Neutrophil ecto-Calreticulin: Implications for Immunity

钙网蛋白是一种多功能内质网驻留蛋白，具有伴侣功能的钙结合蛋白。然而，据报道，钙网蛋白存在于一些细胞的表面，在那里它可以参与多种细胞功能。我们已经证明，钙网织蛋白存在于循环的中性粒细胞表面，由于钙网织蛋白缺乏跨膜区，它必须与驻留在中性粒细胞质膜上的接头蛋白结合。初步数据表明，中性粒细胞上钙网织蛋白的接头位于脂筏上，并且是GPI锚定的。相互免疫沉淀-抗钙网织蛋白和抗CD59免疫印迹鉴定CD59是PMN上的接头蛋白。已知的钙网硬蛋白具有免疫功能和 这项提案将集中在PMN如何利用表面钙网硬蛋白。中性粒细胞作为原始吞噬细胞在先天免疫中起着至关重要的作用。我们假设，PMN使用表面钙网蛋白通过促进抗原的处理来启动适应性免疫反应，并通过促进PMN一旦凋亡后清除来限制错误的适应性反应的机会。 以下目标将提供解决更大的免疫学问题所需的信息：1)确定钙网织蛋白是否利用其KDEL序列向质膜转运，以及应激和细胞凋亡等其他因素是否影响转运和/或脱落；2)利用具有选定突变的钙网蛋白和sCD59的重组结构域来定义钙网织蛋白和CD59上的相互结合结构域。动态的实时结合将通过表面等离子激元共振进行监测；3)确定连接外钙网织蛋白是否诱导独特的细胞内信号。中性粒细胞来源的钙网织蛋白的免疫功能将在很大程度上取决于钙网织蛋白与CD59相互作用的生物学特性。拟议的钙网织蛋白-CD59研究的结果将使未来能够研究PMN外钙网织蛋白在启动获得性免疫方面的潜在双重作用，这一功能与疫苗设计相关；以及增强对凋亡的PMN的清除，这是一种防止自身免疫的功能。

Calreticulin is best known as a multifunctional endoplasmic reticulum resident, calcium binding protein with chaperone function. However, calreticulin has been reported on the surface of some cells where it can participate in a variety cell functions. We have shown that calreticulin is present on the surface of circulating PMN and since calreticulin lacks a transmembrane domain, it must bind to an adaptor protein, that is resident in the PMN plasma membrane. Preliminary data indicate that the adaptor for calreticulin on PMN is resident in lipid rafts and GPI-anchored. Reciprocal immunoprecipitation-and immunoblotting with anti-calreticulin and anti-CD59 identify CD59 as the adaptor protein on PMN. Calreticulin is known to have immune functions and this proposal will focus on how PMN utilize surface calreticulin. PMN have a critical role in innate immunity as the primary phagocyte. We hypothesize that PMN use surface calreticulin to both prime the adaptive immune response by facilitating the processing of antigens; and limit the chance for an errant adaptive response by facilitating the PMN's clearance once it is apoptotic. The following objectives will provide information necessary for addressing the larger immunologic questions: 1) determine whether calreticulin uses its KDEL sequence for transport to the plasma membrane, and whether other factors such as stress and apoptosis affect transport and/or shedding; 2) define the reciprocal binding domains on calreticulin and CD59 using recombinant domains of calreticulin and sCD59 with selected mutations. Kinetic, real time binding will be monitored with surface plasmon resonance; 3) Define if ligating ecto-calreticulin induces unique intracellular signaling. The immune functions of PMN derived calreticulin will be largely determined by the biology of calreticulin-CD59 interaction. The results of the proposed calreticulin-CD59 studies will enable future studies on the potential dual roles of PMN ecto-calreticulin for priming adaptive immunity, a function that is relevant to vaccine design; and for augmenting the clearance of apoptotic PMN, a function that prevents autoimmunity.