ROLE OF N-LINKED GLYCANS AND PROTEIN FOLDING

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Protein folding is a fundamental but still poorly understood process in living cells. The experiments proposed in this application are designed to evaluate the molecular basis of a novel concept in the folding field: the direct role of N-linked glycans as appendages for chaperone-binding during glycoprotein folding and assembly in the ER. Preliminary results indicate that calnexin, a ubiquitous ER chaperone, associates with its ligands by lectin-like affinity to partially glucose-trimmed N-linked oligosaccharides(Glc1Man9-7GlcNAc2). For many glycoproteins, this association (modulated by glucosidases and the lumenal UDP- Glc:glycoprotein glucosyltransferase) is likely to determine the rate of folding and the efficiency of export to the Golgi complex. Our overall goal is to analyze the role of calnexin and its soluble homologue calreticulin in the conformational maturation and quality control of newly synthesized glycoproteins. We will analyze the specificity and structural basis for glycoprotein binding to calnexin and calreticulin, a soluble homologue in the ER lumen. We will also determine the functional significance of the calnexin and calreticulin association in glycoprotein biogenesis. Finally, we will investigate the role of UDP- glucose: glycoprotein glucosyl transferase, and the mechanisms involved in the re-and deglucosylation cycle that it supports in the ER. The experiments will utilize biochemical, cell biological, genetic and structural approaches. The results will throw light on the mechanisms by which a variety of glycoproteins acquire heir transport-competent three-dimensional structure in the ER of living cell. The results will have direct relevance to a variety of diseases with ER storage/quality control etiology including cystic fibrosis and alpha1-antitrypsin deficiency. The need for cotranslational glycosylation and trimming of core glycans may, moreover, find their explanation in the link between glycosylation and folding, oligomeric assembly, degradation and quality control.