Identification of Tyrosine-Sulfated Proteins in the Male Genital Tract

男性生殖道中酪氨酸硫酸化蛋白的鉴定

基本信息

批准号：
7618487
负责人：
KEVIN L MOORE
金额：
$ 31.52万
依托单位：
OKLAHOMA MEDICAL RESEARCH FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7618487
关键词：
Address Affinity Chromatography Animal Model Antibodies Area Binding Biological Biological Models Biology Cell Communication Cell Fractionation Cell Line Cells Complex Detection Enzymes Fertility Foundations G-Protein-Coupled Receptors Goals Golgi Apparatus Human Infertility Inorganic Sulfates Integral Membrane Protein Investigation Knockout Mice L-Selectin Leukocytes Ligands Male Genital Organs Male Infertility Mass Spectrum Analysis Mediating Methodology Methods Molecular Molecular Profiling Monoclonal Antibodies Mus Oocytes P-selectin ligand protein Partner in relationship Pathway interactions Phenotype Play Post-Translational Protein Processing Property Proteins Reproductive Biology Role Sampling Site Site-Directed Mutagenesis Spermatogenesis Structure-Activity Relationship System Tyrosine Unspecified or Sulfate Ion Sulfates Western Blotting Wild Type Mouse analytical method base biological systems cell motility chemokine receptor human disease insight knockout animal male mouse genome novel protein complex protein function protein protein interaction protein-tyrosine sulfotransferase public health relevance sperm cell sperm function sulfation tool tyrosine O-sulfate

项目摘要

DESCRIPTION (provided by applicant): Protein-tyrosine sulfation is mediated by one of only two Golgi tyrosylprotein sulfotransferases in the human and mouse genome (TPST-1 and TPST-2) that sulfate an unknown number of secreted and transmembrane proteins that transit the secretory pathway. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. However, only several dozen tyrosine-sulfated proteins have been described and for many, the functional importance of sulfotyrosine residues remains unknown. Many more tyrosine-sulfated proteins are likely to exist and await description. The pace of discovery of tyrosine-sulfated proteins has been very slow. There have been three major barriers to developing a full understanding of the importance of tyrosine sulfation in biological systems. First, there has been the lack of knockout animal models to study the biology of this enzyme system. Second, there has been the lack of a facile means to both identify additional tyrosine-sulfated proteins and probes to explore the role of tyrosine sulfation in protein function. Finally, a robust analytical method to determine the site of tyrosine sulfation in proteins has not been available. We developed Tpst1 and Tpst2 knockout mice and found that Tpst2-/- males are infertile, whereas Tpst1-/- males have normal fertility. The male infertility phenotype is characterized by normal spermatogenesis, but abnormal sperm function. These findings suggest that one or more proteins expressed in the male genital tract must be tyrosine-sulfated for normal sperm function. However, no proteins directly or indirectly involved in sperm function are known to be tyrosine- sulfated. We have developed a novel anti-sulfotyrosine monoclonal antibody that recognizes sulfotyrosine residues in proteins independent of the sequence context and which allows the detection and purification of tyrosine-sulfated proteins from complex biological samples. In addition, we have developed a novel subtractive mass spectrometry based method to determine the sites of tyrosine sulfation in proteins that is broadly applicable. We will exploit these novel tools and methods to identify tyrosine-sulfated proteins using the male genital tract as a model system in order to fully validate the approach, to refine our methodology, and to explore the role of tyrosine sulfation in male reproductive biology. Three specific aims are proposed. Aim 1 - Identify tyrosine-sulfated proteins expressed in the male genital tract. Expression profiles of tyrosine-sulfated proteins in the male genital tract of wild type, Tpst1-/-, and Tpst2-/- mice will be determined by PSG2 Western blotting in combination with subcellular fractionation. Tyrosine-sulfated proteins will then be purified from wild type mice by PSG2 affinity chromatography and identified by microsequencing. Aim 2 The presence of sulfotyrosine and/or the site(s) of sulfation in the proteins identified in Aim 1 will be determined using our subtractive mass spectrometry strategy and other independent methodology. Aim 3 Identify the protein(s) that are under sulfated in Tpst2-/- mice and the enzymatic basis for defective sulfation of these proteins. PUBLIC HEALTH RELEVANCE: We propose to apply novel tools and methodology to identify tyrosine-sulfated proteins in the male genital tract. Our studies will catalyze studies in many areas of investigation by expanding the known TPST substrate repertoire, providing novel insights into structure-function relationships of proteins expressed in the male genital tract, and providing insights into the molecular basis for male infertility in Tpst2-/- mice. Our methods we will be broadly applicable to other system and/or model organism and facilitate a rapid expansion in our understanding of the role of tyrosine sulfation in biologic systems and human diseases.

描述（由申请人提供）：蛋白质 - 酪氨酸硫酸化是由人和小鼠基因组中仅有的两个高尔基酪蛋白蛋白硫代硫代硫代硫酸盐（TPST-1和TPST-2）介导的，该硫酸硫酸硫酸硫酸硫酸硫酸硫酸盐是一种未知数量的分泌和跨膜蛋白，这些蛋白质与分泌途径交通。酪氨酸硫酸化在几个定义明确的系统中的蛋白质 - 蛋白质相互作用中起作用。但是，仅描述了几十个酪氨酸硫酸蛋白，并且对于许多人来说，磺胺酪氨酸残基的功能重要性仍然未知。更多的酪氨酸硫磺蛋白可能存在并等待描述。发现酪氨酸硫化蛋白的速度非常慢。对酪氨酸在生物系统中的重要性有充分的了解，已经存在三个主要障碍。首先，缺乏敲除动物模型来研究该酶系统的生物学。其次，缺乏识别额外的酪氨酸硫酸蛋白质和探针来探索酪氨酸硫酸化在蛋白质功能中的作用的缺乏便利手段。最后，尚未提供一种确定蛋白质酪氨酸硫酸盐位点的可靠分析方法。我们开发了TPST1和TPST2敲除小鼠，发现TPST2 - / - 雄性不育，而TPST1 - / - 雄性具有正常的生育能力。男性不育症表型的特征是正常的精子发生，但精子功能异常。这些发现表明，在男性生殖道中表达的一种或多种蛋白质必须被酪氨酸降低以达到正常的精子功能。但是，已知没有直接或间接参与精子功能的蛋白质是酪氨酸硫酸的。我们已经开发了一种新型的抗磺基酪氨酸单克隆抗体，该抗体识别蛋白质中的磺胺酪氨酸残基与序列上下文无关，并允许从复杂的生物学样品中检测和纯化酪氨酸硫酸化的蛋白质。此外，我们开发了一种新型的基于质谱的方法，以确定蛋白质中酪氨酸硫酸化的位点，这是广泛适用的。我们将利用这些新颖的工具和方法来使用男性生殖道作为模型系统来识别酪氨酸硫化的蛋白质，以充分验证该方法，完善我们的方法论，并探索酪氨酸硫酸盐在男性生殖生物学中的作用。提出了三个具体目标。 AIM 1-鉴定在男性生殖道中表达的酪氨酸硫化蛋白。野生型，TPST1 - / - 和TPST2 - / - 小鼠在男性生殖道中酪氨酸硫化蛋白的表达分布将由PSG2 Western斑点与亚细胞分级结合使用。然后，将通过PSG2亲和色谱从野生型小鼠中纯化酪氨酸硫酸化蛋白，并通过微钉测序鉴定。 AIM 2使用我们的减法质谱策略和其他独立的方法，确定AIM 1中蛋白质中硫酸盐的存在和/或硫化位点。 AIM 3鉴定在TPST2 - / - 小鼠中硫酸下的蛋白质以及这些蛋白质缺陷的酶基础。公共卫生相关性：我们建议采用新颖的工具和方法来鉴定雄性生殖道中酪氨酸硫化的蛋白质。我们的研究将通过扩展已知的TPST底物库来促进许多研究领域的研究，从而提供对男性生殖道中表达的蛋白质的结构功能关系的新见解，并为TPST2 - / - 小鼠中男性不育症的分子基础提供洞察力。我们的方法将广泛适用于其他系统和/或建模生物体，并促进我们对酪氨酸硫酸在生物系统和人类疾病中的作用的理解的快速扩展。