Kusche M, Bäckström G, Riesenfeld J, Petitou M, Choay J, Lindahl U
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
J Biol Chem. 1988 Oct 25;263(30):15474-84.
The antithrombin-binding region in heparin is a pentasaccharide sequence with the predominant structure GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-IdoA -(2-OSO3)-GlcNSO3(6-OSO3) (where GlcA and IdoA represent D-glucuronic and L-iduronic acid, respectively), in which the 3-O-sulfate residue on the internal glucosaminyl unit is a marker group for this particular region of the polysaccharide molecule. A heparin octasaccharide which contained the above pentasaccharide sequence was N/O-desulfated and re-N-sulfated and was then incubated with adenosine 3'-phosphate 5'-phospho[35S]sulfate in the presence of a microsomal fraction from mouse mastocytoma tissue. Fractionation of the resulting 35S-labeled octasaccharide on antithrombin-Sepharose yielded a high affinity fraction that accounted for approximately 2% of the total incorporated label. Structural analysis of this fraction indicated that the internal glucosamine unit of the pentasaccharide sequence was 3-O-35S-sulfated, whereas both adjacent glucosamine units carried 6-O-[35S]sulfate groups. In contrast, the fractions with low affinity for antithrombin (approximately 98% of incorporated 35S) showed no consistent O-35S sulfation pattern and essentially lacked glucosaminyl 3-O-[35S]sulfate groups. It is suggested that the 3-O-sulfation reaction concludes the formation of the antithrombin-binding region. This proposal was corroborated in a similar experiment using a synthetic pentasaccharide with the structure GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-Id oA (2-OSO3)-GlcNSO3(6-OSO3) as sulfate acceptor. This molecule corresponds to a functional antithrombin-binding region but for the lack of a 3-O-sulfate group at the internal glucosamine unit. The 35S-labeled pentasaccharide recovered after incubation bound with high affinity to antithrombin-Sepharose and contained a 3-O-[35S]sulfate group at the internal glucosamine residue as the only detectable labeled component. The use of this pentasaccharide substrate along with the affinity matrix provides a highly specific assay for the 3-O-sulfotransferase.
肝素中的抗凝血酶结合区域是一个五糖序列，其主要结构为GlcNAc(6-OSO3)-GlcA-GlcNSO3(3,6-二-OSO3)-IdoA-(2-OSO3)-GlcNSO3(6-OSO3)（其中GlcA和IdoA分别代表D-葡萄糖醛酸和L-艾杜糖醛酸），其中内部氨基葡萄糖单元上的3-O-硫酸酯残基是多糖分子这一特定区域的标记基团。一个含有上述五糖序列的肝素八糖进行了N/O-脱硫酸化和重新N-硫酸化，然后在来自小鼠肥大细胞瘤组织的微粒体部分存在的情况下，与腺苷3'-磷酸5'-磷酸[35S]硫酸盐一起孵育。在抗凝血酶-琼脂糖上对所得的35S标记的八糖进行分级分离，得到一个高亲和力级分，其占总掺入标记的约2%。对该级分的结构分析表明，五糖序列的内部氨基葡萄糖单元被3-O-35S-硫酸化，而两个相邻氨基葡萄糖单元带有6-O-[35S]硫酸酯基团。相反，对抗凝血酶亲和力低的级分（约占掺入35S的98%）没有一致的O-35S硫酸化模式，并且基本上缺乏氨基葡萄糖基3-O-[35S]硫酸酯基团。有人提出3-O-硫酸化反应完成了抗凝血酶结合区域的形成。在使用具有结构GlcNSO3(6-OSO3)-GlcA-GlcNSO3(6-OSO3)-IdoA(2-OSO3)-GlcNSO3(6-OSO3)的合成五糖作为硫酸酯受体的类似实验中，这一建议得到了证实。该分子对应于一个功能性抗凝血酶结合区域，但内部氨基葡萄糖单元缺少一个3-O-硫酸酯基团。孵育后回收的35S标记五糖以高亲和力与抗凝血酶-琼脂糖结合，并且在内部氨基葡萄糖残基处含有一个3-O-[35S]硫酸酯基团作为唯一可检测的标记成分。使用这种五糖底物以及亲和基质为3-O-磺基转移酶提供了一种高度特异性的测定方法。
