N-linked glycosylation is the major modification of membrane proteins and most of the reactions in the biosynthesis of the oligosaccharide precursor and its processing to mature forms, are known. Yet, we know relatively little about how N-linked glycosylation is controlled in the cell, and only a few of the animal glycosyltransferases have been purified to homogeneity or characterized. Cultured animal cells treated with protein synthesis inhibitors or energy perturbants, produce truncated lipid-linked oligosaccharides, indicating regulation of one or more of the glycosyltransferases of the lipid-linked oligosaccharide pathway. However, the mechanism of regulation is unknown. We are in a good position to purify key enzymes (i.e., dol-P-man and dol-P-Glc synthases, GlcNAc-1-P transferase, beta-mannosyl-transferase) of the LLO pathway since: 1. We have previously solubilized these enzymes from aorta microsomes and purified several of them extensively, 2. We have substrate photoaffinity probes (such as N3-GDP[32P]-mannose) to identify the catalytic subunits on SDS-PAGE, and 3. We now have much more experience and better purification methods to isolate these proteins. Once we have the proteins purified, we will prepare antibody against each of thee and examine the amounts of enzyme activity, and enzyme protein in cells treated in ways that disturb the LLO pathway (such as with protein synthesis inhibitors or in glucose-starved cells). We will also examine the rate of synthesis and turnover of each enzyme in normal and treated cells. In addition, we will determine whether any of these enzymes are subjected to end-project inhibition or other feedback regulations, or to covalent modification. One function of N-linked glycosylation is to affect folding of some proteins. This process appears to be mediated by recognition and binding of chaperonins to glucosylated-high-mannose structures on unfolded or denatured proteins. Since we have synthesized various radiolabeled glucosylated-proteins and glucosylated-oligosaccharides (such as Glc[3-1]- Man9GlcNAc), and have the purified glucosidase I and II available to study, we will determine the interactions of chaperonins with these enzymes, and the binding of chaperonins to the labeled substrates. We will also look for an inhibitor that specifically blocks the removal of the last glucose by glucosidase II.
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