Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1
Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1
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DOI：
10.1021/bi9903345
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发表时间：
1999-07-20
期刊：
影响因子：
2.9
通讯作者：
Chiu, IM
中科院分区：
文献类型：
--
作者：
Patrie, KM;Botelho, MJ;Chiu, IM
肝素通过增加对其受体的亲和力和延长其生物半衰期来增强成纤维细胞生长因子1的有丝分裂活性。在用(NaI)-I-125和氯胺T标记人成纤维细胞生长因子-1的过程中，观察到该蛋白失去了与肝素结合的能力。相比之下，牛成纤维细胞生长因子-1即使在碘化后仍保持其与肝素的亲和力。为了定位导致肝素亲和力丧失的区域，从人和牛的FGF1表达载体中构建了嵌合的成纤维细胞生长因子-1蛋白，并在碘标记后检测了它们的肝素亲和力。结果表明，人成纤维细胞生长因子-1的C末端区域是导致肝素亲和力丧失的原因。这一区域在人的成纤维细胞生长因子-1中含有单一的酪氨酸残基，而在牛成纤维细胞生长因子-1的这个位置上有苯丙氨酸残基。将人成纤维细胞生长因子-1序列中的酪氨酸残基突变为苯丙氨酸并不能恢复碘化蛋白的肝素亲和力。同样，将牛成纤维细胞生长因子-1中的苯丙氨酸改变为酪氨酸并不会降低碘化蛋白与肝素结合的能力。相比之下，半胱氨酸-131被丝氨酸取代的突变体人成纤维细胞生长因子-1(C131S)即使在加碘后仍能与肝素结合，而牛成纤维细胞生长因子-1(S131C)在加碘后失去了与肝素的结合亲和力。此外，人成纤维细胞生长因子-1C131S突变体在暴露于CuCl2时显示出同源二聚体形成减少。分子模拟表明，成纤维细胞生长因子-1的肝素结合区包括半胱氨酸-131，半胱氨酸-131在碘化过程中氧化为半胱氨酸后，会与赖氨酸-126和赖氨酸-132相互作用。这种相互作用改变了碱性残基的构象，使它们不再与肝素结合。
Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with (NaI)-I-125 and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl2. Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.