GlycoProtein biosynthesis in yeast. protein conformation affects processing of high mannose oligosaccharides on carboxypeptidase Y and invertase.

Abstract

Carboxypeptidase Y and invertase from baker's yeast, Saccharomyces cerevisiae, have two classes of Nlinked high mannose oligosaccharides which may be distinguished on the basis of their susceptibility to hydrolysis by endo-8-N-acetylglucosaminidase H (Endo H).Thus, three of the four oligosaccharides on carboxypeptidase Y and seven of nine of those on invertase are readily released by Endo H when these proteins are in their native state (accessible), while the fourth chain on carboxypeptidase Y and the remaining two on invertase are hydrolyzed by Endo H only when these proteins are denatured (inaccessible).Analysis of the three accessible oligosaccharides from carboxypeptidase Y revealed these to be mostly Manll_lnGlcNAc in size and to account for 80% of the mannose and essentially all of the phosphate associated with this enzyme.By contrast, the fourth chain from carboxypeptidase Y ranged in size from Man8-lzGlcNAc and contained no phosphate.Comparison of peptide maps with the primary sequence of carboxypeptidase Y (Svendsen, I., Martin, B. M., Viswanatha, T., and Johansen, J. T. (1982) Carlsberg Res.Cornnun 47, 15-27) allowed assignment of the resistant fourth oligosaccharide to the N-glycosylation sequon located at Asn8.l.A similar analysis of the accessible oligosaccharide pool from invertase showed that all of the phosphate and over 85% of the mannose was in species larger than ManzoGlcNAc, but the oligosaccharides released after denaturation ranged in size from Man8-12GlcNAc and were devoid of phosphate.The smaller size and lack of peripheral modification found on the oligosaccharides that are initially resistant to Endo H is most easily explained by the hypothesis that as carboxypeptidase Y and invertase fold into their mature configuration, certain glycosylated domains become inaccessible to the mannosyl transferases which catalyze chain extension and phosphomannose addition.The acquisition of carbohydrate by all eucaryotic organisms, including yeast, starts with transfer of the oligosaccharide from the common biosynthetic intermediate, Glcs-MansGlcNAcz-P-P-Doll, and culminates in mature N-linked glycans via a multistep processing pathway with the most