Characterization of Streptococcal IdeS

描述(由申请人提供)：拟议的研究旨在研究一种由某些化脓性链球菌分离株表达的新型半胱氨酸蛋白酶。这种酶被命名为IDES(免疫球蛋白降解酶)，能够与免疫球蛋白、免疫球蛋白和免疫球蛋白结合，但只选择性地裂解免疫球蛋白同型抗体。选择性底物特异性的进一步例证是，据报道，它不能切割合成的多肽，该合成肽由围绕已知的免疫球蛋白夹点周围的精确氨基酸序列组成。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分子和单一底物IgG分子之间形成络合物才能发生催化作用。 研究的重点是更全面地刻画IDE的功能性质。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、半定量的SELDI-TOF试验来测量从M1血清型化脓性链球菌分离株AP1分离或克隆的野生型和重组IDES的活性。这种分析有助于详细分析产生Fc片段的最佳反应物比例。 第二个具体目标将集中于鉴定分泌IDES的分离株，以及IDES对抗原-抗体复合体中含有的游离免疫球蛋白和免疫球蛋白的影响。这些研究旨在帮助阐明IDES在化脓性链球菌发病机制中的潜在作用，以及分泌型免疫球蛋白降解酶对宿主-病原体相互作用的潜在影响。 第三个具体目的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的相关基因，以确定这种潜在的致病因子是否在针对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降解免疫球蛋白的潜力，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差异。 公共卫生相关性：这是一个很有价值的项目，可以向学生介绍跨学科的方法来解决生物学中的研究问题。这些研究涉及分子生物学、蛋白质组学、酶学和生物信息学的技术组合。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的影响

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