Dual Function of VPO1 in Pathogen Recognition and Killing

描述（由申请人提供）：先天免疫被广泛定义为抵抗入侵病原体的第一道防线。先天免疫识别通常由模式识别受体（PRR）和病原体相关分子模式（PAMP）的相互作用介导，触发一系列下游信号传导事件;或者，当酶遇到微生物时，许多酶如溶菌酶、消化酶和防御素不加选择地杀死细菌。在后一种情况下，酶不直接识别微生物，并且入侵微生物的消除是非特异性的。是否存在直接杀死入侵微生物的PRR仍有待阐明。血管过氧化物酶1（VPO 1）是一种新发现的哺乳动物血红素过氧化物酶（hPx）。VPO 1在hPx家族成员中是独特的，因为它在其C-末端包含催化结构域，并且在其N-末端区域包含五个富含亮氨酸的区域（LRR）和四个免疫球蛋白（IG）C2型结构域。VPO 1在心血管系统、肺、肝、胰腺和脾中高度表达，并且以比MPO高1000倍的浓度分泌到血流中。然而，其生物学功能尚未确定。这项研究的核心假设是VPO 1可以识别并直接杀死入侵的微生物。我们的具体目标是（1）确定VPO 1的LRR和IG C2结构域是否与PAMP结合;（2）确定VPO 1通过LRR和IG C2的结合是否通过产生次卤酸介导杀微生物活性。主要的方法有分子克隆与表达、蛋白纯化、荧光偏振技术和表面等离子体共振技术。该方案的成功完成将：（1）为先天免疫反应提供新的见解;（2）鉴定第一个具有病原体识别和杀灭双重功能的哺乳动物蛋白;（3）通过新型双功能hPx的生理作用在维持血流无菌性方面创造新的范例;（4）阐明VPO 1介导的病原体识别和杀伤的分子机制，为涉及这种新型先天免疫途径的诊断和/或治疗的未来发展奠定基础。 公共卫生相关性：本项目旨在发现和表征一种新的先天免疫机制，即病原体识别和杀伤都发生在含血红素的过氧化物酶中，扩展了我们现有的先天免疫概念，填补了先天免疫系统的差距。这种活性的表征和先天免疫应答的这一分支的阐明将导致针对感染性疾病的治疗的潜在新靶点。

DESCRIPTION (provided by applicant): Innate immunity is broadly defined as the first line of defense against invading pathogens. Innate immune recognition is classically mediated by the interaction of pattern-recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs), triggering serial downstream signaling events; alternatively, a number of enzymes such as lysozyme, digestive enzymes and defensins indiscriminately kill bacteria when the enzyme encounters the microbes. In the latter case, the enzyme does not directly recognize microbes, and the elimination of invading microbes is non-specific. Whether there is a PRR to directly kill invading microorganisms remains to be elucidated. Vascular peroxidase 1 (VPO1) is a newly-discovered mammalian heme-containing peroxidase (hPx). VPO1 is unique among the members of hPx family in that it contains a catalytic domain at its C-terminus and a large N-terminal region including five leucine-rich regions (LRRs) and four immunoglobulin (Ig) C2 type domains. VPO1 is highly expressed in the cardiovascular system, lung, liver, pancreas and spleen, and is secreted into bloodstream at a 1000-fold higher concentration than is MPO. However, its biological function has not been established. The central hypothesis of this proposed research is that VPO1 can recognize and directly kill invading microbes. Our specific aims are to (1) determine whether the LRR and Ig C2 domains of VPO1 bind to PAMPs; (2) determine if the binding of VPO1, via LRR and Ig C2, mediates microbicidal activity via generation of hypohalous acids. The major methods for addressing the aims include molecular cloning and expressing, protein purification, fluorescence polarization technology and surface plasmon resonance technology. Successful completion of this proposal will: (1) provide novel insights into innate immune responses; (2) identify the first mammalian protein with dual functions of pathogen recognition and killing; (3) create a new paradigm in the maintenance of bloodstream sterility by the physiological action of a novel dual function hPx; (4) elucidate the molecular mechanisms of VPO1-mediated pathogen recognition and killing, setting the foundation for the future development of diagnostics and/or therapeutics involving this novel innate immune pathway. PUBLIC HEALTH RELEVANCE: This project seeks is to identify and characterize a new mechanism of innate immunity in that both pathogen-recognition and killing occur within a heme-containing peroxidase, expanding our current concepts of innate immunity and filling the gap in innate immune system. The characterization of this activity and elucidation of this arm of the innate immune response will lead to potential new targets for therapies against infectious diseases.