Cleavage of the C-terminal serine of human αA-crystallin produces αA1-172 with increased chaperone activity and oligomeric size
Cleavage of the C-terminal serine of human αA-crystallin produces αA1-172 with increased chaperone activity and oligomeric size
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DOI：
10.1021/bi0618722
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发表时间：
2007-03-06
期刊：
影响因子：
2.9
通讯作者：
Abraham, Edathara C.
中科院分区：
文献类型：
--
作者：
Aziz, Atya;Santhoshkumar, Puttur;Abraham, Edathara C.
本研究旨在研究 C 端截短的人 α A 晶状体蛋白突变体的寡聚体大小、结构、流体动力学特性和伴侣功能，特别关注 α A(1-172)，它是 Ser(172)-Ser(173) 键的裂解产物，是人类晶状体所独有的，构成 α A 晶状体蛋白的主要部分。通过定点诱变制备了各种截短形式的人αA-晶状体蛋白。蛋白质在大肠杆菌 BL21(DE3) pLysS 细胞中表达，并通过尺寸排阻柱层析纯化。通过动态光散射测量确定分子质量和其他流体动力学性质。分别通过远紫外和近紫外圆二色光谱测量来评估二级和三级结构变化。使用 ADH、胰岛素和 β L-晶状体蛋白作为靶蛋白测定伴侣活性。 α Alpha(1-172)表现出寡聚体大小显着增加，即通过光散射测量，与α A-wt中的702 kDa相比，寡聚体大小为866 kDa。 αα(1-172)和αA-wt具有相似的二级结构，但前者的三级结构略有改变。最有趣的观察是，α Alpha(1-172) 的伴侣分子性能比 α A-wt 好 28-46%。 αα(1-168)的寡聚体大小和结构与αA-wt相似，而分子伴侣活性降低了12-23%。另一方面，α Alpha(1-162) 的寡聚体大小为 400 kDa，分子伴侣活性降低 80-100%，并且二级和三级结构显着改变。数据表明，主要截短产物α Alpha(1-172)的存在将显着改善α A-晶状体蛋白的整体伴侣功能。这将有利于晶状体承受氧化应激。由于 alpha Alpha(1-168) 和 alpha Alpha(1-162) 仅少量存在，因此它们的影响很小。
This study aimed to study the oligomeric size, structure, hydrodynamic properties, and chaperone function of the C-terminally truncated human alpha A-crystallin mutants with special emphasis on alpha A(1-172) which is the cleavage product of the Ser(172)-Ser(173) bond, unique to human lenses and constituting a major part of alpha A-crystallin. Various truncated forms of human alpha A-crystallins were prepared by site-directed mutagenesis. The proteins were expressed in Escherichia coli BL21(DE3) pLysS cells and purified by size exclusion column chromatography. Molecular masses and the other hydrodynamic properties were determined by dynamic light scattering measurements. The secondary and tertiary structural changes were assessed by far- and near-UV CD spectra measurements, respectively. Chaperone activity was determined by using ADH, insulin, and beta L-crystallin as the target proteins. alpha Alpha(1-172) exhibited a significant increase in oligomeric size, i.e., 866 kDa by light scattering measurements as compared to 702 kDa in alpha A-wt. alpha Alpha(1-172) and alpha A-wt had similar secondary structure, but the former exhibited slightly altered tertiary structure. The most interesting observation was that alpha Alpha(1-172) behaved as a 28-46% better chaperone than alpha A-wt. The oligomeric size and structure of alpha Alpha(1-168) were similar to those of alpha A-wt, while the chaperone activity was decreased by 12-23%. alpha Alpha(1-162), on the other hand, had an oligomeric size of 400 kDa, a decrease in chaperone activity of 80-100%, and significantly altered secondary and tertiary structures. The data show that the overall chaperone function of alpha A-crystallin will be significantly improved by the presence of the major truncated product alpha Alpha(1-172). This will be beneficial to the lens undergoing oxidative stress. Since alpha Alpha(1-168) and alpha Alpha(1-162) are present only in small amounts, their effect would be minimal.