TCR␣ and -␤ chains.

EXPERIMENTAL PROCEDURES
Cells and Reagents-Two types of parental BW5147 thymoma cells were used in these studies: parent BW cells, which synthesize high levels of all TCR chains except CD3␦ proteins (5,22); and BW cells transfected with the CD3␦ gene (BW␦) which synthesize significant amounts of CD3␦ proteins (kindly provided by Drs. Ken Katz and Alfred Singer, Experimental Immunology Branch, National Institutes of Health). The interaction of calnexin and calreticulin with TCR␣ and -␤ proteins was identical in parental BW and BW␦ cells (data not shown). BW cells (7), 2B4 T hybridoma cells (23), and glucosidase II-deficient BWPHAR2.7 cells (7) were maintained as described previously. Castanospermine (cas) was purchased from Calbiochem and was used at 50 -100 g/ml.

RESULTS
Initially, BW thymoma cells were metabolically labeled with [ 35 S]methionine for 30 min and solubilized in 1% digitonin, and lysates were precipitated with anti-calnexin Ab or anti-calreticulin Ab; precipitates were analyzed on two-dimensional NEPHGE-SDS-PAGE gels. Numerous proteins were associated with calnexin and calreticulin molecules in BW lysates, including proteins that comigrated with clonotypic TCR␣ and -␤ polypeptides (Fig. 1A, top). Interestingly, when compared with anti-calnexin precipitates, the ratio of TCR␣:TCR␤ proteins coprecipitating with calreticulin was markedly skewed toward TCR␤ proteins (Fig. 1A, top). As shown in experiments using the glucosidase inhibitor cas (13), assembly of nascent TCR␣ and -␤ glycoproteins with both calnexin and calreticulin chaperones was dependent on processing of glycan chains by ER glucosidase enzymes (Fig. 1A, bottom). These results were confirmed in the glucosidase II-deficient BW variant BWPHAR2.7 (22), in which calreticulin assembly with TCR␣ and -␤ glycoproteins, as well as numerous unidentified proteins, was severely decreased relative to parental BW cells (Fig. 1B). Finally, as determined by immunoprecipitation and immunoblotting experiments, the anti-calnexin and anti-calreticulin Abs used in these studies were not cross-reactive ( Fig. 1C), indicating that TCR␣ and -␤ proteins were specifically associated with calnexin and calreticulin molecules in BW lysates. Thus, we conclude that newly synthesized TCR␣ and -␤ proteins associate with both calnexin and calreticulin, and that these interactions require processing of glycan chains by ER glucosidase enzymes.
In our next set of studies, the stability of calnexin-TCR␣ and -␤ and calreticulin-TCR␣ and -␤ interactions was evaluated. To specifically examine association of TCR␣ proteins with calnexin and calreticulin chaperones, BW cells were pulse labeled with [ 35 S]methionine for 5 min and chased for various periods in medium containing excess nonradioactive methionine. Digitonin lysates were precipitated with anti-calnexin and anticalreticulin Abs; precipitates were boiled in 1% SDS to release bound material, Nonidet P-40 detergent was added to counteract the SDS, and TCR␣ proteins were recaptured by precipitation with anti-TCR␣ mAb (2). As demonstrated, the vast majority of TCR␣-calnexin complexes formed during a short 5-min pulse were stable during a 30-min chase period (Fig. 2). In contrast, most nascent TCR␣ proteins associated with calreti- culin during the pulse period rapidly dissociated during the chase, with only trace amounts of TCR␣ proteins coprecipitating with calreticulin after 30 min (Fig. 2). These data demonstrate that newly formed calreticulin-TCR␣ protein complexes are much less stable than calnexin-TCR␣ protein complexes. As previously established, the initial association of TCR␣ chains with calreticulin required removal of Glc residues from nascent glycan chains by ER glucosidase enzymes (Fig. 1, A  and B). To determine whether rapid disassembly of TCR␣ proteins from calreticulin was also dependent on glucosidase activity, experiments were performed in which cas was present only during the chase period, subsequent to formation of TCR␣calreticulin protein complexes. As is evident, disassembly of calreticulin-TCR␣ protein complexes was markedly impaired in cas-treated groups relative to control groups (Fig. 3), indicating that dissociation of TCR␣ proteins from calreticulin is facilitated by removal of Glc residues from glycan chains.
To examine the stability of calreticulin-TCR␤ protein complexes, anti-calreticulin precipitates of pulse-chase BW lysates were analyzed directly on two-dimensional NEPHGE-SDS-PAGE, since the anti-TCR␤ mAb used in these studies does not recapture denatured TCR␤ chains. 2 Unlike calreticulin association with newly synthesized TCR␣ proteins, which was exceedingly transient, calreticulin interactions with nascent TCR␤ proteins were relatively stable during this time period (Fig. 4). The rapid disassembly of calreticulin-TCR␣ protein complexes relative to calreticulin-TCR␤ protein complexes provides a molecular basis for our previous observation that the ratio of TCR␣:TCR␤ proteins coprecipitating with calreticulin during a 30-min pulse period was markedly skewed toward TCR␤ (Fig. 1A). In experiments using extended chase periods, it can be seen that most calreticulin-TCR␤ protein complexes eventually dissociated (Fig. 5); as is evident, however, both TCR␣ and TCR␤ proteins remained stably associated with calnexin during this period (Fig. 5). Taken together, these results demonstrate that calreticulin-TCR␤ protein complexes are more stably associated than calreticulin-TCR␣ protein complexes and that TCR␣ and -␤ proteins interact more steadily with calnexin than with calreticulin.
Finally, we wished to determine whether calreticulin assembled with both clonotypic TCR␣ and -␤ proteins and invariant CD3 chains, as has been reported for calnexin (3,5,16,17). For these studies BW cells that had been stably transfected with the CD3␦ gene were used (BW␦ cells), which synthesize high amounts of CD3␦ proteins relative to parental BW cells, which make few, if any, CD3␦ proteins (5); experiments were also performed using 2B4 T hybridoma cells. BW␦ and 2B4 T cells were metabolically labeled with [ 35 S]methionine for 30 min and solubilized in 1% digitonin, and lysates were precipitated with anti-calnexin Ab or anti-calreticulin Ab; bound material was released by boiling in SDS, and CD3 proteins were recaptured with various anti-CD3-specific Abs. As demonstrated, CD3␦ and CD3⑀ proteins were recaptured from anti-calnexin precipitates, but not from anti-calreticulin precipitates, of BW␦ and 2B4 T cell lysates (Fig. 6). Identical results were observed in splenic T cells; and association of CD3␦ and -⑀ components with calnexin but not calreticulin in T cells was confirmed by immunoblotting (data not shown). Therefore, we conclude that calreticulin associates specifically with nascent, clonotypic TCR␣ and -␤ proteins but not with newly synthesized, invariant CD3␦ and -⑀ chains. DISCUSSION In the current report we examined the association of newly synthesized TCR proteins with calreticulin. These studies demonstrate that, analogous to calnexin, glycan processing is required for assembly of calreticulin with TCR␣ and -␤ proteins in the ER, that nascent TCR␣ and -␤ proteins are more stably associated with calnexin than with calreticulin, and finally, 2 J. E. M. Van Leeuwen and K. P. Kearse, unpublished observations. FIG. 2. Nascent TCR␣ proteins rapidly dissociate from calreticulin but not calnexin. BW cells were metabolically pulse labeled for 5 min with [ 35 S]methionine and chased for the periods indicated. Cells were solubilized in 1% digitonin, and lysates were immunoprecipitated (Immpt.) with anti-calnexin or anti-calreticulin Abs; precipitates were boiled in 1% SDS to release bound material, Nonidet P-40 detergent was added, and TCR␣ proteins were specifically recaptured with anti-TCR␣ mAb.
FIG. 3. Deglucosylation is an important step in the dissociation of TCR␣ proteins from calreticulin. BW cells were metabolically pulse labeled for 5 min with [ 35 S]methionine (in the absence of cas) and chased in the presence (MED) or absence (CAS) of cas for the periods indicated. Cells were solubilized in 1% digitonin, and lysates were precipitated (Immpt.) with anti-calreticulin (Anti-Crt) Abs; bound material was released by boiling in 1% SDS, and TCR␣ proteins were recaptured as in Fig. 2. The position of TCR␣ proteins is indicated.
FIG. 4. Stability of calreticulin-TCR␣ and calreticulin-TCR␤ protein complexes. BW cells were metabolically pulse labeled for 5 min with [ 35 S]methionine and chased for the periods indicated. Cells were solubilized in 1% digitonin, and lysates were immunoprecipitated (Immpt.) with anti-calreticulin Ab; precipitates were analyzed on twodimensional NEPHGE/SDS-PAGE under reducing conditions. The positions of calreticulin (arrow) and TCR␣ and TCR␤ proteins are indicated.
that calreticulin associates specifically with clonotypic TCR␣ and -␤ proteins but not with invariant CD3␦ and -⑀ chains.
Similar to what has been observed with calnexin (5), we found that Glc trimming was necessary for efficient association of newly synthesized TCR␣ and -␤ proteins with calreticulin. Thus, as recently proposed by several laboratories (11, 12, 29 -32), calreticulin represents the second member of a family of endogenous lectin molecules specific for glycoproteins containing partially glucosylated glycans, with calnexin being the first (10). Despite intensive investigation over the past few years, the precise function of calnexin in the assembly of nascent TCR complexes within the ER remains to be delineated. Current hypotheses suggest that calnexin interacts with individual newly synthesized TCR proteins to facilitate their folding within the ER, prevent their escape to the Golgi compartment, and catalyze their oligomerization into complete ␣␤␦⑀␥⑀ TCR complexes (3-5, 16, 17). The identification of both calnexin and calreticulin as chaperones for TCR␣ and -␤ glycoproteins containing incompletely trimmed glycan chains raises the intriguing possibility that calnexin and calreticulin perform distinct molecular functions in the folding and assembly of nascent TCR␣ and -␤ proteins within the ER. It is noteworthy to mention that monoglucosylated (Glc 1 Man 9 (GlcNAc) 2 ) glycans can be generated in the ER by two different mechanisms: either directly via the Glc-trimming pathway or indirectly via reglucosylation of processed Man [8][9] (GlcNAc) 2 species by ER glucosyltransferase enzymes (11,14,33,34). Thus, it is interesting to speculate that calreticulin and calnexin may differentially associate with monoglucosylated glycoproteins derived via the Glc-trimming and Glc-trimming and reglucosylation pathways in the ER. Alternatively, calnexin and calreticulin chaperones may function redundantly in the ER quality control system to maximize the probability that nascent TCR␣ and -␤ chains (and other neoglycoproteins) are correctly folded. Regarding this issue, recent studies by Scott and Dawson (35) have demonstrated that major histocompatibility complex class I proteins are efficiently assembled and expressed in a human cell line deficient in calnexin expression (35), indicating that "backup" mechanisms exist within the ER quality control system for correct folding and assembly of oligomeric protein complexes.
In contrast to calnexin association with TCR␣ and -␤ proteins and calreticulin association with TCR␤ proteins, the association of calreticulin with nascent TCR␣ proteins was remarkably unstable, with most TCR␣ proteins rapidly dissociating from calreticulin molecules within 15 min of their assembly. Blockade of glucosidase activity by cas treatment stabilized calreticulin-TCR␣ protein interactions, indicating that removal of Glc residues from nascent glycan chains is an important step in the disassembly of TCR␣ proteins from calreticulin. In contrast, calnexin-TCR␣ protein complexes were not stabilized by cas treatment, 2 providing another interesting difference between calnexin and calreticulin and their function as molecular chaperones for nascent TCR proteins.
Finally, the current report shows that, in contrast to calnexin, which associates with both clonotypic TCR␣ and -␤ proteins and invariant CD3␦ and -⑀ proteins, calreticulin association is restricted to clonotypic, antigen-reactive chains. Although these studies do not formally exclude the possibility that weak interactions between invariant CD3␦ and -⑀ chains and calreticulin molecules may exist, our data clearly show that protein complexes of calnexin-CD3␦ and -⑀ chains and calreticulin-TCR␣ and -␤ chains were stably isolated under conditions in which association of calreticulin with CD3 chains was not detected. It is conceivable that, unlike the extracellular regions of TCR␣ and -␤ molecules, the extracellular domains of CD3␦ and -⑀ chains do not sufficiently extend into the ER lumen, thereby precluding their (stable) association with soluble calreticulin molecules (1). Alternatively, it is possible that invariant CD3␦ and -⑀ chains lack the structural domains necessary for their assembly with calreticulin molecules. Elucidation of the structural determinants on TCR proteins important for their interaction with calnexin and calreticulin molecules should provide valuable information regarding these issues. FIG. 5. Prolonged association of TCR␣ and -␤ proteins with calnexin but not calreticulin. BW cells were metabolically pulse labeled for 5 min with [ 35 S]methionine and chased for the periods indicated. Cells were solubilized in 1% digitonin, lysates were immunoprecipitated with anti-calnexin or anti-calreticulin Ab, and precipitates were analyzed on two-dimensional NEPHGE/SDS-PAGE under reducing conditions. The positions of TCR␣ and TCR␤ proteins are indicated.
FIG. 6. Differential assembly of newly synthesized, invariant CD3-␦ and -⑀ proteins with calnexin and calreticulin. Digitonin lysates of radiolabeled BW␦ cells and 2B4 T hybridoma cells were immunoprecipitated with anti-calnexin (Anti-Cx) or anti-calreticulin (Anti-Crt) Abs, precipitates were boiled in 1% SDS to release bound material, Nonidet P-40 detergent was added, and TCR proteins were recaptured with anti-CD3␦ (R9) antiserum or anti-CD3⑀ mAb (HMT3.1). The positions of CD3␦ and -⑀ proteins are marked. ‫,ء‬ unknown protein that is specifically recaptured with anti-CD3⑀ mAb; this protein is not CD3␦, as it is not glycosylated (data not shown). Note that radiolabeled CD3␦ and -⑀ proteins were not visible in recaptures from anti-calreticulin precipitates, even on prolonged exposure of autoradiographs.