Determination of the reactive site (s) of kininogens for cysteine proteases using technique of site-directed mutagenesis

高分子量和低分子量激肽原以非共价和非竞争性的方式强烈灭活半胱氨酸蛋白酶，如组织蛋白酶B、H和L、钙蛋白酶I和钙蛋白酶II、木瓜蛋白酶和非竞争性蛋白酶。这两种激肽原在半胱氨酸蛋白酶的调控中都起着重要的生理作用。在这两种激肽原的共同重链上有两个抑制结构域。据推测，结构域2和3包含两个区域作为半胱氨酸蛋白酶活性位点的潜在候选区域：Gln-Val-Val-Ala-Gly （QVVAG）位于距结构域n端约三分之二距离的位置，而Gly位于QVVAG区域上游44个氨基酸残基的位置。在本项目中，我们利用编码人类低分子量激肽原的cDNA，试图在真核细胞如BHK、COS和CHO中表达野生型蛋白和2、3结构域缺失以及QVVAG和Gly区域缺失或取代的蛋白。虽然地塞米松诱导的野生型蛋白和2、3结构域缺失蛋白（<50ng/ml）在BHK细胞中的表达量很低，但我们从细胞中收集了这三种蛋白（各约5l），用Red-Sepharose和Mono Q柱层色谱对这三种蛋白进行了部分纯化，最终得到每种蛋白约3ug。现在，我们正试图分析它们的动力学参数，并确定半胱氨酸蛋白酶激原的真正活性位点。

High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B, H and L, calpains I and II, papain, and ficin in noncovalent and noncompetitive manners. It is thought that both kininogens play important physiological roles in the regulation of cysteine proteases. There are two inhibitory domains on the common heavy chain of both kininogens. It is speculated that domains 2 and 3 contain two regions as potential candidates for the reactive site of cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at approximately two-thirds of the distance from the N-terminus of the domains and Gly located at 44 amino acid residues upstream from the QVVAG region.In this project, using a cDNA coding human low molecular weight kininogen, we have attemed to express the wild type protein and proteins with deletions of domains 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in eukaryotic cells such as BHK, COS and CHO.Although the amounts of the wild type protein and two proteins with deletions of domains 2 or 3 (<50ng/ml) expressed by the induction with dexamethasone in BHK cells were very low, we collected these mediums from the cells (totally about 5 L each), partially purified these three proteins by using Red-Sepharose and Mono Q column chromatographies, and finally obtained about 3 ug of each protein. Now, we are attempting to analyze their kinetic parameters, and determine the real reactive site (s) of kininogens for cysteine proteases.