Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae.
A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for alpha-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro alpha-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 alpha-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.
Chiba
Y
Y
Central Laboratories for Key Technology, KIRIN Brewery Co., Ltd., Yokohama, Kanagawa 236-0004, Japan.
Suzuki
M
M
Yoshida
S
S
Yoshida
A
A
Ikenaga
H
H
Takeuchi
M
M
Jigami
Y
Y
Ichishima
E
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D017931:DNA Primers; D006801:Humans; D008358:Mannose / Q000737:chemistry*; D008361:Mannosidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D043323:alpha-Mannosidase
D017931:DNA Primers; D009844:Oligosaccharides; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose