Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.
Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.
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人 β2-糖蛋白 I 重组第五结构域的结构和功能：Lys77 和 Thr78 之间特异性切割的影响。
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发表时间：
1997
期刊：
影响因子：
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通讯作者：
Y. Goto
中科院分区：
文献类型：
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作者：
Y. Hagihara;K. Enjyoji;T. Omasa;Y. Katakura;K. Suga;M. Igarashi;E. Matsuura;H. Kato;T. Yoshimura;Y. Goto
为了阐明β2-糖蛋白I（β2-GPI）与心磷脂（CL）的结合机制，我们利用毕赤酵母构建了β2-GPI C末端结构域（Domain V）的高水平表达系统，并研究了其构象和脂质体结合活性。发现纯化的结构域 V 具有天然二硫键。从圆二色光谱来看，它具有紧凑的折叠构象，并表现出由pH或尿素诱导的协同展开转变。此外，它还结合含有 CL 的脂质体。已知市售的人β2-GPI在Lys 317和Thr 318之间被选择性切割。我们发现牛因子Xa弱但特异性地切割重组结构域V的相应位点，即Lys 77和Thr 78之间的肽键。通过圆二色性和荧光测量检查了在该位点切割的“切口”结构域V的构象，并得出结论与重组域V的构象相似。完整的蛋白质。带切口的结构域 V 对尿素的稳定性略低于完整蛋白。尽管两个结构域 V 均与含有 CL 的脂质体结合，但与完整蛋白相比，带切口的结构域 V 的亲和力大大降低，表明 Lys 77 和 Thr 78 之间肽键的断裂控制了与 CL 的结合。此外，在存在和不存在 CL 脂质体的情况下对荧光光谱的分析表明，Trp 76 参与了结合位点。这些结果表明，包括Trp 76、Lys 77 和Thr 78 的区域在与CL 的结合中具有关键作用。
In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.