Shido K, Ahmad G, Hsu L, Kamiyama M
Department of Biology, Seton Hall University, South Orange, N.J. 07079-2689, USA.
J Clin Lab Immunol. 1995;46(1):25-33.
Human platelets are known to carry Fc receptors (Fc R), but the binding characteristics between ligands and Fc gamma R has not been well elucidated. In this study, we investigated the binding kinetics of IgG Fc fragments (Fc) to Fc R, the association and dissociation characteristics of the ligands to and from Fc gamma R using enzymatically modified Fc fragment derivatives. Approximately 60 minutes and 90 minutes were needed at 37 degrees C and 22 degrees C, respectively, for complete saturation of the Fc binding sites with horseradish peroxidase-conjugated Fc (HPO-Fc). Heat aggregated IgG (HAG) had a greater affinity for the Fc gamma R than Fc monomers. Additional binding of HAG was observed even after the binding sites were saturated with Fc monomers. This could be explained by different binding sites available only for immune complexes or by the partial dissociation of binding sites saturated with Fc by HAG. Further, we noted partial dissociation of HPO-Fc, when HAG was added after saturation of the binding sites with HPO-Fc. In a subsequent experiment, we compared the relative affinities of chemically or enzymatically modified Fc derivatives for Fc gamma R. HAG, which was used as a model for CIC, had a greater affinity for platelet Fc gamma R than IgG monomer and Fc derivatives. Pepsin-digestion of Fc caused a total loss of its affinity for the Fc gamma R, whereas b-mercaptoethanol-treated Fc fragments demonstrated substantial binding to the Fc gamma R. These results indicate that the pepsin digestion affects the Fc portion and causes a disruption in the area of the Fc which is essential for the recognition by the platelet Fc gamma R. On the other hand, cleavage of disulfide bridges by beta-mercaptoethanol resulted in a marked increase in affinity for the Fc gamma R. On the other hand, enzymatic cleavage of the carbohydrate moieties of Fc did not alter the affinity of Fc fragments for the Fc gamma R, indicating that the carbohydrates play an insignificant role or are not involved in their binding to the Fc gamma R.
已知人类血小板携带Fc受体（Fc R），但配体与FcγR之间的结合特性尚未得到充分阐明。在本研究中，我们使用酶修饰的Fc片段衍生物研究了IgG Fc片段（Fc）与Fc R的结合动力学，以及配体与FcγR的结合和解离特性。在37℃和22℃下，分别需要约60分钟和90分钟，辣根过氧化物酶偶联的Fc（HPO-Fc）才能完全饱和Fc结合位点。热聚集IgG（HAG）对FcγR的亲和力高于Fc单体。即使Fc单体使结合位点饱和后，仍观察到HAG的额外结合。这可以通过仅对免疫复合物可用的不同结合位点，或通过HAG使Fc饱和的结合位点的部分解离来解释。此外，我们注意到，在用HPO-Fc使结合位点饱和后加入HAG时，HPO-Fc会发生部分解离。在随后的实验中，我们比较了化学或酶修饰的Fc衍生物对FcγR的相对亲和力。用作CIC模型的HAG对血小板FcγR的亲和力高于IgG单体和Fc衍生物。Fc经胃蛋白酶消化后，其对FcγR的亲和力完全丧失，而经β-巯基乙醇处理的Fc片段显示出与FcγR的大量结合。这些结果表明，胃蛋白酶消化会影响Fc部分，并导致Fc区域的破坏，而该区域对于血小板FcγR的识别至关重要。另一方面，β-巯基乙醇对二硫键的切割导致对FcγR的亲和力显著增加。另一方面，Fc碳水化合物部分的酶促切割并未改变Fc片段对FcγR的亲和力，这表明碳水化合物在它们与FcγR的结合中起的作用微不足道或不参与其中。
