STRUCTURES OF THE GLYCOSYL-PHOSPHATIDYLINOSITOL ANCHORS OF PORCINE AND HUMAN RENAL MEMBRANE DIPEPTIDASE - COMPREHENSIVE STRUCTURAL STUDIES ON THE PORCINE ANCHOR AND INTERSPECIES COMPARISON OF THE GLYCAN CORE STRUCTURES
STRUCTURES OF THE GLYCOSYL-PHOSPHATIDYLINOSITOL ANCHORS OF PORCINE AND HUMAN RENAL MEMBRANE DIPEPTIDASE - COMPREHENSIVE STRUCTURAL STUDIES ON THE PORCINE ANCHOR AND INTERSPECIES COMPARISON OF THE GLYCAN CORE STRUCTURES
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DOI：
10.1074/jbc.270.39.22946
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发表时间：
1995-09-29
影响因子：
4.8
通讯作者：
HOOPER, NM
中科院分区：
文献类型：
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作者：
BREWIS, IA;FERGUSON, MAJ;HOOPER, NM
通过结合液相色谱、外切糖苷酶消化和甲基化分析，在脱氨和还原后确定猪和人肾膜二肽酶 (EC 3.4.13.19) 上糖基磷脂酰肌醇 (GPI) 锚定的聚糖核心结构。发现聚糖核心表现出微观异质性，在猪 GPI 锚中观察到三种结构：Man α 1-2Man α 1-6Man α 1-4GlcN（占总群体的 29%）、Man α 1-2Man α 1-6(GalNAc beta 1-4)Man α 1-GlcN (33%) 和 Man α 1-2Man α 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%)。在人类锚中也发现了相同的聚糖核心结构，但比例略有不同（分别为 25%、52% 和 17%）。此外，在人膜二肽酶 GPI 锚中还观察到少量 (6%) 的第二结构，其具有与非还原末端甘露糖连接的额外甘露糖 α(1-2)。发现一小部分（最多 9%）的猪 GPI 锚定结构含有唾液酸，可能与 GalNAc 残基有关。发现猪 GPI 锚含有 2.5 mol 乙醇胺/mol 锚。负离子电喷雾质谱分析显示猪膜二肽酶锚中仅存在二酰基磷脂酰肌醇（主要是二硬脂酰基磷脂酰肌醇和少量硬脂酰基棕榈酰磷脂酰肌醇）。用胰蛋白酶消化猪膜二肽酶，并通过去除脱水胰蛋白酶-琼脂糖上的其他胰蛋白酶肽来将C末端肽附着在GPI锚定点上60°。该肽的序列确定为 Thr-Asn-Tyr-Gly-Tyr-Ser，从而将 GPI 锚定点的附着位点鉴定为 Ser(368)。这项工作代表了对猪膜二肽酶 GPI 锚定结构的全面研究，也是对同一蛋白质上哺乳动物 GPI 锚定结构的首次种间比较。
The glycan core structures of the glycosyl-phosphatidylinositol (GPI) anchors on porcine and human renal membrane dipeptidase (EC 3.4.13.19) were determined following deamination and reduction by a combination of liquid chromatography, exoglycosidase digestions, and methylation analysis. The glycan core was found to exhibit microheterogeneity with three structures observed for the porcine GPI anchor: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN (29% of the total population), Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-GlcN (33%), and Man alpha 1-2Man alpha 1-6(Gal beta 1-3GalNAc beta 1-4)Man alpha 1-4GlcN (38%). The same glycan core structures were also found in the human anchor but in slightly different proportions (25, 52, and 17%, respectively). Additionally, a small amount (6%) of the second structure with an extra mannose alpha(1-2)-linked to the non-reducing terminal mannose was also observed in the human membrane dipeptidase GPI anchor. A small proportion (maximally 9%) of the porcine GPI anchor structures was found to contain sialic acid, probably linked to the GalNAc residue. The porcine GPI anchor was found to contain 2.5 mol of ethanolamine/mol of anchor. Negative-ion electrospray-mass spectrometry revealed the presence of exclusively diacyl-phosphatidylinositol (predominantly distearoyl-phosphatidylinositol with a minor amount of stearoyl-palmitoyl-phosphatidylinositol) in the porcine membrane dipeptidase anchor. Porcine membrane dipeptidase was digested with trypsin and the C-terminal peptide attached 60 the GPI anchor isolated by removal of the other tryptic peptides on anhydrotrypsin-Sepharose. The sequence of this peptide was determined as Thr-Asn-Tyr-Gly-Tyr-Ser, thereby identifying the site of attachment-of the GPI anchor as Ser(368). This work represents a comprehensive study of the GPI anchor structure of porcine membrane dipeptidase and the first interspecies comparison of mammalian GPI anchor structures on the same protein.