Deletion mutagenesis of high molecular weight kininogen light chain. Identification of two anionic surface binding subdomains.
Deletion mutagenesis of high molecular weight kininogen light chain. Identification of two anionic surface binding subdomains.
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DOI：
10.1016/s0021-9258(18)53802-3
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发表时间：
1993-02
期刊：
影响因子：
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通讯作者：
S. Kunapuli;R. D. Dela Cadena;R. Colman
中科院分区：
文献类型：
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作者：
S. Kunapuli;R. D. Dela Cadena;R. Colman
裂解的高分子量激肽原 (HK) 的轻链 (LC) 与阴离子表面以及酶原激肽释放酶前体和因子 XI 结合，从而加速激肽释放酶-激肽、纤溶和凝血途径的激活。 HK LC 上因子 XI（氨基酸残基 574-631）和前激肽释放酶（残基 583-613）的结合位点已定位于结构域 6。结构域 5（残基 438-520）已被假定包含阴离子表面结合子结构域。为了定义这个子结构域，我们在大肠杆菌中将 HK LC（残基 Lys438-Ser644）表达为与谷胱甘肽-S-转移酶（GST）的融合蛋白，并生成了各种 HK LC 缺失突变体。通过谷胱甘肽-琼脂糖亲和层析从细菌细胞提取物中纯化重组 HK LC (rHK LC) 和各种 HK LC 片段作为 GST 融合蛋白。 rHK LC 和重组片段 His459-Ser644、Glu466-Ser644、Leu483-Ser644、His493-Ser644、Lys438-Asp492、Lys438-Ser531 和 His493-Lys520 抑制 125I-HKa 与高岭土（接触中使用的模型阴离子表面）的结合系统，以浓度依赖性方式。缺少结构域 5、Thr521-Ser644 和 Ser583-Ser644 的缺失突变蛋白不会抑制放射性标记的 HKa 与高岭土的结合。 rHK LC 和重组片段 Lys438-Asp492、Lys438-Ser531、His493-Ser644、His493-Lys520、Thr521-Ser644 和 Ser583-Ser644 用 125I 进行放射性标记，然后测试它们在纤维蛋白原和白蛋白存在下与高岭土结合的能力。除Thr521-Ser644和Ser583-Ser644片段外，所有其他放射性标记的HK LC缺失突变蛋白和rHK LC均以浓度依赖性方式与高岭土结合。这种与高岭土的结合是特异性的，因为它会被添加过量的未标记 HKa 所抑制。 rHK LC、His493-Ser644 和 delta 493-520 HK LC 具有凝血活性，而其他缺失突变蛋白不表现出凝血特性。我们得出结论，HK LC 的结构域 5 中至少存在两个阴离子表面结合子结构域，一个位于富含组氨酸-甘氨酸的区域 (Lys438-Asp492)，另一个位于富含组氨酸-甘氨酸-赖氨酸的区域 (His493-Lys520)。在酶原结合结构域6存在的情况下，任一子结构域都足以赋予HK LC凝血活性，而两者的存在并不额外增加HK LC的凝血活性。
The light chain (LC) of cleaved high molecular weight kininogen (HK) binds to anionic surfaces as well as the zymogens prekallikrein and factor XI and thus accelerates activation of the kallikrein-kinin, fibrinolytic, and coagulation pathways. The binding sites on HK LC for factor XI (amino acid residues 574-631) and prekallikrein (residues 583-613) have been localized to domain 6. Domain 5 (residues 438-520) has been postulated to contain the anionic surface binding subdomain. In order to define this subdomain we have expressed HK LC (residues Lys438-Ser644) as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and generated various HK LC deletion mutants. The recombinant HK LC (rHK LC) and various HK LC fragments were purified as GST fusion proteins by glutathione-Sepharose affinity chromatography from bacterial cell extracts. The rHK LC and recombinant fragments His459-Ser644, Glu466-Ser644, Leu483-Ser644, His493-Ser644, Lys438-Asp492, Lys438-Ser531, and His493-Lys520 inhibited 125I-HKa binding to kaolin, a model anionic surface used in the contact system, in a concentration-dependent manner. Deletion mutant proteins lacking domain 5, Thr521-Ser644 and Ser583-Ser644, did not inhibit the radiolabeled HKa binding to kaolin. The rHK LC and recombinant fragments Lys438-Asp492, Lys438-Ser531, His493-Ser644, His493-Lys520, Thr521-Ser644, and Ser583-Ser644 were radiolabeled with 125I and were then tested for their ability to bind to kaolin in the presence of fibrinogen and albumin. Except for the Thr521-Ser644 and Ser583-Ser644 fragments, all other radiolabeled HK LC deletion mutant proteins and rHK LC bound to kaolin in a concentration-dependent manner. This binding to kaolin was specific since it was inhibited by the addition of excess unlabeled HKa. The rHK LC, His493-Ser644 and delta 493-520 HK LC have coagulant activity, while other deletion mutant proteins did not exhibit coagulant properties. We conclude that there are at least two anionic surface binding subdomains, one in the histidine-glycine-rich region (Lys438-Asp492) and the other in the histidine-glycine-lysine-rich region (His493-Lys520), in the domain 5 of HK LC. Either subdomain, in the presence of the zymogen binding domain 6, is sufficient to impart coagulant activity to HK LC, while the presence of both did not increase the coagulant activity of HK LC additively.