Characterization of Streptococcal IdeS

描述（由申请方提供）：拟定研究旨在研究由某些化脓链球菌分离株表达的新型半胱氨酸蛋白酶。这种酶，称为IdeS（链球菌的免疫球蛋白降解酶），具有结合IgG、伊加和IgM的能力，但仅选择性地切割IgG同种型的抗体。选择性底物特异性的进一步例证是其报道的不能切割由IgG中已知剪切位点周围的精确氨基酸序列组成的合成肽。最近的研究表明，这些不寻常的性质中的一些可能是由于需要在两个IdeS分子和单个底物IgG分子之间形成复合物以进行催化。 建议的研究的重点是更充分地表征IdeS的功能特性。最初的重点（具体目标#1）将是使用我们实验室开发的快速、灵敏、半定量SELDI-TOF测定法来测量从M1血清型S中分离或克隆的野生型和重组形式IdeS的活性。化脓性链球菌分离株，AP 1。该测定将有助于详细分析用于产生Fc片段的反应物的最佳比率。 第二个具体目标将集中于表征分泌IdeS的分离株以及IdeS对游离IgG与抗原-抗体复合物内所含IgG的影响。这些研究旨在帮助阐明IdeS在S.化脓性链球菌和分泌的IgG降解酶对宿主-病原体相互作用的潜在影响。 第三个具体目标是利用生物信息学数据挖掘方法，克隆和表达感染马和其他动物的其他链球菌物种的相关基因，以确定这种潜在的致病因子是否在靶向不同宿主的相关链球菌物种中保守。这些研究的重点将是确定相关基因产物是否具有IgG降解潜力，如果是，则证明它们优先感染的宿主对免疫球蛋白的底物偏好是否存在任何差异。 公共卫生相关性：这是一个有价值的项目，为学生介绍跨学科的方法来研究生物学问题。这些研究涉及分子生物学、蛋白质组学、酶学和生物信息学技术的结合。这些研究与基础免疫化学相关，并对一种重要的人类病原体的宿主-病原体相互作用具有潜在意义

DESCRIPTION (provided by applicant): The proposed studies are designed to study a novel cysteine protease, expressed by certain Streptococcus pyogenes isolates. This enzyme, designated IdeS (immunoglobulin degrading enzyme of streptococcus), has the ability to bind to IgG, IgA and IgM but only selectively cleave antibodies of the IgG isotype. The selective substrate specificity is further exemplified by its reported inability to cleave a synthetic peptide consisting of the precise amino acid sequence surrounding the known clip site in IgG. Recent studies suggest that some of these unusual properties may be due to the need to form a complex between two IdeS molecules and a single substrate IgG molecule for catalysis to occur. The focus of the proposed research is to characterize the functional properties of IdeS more fully. The initial focus (specific aim #1) will be to use a rapid, sensitive, semi-quantitative SELDI-TOF assay developed in our laboratory to measure the activity of a wild type and recombinant form of IdeS, isolated or cloned from a M1 serotype S. pyogenes isolate, AP1. This assay will facilitate detailed analysis of the optimal ratio of reactants for generation of Fc fragments. The second specific aim will focus on characterizing isolates that secrete IdeS and the effects of IdeS on free IgG versus IgG contained within an antigen-antibody complex. These studies are designed to help elucidate the potential roles of IdeS in the pathogenesis of S. pyogenes and the potential impact of a secreted IgG-degrading enzyme on the host-pathogen interaction. The third specific aim seeks to use bioinformatics data mining approaches, cloning and expression of related genes from other species of streptococci that infect horses and other animals to determine if this potential pathogenic factor is conserved among related streptococcal species that target different hosts. The focus of these studies will be to determine if the related gene products have IgG-degrading potential and, if so, demonstrate any difference in substrate preference towards immunoglobulin from the hosts they preferential infect. PUBLIC HEALTH RELEVANCE: This is valuable project for introducing students to cross-disciplinary approaches to a research problem in biology. The studies involve a combination of techniques in molecular biology, proteomics, enzymology and bioinformatics. The studies have relevance to basic immunochemistry, as well as having potential implications for host-pathogen interactions of an important human pathogen