The αA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with αβ-crystallin
The αA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with αβ-crystallin
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DOI：
10.1021/bi011010v
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发表时间：
2002-01-08
期刊：
影响因子：
2.9
通讯作者：
Abraham, EC
中科院分区：
文献类型：
--
作者：
Bera, S;Abraham, EC
人类常染色体显性遗传性先天性白内障与α A-晶状体蛋白（α A-R116 C）中Arg-116突变为Cys相关通过凝胶渗透色谱、SDS-聚丙烯酰胺凝胶电泳、荧光光谱和圆二色光谱研究了不同比例的野生型α B-晶状体蛋白（alphaB-wt）和α A-R116 C-晶状体蛋白重构的α-晶状体蛋白的分子伴侣活性和生物物理性质，并与野生型α A-晶状体蛋白（alphaA-wt）和野生型α B-晶状体蛋白重构的α-晶状体蛋白的分子伴侣活性和生物物理性质进行了比较。含有α A-R116 C和α B-wt的重构α-晶状体蛋白具有比含有α A-wt和α B-wt的α-晶状体蛋白更高的分子量、对Trp侧链暴露更高的热敏性、更少的可用疏水表面和更低的伴侣蛋白活性。二级结构表现出非常小的变化，而对于由α A-R116 C和α B-wt形成的α-晶状体蛋白，三级结构明显不同。最重要的是，通过荧光共振能量转移进行的亚基交换研究表明，alphaA-R116 C形成异源聚集体的速度比alphaA-wt与alphaB-wt更快，并且重构的α-晶体蛋白是两个相互作用的亚基的真正异源聚集体。这些发现表明，具有α A-R116 C突变的先天性白内障的分子基础是形成具有修饰结构的α-晶状体蛋白的高度寡聚化异源聚集体。然而，与基于同质聚集体研究的早期结论相反，具有α A-R116 C的异质聚集体的伴侣蛋白活性的损失似乎没有大到足以成为在受影响个体中引发白内障发展的主要因素。
An autosomal dominant congenital cataract in humans is associated with mutation of Arg-116 to Cys in alphaA-crystallin (alphaA-R116C ). The chaperone activity and biophysical properties of reconstituted alpha-crystallin from different proportions of wild-type alphaB-crystallin (alphaB-wt) and alphaA-R116C-crystallin were studied by gel permeation chromatography, SDS-polyacrylamide gel electrophoresis, and fluorescence and circular dichroism spectroscopy and compared with those of reconstituted a-crystallin from alphaB-wt and wild-type alphaA-crystallin (alphaA-wt). The reconstituted alpha-crystallin containing alphaA-R116C and alphaB-wt had a higher molecular mass, a higher thermal sensitivity to exposition of Trp side chains, fewer available hydrophobic surfaces, and lower chaperone activity than the alpha-crystallin containing alphaA-wt and alphaB-wt. The secondary structure exhibited very small changes, whereas the tertiary structure was distinctly different for alpha-crystallin formed from alphaA-R116C and alphaB-wt. Most importantly, subunit exchange studies by fluorescence resonance energy transfer showed that alphaA-R116C forms heteroaggregates faster than alphaA-wt with alphaB-wt, and the reconstituted alpha-crystallins were true heteroaggregates of two interacting subunits. These findings suggest that the molecular basis for the congenital cataract with the alphaA-R116C mutation is the formation of highly oligomerized heteroaggregates of alpha-crystallin with modified structure. However, contrary to the earlier conclusions based on the studies of homoaggregates, the loss in chaperone activity of the heteroaggragates having alphaA-R116C does not appear to be large enough to become the main factor in initiating cataract development in the affected individuals.