STRUCTURE AND FUNCTION OF HEPARIN COFACTOR II

肝素辅因子 II 的结构和功能

• 批准号: 6184419
• 负责人: DOUGLAS M TOLLEFSEN
• 金额: $ 46.09万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6184419
• 关键词: X ray crystallography; binding proteins; carbohydrate structure; chemical association; chondroitin sulfate B; cofactor; embryonic stem cell; enzyme activity; enzyme inhibitors; enzyme structure; enzyme substrate complex; genetically modified animals; heparin; human tissue; immunocytochemistry; laboratory mouse; mucopolysaccharides; mutant; protein structure function; synthetic peptide; thrombin

DESCRIPTION: Six specific aims are identified. These include: 1) investigating the mechanism by which heparin facilitates dissociation between thrombin the HCII mutant Leu444 Arg; 2) to determine the affects of synthetic reactive site peptides on the ability of heparin cofactor 2 to serve as an inhibitor or substrate; 3) to identify structures in dermatan sulfate required for high-affinity binding to heparin cofactor II; 4) to crystallize heparin cofactor II and its complexes with dermatan sulfate for x-ray analysis; 5) to determine the location of endogenous heparin cofactor II in human and murine tissues by immunohistochemistry; 6) to prepare by homologous recombination, homozygous heparin cofactor II-deficient mice. The first Specific Aim is based on an observation by the investigator that for HCII Leu444 Arg, the complex with thrombin can be reversed if heparin is present, while the complex is stable in the presence of dermatan sulfate or in the absence of the added cofactor. The data obtained thus far suggests a model which involves the initial reversible complex of inhibitor and enzyme to an initially reversible covalent bond and subsequently to an SDS irreversible covalent bond. The studies anticipated with this mutant will allow elucidation and confirmation of this attractive mechanism. In the second Specific Aim, peptides which mimic the reactive site loop present in all serpins will be evaluated with respect to their ability to influence HCII inhibitory functions. Various loop peptides incorporating radiolabels with different sequences will be used to evaluate the interaction between the reactive site loop and the beta sheet will be permissible. Using binding and activity measurement. The peptide will also permit evaluation of the influence of glycosaminoglycans on the conformational-linkage between the various states of HCII. An evaluation of thrombin inhibition by binary complexes of synthetic peptide and HCII in the presence and absence of glycosaminoglycans will be evaluated using activity for the formation of covalent complexes with 125[I] thrombin. The third Specific Aim, tetra- and penta-sulfated hexasacchrides from porcine skin dermatan sulfate will be evaluated with respect to their structure and their ability to influence HCII binding and function. The techniques of approach are fairly common in the identification of glycosaminoglycan structure involved classical carbohydrate chemistry. In the further Specific Aim HCII and its complexes will be subjected to hanging drop crystallization techniques in an attempt to produce crystals of both the protein and its complexes suitable for crystallographic evaluation. The fifth and sixth Specific Aims involve the generation of a murine HCII deficiency model. Studies involve histochemistry in both human and murine systems to identify the extravascular sites of HCII. The development of a murine deletion model by homologous recombination in embryonic stem cells using the now standard techniques.

描述：确定了六个具体目标。 这些措施包括：1） 研究肝素促进解离的机制 凝血酶与HCII突变体Leu 444 Arg之间的关系; 2）以确定凝血酶与HCII突变体Leu 444 Arg之间的关系。 人工合成的活性部位肽对肝素抗凝能力的影响 辅因子2作为抑制剂或底物; 3）鉴定 高亲和力结合所需的硫酸皮肤素结构 肝素辅因子II; 4）使肝素辅因子II及其 与硫酸皮肤素的复合物用于X射线分析; 5） 确定内源性肝素辅因子II在人体内的位置， 小鼠组织免疫组化; 6）制备同源 重组，纯合子肝素辅因子II缺陷小鼠。 第一个具体目标是基于研究者的观察 对于HCII Leu 444， Arg，与凝血酶的复合物可被逆转 如果存在肝素，则复合物在肝素存在下是稳定的， 硫酸皮肤素或不存在添加的辅因子。 数据 到目前为止获得的建议，其中涉及初始模型 抑制剂和酶的可逆复合物， 共价键，随后转化为SDS不可逆共价键。 对这种突变体的预期研究将允许阐明和 证实了这种吸引人的机制。在第二个具体目标中， 模拟存在于所有丝氨酸蛋白酶抑制剂中的反应位点环的肽将 评价其影响HCII抑制的能力 功能协调发展的 掺入放射性标记的各种环肽， 将使用不同的序列来评估 反应位点环和β折叠将是允许的。使用 结合和活性测量。肽还将允许评估 糖胺聚糖对构象连接的影响 在HCII的不同状态之间。凝血酶抑制的评价 通过合成肽和HCII的二元复合物， 和不存在糖胺聚糖将使用活性 用于与125[I]凝血酶形成共价复合物。 第三 特定目标，来自猪的四硫酸化和五硫酸化六糖 将评价硫酸皮肤素 结构及其影响HCII结合和功能的能力。 方法的技术是相当常见的识别 糖胺聚糖结构涉及经典的碳水化合物 化学.在进一步的具体目标中，HCII及其复合物将 进行悬滴结晶技术， 以产生蛋白质及其复合物的晶体， 晶体学评价 第五和第六项具体目标涉及 鼠HCII缺陷模型的产生。 研究涉及 在人和鼠系统中进行组织化学，以鉴定 HCII的血管外部位。 小鼠基因缺失的发展 在胚胎干细胞中同源重组的模型， 现在是标准技术。