BBB : Vol. 70 (2006) , No. 11 pp.2662-2668

In Vivo Expression of UDP-N-Acetylglucosamine: α-3-D-Mannoside β-1,2-N-Acetylglucosaminyltransferase I (GnT-1) in Aspergillus oryzae and Effects on the Sugar Chain of α-Amylase

Yuya KASAJIMA¹⁾, Masako YAMAGUCHI¹⁾, Nobuaki HIRAI¹⁾, Tetsuo OHMACHI¹⁾ and Takashi YOSHIDA¹⁾

1) Laboratory of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University

(Received May 12, 2006)
(Accepted July 21, 2006)

UDP-N-Acetylglucosamine: α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is an essential enzyme in the conversion of high mannose type oligosaccharide to the hybrid or complex type. The full length of the rat GnT-I gene was expressed in the filamentous fungus Aspergillus oryzae. A microsomal preparation from a recombinant fungus (strain NG) showed GnT-I activity that transferred N-acetylglucosamine residue to acceptor heptaose, Man₅GlcNAc₂. The N-linked sugar chain of α-amylase secreted by the strain showed a peak of novel retention on high performance liquid chromatography that was same as a reaction product of in vitro GnT-1 assay. The peak of oligosaccharide disappeared on HPLC after β-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan₅GlcNAc₂ as a sugar chain of α-amylase from strain NG. Chimera of GnT-I with green fluorescent protein (GFP) showed a dotted pattern of fluorescence in the mycelia, suggesting localization at Golgi vesicles. We concluded that GnT-1 was functionally expressed in A. oryzae cells and that N-acetylglucosamine residue was transferred to N-glycan of α-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.

Key words:

N-acetylglucosaminyltransferase I; glycosylation; α-amylase; Aspergillus oryzae; fungi

	To cite this article:
	Yuya KASAJIMA, Masako YAMAGUCHI, Nobuaki HIRAI, Tetsuo OHMACHI and Takashi YOSHIDA, “In Vivo Expression of UDP-N-Acetylglucosamine: α-3-D-Mannoside β-1,2-N-Acetylglucosaminyltransferase I (GnT-1) in Aspergillus oryzae and Effects on the Sugar Chain of α-Amylase”, Biosci. Biotechnol. Biochem., Vol. 70, 2662-2668 (2006) .

	doi:10.1271/bbb.60265
	JOI JST.JSTAGE/bbb/60265