Inhibition of hybrid-and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

被引:52
作者
Crispin, Max
Harvey, David J.
Chang, Veronica T.
Yu, Chao
Aricescu, A. Radu
Jones, E. Yvonne
Davis, Simon J.
Dwek, Raymond A.
Rudd, Pauline M.
机构
[1] Wellcome Trust Ctr Human Genet, Div Struct Biol, Canc Res UK Receptor Struct Res Grp, Oxford OX3 7BN, England
[2] Univ Oxford, John Radcliffe Hosp, Weatherall Inst Mol Med, Oxford OX3 9DS, England
基金
英国惠康基金; 英国医学研究理事会;
关键词
electrospray ionization mass spectrometry; fucosyltransferase; matrix-assisted laser desorption; ionization (MALDI)/N-linked glycosylation;
D O I
10.1093/glycob/cwj119
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A mammalian N-acetylglucosamine (GIcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man(5)GlcNAC(2) glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man(5)GlcNAc(2) glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc(1)Man(7)GlcNAc(2)Fuc. Furthermore, no fucosylated Man(9)GlcNAc(2) glycans were detected following inhibition of (x-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha 1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core, Fucosylated Man(5)GlcNAc(2) glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.
引用
收藏
页码:748 / 756
页数:9
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