TYROSYLPROTEIN SULFOTRANSFERASES

酪氨酰蛋白磺基转移酶

• 批准号: 6390450
• 负责人: KEVIN L MOORE
• 金额: $ 32.61万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6390450
• 关键词: CHO cells; Golgi apparatus; chemical kinetics; enzyme mechanism; enzyme substrate; immunoelectron microscopy; immunofluorescence technique; inflammation; isozymes; laboratory mouse; monoclonal antibody; posttranslational modifications; protein localization; recombinant proteins; sulfotransferase; transfection; tyrosine

Tyrosine O-sulfation, a common post-translational modification of secretory and membrane proteins in higher eukaryotes, is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST). TPST catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to tyrosines within acidic motifs of polypeptides. Several proteins involved in different aspects of vascular biology contain tyrosine sulfate, including adhesion molecules, coagulation factors, complement components, serpins, and extracellular matrix proteins. Tyrosine O-sulfation is known to be important for the biological function of P-selectin glycoprotein ligand-1, glycoprotein Ibalpha, coagulation factors V and VIII, complement factor C4, and others. The kinetics of the TPST reaction has been extensively studied using crude and partially purified enzyme preparations. However, the kinetic properties of the enzyme(s) in purified systems have not been explored, little is known about the regulation of TPST, and until recently nothing was known about the structure of TPST(s). We recently identified and cloned a family of two mammalian tyrosylprotein sulfotransferases, designated TPST-1 and TPST-2. The specific aims of this proposal are designed to compare the function of these two enzymes and to begin to address the broad question as to why more than one such enzyme exists. We hypothesize that TPST-1 and TPST-2 have distinct substrate specificities and may differentially modulate the biological function of a variety of proteins involved in diverse physiological functions. To explore this hypothesis the following specific aims are proposed. Aim 1 Determine the kinetic mechanism of TPST-1 and TPST-2. Aim 2 - Determine the substrate specificities of TPST-1 and TPST-2 toward various synthetic peptide and macromolecular substrates in vitro. Aim 3 - Determine the subcellular localization of TPST-1 and TPST-2. Aim 4. Define the pattern of TPST protein and mRNA expression in tissues and how TPST expression is regulated during inflammation and development. These studies should provide fundamental and new information on the function and regulation of TPSTs and provide insights into how the two known TPSTs might interact in biological systems.

酪氨酸O-硫化是高等真核生物中分泌蛋白和膜蛋白的常见后翻译后修饰，是由称为酪糖基蛋白质硫代转移酶（TPST）的高尔基酶活性介导的。 TPST催化硫酸盐从3'-磷酸腺苷5'-磷硫酸盐（PAPS）转移到多肽酸性基序内的酪氨酸。 血管生物学不同方面涉及的几种蛋白质含有硫酸酪氨酸，包括粘附分子，凝结因子，补体成分，SERPINS和细胞外基质蛋白。 已知酪氨酸O-硫化对于P-选择蛋白糖蛋白配体1，糖蛋白IBALPHA，凝结因子V和VIII，补体C4等很重要。 TPST反应的动力学已通过粗制和部分纯化的酶制剂进行了广泛的研究。 然而，尚未探索纯化系统中酶的动力学特性，对TPST的调节知之甚少，直到最近，对TPST的结构尚无任何了解。 我们最近确定并克隆了两个指定为TPST-1和TPST-2的两个哺乳动物酪蛋白硫代硫代硫代硫代酶的家族。 该提案的具体目的旨在比较这两种酶的功能，并开始解决为什么不止一种这样的酶存在的广泛问题。我们假设TPST-1和TPST-2具有不同的底物特异性，并且可能会差异地调节参与多种生理功能的多种蛋白质的生物学功能。为了探讨这一假设，提出了以下特定目标。 AIM 1确定TPST-1和TPST-2的动力学机制。 AIM 2-在体外，确定TPST-1和TPST-2的底物特异性对各种合成肽和大分子底物。 AIM 3-确定TPST-1和TPST-2的亚细胞定位。 AIM 4。定义组织中TPST蛋白的模式和mRNA表达，以及在炎症和发育过程中如何调节TPST表达。这些研究应提供有关TPST功能和调节的基本和新信息，并提供有关两个已知TPST如何在生物系统中相互作用的见解。