PRODUCTION OF HUMAN COMPATIBLE HIGH MANNOSE-TYPE (MAN(5)GLCNAC(2)) SUGAR CHAINS IN SACCHAROMYCES-CEREVISIAE

A yeast mutant capable of producing Man(5)GlNAc(2) human compatible su gar chains ore glycoproteins was constructed. An expression vector for alpha-1,2-mannosidase with the ''HDEL'' endoplasmic reticulum retenti on/retrieval tag was designed and expressed in Saccharomyces cerevisia e. An in vitro alpha-1,2-mannosidase assay and Western blot analysis s howed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities wa s then transformed with an alpha-1,2-mannosidase expression vector. Th e oligosaccharide structures of carboxypeptidase Y as well as cell sur face glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man(5) GlcNAc(2). This is the first report of a recombinant S. cerevisiae abl e to produce Man(5)GlcNAc(2)-oligosaccharides, the intermediate for hy brid-type and complex-type sugar chains.