CONFORMATIONAL TRANSITIONS IN THE CELL-BINDING DOMAIN OF FIBRONECTIN
CONFORMATIONAL TRANSITIONS IN THE CELL-BINDING DOMAIN OF FIBRONECTIN
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DOI：
10.1021/bi00013a039
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发表时间：
1995-04-04
期刊：
影响因子：
2.9
通讯作者：
PLOW, EF
中科院分区：
文献类型：
--
作者：
UGAROVA, TP;ZAMARRON, C;PLOW, EF
血浆纤维连接蛋白很容易随着环境条件的改变而改变形状，这反过来可能导致其多种功能位点的差异表达。为了测试这种可能性，用单抗(MAb)评估了纤维连接蛋白细胞结合域中的两个III型模块的表达。利用血浆纤维连接蛋白的蛋白分解片段和重组片段，将mAbIII-9和mAbIII-10识别的表位分别定位于纤维连接蛋白的第9和第10(含RGD)III型重复序列。两种单抗均可抑制血小板与固定化纤维连接蛋白的黏附，提示识别的表位与细胞结合部位的空间位置非常接近。放射免疫分析和Scatchard分析表明，在溶液中，每个二聚体纤维连接蛋白分子结合两个mAbIII-9，但只结合一个mAbIII-10分子(离子强度0.15，pH 7.4)。通过电子显微镜证实每个纤维连接蛋白分子都有一个mAbIII-10的结合。肝素、硫酸肝素、神经节苷脂(但不包括硫酸软骨素A和B以及透明质酸)和自结合增加了mAbIII-10对可溶性纤维连接蛋白的表观亲和力。纤维连接蛋白在聚苯乙烯表面的吸附导致了mAbIII-10的额外结合位点的出现。纤维连接蛋白的固定化也改变了MAbIII-9的结合。这些结果表明，纤维连接蛋白的沉积及其与细胞外基质成分的相互作用可以调节细胞结合域的表达，包括包含RGDS的III型重复序列。由于纤维连接蛋白二聚体在表面展开，暴露在纤维连接蛋白二聚体内的第二个第十型III重复序列，可能有助于增强吸附的纤维连接蛋白的粘附性。
Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self-association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin.