Kusche M, Hannesson H H, Lindahl U
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Biochem J. 1991 Apr 1;275 ( Pt 1)(Pt 1):151-8. doi: 10.1042/bj2750151.
A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)----(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.
先前发现，来自大肠杆菌K5的荚膜多糖具有与肝素生物合成中未硫酸化的前体多糖相同的结构，即[-(4)βGlcA(1)----(4)αGlcNAc(1)-]n[范恩、施密特、扬恩和扬恩（1981年），《欧洲生物化学杂志》116卷，359 - 364页]。将K5多糖进行N - 脱乙酰化（通过肼解）和N - 硫酸化，然后在3'-磷酸5'-磷酰[35S]硫酸腺苷（[35S]PAPS）存在的情况下，与来自产生肝素的小鼠肥大细胞瘤的去污剂溶解酶一起孵育。对所得的35S标记多糖的结构分析表明，形成了肝素中发现的所有主要二糖单元。鉴定出2 - O - [35S]硫酸化的艾杜糖醛酸（L - 艾杜糖醛酸）以及6 - O - [35S]硫酸化的葡糖胺硫酸酯单元，这表明修饰后的K5多糖在肝素生物合成中参与的己糖醛酸C - 5 - 表异构酶和主要的O - 硫酸转移酶反应中作为底物。只有当孵育中存在PAPS时，天然（N - 乙酰化）大肠杆菌多糖的GlcA单元才会被表异构酶作用，而化学N - 硫酸化多糖的GlcA单元在没有PAPS的情况下也会发生表异构化，这与表异构化需要N - 硫酸基团的观点一致。随着PAPS浓度的增加，单O - 硫酸化二糖单元 - IdoA(2 - OSO3)-GlcNSO3逐渐转化为双O - 硫酸化物种 - IdoA(2 - OSO3)-GlcNSO3(6 - OSO3)-。发现一小部分35S标记的多糖与蛋白酶抑制剂抗凝血酶具有高亲和力结合。这一比例随着PAPS浓度的增加而增加，直至达到相当于总掺入35S的约1 - 2%的水平。因此，溶解的酶催化了产生功能性抗凝血酶结合位点所需的所有反应。
