BIOSYNTHESIS OF HEPARIN AND HEPARAN SULFATE

肝素和硫酸乙酰肝素的生物合成

• 批准号: 2130026
• 负责人: LENNART RODEN
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2130026
• 关键词: animal tissue; carbohydrate biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; fibroblasts; glucuronosyltransferase; heparan sulfate; heparin; isomerase; liver cells; protein purification; tissue /cell culture

Heparin and heparan sulfate proteoglycans are biologically active macromolecules composed of a core protein and one or more polysaccharide chains attached via an O-glycosidic linkage between xylose and serine. Among several distinct biological activities, the anticoagulant activity displayed by some heparins and heparan sulfates is the most conspicuous, and this property is now known to reside in an antithrombin-binding pentasaccharide segment of unique structure. The biosynthesis of heparins and heparan sulfates encompasses 15 enzymatic steps, which take place in three phases: a) formation of a specific carbohydrate-protein linkage region; b) assembly of repeating disaccharides (GlcUA-GlcNAc); and c) polymer modifications leading to the generation of the biological activities of the polysaccharides. Basic knowledge about the properties of the enzymes catalyzing these reactions is meager, and, to date, only two of the enzymes have been purified to homogeneity. Our long-term objectives are to establish the structures and mechanisms of action of some of these enzymes, to determine how the synthetic apparatus is organized in the intracellular membranes, and to elucidate how the synthesis of the polysaccharides is regulated by intrinsic mechanisms at the molecular and cellular levels as well as by extrinsic influences on the intact cells. In the immediate future, our investigations will be focused on two reactions, glucuronysyl transfer to galactose residues during linkage region formation, which may be a rate-limiting step in the overall assembly process, and uronosyl 5-epimerization, which is a polymer modification essential to the formation of the antithrombin- binding sites. The following specific aims will be pursued: 1) purification of heparosan N-sulfate D-glucuronosyl 5-epimerase; 2) characterization of the purified enzyme with respect to molecular and catalytic properties; 3) development of simplified methodology for the assay of glucuronosyltransferase I and the closely related transferase involved in the biosynthesis of the HNK-1 antigen; 4) purification of the two glucuronosyltransferases; and 5) investigations of the metabolism of the 5-epimerase, with initial emphasis on its turnover in cultured skin fibroblasts and hepatocytes. Research in this general area has an intrinsic value, inasmuch as it advances our basic knowledge of important processes in the human body, but it also has a direct and proven clinical value in that it has led to improved forms of antithrombotic therapy and has aided in the diagnosis of genetic metabolic deficiencies.

肝素和硫酸乙酰肝素蛋白聚糖具有生物活性 由核心蛋白和一种或多种多糖组成的大分子 通过木糖和丝氨酸之间的O-糖苷连接连接的链。 在几种不同的生物学活动中，抗凝活性 某些肝素和乙酰硫酸盐展示是最明显的， 现在已知该特性居住在抗凝血酶结合中 独特结构的五糖段。 生物合成 肝素和乙酰硫酸硫酸盐包含15个酶促步骤，该步骤采用 分为三个阶段：a）特定碳水化合物蛋白的形成 连锁区域； b）重复二糖（Glcua-glcnac）的组装； c）聚合物修饰导致生物的产生 多糖的活动。 关于属性的基本知识 催化这些反应的酶是微薄的，迄今为止仅 两个酶已被纯化为均匀性。 我们的长期 目标是建立作用的结构和机制 这些酶中的一些，以确定合成设备的方式 在细胞内膜中组织，并阐明 多糖的合成受到固有机制的调节 分子和细胞水平以及外在影响 完整的细胞。 在不久的将来，我们的调查将是 专注于两个反应，葡萄糖醛酸转移到半乳糖残基 在连锁区域的形成期间，这可能是限制步骤 整体组装过程和uronosyl 5-滴定，这是一个 聚合物修饰对于抗凝血酶形成至关重要 绑定位点。 将追求以下具体目标：1） 肝素N-硫酸盐d-葡萄糖基5-二二聚体酶的纯化； 2） 相对于分子和 催化特性； 3）开发简化的方法论 葡萄糖基转移酶I和密切相关的转移酶的测定 参与HNK-1抗原的生物合成； 4）纯化 两个葡萄糖糖基转移酶； 5）调查 5- epimerase，最初强调其在培养的皮肤中的周转 成纤维细胞和肝细胞。 在这个一般领域的研究有 固有的价值，因为它可以提高我们对重要的基本知识 人体中的过程，但也有直接且经过验证的临床 价值导致改善形式的抗血栓疗法和 有助于诊断遗传代谢缺陷。