Binding of heparin by type III domains and peptides from the carboxy terminal hep-2 region of fibronectin.
Binding of heparin by type III domains and peptides from the carboxy terminal hep-2 region of fibronectin.
复制标题
肝素与纤连蛋白羧基末端 hep-2 区域的 III 型结构域和肽的结合。
DOI：
10.1021/bi00097a035
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Busby,TF
中科院分区：
文献类型：
--
作者：
Ingham,KC;Brew,SA;Migliorini,MM;Busby,TF
修订稿于 19939 年 9 月 7 日收到 摘要：纤连蛋白与肝素结合的主要位点位于 30 或 40 kDa 的片段中，其中包含 III 型模块 12 至 14 或 15。已比较了源自该区域的各种蛋白水解或重组子片段以及几种合成肽与荧光素标记的肝素溶液的结合。通过在 25°C 和 pH 7.4 下单独使用 (TB) 或与 0.15 M NaCl (TBS) 一起使用 0.02 M Tris 缓冲液中荧光各向异性的变化来监测结合。包含 III13 和 III14 但缺乏 III12 的 23 kDa 片段在 TB 和 TBS 中的 K& 值分别为 0.3 和 1.8，与 30 kDa 亲本无法区分。仅包含模块 III13 的片段的结合力比母体弱 2-3 倍，而仅包含 III14 的片段的结合力弱 6-50 倍，具体取决于离子强度。仅包含 III12 或 IIIj5 的片段在 TBS 中根本无法结合。衍生自 III13 氨基末端并含有 Arg-Arg-Ala-Arg 共有序列的阳离子肽，Barkalow 和 Schwarzbauer 显示了其完整性 [Barkalow, FJ, & Schwarzbauer, J. E.(1991) J. Biol.化学。 266, 7812-7818] 至关重要的是，未能在 TBS 中结合，但在 TB 中结合较弱。来自 III14 的中间和 C 末端区域的另外两种阳离子肽显示出类似的行为。因此，虽然肝素结合的主要决定簇位于 III 13，但这些决定簇仅在正确折叠结构的一部分时才具有活性。此外，模块 III13 在分离时具有比同时含有 III13 和 III14 的片段稍低的亲和力。结论是，这两个模块之间的相互作用对于排列两个模块的带正电残基以实现肝素的最佳识别可能很重要。纤连蛋白是一种大糖蛋白，存在于细胞表面、结缔组织基质和细胞外液中（Hynes，1990）。它由两个非常相似的分子量为 250 000 道尔顿的亚基组成，在 C 末端区域通过二硫键连接在一起。每个子单元包含
Revised Manuscript Received September 7, 19939 abstract: The major sites of heparin binding by fibronectin are located in fragments of 30 or 40 kDa that contain type III modules 12through 14or 15. Various proteolytic or recombinant subfragments andseveral synthetic peptides derived from this region have been compared with respect to their binding to fluoresceinlabeled heparinin solution. Binding was monitored by the change in fluorescence anisotropy at 25 C and pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15 M NaCl (TBS). A 23-kDa fragment containing III13 and III14 but lacking III12had K& values of 0.3 and 1.8 in TB and TBS, respectively, indistinguishable from the 30-kDa parent. Fragments containing only module III13 bound 2-3-fold weaker than the parent while those containing only III14 bound 6-50-fold weaker depending on the ionic strength. Fragments containing only III12 or IIIj5 failed to bind at all in TBS. A cationic peptide derived from the amino terminus of III13 and containing the Arg-Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow and Schwarzbauer [Barkalow, FJ, & Schwarzbauer, J. E.(1991) J. Biol. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bound weakly in TB. Two additional cationic peptides derived from the middle and C-terminal regions of III14 showed similar behavior. Thus while the major determinant (s) of heparin binding are located in III 13, those determinants are only active when part of a properly folded structure. Furthermore, module III13 when isolated had a slightly lower affinity thanfragments containing both III13 and III14. It is concluded that interactions between these two modules may be important to arrange positively charged residues from both modules for optimal recognition by heparin.Fibronectin is a large glycoprotein which occurs on cell surfaces, in the connectivetissue matrix, and in extracellular fluids (Hynes, 1990). It consists of two very similar subunits of molecular mass 250 000 daltons, held together at the C-terminal region by disulfide bonds. Eachsubunit contains