含抗凝血酶III结合位点的肝素寡糖的酶法制备。

Enzymatic preparation of heparin oligosaccharides containing antithrombin III binding sites.

作者信息

Toida T, Hileman R E, Smith A E, Vlahova P I, Linhardt R J

机构信息

Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.

出版信息

J Biol Chem. 1996 Dec 13;271(50):32040-7. doi: 10.1074/jbc.271.50.32040.

DOI:10.1074/jbc.271.50.32040

PMID:8943254

Abstract

Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.

摘要

为了制备具有完整抗凝血酶III结合位点的寡糖，使用肝素裂解酶I（EC 4.2.2.7）对肝素进行部分解聚，从而制备出两种新的寡糖。这些寡糖通过基于大小和电荷的色谱法进行纯化，并通过毛细管电泳显示出高纯度。在500 MHz下进行的一维和二维1H NMR光谱揭示了每种寡糖的结构。八糖和十糖分别为ΔUAp2S(1→4)-α-DGlcNpS6S(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpAc6S(1→4)-β-D-GlcAp(1→4)-α-D-GlcNpS 3S6S(1→4)-α-L-IdoAp2S(1→4)α-D-GlcNpS6S（其中ΔUAp为4-脱氧-α-L-苏式-己烯吡喃糖醛酸，GlcNp为2-氨基-2-脱氧-吡喃葡萄糖，GlcAp为吡喃葡萄糖醛酸，S为硫酸根，Ac为乙酰基）和ΔUAp2S(1→4)-α-D-GlcNpS6S(1→4)-α-L-IdoAp++ +(1→4)-α- D-GlcNpAc6S (1→4)-β-D-GlcAp(1→4)-α-D-GlcNpS3S6S(1→4)-α-L-IdoAp2S (1→4)-α-D-GlcNpS6S(1→4)-α-L-IdoAp2S(1→4)-α -D-GlcNpS 6S。还分离出一种六糖，其含有与抗凝血酶III结合位点中发现的结构基序相似的结构基序，并且具有大大降低的抗凝血活性。该六糖的结构为ΔUAp2S(1→4)-α-D-GlcNpAc6S(1→4)-β-D-GlcAp++ +(1→4)-α- D-GlcNpS3S6S(1→4)-α-L-IdoAp(1→4)-α-D-GlcNpS6S。获得了足够量的十糖，以使用微量滴定热法研究其与抗凝血酶III的相互作用。该十糖与抗凝血酶III的结合亲和力与肝素相似，并显示出相当的抗凝血活性，这是使用抗凝血酶III依赖性抗因子Xa测定法确定的。有趣的是，虽然十糖和肝素都以纳摩尔亲和力与抗凝血酶结合，但观察到的结合热非常少。