Persistence of glucose residues on core oligosaccharides prevents association of TCR alpha and TCR beta proteins with calnexin and results specifically in accelerated degradation of nascent TCR alpha proteins within the endoplasmic reticulum.

Abstract

The ac T-cell antigen receptor (TCR) is a multisubunit transmembrane complex composed of at least six different proteins (a, f, y, 8, e and 4) that are assembled in the endoplasmic reticulum (ER). In this report we have examined the role of oligosaccharide processing on survival and assembly of nascent TCR proteins within the ER and their associations with molecular chaperone proteins important in TCR assembly. We found that treatment of BW5147 T cells with the glucosidase inhibitor castanospermine resulted in markedly accelerated degradation of nascent TCRa proteins with a half-life of -20 min. Accelerated degradation was unique to TCRax proteins, as the stability of nascent TCRfi and CD3y,e chains was unaltered. Consistent with a requirement for glucose (Glc) trimming for survival of nascent TCRax proteins within the ER, we found that newly synthesized TCRa chains were innately unstable in the glucosidase II-deficient BW5147 mutant cell line PHAR2.7. In addition to destabilizing nascent TCRa proteins we found that persistence of Glc residues on core oligosaccharides markedly interfered with association of both TCRax and TCR, glycoproteins with the molecular chaperone calnexin. Finally, using 2B4 T hybridoma cells in which TCR complexes are efficiently assembled, we found that rapid degradation of nascent TCRa proteins induced by impaired Glc trimming severely limits assembly of TCRa proteins with TCRP proteins. These results demonstrate that the persistence of Glc residues on asparagine-linked (N-linked) oligosaccharide side chains (i) disrupts associations of calnexin with both TCRa and TCR, proteins, (ii) results in destabilization of nascent TCRa proteins, and (iii) restricts the ability of TCRa and TCRI proteins to assemble together. Thus, association with calnexin is uniquely important for the stabilization of nascent TCRa proteins within the ER.

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

“…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.…”

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones

“…In particular, we thought it possible that molecular chaperones continued to associate with glycoproteins even after escape from the ER, thereby blocking accessibility to processing enzymes during transit through the Golgi. Indeed, the exodomain of calnexin can bind glycoproteins via their N-linked oligosaccharide side chains, but this binding is restricted to oligosaccharide chains containing a terminal glucose moiety, a modification that is normally removed from nascent glycoproteins before their exit from the ER (49)(50)(51). Interestingly, we found that the surface CD3 complexes expressed by immature thymocytes could be subdivided into two populations based upon the availability of their carbohydrate side chains to binding by the mannose and glucose reactive-lectin, ConA (Fig.…”

“…It is also tempting to speculate that the ER resident molecular chaperones that have been released to the surface of immature thymocytes may themselves play an important role in thymocyte development. In particular, it is possible that molecular chaperones like calnexin and CRT, through their ability to bind carbohydrate ligands (49)(50)(51)(52), may promote interactions between developing thymocytes and thymic stromal cells, an important aspect of their codependent differentiation.…”

Calnexin Interaction with N-Glycosylation Mutants of a Polytopic Membrane Glycoprotein, the Human Erythrocyte Anion Exchanger 1 (Band 3)

“…Calnexin and calreticulin are lectins that interact with the Glc 1 Man 9 -5 GlcNAc 2 oligosaccharide (21-26), and calnexin-bound monoglucosyl glycoproteins are protected from ␣-glucosidase II removal of the final glucose (25). Inhibition of ␣-glucosidase I and II with castanospermine (CST) prevents calnexin binding (27,28), and calnexin associations are absent in glucosidase-deficient cell lines (29). The presence of a single oligosaccaride moiety on a protein is sufficient for the interaction with calnexin; however, the interaction is enhanced when multiple N-glycosylated sites are present (30,31).…”