ENHANCED PLASMIN INHIBITION BY A REACTIVE CENTER LYSINE MUTANT OF THE KUNITZ-TYPE PROTEASE INHIBITOR DOMAIN OF THE AMYLOID BETA-PROTEIN PRECURSOR
ENHANCED PLASMIN INHIBITION BY A REACTIVE CENTER LYSINE MUTANT OF THE KUNITZ-TYPE PROTEASE INHIBITOR DOMAIN OF THE AMYLOID BETA-PROTEIN PRECURSOR
复制标题
DOI：
10.1074/jbc.270.39.22827
复制
发表时间：
1995-09-29
影响因子：
4.8
通讯作者：
RASCHKE, WC
中科院分区：
文献类型：
--
作者：
VANNOSTRAND, WE;SCHMAIER, AH;RASCHKE, WC
阿尔茨海默病相关蛋白，淀粉样β蛋白前体（A β PP），含有与Kunitz型丝氨酸蛋白酶抑制剂（KPI）同源的结构域。A β PP的重组KPI结构域是凝血因子XIa和IXa的有效抑制剂，并在体外起抗凝剂的作用。在这里，我们报告了A β PP的KPI结构域的反应中心赖氨酸突变体（KPI-Lys（17））的表达、纯化和表征。A β PP的KPI-Lys（17）结构域的表达质粒编码A β PP cDNA的氨基酸285-345，其在KPI结构域中的精氨酸17处含有赖氨酸取代。将分泌的61-氨基酸产物纯化至均一并进行功能表征。将KPI-Lys（17）结构域的蛋白酶抑制性质与A β PP的天然KPI结构域的蛋白酶抑制性质进行比较。两个KPI结构域同等地抑制胰蛋白酶、胰凝乳蛋白酶和凝血因子IXa和Xa。然而，与A β PP的天然KPI结构域相比，KPI-Lys（17）结构域是凝血因子XIa的有效抑制剂，约低25倍，导致活化部分凝血活酶时间的延长显著减少。另一方面，在显色底物测定和纤溶测定中，KPI-Lys（17）结构域分别是A β PP的天然KPI结构域的约10倍和5倍更好的纤溶酶抑制剂。总之，这些研究表明，与A β PP的天然KPI结构域相比，KPI-Lys（17）结构域具有增强的抗纤维蛋白溶解和减弱的因子XIa抑制特性。
The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys(17)). An expression plasmid for the KPI-Lys(17) domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys(17) domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys(17) domain was an approximate to 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys(17) domain was an approximate to 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys(17) domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.