Makogonenko Evgeny, Ingham Kenneth C, Medved Leonid
Center for Vascular and Inflammatory Diseases and the Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Biochemistry. 2007 May 8;46(18):5418-26. doi: 10.1021/bi7001373. Epub 2007 Apr 11.
Incorporation of fibronectin into fibrin clots is important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. Previous studies revealed that this incorporation occurs through noncovalent interaction between two NH2-terminal Fib-1 regions of fibronectin (one on each chain) and the alphaC-regions of fibrin, and is further reinforced by factor XIIIa-mediated covalent cross-linking of fibronectin to the fibrin matrix. To clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin, we prepared by limited proteolysis a dimeric 140 kDa (Fib-2)2 fragment containing both Fib-2 regions and tested its interaction with recombinant fragments corresponding to the alphaC regions of fibrin(ogen). In both ELISA and surface plasmon resonance (SPR) experiments 140 kDa (Fib-2)2 bound to the immobilized Aalpha221-610 alphaC-fragment. However, the affinity of binding was substantially lower than that for Fib-1. Ligand blotting and ELISA established that the Fib-2 binding site is located in the connector part of the alphaC region including residues Aalpha221-391. Analysis of the SPR-detected binding of fibronectin to the immobilized Aalpha221-610 alphaC-fragment revealed two types of fibronectin-binding sites, one with high affinity and another one with much lower affinity. Competition experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment suggesting partial overlap of the two sets of binding sites. Based on these results and our previous studies we propose a mechanism of interaction of fibronectin with fibrin in which both Fib-1 and Fib-2 play a role.
纤连蛋白掺入纤维蛋白凝块对于形成临时基质很重要，该临时基质在伤口愈合过程中促进细胞黏附和迁移。先前的研究表明，这种掺入是通过纤连蛋白两个NH2末端Fib-1区域（每条链上一个）与纤维蛋白的αC区域之间的非共价相互作用发生的，并且通过因子XIIIa介导的纤连蛋白与纤维蛋白基质的共价交联进一步增强。为了阐明位于纤连蛋白二硫键连接的COOH末端的另一对纤维蛋白结合区域Fib-2的作用，我们通过有限蛋白酶解制备了一个包含两个Fib-2区域的140 kDa二聚体（Fib-2）2片段，并测试了其与对应于纤维蛋白（原）αC区域的重组片段的相互作用。在酶联免疫吸附测定（ELISA）和表面等离子体共振（SPR）实验中，140 kDa（Fib-2）2均与固定化的Aα221-610αC片段结合。然而，结合亲和力明显低于Fib-1。配体印迹和ELISA确定Fib-2结合位点位于αC区域的连接部分，包括残基Aα221-391。对SPR检测的纤连蛋白与固定化的Aα221-610αC片段的结合分析显示出两种类型的纤连蛋白结合位点，一种具有高亲和力，另一种具有低得多的亲和力。竞争实验表明，随着Fib-1片段浓度增加，Fib-2介导的结合受到约30%的抑制，表明两组结合位点部分重叠。基于这些结果和我们先前的研究，我们提出了纤连蛋白与纤维蛋白相互作用的机制，其中Fib-1和Fib-2都发挥作用。
