Barkalow F J, Schwarzbauer J E
Department of Molecular Biology, Princeton University, New Jersey 08544.
J Biol Chem. 1991 Apr 25;266(12):7812-8.
We have identified the major site required for the interaction of fibronectin (FN) with heparin. Affinity chromatography was used to test the binding ability of a library of truncated, monomeric forms of fibronectin (deminectins) containing deletions or two point mutations in the heparin-binding domain. This domain consists of type III repeats 12, 13, and 14. Deletions of individual repeats showed that both III13 and III14 are required for complete binding. Small deletions within these repeats localized a major site of heparin interaction to the amino-terminal half of III13. Site-directed mutagenesis of adjacent arginines within this sequence to uncharged residues reduced heparin binding by 98%, identifying these positively charged amino acids as essential for the interaction. A significant role for the flanking alternatively spliced regions and for repeat III12 was not found. We conclude that, while both repeats III13 and III14 participate in heparin binding, there is a major site of interaction in repeat III13 that accounts for nearly all of the activity. The significance of multiple heparin-binding sites within this domain is discussed and a model is proposed to account for how these sites may function in vivo.
我们已经确定了纤连蛋白（FN）与肝素相互作用所需的主要位点。采用亲和色谱法测试了一系列截短的、单体形式的纤连蛋白（去明胶纤连蛋白）与肝素的结合能力，这些去明胶纤连蛋白在肝素结合结构域存在缺失或两个点突变。该结构域由III型重复序列12、13和14组成。单个重复序列的缺失表明，III13和III14对于完全结合都是必需的。这些重复序列内的小缺失将肝素相互作用的主要位点定位到III13的氨基末端一半。将该序列内相邻的精氨酸定点突变为不带电荷的残基，使肝素结合减少了98%，表明这些带正电荷的氨基酸对于相互作用至关重要。未发现侧翼可变剪接区域和重复序列III12有显著作用。我们得出结论，虽然重复序列III13和III14都参与肝素结合，但III13中存在一个主要的相互作用位点，几乎占了所有活性。本文讨论了该结构域内多个肝素结合位点的意义，并提出了一个模型来解释这些位点在体内可能的功能。
