Analysis of the funcional domains of the heavy chain of kininogens by using the technique of site directed mutagenesis

利用定点诱变技术分析激肽原重链的功能域

• 批准号: 05670118
• 负责人: OHKUBO Iwao
• 金额: $ 1.34万
• 依托单位: Shiga University of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670118/
• 关键词: Mutagenesis; Functional domains; Kininogens; Bacuro virus; Eukaryotic cells; Prokaryotic cells; 重鎖; ミュタジェネシス; 発現

High and low molecular weight kininogens strongly inactivate cysteine proteases such as cathepsin B,H and L,calpain 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 region as potential candidates for the reactive site toward cysteine proteases : Gln-Val-Val-Ala-Gly (QVVAG) located at two-thirds of the distance from the N-terminus of the domains and Glycine (Gly) located at 44 amino acid residues upstream from the GVVAG region.In this project, by using a cDNA coding for human low molecular weight kininogen, we had attempted to express the wild type protein and proteins with deletions of domain 2 and 3, and with deletions or substitutions of the QVVAG and Gly regions, in the eukaryotic cells such as BHK and CHO,in Bacureo virus system cells and in the prokaryotic cells such as E.coli.Although the amounts of the wild type of low molecular weight kininogen with deletion of domains 2 or 3 (<less than or equal>50ng/ml) expressed by the induction with dexamethsone in BHK cells were very low. As the next steps, using site-directed cDNA for low molecular weight kininogen, we have been attemptting to express the wild type and other proteins with deletion and/or substitution in Bacuro virus system and in E

高、低分子激肽原以非共价和非竞争性方式强烈失活组织蛋白酶B、H和L、钙蛋白酶I和II、木瓜酶和无花果蛋白等半胱氨酸蛋白酶。这两种激肽原都被认为在半胱氨酸蛋白酶的调节中起着重要的生理作用。在两种激肽原的共同重链上都有两个抑制域。推测结构域2和3含有两个可能的半胱氨酸蛋白酶反应位点的候选区域：位于结构域N末端三分之二的Gln-Val-Val-Ala-Gly(QVVAG)和位于GVVAG区上游44个氨基酸残基的甘氨酸(Gly)。在本项目中，我们试图通过编码人低分子激肽原的cDNA2和3，以及QVVAG和Gly区的缺失或替换，在BHK和CHO等真核细胞中表达野生型和野生型蛋白在Bacureo病毒系统细胞和原核细胞中，虽然野生型低分子激肽原的量与结构域2或3的缺失(&lt；用地塞米松诱导BHK细胞表达的-GT；50 ng/ml)很低。下一步，我们利用低分子激肽原的定点cdna，尝试在杆状病毒系统和E。

项目成果

Ohkubo I, Tada T, Ochiai Y, Ueyama H, Eimoto T and Sasaki M: "Human seminal plasma β-microseminoprotein: Its purification, characterization, and immunohistochemical localization." Int. J. Biochem&Cell Biol. in press.(1995)

Ohkubo I、Tada T、Ochiai Y、Ueyama H、Eimoto T 和 Sasaki M：“人精浆 β-微精蛋白：其纯化、表征和免疫组织化学定位”，《生物化学与细胞生物学杂志》出版。

Maruo K,Akaike T,Ohkubo I,Ono T and Maeda H: "Effect of microbial and mite proteases on low and high molecular weight kininogens : Generation of kinin and inactivation of thiol protease inhibitory activity." J.Biol.Chem. 268. 17711-17715 (1993)

Maruo K、Akaike T、Ohkubo I、Ono T 和 Maeda H：“微生物和螨蛋白酶对低分子量和高分子量激肽原的影响：激肽的生成和硫醇蛋白酶抑制活性的失活。”

Ohkubo I,Tada T,Ochiai Y,Ueyama H,Eimoto T and Sasaki M: "Human seminal plasma beta-micro-seminoprotein : Its purification, characterization, and immunohistochemical localization." Int.J.Biochem & Cell Biol. (in press.).

Ohkubo I、Tada T、Ochiai Y、Ueyama H、Eimoto T 和 Sasaki M：“人精浆 β-微精蛋白：其纯化、表征和免疫组织化学定位。”

Yasuhara O, Hanai K, Ohkubo I, McGeer PL and Kimura H.: "Expression of cystatin C in rat,monkey and human brains." Brain Res.628. 85-92 (1993)

Yasuhara O、Hanai K、Ohkubo I、McGeer PL 和 Kimura H.：“胱抑素 C 在大鼠、猴子和人脑中的表达。”

Sasaki M and Ohkubo I: "Molecular recogintion of proteases associated with cascade reaction." Biological Functions of Proteases and Inhibitors (edt.by Katunuma N,Suzuki K and Fritz H). Scientific Societies Press, Tokyo, Japan. 61-71 (1994)

Sasaki M 和 Ohkubo I：“与级联反应相关的蛋白酶的分子识别。”