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Structural and Functional Studies of a Protease That Preferentially Degrades Oxidized Proteins

优先降解氧化蛋白的蛋白酶的结构和功能研究

基本信息

批准号：
09672256
负责人：
BEPPU Masatoshi
金额：
$ 1.92万
依托单位：
Tokyo University of Pharmacy and Life Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672256/
关键词：
Protease Serine protease Oxidative stress ACPH Proteolysis Erythrocyte protease Glycation Oxidized protein グリケーション

项目摘要

In this project, we have carried out studies on the primary structure and the function of a serine protease of human erythrocytes that preferentially degrades oxidatively modified proteins (Oxidized Protein Hydrolase : OPH), and obtained the following findings.1) STUDY ON THE PRIMARY STRUCTUREThe enzyme OPH was purified from the cytosol of human erythrocytes according the method we have previously established, and some peptide fragments were separated after degradation of the purified enzyme by endopeptidase. The amino acid sequence of these peptides were found to be highly homologous to the sequence of human acylpeptide hydrolase (ACPH), an enzyme that hydrolyzes an acylated N-terminal amino acid residue of protein. We, then, prepared recombinant human ACPH (rACPH), by cloning of human ACPH cDNA from cDNAs of human erythroblasts based on the known nucleotide sequence of human ACPH.The rACPH exhibited proteolytic activity on oxidized and glycated proteins, and reacted with specific antibody to OPH.Conversely, OPH exhibited ACPH activity. We concluded that OPH is ACPH.This is the first work that elucidated the structure of oxidized protein-selective protease, although presence of several kinds of such proteases have been reported.2) STUDY ON ITS FUNCTIONOPH purified from human erythrocyte cytosol strongly bound to oxidized membranes of human erythrocytes. This enzyme was found to be present more on oxidized erythrocyte membranes than on unoxidized membranes. These results suggested that OPH plays an important role in degrading oxidized proteins of cell membrane.

在本项目中，我们对优先降解氧化修饰蛋白的人红细胞丝氨酸蛋白酶的一级结构和功能进行了研究（氧化蛋白水解酶：OPH），并获得了以下结果：1）一级结构的研究OPH酶是根据我们以前建立的方法从人红细胞胞质溶胶中纯化的，纯化的酶经内肽酶降解后，分离出部分肽段。发现这些肽的氨基酸序列与人酰基肽水解酶（ACPH）的序列高度同源，该酶水解蛋白质的酰化N-末端氨基酸残基。根据已知的人ACPH的核苷酸序列，从人成红细胞cDNA中克隆了人ACPH cDNA，制备了重组人ACPH（rACPH），rACPH对氧化蛋白和糖基化蛋白具有蛋白水解活性，并能与OPH特异性抗体反应，而OPH则具有ACPH活性。本文首次阐明了氧化蛋白选择性蛋白酶的结构，虽然目前还没有几种氧化蛋白选择性蛋白酶的存在。2）功能研究从人红细胞胞浆中纯化的OPH与人红细胞氧化膜有很强的结合。发现这种酶在氧化红细胞膜上比在未氧化膜上存在更多。这些结果表明，OPH在降解细胞膜氧化蛋白中起重要作用。