Structure/Function Studies of LILRs Enabled by a Bacterially-Derived Ligand

项目摘要 白细胞免疫球蛋白样受体（LILR）是一类I型糖蛋白 受体只由造血谱系细胞表达。而11名人类的细胞外区域 LILR共享相似的重复Ig结构域结构，它们的细胞内区域可以根据其结构进行分类。 LILRs A1-A6通常与Fc γ R结合，并利用其信号激活ITAM， LILR B1-B5含有它们自己的信号抑制性ITIM基序。LILR本身是灵长类动物- 特异性，但对它们的鼠旁系同源物（称为PIR-A和PIR-B）或分离的人白细胞的研究 显示配体结合触发信号传导事件，激活或抑制各种免疫细胞，主要是 骨髓来源的。已知的LILR配体包括细胞表面（例如HLA I类）和寡聚蛋白 (e.g.β-淀粉样蛋白聚合物），而11种人LILR中的5种（即LILR-A2、-A5、-A6、-B3和-B4）的配体 仍然身份不明对于那些已知的，几乎没有详细的生化信息， 识别这些配体。此外，迄今为止还没有报道配体结合LILR的共晶结构。这些 这些缺点代表了LILR基础生物学知识的关键差距。他们还阻止了一个更 深入了解LILR信号传导及其对健康和疾病中白细胞功能的贡献。 我们的实验室多年来一直致力于了解细菌感染的机制， 补体/中性粒细胞轴的逃避。在这些研究的过程中，我们最近做出了意想不到的结果， 发现B族链球菌细胞与LILR-A6和LILR-B3的细胞外区域结合。这两 LILR由吞噬细胞（包括中性粒细胞和单核细胞）表达，其在先天免疫应答中起关键作用。 对细菌病原体的免疫力。此后，我们确定β-抗原C蛋白（β AC）为细菌细胞- 负责LILR-A6和LILR-B3与B组链球菌细胞结合的表面分子。因为β AC是 作为这两种LILR的第一个已知配体，我们相信β AC蛋白代表了获得 LILR如何识别其配体以及配体结合如何诱导信号传导的新见解。我们相信这些 这些原则可能与LILR作为一个系列广泛相关，而不仅仅是具体涉及这个系统。 在这个项目中，我们将使用β AC蛋白作为定义基本结构/功能的范例 LILR的关系。我们将开始定义β AC与 人LILR-A6和LILR-B3的胞外区。然后，我们将使用基于嵌合T细胞的报告系统 以确定β AC如何影响这些LILR的信号传导。最后，我们将定义 β AC与人补体因子H相互作用作为检查这种内源性 补体调节因子可能影响β AC诱导的LILR信号转导。总之，我们提出的研究将 揭示了免疫细胞表面受体这一至今仍知之甚少的家族的新机制。

PROJECT ABSTRACT The Leukocyte Immunoglobulin-Like Receptors (hereafter “LILRs”) are a family of type-I glycoprotein receptors expressed exclusively by hematopoietic lineage cells. While the extracellular regions of the 11 human LILRs share a similar architecture of repeating Ig-domains, their intracellular regions can be classified according to their signaling capacities: LILRs A1-A6 generally associate with FcγR and utilize its signal-activating ITAM motif, whereas LILRs B1-B5 contain their own signal-inhibitory ITIM motif. LILRs themselves are primate- specific, but studies on their murine paralogs (known as PIR-A and PIR-B) or isolated human leukocytes have shown that ligand binding triggers signaling events that either activate or inhibit various immune cells, primarily those of myeloid origin. Known LILR ligands include both cell-surface (e.g. HLA Class-I) and oligomeric proteins (e.g. β-amyloid polymers), yet the ligand(s) of 5 of the 11 human LILRs (i.e. LILR-A2, -A5, -A6, -B3, and -B4) remain unidentified. For those that are known, there is little detailed biochemical information as to how LILRs recognize these ligands. Moreover, no co-crystal structures of a ligand-bound LILR been reported thus far. These shortcomings represent a critical gap in knowledge of basic LILR biology. They have also prevented a more thorough understanding of LILR signaling and its contributions to leukocyte function in health and disease. Our laboratory has worked for many years toward understanding the mechanisms that underlie bacterial evasion of the complement/neutrophil axis. In the course of these studies, we recently made the unexpected discovery that Group B streptococcal cells bind to the extracellular regions of LILR-A6 and LILR-B3. These two LILRs are expressed by phagocytes, including neutrophils and monocytes, which serve critical roles in innate immunity to bacterial pathogens. We have since identified the β-Antigen C Protein (βAC) as the bacterial cell- surface molecule responsible for LILR-A6 and LILR-B3 binding to Group B streptococcal cells. Since βAC is the first known ligand for either of these LILRs, we believe the βAC protein represents a powerful tool for obtaining new insights into how LILRs recognize their ligands and how ligand-binding induces signaling. We believe these principles are likely broadly relevant to LILRs as a family, rather than pertaining to only this system specifically. In this project, we will use the βAC protein as a paradigm for defining the fundamental structure/function relationships of LILRs. We will begin by defining the molecular basis for the interaction of βAC with the extracellular regions of human LILR-A6 and LILR-B3. We will then use a chimeric T-cell based reporter system to determine how βAC influences signaling by these LILRs. Finally, we will define the molecular basis for interaction of βAC with human complement Factor H as a prelude for examining how this endogenous complement regulator might affect LILR signaling in response to βAC. Together, the studies we propose will shed new mechanistic light on this as yet poorly understood family of immune cell surface receptors.