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RECOMBINANT HUMAN MAST CELL TRYPTASES

重组人肥大细胞类胰蛋白酶

基本信息

批准号：
6159344
负责人：
David Andrew Johnson
金额：
$ 12.7万
依托单位：
EAST TENNESSEE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-01 至 2004-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6159344
关键词：
Saccharomyces biotechnology enzyme activity fibronectins mast cell recombinant proteins site directed mutagenesis tryptase vitronectin

项目摘要

DESCRIPTION (from applicant's abstract): Human mast cell tryptases (trypsin-like serine proteases) are the major protein products of mast cells suggesting that they play an important role in the function of mast cells. Based on published cDNA sequences there are four different human tryptases and the principal investigator hypothesizes that these different mast cell tryptases have different functional characteristics that allow for different functions. The presence of mast cells in almost all tissues indicate that tryptases have significant functions in native an pathological conditions and the general lack of knowledge concerning how these enzymes differ in function provide the rationale for the proposal. The central hypothesis is that the different mast cell tryptases have evolved to serve different functions and that knowledge of the structures and functions of these enzymes is critical to understanding the roles of mast cells in health and disease. In preliminary studies the principal investigator has expressed and purified fully active recombinant human skin tryptase 1 (rHST1) in Pichia pastoris. In Aim 1 site-directed mutagenesis of rHST1 cDNA will be used to express human tryptase beta/2 and tryptase alpha in Pichia pastoris. In Aim 2 Tryptases 1, beta/2 and alpha will be characterized and compared with regard to heparin binding and kinetics on synthetic and natural substrates to define their differences and similarities. In Aim 3 based on the finding that human lung tryptase rapidly and specifically cleaves the single RGD motif in fibrinogen that is bound by alphav-beta-3 integrins, other proteins with RGD sites, including vitronectin and fibronectin, will be examined for cleavage by the three rHTs. This work will provide needed comparative data on tryptases of known sequence and should yield insight as to why humans have such similar, yet different, enzymes in mast cells. Support will allow this laboratory to continue the study of tryptases and the mentoring of undergraduate students, while gaining knowledge that could improve our understanding of the roles of mast cells in diseases, such as asthma, arthritis, cancer, and cardiovascular diseases.

描述(摘自申请者摘要)：人肥大细胞类胰蛋白酶 (类胰蛋白酶)是肥大细胞的主要蛋白质产物提示它们在肥大细胞的功能中起着重要作用。根据已发表的cdna序列，有四种不同的人类类胰蛋白酶和主要研究者假设这些不同的肥大细胞类胰蛋白酶具有不同的功能特性，从而允许不同的功能。肥大细胞在几乎所有组织中的存在表明类胰蛋白酶在自然和病理条件下具有重要功能普遍缺乏关于这些酶如何在功能上不同的知识提供这项建议的理由。中心假设是不同的肥大细胞类胰蛋白酶已经进化为服务于不同的功能和对这些酶的结构和功能的了解对了解肥大细胞在健康和疾病中的作用。在预赛中主要研究人员表达和纯化了完全有效的研究重组人皮肤类胰蛋白酶1(RHST1)在毕赤酵母中的表达。在目标1中 RHST1基因的定点突变将用于表达人类胰蛋白酶在巴斯德毕赤酵母中表达β/2和类胰蛋白酶α。在Aim 2中，类胰蛋白酶1、β/2和阿尔法将表征和比较肝素结合和在合成底物和天然底物上的动力学以确定它们的差异和相似之处。在目标3中，基于发现人肺类胰蛋白酶迅速并特异性地切割纤维蛋白原中的单个RGD基序，该基序由 α-β-3整合素，其他带有RGD位点的蛋白质，包括Vitronectin 和纤维连接蛋白，将由三个rHTs进行切割检查。这部作品将提供已知序列的类胰蛋白酶所需的比较数据，并应洞察为什么人类体内有如此相似但又不同的酶肥大细胞。支持将使本实验室能够继续研究胰酶和本科生的指导，同时获得知识这可能会提高我们对肥大细胞在疾病中的作用的理解，如哮喘、关节炎、癌症和心血管疾病。