A unique form of nucleoplasmic and cytoplasmic protein glycosylation, 0-linked GlcNAc, (O-GlcNAc) is present on proteins ranging from those of yeast to man, including many chromatin proteins, transcription factors, nuclear pore proteins, and certain types of cytoskeletal proteins.In this report we have studied the effects of cellular activation on O-GlcNAc-modified proteins, using T lymphocytes as a model system. Results indicate that the apparent levels of O-GlcNAc on many nuclear proteins increases rapidly after lymphocyte activation, returning to control levels after a few hours. In contrast, the apparent levels of O-GkcNAc on a distinct population of cytosolic proteins decreases rapidly after cellular activation and also returns to control levels after a few hours. These data are consistent with the hypothesis that O-GlcNAc is a regulatory modification and suggest that O-GlcNAc modification may play an important role in the early stages of T-lymphocyte activation.A unique form of nucleoplasmic and cytoplasmic protein glycosylation in which N-acetylglucosamine monosaccharides are O-glycosidically linked to serine or threonine residues (O-GlcNAc) has been described in organisms including viruses, yeast, protozoa, insects, frogs, rodents, and man but not bacteria (for reviews, see refs. 1 and 2). O-GlcNAcmodified proteins are enriched in nuclei (3) and nuclear pores and are particularly dense in chromatin (4). Subsets of the family of proteins compromising RNA polymerase II transcription factors, but not RNA polymerase I or III transcription factors, appear to be glycosylated by O-GlcNAc (5, 6). One such glycosylated transcription factor, c-fos, is important for the activation of the interleukin 2 gene, which encodes a lymphokine critical to T-cell activation and commitment (7).Aside from the apparent role of O-GlcNAc-bearing nuclear pore glycoproteins in nuclear transport (8-11) and the possible involvement of O-GlcNAc in enhancing the activities of transcription factors (5), virtually nothing is known concerning the functions of these monosaccharide moieties. It has been suggested that O-GlcNAc might be required for the proper assembly of multimeric protein complexes (12) or that it might serve as a nuclear targeting signal (13) analogous to the mannose 6-phosphate-mediated targeting of proteins into lysosomes (14). Based upon the sequences surrounding sites of attachment of O-GlcNAc on three proteins (15), it also has been suggested that O-GlcNAc might reversibly block potential phosphorylation sites on proteins (1, 12). The sites of attachment of O-GlcNAc are similar to the phosphorylation sites of several known serine/threonine kinases (for a review, see ref. 16) and a recently described growth factor-sensitive proline-directed serine/threonine kinase (17, 18). In addition, the sites of attachment of O-GlcNAc are strikingly homologous to previously defined proline-acidic residue-(serine or threonine) regions, which are believed to regulate proteolysis of rapidly turning-over regulatory proteins (19...

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.

Immature thymocytes express clonotype‐independent CD3 complexes that, when engaged by anti‐CD3 antibodies, can signal CD4‐CD8‐ thymocytes to differentiate into CD4+CD8+ cells. Clonotype‐independent CD3 complexes consist of CD3 components associated with an unknown 90 kDa surface protein. We now report the surprising finding that this 90 kDa surface protein is the molecular chaperone calnexin, an integral membrane protein previously thought to reside only in the endoplasmic reticulum (ER). We found that calnexin‐CD3 complexes escaping to the cell surface utilize interchain associations distinct from those utilized by calnexin‐CD3 complexes remaining within the ER. Specifically, we demonstrate that carbohydrate‐mediated luminal domain interactions that are necessary for formation of most internal calnexin‐CD3 complexes destined to be expressed on the cell surface, and we provide evidence that cytoplasmic domain interactions between calnexin and CD3 epsilon chains mask calnexin's ER retention signal, permitting calnexin and associated proteins to escape ER retention. Thus, the present study demonstrates that partial T cell antigen receptor complexes can escape the ER of immature thymocytes in association with their molecular chaperone to be expressed at low levels on the cell surface where they may function as a signaling complex to regulate thymocyte maturation.