YEAST OLIGOSACCHARYLTRANSFERASE - GLYCOSYLATION OF PEPTIDE-SUBSTRATES AND CHEMICAL CHARACTERIZATION OF THE GLYCOPEPTIDE PRODUCT

The product of the reaction catalyzed by yeast oligosaccharyltransferase was examined in order to determine the nature of the chemical linkage between the sugar and peptide. Biosynthetic donor lipid [H-3]oligosaccharide was prepared and used as a substrate for yeast oligosaccharyltransferase together with a chemically synthesized peptide acceptor, N-benzoyl-Asn-Leu-Thr-NH2. the glycosylated peptide product of the in vitro reaction was isolated and hydrolyzed with endo-beta-N-acetylglucosaminidase-H to yield a large oligosaccharide and the glycotripeptide, N-benzoyl-Asn(GlcNAc)-Leu-Thr-Nh2. This glycopeptide was purified using gel filtration, affinity binding, and reverse-phase high-performance liquid chromatography. The biosynthetic glycopeptide was compared with chemically synthesized glycopeptides in which a 1-amino-GlcNAc moiety was linked to either the alpha- or beta-carboxyl of aspartate. It was determined that the sole biosynthetic product has the structure in which the carbohydrate is linked to the peptide through the beta-carbonyl of asparagine, i.e., a normal alpha-peptide. These experiments provide an unambiguous structural proof of the protein-carbohydrate linkage in the glycoprotein product of the oligosaccharyltransferase-catalyzed reaction.