Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae.
The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP- N- acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N- acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N- glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c-Myc epitope tag at the C-terminus elucidates that the N - terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.
Yoshida
S
S
KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan.
Suzuki
M
M
Yamano
S
S
Takeuchi
M
M
Ikenaga
H
H
Kioka
N
N
Sakai
H
H
Komano
T
T
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015870:Gene Expression; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000201:enzymology; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology
D018076:DNA, Complementary; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D014357:Trypsin