Comprehensive glyco-proteomic analysis of human α1-antitrypsin and its charge isoforms
Comprehensive glyco-proteomic analysis of human α1-antitrypsin and its charge isoforms
复制标题
DOI：
10.1002/pmic.200500751
复制
发表时间：
2006-06-01
期刊：
影响因子：
3.4
通讯作者：
Altmann, Friedrich
中科院分区：
文献类型：
--
作者：
Kolarich, Daniel;Weber, Alfred;Altmann, Friedrich
人α(1)-抗胰蛋白酶(A1PI)是人血浆中一种已知的糖蛋白，对保护组织免受蛋白水解酶的影响很重要。A1PI的三个N-糖基化位点含有二天线N-糖聚糖，但也有三天线甚至四天线结构的痕迹，导致了A1PI典型的IEF图谱。本文提出了一种用LC-ESI-MS和LC-ESI-MS/MS鉴定人血浆及其PTMS中A1PI异构体的方法。A1PI的单个半胱氨酸残基与游离半胱氨酸形成二硫键。触角数量的可变性，因此糖基化位点N107上的唾液酸，甚至包含微量的四天线结构，出现了A1PI的IEF模式的主要原因。在所分析的每种异构体中，只有少量的三触角结构附着在N70上，而N271位上只存在二触角结构。胞外糖苷酶消化揭示了二天线N-糖链上的α-2，6-神经氨基酸，而三天线在N-糖链上还含有一个α-2，3-神经氨基酸，它与岩藻糖一起形成了β-1，4-连接N-乙酰氨基葡萄糖上的唾液酸路易斯X决定簇，如吡啶层叠去乙酰葡聚糖的2-D-高效液相色谱所示。二触角结构的岩藻糖基化是边缘的和核心的α1，6型。
Human alpha(1)-antitrypsin (A1PI) is a well-known glycoprotein in human plasma important for the protection of tissues from proteolytic enzymes. The three N-glycosylation sites of A1PI contain diantennary N-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for A1PI. Here we present an approach to characterize A1PI isoforms from human plasma and its PTMs by LC-ESI-MS and LC-ESI-MS/MS of peptides obtained by proteolytic digestion. The single cysteine residue of A1PI formed a disulfide bridge with free cysteine. The variability of the number of antennae and hence sialic acids on glycosylation site N107, which even contained minute amounts of tetraantennary structures, emerged as a major cause for the IEF pattern of A1PI. Only negligible amounts of triantennary structures were identified attached to N70, and exclusively diantennary structures were present on site N271 in each of the isoforms analyzed. Exoglycosidase digests revealed alpha 2,6-linked neuraminic acids on diantennary N-glycans, and triantennary contained additionally one single alpha 2,3-neuraminic acid per N-glycan, which, together with a fucose, formed a sialyl Lewis X determinant on the beta 1,4-linked N-acetylglucosamine, as shown by 2-D-HPLC of pyridylaminated asialoglycans. Fucosylation of diantennary structures was marginal and of the core alpha 1,6 type.