The objective of our research includes three related areas that deal with the synthesis and maturation of asparagine-linked oligosaccharide chains. One project involves the dependence of glucose on the glycosylation of proteins in mammalian cells. Expanding on our previous findings, an in vitro rat liver microsomal system will be used to test a variety of glucose metabolites in affecting the assembly of the dolichol-linked oligosaccharide intermediates; particularly, the two mannosyltransferases that participate in the conversion of dolichol-linked Man5GlcNAc2 to the Man6GlcNAc2 species. We will also measure the cellular concentrations of these metabolites and of the lipid-linked oligosaccharide intermediates by either a sodium (3H)borohydride quantitation procedure or by a fluorescence derivatization method as a function of glucose starvation of BHK fibroblasts. The information generated by these experiments may elucidate the molecular basis that leads to aberrant protein glycosylation in fibroblasts starved for glucose. Another project that we are working on is the basis for the alterations in the structure of erythroglycan carbohydrate chains of K-562 cells caused by ouabain and other reagents that induce differentiation. We will measure the activities of the Beta 1,3 and Beta 1,6 N-acetylglucosaminyl transferases from cell-free membrane extracts prepared from untreated and ouabain-treated cells. This work involves the preparation of oligosaccharide substrates from erythrocytes. A recent project that we have undertaken is the investigation of the structure and biosynthesis of complex carbohydrates in the human parasite Leishmania donovani. These eukaryotic organisms do not glycosylate proteins via the Glc3Man9GlcNAc2-P-P-Dol intermediate typical of all other eukaryotic cells. We are examining the structure of the oligosaccharides as well as the lipid carrier assembled in these cells. These cells also express a major polysaccharide that we are characterizing by methylation linkage analysis, gas-liquid chromatography, lectin-affinity chromatography, and HPLC.
