Functional Mapping and Protein Engineering of Thrombin

描述(由申请人提供)：骨桥蛋白(OPN)的凝血酶裂解是一种含有RGD的促炎细胞因子，通过在其C端暴露新的整合素α4beta1和alpha9beta1整合素结合位点SVVYGLR，大大增强了其细胞相互作用特性。OPN和α4beta1整合素在实验性自身免疫性脑炎(EAE)中都很重要，EAE是多发性硬化症的小鼠模型。我们推测，OPN的凝血酶裂解及其与α4β1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OPN的凝血酶切割显著增加了Jurkat细胞(表达α4β1)的结合，激活的凝血酶激活的纤溶抑制物(TAF1a)通过裂解C端精氨酸而取消了这种增强的细胞结合。TAFI是一种潜在的血浆羧肽酶，由内皮细胞上的凝血酶-血栓调节蛋白(TM)复合体激活。我们发现TAF1a在灭活缓激肽(BK)和激活补体C5a方面比血浆羧肽酶N更有效。我们假设TAF1a是一种抗炎分子，它与APC一起抵消凝血酶的促炎作用。E229K凝血酶是一种具有最小促凝血功能的工程化人类凝血酶，可激活小鼠的PC和TAFI，并阻断BK诱导的降压。目的#1凝血酶-骨桥蛋白相互作用和SVVYGLR位点TAFla失活的结构-功能分析。我们将对可溶性和固定化OPN以及磷酸化和非磷酸化OPN的凝血酶裂解进行表征。我们将确定OPN裂解对凝血酶的结构要求，并表征SVVYGLR和其他相关底物的TAFla裂解效率。目的#2凝血酶裂解OPN的细胞生物学及TAF1a对其的调节我们假设凝血酶裂解的OPN片段上的RGD和SVVYGLR位点分别与RGD依赖和RGD非依赖的整合素结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，它们单独或组合代表这两个结合位点。我们将从亲和力、金属蛋白酶分泌、细胞因子释放和基因转录谱等方面研究它们对Jurkat细胞的影响。我们将测试SDF-1α转化生长因子-β1和凝血酶受体激活肽是否激活Jurkat细胞上的α4β1。我们将测试凝血酶裂解的OPN是否诱导内皮细胞血栓前表型。目的#3TAF1a在小鼠炎症模型中的体内作用为了验证TAF1a是否在调节凝血酶裂解的OPN的促炎效应中起重要作用，我们将比较WT和Tafi缺失小鼠的EAE表型，并在OPN诱导的腹膜炎模型中检测OPN融合蛋白。我们将证实E229K凝血酶阻断BK诱导的低血压是通过激活TAFI介导的。我们将在中性粒细胞肺泡炎模型中测试TAFla灭活C5a的作用。

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.