Kasajima Yuya, Yamaguchi Masako, Hirai Nobuaki, Ohmachi Tetsuo, Yoshida Takashi
Laboratory of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori, Japan.
Biosci Biotechnol Biochem. 2006 Nov;70(11):2662-8. doi: 10.1271/bbb.60265. Epub 2006 Nov 7.
UDP-N-Acetylglucosamine: alpha-3-D-mannoside beta-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, Man(5)GlcNAc(2). The N-linked sugar chain of alpha-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 beta-N-acetylglucosaminidase treatment. Mass analysis supported the presence of GlcNAcMan(5)GlcNAc(2) as a sugar chain of alpha-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 alpha-amylase in vivo. A. oryzae is expected to be a potential host for the production of glycoprotein with a genetically altered sugar chain.
UDP-N-乙酰葡糖胺：α-3-D-甘露糖苷β-1,2-N-乙酰葡糖胺基转移酶I（GnT-I）是高甘露糖型寡糖转化为杂合型或复合型寡糖过程中的一种关键酶。大鼠GnT-I基因的全长在丝状真菌米曲霉中表达。来自重组真菌（菌株NG）的微粒体制剂显示出GnT-I活性，该活性可将N-乙酰葡糖胺残基转移至受体庚糖Man(5)GlcNAc(2)。该菌株分泌的α-淀粉酶的N-连接糖链在高效液相色谱上显示出一个新的保留峰，该峰与体外GnT-1测定的反应产物相同。用β-N-乙酰葡糖胺酶处理后，寡糖峰在高效液相色谱上消失。质谱分析支持存在GlcNAcMan(5)GlcNAc(2)作为菌株NG的α-淀粉酶的糖链。GnT-I与绿色荧光蛋白（GFP）的嵌合体在菌丝体中显示出点状荧光模式，表明其定位于高尔基体小泡。我们得出结论，GnT-1在米曲霉细胞中功能性表达，并且N-乙酰葡糖胺残基在体内被转移至α-淀粉酶的N-聚糖上。米曲霉有望成为生产糖链经基因改造的糖蛋白的潜在宿主。
