Mechanisms of a Novel Chemotactic Cofactor for C5a

描述（由申请人提供）：白细胞对各种细胞的趋化性 已知组织的损伤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C5 a和C5 a des Arg还与疾病的发病机制有关。C5衍生肽是 对多种细胞类型非常有效的化学引诱剂。多已知 关于C5衍生肽的生物活性，但这些肽的调节 功能了解不多。以前，我们是第一批 证明维生素D结合蛋白（DBP），也称为 GC-球蛋白，可以增强C5 a和C5 a des Arg的趋化活性，即， 作为协同趋化因子发挥作用。此外，DBP的共趋化活性是 对C5衍生肽具有特异性。虽然DBP似乎是一个 生理上重要的调节剂的趋化活性的活化 补体，DBP增强趋化性的机制尚不清楚。 最近，我们报告了几个重要的观察结果， 定义DBP作为C5 a的共趋化因子的机制。（一） DBP需要与细胞表面结合，以发挥其作为一种 C5 a的共趋化因子。(2)中性粒细胞DBP结合位点是软骨素 硫酸化蛋白聚糖。(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，其切割和脱落 蛋白聚糖(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)来自患者的临床样本 与炎症性疾病（ARDS）含有修饰的共趋化形式的 DBP。我们的假设是DBP的修饰形式结合到细胞表面， 并启动对C5 a的增强的趋化反应。在本提案中，我们 奋进探讨DBP增强白细胞增殖的机制 利用人中性粒细胞和U937细胞的C5 a趋化活性 用C5 a受体（U937-C5 aR）转染的细胞系。的过程 共趋化性将被分成组成部分并单独检查。 首先，确定活化的血小板如何通过关注最重要的 可能的改变：普遍存在的蛋白激酶引起的细胞外磷酸化。 其次，通过检测结合来研究修饰的DBP如何与细胞相互作用。 并在细胞表面脱落。第三，确定细胞如何终止 通过集中于磷酸酶的去磷酸化作用来调节共趋化信号。 最后，将从患有炎性疾病的患者中收集临床样品。 使用蛋白质组学方法进行分析，以确定修饰的DBP是否与 疾病的结果。这项研究将桥梁基础知识， 体外生物化学方法，并将其应用于检查从 炎症性疾病患者。这项研究的结果将证明， 一种趋化辅因子的新机制，可以作为一种原型， 其他辅助因子还有待发现。

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.