Van Strijp J A, Russell D G, Tuomanen E, Brown E J, Wright S D
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
J Immunol. 1993 Sep 15;151(6):3324-36.
We have purified CR3 to homogeneity by affinity chromatography on C3bi-Sepharose and elution with EDTA. C3bi-coated erythrocytes bound to this purified CR3, and binding was dependent on the concentration of both C3bi and CR3, as well as on temperature and the presence of divalent cations. Moreover, binding could be blocked by mAb against CR3 or C3bi and could be enhanced by the addition of integrin modulating factor-1. We used the purified CR3 to test whether several putative ligands of CR3 directly bound the receptor. The interaction of purified CR3 with fibrinogen, filamentous hemagglutinin of Bordetella pertussis, lipophosphoglycan and glycoprotein 63 of Leishmania mexicana, and lipopolysaccharide from Escherichia coli was confirmed. However the interaction of CR3 with zymosan or its major component, beta-glucan, was not observed in these assays. Previous studies showed that binding of C3bi to PMN could be blocked with Arg-Gly-Asp (RGD) containing peptides and were interpreted to indicate that the RGD sequence in C3bi interacts directly with CR3. We found, however, that RGD containing peptides were unable to block the interaction of C3bi with purified CR3, yet retained the ability to block binding of C3bi to cells. We conclude that RGD-peptides do not directly bind CR3, but instead indirectly effect CR3 function. Inasmuch as the effect of RGD-peptides could be mimicked with antibodies against leukocyte response integrin, we suggest that RGD-peptides may bind to leukocyte response integrin on polymorphonuclear leukocytes and influence CR3 activity via this receptor.
我们通过在C3bi-琼脂糖凝胶上进行亲和层析并用EDTA洗脱，将CR3纯化至同质状态。C3bi包被的红细胞与这种纯化的CR3结合，且结合依赖于C3bi和CR3的浓度，以及温度和二价阳离子的存在。此外，结合可被抗CR3或C3bi的单克隆抗体阻断，并可通过添加整联蛋白调节因子-1增强。我们使用纯化的CR3来测试几种假定的CR3配体是否直接结合该受体。证实了纯化的CR3与纤维蛋白原、百日咳博德特氏菌的丝状血凝素、墨西哥利什曼原虫的脂磷壁酸聚糖和糖蛋白63以及大肠杆菌的脂多糖之间的相互作用。然而，在这些试验中未观察到CR3与酵母聚糖或其主要成分β-葡聚糖之间的相互作用。先前的研究表明，含精氨酸-甘氨酸-天冬氨酸（RGD）的肽可阻断C3bi与中性粒细胞的结合，并被解释为表明C3bi中的RGD序列直接与CR3相互作用。然而，我们发现含RGD的肽无法阻断C3bi与纯化的CR3的相互作用，但仍保留阻断C3bi与细胞结合的能力。我们得出结论，RGD肽不直接结合CR3，而是间接影响CR3的功能。由于RGD肽的作用可被抗白细胞反应整联蛋白的抗体模拟，我们认为RGD肽可能与多形核白细胞上的白细胞反应整联蛋白结合，并通过该受体影响CR3的活性。
