Huff S, Matsuka Y V, McGavin M J, Ingham K C
Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
J Biol Chem. 1994 Jun 3;269(22):15563-70.
The N-terminal 29-kDa fragment of fibronectin (Fn29K) contains five type I "finger" modules. It binds to heparin, fibrin, and bacteria and is involved in fibronectin (Fn) matrix assembly. Binding to Staphylococcus aureus involves a cell wall-associated protein that contains approximately three repeats of a 38-residue D motif (Signäs, C., Raucci, G., Jönsson, K., Lindgren, P.-E., Anantharamaiah, G.M., Höök, M., and Lindberg, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 699-703). Synthetic peptides representing D1, D2, and D3, when labeled with fluorescein isothiocyanate (FITC), exhibited increases in fluorescence anisotropy upon addition of Fn29K but not other Fn fragments. The response could be reversed by titration with unlabeled peptides to yield inhibition constants that agreed with the dissociation constants obtained by fitting the initial response. Values of Kd ranged between 2 and 12 microM, with D3 having the highest affinity. Specificity of D3 for Fn29K was further illustrated by the fact that its C-terminal half (D3b, Lys801 to Lys821), when immobilized, selectively adsorbed Fn29K from a thermolysin digest of fibronectin. The binding site in Fn was further localized within Fn29K by analyzing smaller proteolytic or recombinant subfragments. Those containing fingers, F3-5 and F4-5, were purified on D3b-Sepharose and bound FITC-D3b with Kd values of 4-6 microM. Subfragments containing pairs of fingers 1-2, 2-3, or single fingers 1, 4, or 5 were inactive. Whole D1-3, expressed in Escherichia coli and labeled with fluorescein, bound 1.9 mol/mol of Fn29K with Kd = 1.5 nM. F4-5 and F2-3 bound with respective Kd values of 0.35 and 4.4 microM. These and other results indicate that binding of the individual D region peptides is mediated through their C-terminal halves, primarily to fingers 4 and 5 of fibronectin. The possible basis of the much higher affinity of D1-3 is discussed.
纤连蛋白的N端29 kDa片段（Fn29K）包含五个I型“指状”模块。它能与肝素、纤维蛋白和细菌结合，并参与纤连蛋白（Fn）基质的组装。与金黄色葡萄球菌的结合涉及一种细胞壁相关蛋白，该蛋白含有约三个38个残基的D基序重复序列（西尼亚斯，C.，劳奇，G.，琼森，K.，林德格伦，P.-E.，阿南塔拉马亚，G.M.，胡克，M.，和林德伯格，M.（1989年）美国国家科学院院刊86，699 - 703）。当用异硫氰酸荧光素（FITC）标记时，代表D1、D2和D3的合成肽在加入Fn29K后荧光 anisotropy增加，但加入其他Fn片段后则没有。通过用未标记的肽滴定可以使这种反应逆转，从而得到与通过拟合初始反应获得的解离常数一致的抑制常数。Kd值在2至12 microM之间，其中D3的亲和力最高。D3对Fn29K的特异性进一步体现在其C端一半（D3b，赖氨酸801至赖氨酸821）固定后能从纤连蛋白的嗜热菌蛋白酶消化物中选择性吸附Fn29K这一事实上。通过分析更小的蛋白水解或重组亚片段，Fn中的结合位点在Fn29K内进一步定位。那些包含指状结构的片段，F3 - 5和F4 - 5，在D3b - 琼脂糖上纯化，并以4 - 6 microM的Kd值结合FITC - D3b。包含指状结构对1 - 2、2 - 3或单个指状结构1、4或5的亚片段没有活性。在大肠杆菌中表达并用荧光素标记的完整D1 - 3，以Kd = 1.5 nM的亲和力结合1.9 mol/mol的Fn29K。F4 - 5和F2 - 3的结合Kd值分别为0.35和4.4 microM。这些以及其他结果表明，各个D区域肽的结合是通过它们的C端一半介导的，主要与纤连蛋白的指状结构4和5结合。文中讨论了D1 - 3亲和力高得多的可能原因。
