Mechanisms of a Novel Chemotactic Cofactor for C5a

描述(申请人提供)：白血球对多种细胞的趋化 已知组织是几种疾病发病机制中的关键步骤 炎症性疾病。补体促炎肽C5a和C5a Des 精氨酸也参与了疾病的发病机制。C5衍生的多肽是 对多种细胞类型都有很强的化学诱导剂。很多事情都是众所周知的 关于C5衍生肽的生物活性及其调节 人们对其功能知之甚少。此前，我们是几个小组中的第一个 为了证明维生素D结合蛋白(DBP)，也被称为 GC-球蛋白可增强C5a和C5a Des Arg的趋化活性，即 起到协同趋化因子的作用。此外，DBP的协同趋化活性是 专用于C5衍生的多肽。尽管DBP似乎是一种 激活的趋化活性的重要生理调节因子 补体，DBP增强趋化作用的机制尚不清楚。 最近，我们报道了几个重要的观察结果，这应该会有所帮助 明确DBP作为C5a的协同趋化因子的机制。(1) DBP需要结合到细胞表面才能发挥作用 对C5a的协同趋化作用。(2)中性粒细胞DBP结合部位为软骨素 硫酸盐蛋白多糖。(3)DBP结合位点的表达受 细胞表面结合的中性粒细胞弹性蛋白酶，它裂解和脱落 蛋白多糖。(4)初步研究表明，活化的血小板 将DBP修饰为活性共趋化形式。(5)患者临床标本 与炎症性疾病(ARDS)包含改良的共趋化形式 DBP。我们的假设是，一种修饰形式的DBP结合到细胞表面 并启动对C5a的增强趋化反应。在这项提案中，我们 努力研究DBP增加白细胞的机制 人中性粒细胞和U937细胞对C5a趋化活性的研究 C5a受体(U937-C5aR)转基因株。这一过程 共趋化性将被分成不同的部分，并分别进行检查。 首先，确定激活的血小板如何通过关注最多的 可能的变化：普遍存在的蛋白激酶对细胞外的磷酸化。 其次，通过检查结合来研究修饰后的DBP如何与细胞相互作用 并在细胞表面脱落。第三，确定单元格如何终止 共同趋化信号通过关注磷酸酶的去磷酸化作用。 最后，来自炎症性疾病患者的临床样本将是 使用蛋白质组学方法分析以确定修饰的DBP是否相关 与疾病结局有关。这项研究将架起基础知识的桥梁 体外生化方法及其在样品检测中的应用 患有炎症性疾病的患者。这项研究的结果将证明 一种趋化辅因子的新机制，并可作为 其他尚未发现的辅因。

DESCRIPTION (provided by applicant): Chemotaxis of leukocytes into various tissues is known to be a critical step in the pathogenesis of several inflammatory disorders. Complement pro-inflammatory peptides C5a and C5a des Arg also have been implicated in disease pathogenesis. C5-derived peptides are very potent chemoattractants for a wide variety of cell types. Much is known about the bioactivities of C5-derived peptides but the regulation of these functions is poorly understood. Previously, we were the first of several groups to demonstrate that the vitamin D binding protein (DBP), also known as Gc-globulin, can enhance the chemotactic activity of C5a and C5a des Arg, i.e., function as a co-chemotaxin. Moreover, the co-chemotactic activity of DBP is specific for the C5-derived peptides. Although DBP appears to be a physiologically important regulator of the chemotactic activity for activated complement, the mechanism of chemotaxis enhancement by DBP is not known. Recently, we have reported several important observations that should help define the mechanism by which DBP acts as a co-chemotactic factor for C5a. (1) DBP needs to be bound to the cell surface in order to function as a co-chemotaxin for C5a. (2) The neutrophil DBP binding site is a chondroitin sulfate proteoglycan. (3) Expression of the DBP binding site is regulated by cell surface-bound neutrophil elastase, which cleaves and sheds the proteoglycan. (4) Preliminary studies have shown that activated platelets modify DBP to an active co-chemotactic form. (5) Clinical samples from patients with inflammatory disorder (ARDS) contain the modified co-chemotactic form of DBP. It is our hypothesis that a modified form of DBP binds to the cell surface and initiates an enhanced chemotactic response to C5a. In this proposal, we endeavor to investigate the mechanism by which DBP augments the leukocyte chemotactic activity of C5a by utilizing human neutrophils and the U937 cell line transfected with the C5a receptor (U937-C5aR). The process of co-chemotaxis will be divided into component parts and examined individually. First, determine how activated platelets modify DBP by focusing on the most likely alteration: extracellular phosphorylation by ubiquitous protein kinases. Second, investigate how modified DBP interacts with cells by examining binding and shedding on the cell surface. Third, determine how cells terminate the co-chemotactic signal by focusing on dephosphorylation by phosphatases. Finally, clinical samples from patients with inflammatory disorders will be analyzed using a proteomic approach to determine if modified DBP is correlated with disease outcome. This study will bridge basic knowledge derived from in vitro biochemical approaches and apply it to examine samples obtained from patients with inflammatory disorders. Results of this study will demonstrate a novel mechanism for a chemotactic cofactor and could serve as a prototype for other cofactors yet to be discovered.