与人类凝血因子IX第61位丝氨酸O-连接的四糖NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→3)Fucα1的鉴定及结构分析

Identification and structural analysis of the tetrasaccharide NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-linked to serine 61 of human factor IX.

作者信息

Harris R J, van Halbeek H, Glushka J, Basa L J, Ling V T, Smith K J, Spellman M W

机构信息

Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.

出版信息

Biochemistry. 1993 Jul 6;32(26):6539-47. doi: 10.1021/bi00077a007.

DOI:10.1021/bi00077a007

PMID:8329384

Abstract

O-Linked fucose has been found attached to Thr/Ser residues within the sequence Cys-X-X-Gly-Gly-Thr/Ser-Cys in the N-terminal EGF domains of several coagulation/fibrinolytic proteins. Carbohydrate composition and mass spectrometric analyses of tryptic and thermolytic peptides containing the corresponding site (Ser-61) in the first EGF domain of human factor IX indicated the presence of a tetrasaccharide containing one residue each of sialic acid, galactose, N-acetylglucosamine, and fucose. The Ser-61 tetrasaccharide was not susceptible to alpha-fucosidase digestion. Fragments generated during mass spectrometric analysis indicated that fucose was the attachment sugar residue. The involvement of fucose in the carbohydrate-peptide linkage was confirmed by two-dimensional 1H NMR spectroscopic analysis of the glycopeptide containing factor IX residues 57-65. The complete structure of the tetrasaccharide was obtained by methylation analysis and two-dimensional 1H TOCSY and ROESY experiments as NeuAc alpha(2-->6)Gal beta(1-->4)GlcNAc beta(1-->3)Fuc alpha 1-->O-Ser61.

摘要

在几种凝血/纤维蛋白溶解蛋白的N端表皮生长因子（EGF）结构域中，已发现O-连接的岩藻糖连接在序列Cys-X-X-Gly-Gly-Thr/Ser-Cys中的Thr/Ser残基上。对人因子IX第一个EGF结构域中含有相应位点（Ser-61）的胰蛋白酶和热解肽进行碳水化合物组成和质谱分析，结果表明存在一种四糖，其分别含有一个唾液酸、半乳糖、N-乙酰葡糖胺和岩藻糖残基。Ser-61四糖不易被α-岩藻糖苷酶消化。质谱分析过程中产生的片段表明岩藻糖是连接糖残基。通过对含有因子IX残基57-65的糖肽进行二维1H NMR光谱分析，证实了岩藻糖参与了碳水化合物-肽键的形成。通过甲基化分析以及二维1H TOCSY和ROESY实验确定了该四糖的完整结构为NeuAcα(2→6)Galβ(1→4)GlcNAcβ(1→3)Fucα1→O-Ser61。