Rostagno A, Williams M, Frangione B, Gold L I
Department of Pathology, New York University Medical School, New York, NY 10016, USA.
Mol Immunol. 1996 Apr;33(6):561-72. doi: 10.1016/0161-5890(95)00161-1.
Fibronectin (Fn), a mosaic protein composed of multiple copies of three different module types (Fl, F2 and F3), has been found associated with circulating immune complexes (ICs) and immunoglobulin (Ig) aggregates in a variety of IC diseases and myeloproliferative disorders. We have previously shown that a proteolytic fragment of Mr = 25,900 Da, from the NH2-terminal domain of Fn, composed of five type 1 modules (1Fl -5Fl) binds to the major Ig classes under physiologic conditions, suggesting that the presence of Fn in ICs and cryoglobulins results from a physicochemical binding interaction between these two molecules. Using an ELISA, we now show that the interaction between Fn and IgG is: (1) not influenced by any other constituent of plasma; (2) unaffected by temperature; and (3) has an estimated Kd of 3.77 x 10(-9) M. In addition, we have further delineated the respective sites involved in the interaction between Fn and IgG. Recombinant type l module pairs (1Fl.2Fl and 4Fl.5Fl) from the NH2-terminus of Fn, expressed in yeast, were employed in an ELISA and affinity chromatography and compared with the 25.9 kDa (1Fl - 5Fl) fragment and intact Fn for binding to IgG. The 4Fl.5Fl and the 25.9 kDa fragment bound to immobilized IgG and inhibited Fn binding to IgG to nearly the same extent as the intact molecule (IC50: Fn = 6.77 x 1O(-9) M; 25.9 kDa fragment = 5 x 10(-9) M; 4Fl.5Fl = 7.6 x 10(-9) M). Thus, the binding site for IgG on the Fn molecule is localized to and completely conferred by the 4Fl.5Fl module pair (residues 151-244). Similar experiments using papain-generated Fab and Fc fragments of IgG localized the Fn binding site on IgG to the Fe region of the IgG molecule. Fn bound to the Fc fragment with a nearly identical Kd of 3.69 x 10(-9) M, as to intact IgG (3.77 x 10(-9) M). These studies support the hypothesis that the interaction between Fn and Ig may contribute to the pathophysiology of immune complex related disorders.
纤连蛋白（Fn）是一种由三种不同模块类型（F1、F2和F3）的多个拷贝组成的镶嵌蛋白，已发现在多种免疫复合物（IC）疾病和骨髓增殖性疾病中与循环免疫复合物（ICs）和免疫球蛋白（Ig）聚集体相关。我们之前已经表明，来自Fn NH2末端结构域的一个Mr = 25,900 Da的蛋白水解片段，由五个1型模块（1F1 - 5F1）组成，在生理条件下能与主要的Ig类别结合，这表明ICs和冷球蛋白中Fn的存在是这两种分子之间物理化学结合相互作用的结果。现在我们使用酶联免疫吸附测定（ELISA）表明，Fn与IgG之间的相互作用：（1）不受血浆中任何其他成分的影响；（2）不受温度影响；（3）估计解离常数（Kd）为3.77×10^(-9) M。此外，我们进一步确定了Fn与IgG相互作用中各自涉及的位点。在酵母中表达的来自Fn NH2末端的重组1型模块对（1F1.2F1和4F1.5F1）用于ELISA和亲和色谱，并与25.9 kDa（1F1 - 5F1）片段和完整的Fn进行IgG结合比较。4F1.5F1和25.9 kDa片段与固定化的IgG结合，并抑制Fn与IgG的结合，其程度与完整分子几乎相同（半数抑制浓度（IC50）：Fn = 6.77×10^(-9) M；25.9 kDa片段 = 5×10^(-9) M；4F1.5F1 = 7.6×10^(-9) M）。因此，Fn分子上IgG的结合位点定位于4F1.5F1模块对（第151 - 244位氨基酸残基）并完全由其赋予。使用木瓜蛋白酶产生的IgG Fab和Fc片段进行的类似实验将Fn在IgG上的结合位点定位于IgG分子的Fc区域。Fn与Fc片段结合的Kd与完整IgG的Kd几乎相同，为3.69×10^(-9) M（完整IgG的Kd为3.77×10^(-9) M）。这些研究支持了Fn与Ig之间的相互作用可能导致免疫复合物相关疾病病理生理学的假说。
