PRODUCTION OF HUMAN COMPATIBLE HIGH MANNOSE-TYPE (MAN5GLCNAC2) SUGAR CHAINS IN SACCHAROMYCES CEREVISIAE

Abstract A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for α-1,2-mannosidase with the “HDEL” endoplasmic reticulum retention/retrieval tag was designed and expressed inSaccharomyces cerevisiae. An in vitroα-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an α-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.