Functional Mapping and Protein Engineering of Thrombin

凝血酶的功能图谱和蛋白质工程

• 批准号: 6991198
• 负责人: LAWRENCE L LEUNG
• 金额: $ 39.19万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6991198
• 关键词: CHO cells; antifibrinolytic agents; biotechnology; clinical research; experimental allergic encephalomyelitis; fibrin; gene targeting; genetic mapping; genetically modified animals; green fluorescent proteins; human genetic material tag; human tissue; hydrolysis; laboratory mouse; membrane fusion; microarray technology; multiple sclerosis; osteopontin; pathologic process; protein C; protein engineering; protein protein interaction; protein structure function; prothrombin; thrombin; thrombomodulin

DESCRIPTION (provided by applicant): Thrombin cleavage of osteopontin (OPN), an RGD-containing proinflammatory cytokine, greatly augments its cell interactive properties by the exposure of a new alpha4beta1and alpha9beta1 integrin binding site SVVYGLR at its C-terminus. Both OPN and alpha4beta1 integrin are important in experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis. We hypothesize that thrombin-cleavage of OPN, with its resultant enhanced interaction with alpha4beta1 inteqrin, is important in the pathogenesis of EAE. We showed that thrombin cleavage of recombinant OPN markedly increased the binding of Jurkat cells (which express alpha4beta1), and activated thrombin-activatable fibrinolysis inhibitor (TAFla) abolished this enhanced cell binding by cleaving the C-terminal arginine. TAFI is a latent plasma carboxypeptidase, which is activated by the thrombin-thrombomodulin (TM) complex on endothelial cells. We showed that TAFla is more effective than plasma carboxypeptidase N in inactivating bradykinin (BK) and activated complement C5a. We hypothesize that TAFla is an anti-inflammatory molecule, which, together with aPC, counteract the proinflammatory effects of thrombin. E229K thrombin, an engineered human thrombin with minimal procoagulant functions, activated PC and TAFI in mice, and blocked BK-induced hypotension. Aim #1 Structure-function analysis of the thrombin-OPN interaction and TAFla inactivation of the SVVYGLR site. We shall characterize thrombin cleavage of soluble vs immobilized OPN and phosphorylated vs non-phosphorylated OPN. We will determine the structural requirements on thrombin for OPN cleavage, and characterize the efficiency of TAFla cleavage of SVVYGLR and other relevant substrates. Aim #2 Cell biology of thrombin cleavage of OPN and its modulation by TAFla We hypothesize that the engagement of the RGD and the SVVYGLR sites on the thrombin-cleaved OPN fragment to RGD-dependent and RGD-independent integrins respectively will lead to different cellular effects. We have generated various GST-OPN fusion proteins that represent the two binding sites either singly or in combination. We will study their effects on Jurkat cells in terms of haptotaxis, metalloprotease secretion, cytokine release, and gene transcription profiles. We will test whether SDF-1alpha TGF-beta1, and the thrombin receptor activation peptide activate alpha4beta1on Jurkat cells. We will test whether thrombin-cleaved OPN induces a prothrombotic phenotype in endothelial cells. Aim #3 The in vivo role of TAFla in mouse inflammation model To test whether TAFla is important in modulating the proinflammatory effect of thrombin-cleaved OPN, we will compare the EAE phenotype in WT and TAFI-null mice and test the OPN-fusion proteins in an OPN-induced peritonitis model. We will confirm that E229K thrombin blockage of BK-induced hypotension is mediated by TAFI activation. We will test the role of TAFla inactivation of C5a in a neutrophil alveolitis model.

描述（由申请方提供）：骨桥蛋白（OPN）（一种含RGD的促炎细胞因子）的凝血酶裂解通过暴露其C末端的新α 4 β 1和α 9 β 1整联蛋白结合位点SVVYGLR而大大增强其细胞相互作用特性。骨桥蛋白和α 4 β 1整合素在实验性自身免疫性脑炎（EAE）（多发性硬化症的小鼠模型）中均很重要。我们推测凝血酶对骨桥蛋白的切割，以及其与α 4 β 1整合蛋白的相互作用增强，在EAE的发病机制中是重要的。我们发现，重组骨桥蛋白的凝血酶切割显著增加了Jurkat细胞（表达α 4 β 1）的结合，活化的凝血酶活化纤维蛋白溶解抑制剂（TAFla）通过切割C-末端精氨酸消除了这种增强的细胞结合。TAFI是一种潜在的血浆羧肽酶，其被内皮细胞上的凝血酶-血栓调节蛋白（TM）复合物激活。我们表明TAFla在失活缓激肽（BK）和活化补体C5 a方面比血浆羧肽酶N更有效。我们假设TAFla是一种抗炎分子，其与aPC一起抵消凝血酶的促炎作用。E229 K凝血酶，一种具有最小促凝血功能的工程化人凝血酶，在小鼠中激活PC和TAFI，并阻断BK诱导的低血压。目的#1凝血酶-OPN相互作用和SVVYGLR位点的TAFla失活的结构-功能分析。我们将表征可溶性与固定化OPN以及磷酸化与非磷酸化OPN的凝血酶裂解。我们将确定OPN切割对凝血酶的结构要求，并表征TAFla切割SVVYGLR和其他相关底物的效率。目的#2凝血酶切割OPN及其通过TAFla调节的细胞生物学我们假设凝血酶切割的OPN片段上的RGD和SVVYGLR位点分别与RGD依赖性和RGD非依赖性整联蛋白的接合将导致不同的细胞效应。我们已经产生了各种GST-OPN融合蛋白，其代表单独或组合的两个结合位点。我们将研究它们对Jurkat细胞的趋触性、金属蛋白酶分泌、细胞因子释放和基因转录谱的影响。我们将测试SDF-1 α TGF-β 1和凝血酶受体激活肽是否激活Jurkat细胞上的α 4 β 1。我们将测试凝血酶裂解的OPN是否在内皮细胞中诱导促血栓形成表型。目的#3 TAFla在小鼠炎症模型中的体内作用为了测试TAFla在调节凝血酶切割的OPN的促炎作用中是否重要，我们将比较WT和TAFI缺失小鼠中的EAE表型，并在OPN诱导的腹膜炎模型中测试OPN融合蛋白。我们将证实E229 K凝血酶阻断BK诱导的低血压是由TAFI激活介导的。我们将测试C5 a的TAFla失活在中性粒细胞肺泡炎模型中的作用。