Rostagno A, Williams M J, Baron M, Campbell I D, Gold L I
Department of Pathology, New York University Medical School, New York 10016.
J Biol Chem. 1994 Dec 16;269(50):31938-45.
The fibronectin (Fn) monomer contains two major sites of fibrin binding affinity present within the NH2-terminal and COOH-terminal domains; they consist of five (1F1-5F1) and three (10F1-12F1) consecutive type 1 modules, respectively. Recently, we have reported that the fourth and fifth type 1 module pair (4F1.5F1) of the NH2-terminal domain of fibronectin demonstrated fibrin binding ability (Williams, M. J., Phan, I., Harvery, T. S., Rostagno, A., Gold, L. I., and Campbell, I. D. (1994) J. Mol. Biol. 235, 1303-1311). In an attempt to further localize fibrin binding activity and to characterize the nature of the interaction between different type 1 modules of Fn and fibrin, we have tested a range of recombinant proteins and subtilisin generated proteolytic fragments of Fn in an enzyme-linked immunosorbent assay (ELISA) and by fibrin affinity chromatography. Of the recombinant proteins, we found that only the 4F1.5F1 exhibited significant fibrin binding activity, while 1F1, 1F1.2F1, 7F1, and 10F1 had little to no affinity for fibrin. On a molar basis, 4-5 times more 4F1.5F1 than a proteolytic fragment, corresponding to 1F1-5F1 (25.9 kDa) was required to cause 50% inhibition (IC50) of intact biotinylated Fn binding to fibrin in a competitive ELISA. This suggests that all five type 1 modules in tandem engender higher fibrin binding activity than the 4F1.5F1 alone. Furthermore, since fibrin binding activity of the intact Fn molecule was inhibited, by 70-80%, by the 4F1.5F1, the 25.9-kDa fragment, and a MoAb mapped to an epitope on the 4F1.5F1, the fibrin-binding site within the 4F1.5F1 contributes greatly to the non-covalent interaction of intact Fn with fibrin. These results provide significant insight into the Fn/fibrin interaction, a major component of the processes of wound repair and fibrin matrix assembly.
纤连蛋白（Fn）单体在NH2末端和COOH末端结构域内含有两个主要的纤维蛋白结合亲和力位点；它们分别由五个（1F1 - 5F1）和三个（10F1 - 12F1）连续的1型模块组成。最近，我们报道了纤连蛋白NH2末端结构域的第四和第五个1型模块对（4F1.5F1）表现出纤维蛋白结合能力（Williams，M. J.，Phan，I.，Harvery，T. S.，Rostagno，A.，Gold，L. I.，和Campbell，I. D.（1994）J. Mol. Biol. 235，1303 - 1311）。为了进一步定位纤维蛋白结合活性并表征Fn不同1型模块与纤维蛋白之间相互作用的性质，我们在酶联免疫吸附测定（ELISA）和纤维蛋白亲和色谱中测试了一系列重组蛋白以及枯草杆菌蛋白酶产生的Fn蛋白水解片段。在重组蛋白中，我们发现只有4F1.5F1表现出显著的纤维蛋白结合活性，而1F1、1F1.2F1、7F1和10F1对纤维蛋白几乎没有或没有亲和力。在摩尔基础上，在竞争性ELISA中，与对应于1F1 - 5F1（25.9 kDa）的蛋白水解片段相比，需要4 - 5倍摩尔量的4F1.5F1才能导致完整的生物素化Fn与纤维蛋白结合的50%抑制（IC50）。这表明串联的所有五个1型模块产生的纤维蛋白结合活性高于单独的4F1.5F1。此外，由于完整Fn分子的纤维蛋白结合活性被4F1.5F1、25.9 kDa片段以及映射到4F1.5F1上一个表位的单克隆抗体抑制了70 - 80%，4F1.5F1内的纤维蛋白结合位点对完整Fn与纤维蛋白的非共价相互作用有很大贡献。这些结果为Fn/纤维蛋白相互作用提供了重要的见解，这是伤口修复和纤维蛋白基质组装过程的一个主要组成部分。
