Characterization of an N-terminal mutant of αA-crystallin αA-R21Q associated with congenital cataract.
Characterization of an N-terminal mutant of αA-crystallin αA-R21Q associated with congenital cataract.
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DOI：
10.1016/j.exer.2018.05.016
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发表时间：
2018-09
影响因子：
3.4
通讯作者：
Sharma KK
中科院分区：
文献类型：
--
作者：
Phadte AS;Santhoshkumar P;Sharma KK
与人类先天性白内障相关的几种突变靶向α A-晶状体蛋白中的保守精氨酸残基。α A-晶状体蛋白的N-末端区域是一个“突变热点”，在该区域报告了多个白内障相关突变。αA-crystallin -α A-R21 L和α A-R21 W N端21位精氨酸突变与先天性白内障有关。R21的第三种突变体α A-R21 Q最近被确定与南澳大利亚一个家族的先天性白内障有关。据报道，该点突变通过阻止α A-晶状体蛋白组装成更高级的寡聚体而损害α A-晶状体蛋白的四级结构。为了评估α A-R21 Q突变对α A-晶状体蛋白功能的影响，在体外表达、纯化和表征重组α A-R21 Q。与野生型α Accelalin相比，重组α A-R21 Q表现出增强的分子伴侣样活性、增加的表面疏水性、在尿素中较低的稳定性和增加的对胰蛋白酶消化的敏感性。α A-R21 Q表现出对展开的ADH和牛透镜纤维细胞膜的结合亲和力增加。α A-R21 Q同源寡聚体和异源寡聚体也可抑制H2 O2诱导的ARPE-19细胞凋亡。总之，与野生型α A-晶状体蛋白相比，尽管存在细微的结构差异，但α A-R21 Q表现出功能的增加。本研究进一步证实了精氨酸21参与调节α A-晶体蛋白的结构和功能。
Several mutations associated with congenital cataracts in human beings target conserved arginine residues in αA-crystallin. The N-terminal region of αA-crystallin is a “mutational hotspot,” with multiple cataract-related mutations reported in this region. Two mutations at arginine 21 in the N-terminal domain of αA-crystallin —αA–R21L and αA–R21W have been associated with congenital cataract. A third mutant of R21, αA–R21Q, was recently identified to be associated with congenital cataract in a South Australian family. The point mutation was reported to compromise the quaternary structure of αA-crystallin by preventing its assembly into higher ordered oligomers. To assess the effect of the αA–R21Q mutation on αA-crystallin function, recombinant αA–R21Q was expressed, purified and characterized in vitro. Compared to wild-type αAcrystallin, the recombinant αA–R21Q exhibits enhanced chaperone-like activity, increased surface hydrophobicity, lesser stability in urea and increased susceptibility to digestion by trypsin. αA–R21Q demonstrated increased binding affinity towards unfolding ADH and bovine lens fiber cell membranes. αA–R21Q homo-oligomers and hetero-oligomers also prevented H2O2–induced apoptosis in ARPE–19 cells. Taken together, αA–R21Q exhibited a gain of function despite subtle structural differences as compared to wild-type αA-crystallin. This study further validates the involvement of arginine 21 in regulating αA-crystallin structure and function.
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