摘要
内切-β-N-乙酰氨基葡萄糖苷内切酶(Endo-β-N-acetylglucosaminidase,ENGase)是一类可以水解N-糖链中核心壳二糖之间β-1,4-糖苷键的糖苷内切酶,其中糖苷水解酶85家族(glycoside hydrolasefamilies 85,GH85)的ENGase除了水解活性之外还具有转糖基活性,能够以切除后再将均一的寡糖链转移到N-糖蛋白的β-N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)上的方式,改造医药糖蛋白的N-糖基化修饰。通过数据库检索,在布氏锥虫(Trypanosoma brucei)的基因组中找到了GH85家族ENGase的同源序列,并命名为Endo-Tb基因,对其克隆后在带有Nus融合蛋白的原核(E.coli)表达载体上成功表达并纯化。检测到融合蛋白Nus-Endo Tb能够水解高甘露糖型和双天线的复合型糖链,但不能作用于三天线的复合型糖链。同时Nus-Endo Tb可以水解核糖核酸酶B和人类转铁蛋白上的N糖链。当用唾液酸糖肽(SGP)作为糖基供体,MU-GlcNAc作为糖基受体时,通过荧光基质底物四甲基伞形酮(MU)检测到Nus-Endo Tb具有转糖基活性。
Endo-β-N-acetylglucosaminidase(ENGase)belonging to the glycoside hydrolase family 85(GH85)is a series of dual-functional enzymes that not only hydrolyze the glycosidic bonds of the N,N′-diacetylchitobiose moieties of N-glycan cores but also transfer chemically synthesized-homogeneous glycans onto glycoproteins.This unique transglycosylation activity enables them to modify proteins with a defined N-glycan structure.In this study,we identified ENGase homolog in Trypanosoma brucei genome by database search.The gene encoding ENGase was cloned into an expression vector with an N-terminal Nus tag,and expressed in Escherichia coli cells.The recombinant ENGase(Nus-Endo Tb)exhibited hydrolytic activity for high-mannose,bi-antennary N-linked oligosaccharides.Such hydrolytic activity can be applied to hydrolyze N-glycans attached to RNase B and human transferrin.Moreover,Nus-Endo Tb also can transfer a sialobiantennary type complex oligosaccharide onto MU-GlcNA,which indicated its transglycosylation activity.
作者
崔娟
喜多岛敏彦
王宁
中西秀树
高晓冬
CUI Juan;KITAJIMA Toshihiko;WANG Ning;NAKANISHI Hideki;GAO Xiao-Dong(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2018年第8期8-13,共6页
Food and Fermentation Industries
基金
国家自然科学基金(21778023)
国家自然科学基金(21576118)
