UV-A-induced structural and functional changes in human lens deamidated αB-crystallin
UV-A-induced structural and functional changes in human lens deamidated αB-crystallin
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发表时间：
2008-02
期刊：
影响因子：
2.2
通讯作者：
Kerri Mafia;R. Gupta;M. Kirk;L. Wilson;O. P. Srivastava;S. Barnes
中科院分区：
文献类型：
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作者：
Kerri Mafia;R. Gupta;M. Kirk;L. Wilson;O. P. Srivastava;S. Barnes
目的比较紫外线（UV）-A照射对野生型α B-晶状体蛋白及其3种脱酰胺突变体蛋白（α B-Asn 78 Asp、α B-Asn 146 Asp和α B-Asn 78/146 Asp）结构和功能的影响。方法使用了三种先前使用如前所述的位点特异性诱变程序从重组WT α B-晶状体蛋白产生的脱酰胺突变体[32]。将WT α B-晶状体蛋白及其三种脱酰胺物质暴露于强度为20或50 J/cm 2的UV-A光（320-400 nm）。检测了UV-A未暴露和UV-A暴露制剂的分子伴侣活性，并且它们的活性与UV-A诱导的结构变化相关。研究的结构特性包括二聚化和降解，内在色氨酸（Trp）荧光，ANS（8-苯胺基-1-萘硫酸盐）结合，远紫外圆二色性（UV-CD）光谱分析，分子大小的动态光散射，和氧化的色氨酸和蛋氨酸（Met）残基。结果WT α B-晶状体蛋白及其3个脱酰胺突变体蛋白在UV-A照射过程中，随着剂量的增加，其二聚体化程度增加，部分降解为40 kDa。与天冬酰胺（Asn）78残基脱酰胺为天冬氨酸（Asp）或Asn 78和Asn 146残基同时脱酰胺为Asp相比，Asn 146残基脱酰胺为Asp导致更大的伴侣活性损失。研究了UV-A诱导的分子伴侣活性的丧失。ANS结合数据表明，与WT和其他两种脱酰胺蛋白相比，α B-Asn 146 Asp突变蛋白具有相对紧凑的结构和表面疏水斑块的增加。同样，与WT α B-晶状体蛋白相比，UV-A暴露改变了脱酰胺突变蛋白中的Trp微环境。远紫外CD光谱分析显示，在UV-A暴露下，WT和脱酰胺物质之间几乎没有变化，除了α B-Asn 146 Asp突变蛋白相对于WT α B-晶状体蛋白和其他两种脱酰胺蛋白在无规卷曲结构中显示出最大变化。UV-A暴露还导致WT和三种脱酰胺突变蛋白的聚集，与非UV-A暴露的物质相比，这些物质具有更大的质量。在WT和3种脱酰胺蛋白的二维（2D）凝胶电泳后回收的4个斑点中，α B-Asn 146 Asp突变体的Met和Trp残基在UV-A暴露后显示出最大氧化，这可能是其分子伴侣活性损失大于WT α B-晶状体蛋白和其他两种脱酰胺蛋白的原因。结论与野生型αB、α B-Asn 78 Asp和α B-Asn 78/146 Asp脱酰胺蛋白相比，α B-Asn 146 Asp脱酰胺蛋白在UV-A照射后的分子伴侣活性完全丧失。显然，这种伴侣活性的丧失是由于氧化变化导致其结构改变大于其他α B-物质。
Purpose To determine comparative effects of ultraviolet (UV)-A irradiation on structural and functional properties of wild type (WT) αB-crystallin and its three deamidated mutant proteins (αB-Asn78Asp, αB-Asn146Asp, and αB-Asn78/146Asp). Methods Three deamidated mutants previously generated from recombinant WT αB-crystallin, using a site-specific mutagenesis procedure as previously described [32], were used. The WT αB-crystallin and its three deamidated species were exposed to UV-A light (320–400 nm) at intensities of 20 or 50 J/cm2. The UV-A-unexposed and UV-A-exposed preparations were examined for their chaperone activity, and their activities were correlated with the UV-A-induced structural changes. The structural properties studied included dimerization and degradation, intrinsic tryptophan (Trp) fluorescence, ANS (8-anilino-1-naphthalenesulfate)-binding, far ultraviolet circular dichroism (UV-CD) spectral analysis, molecular sizes by dynamic light scattering, and oxidation of Trp and methionine (Met) residues. Results The WT αB-crystallin and its three deamidated mutant proteins showed enhanced dimerization to 40 kDa species and partial degradation with increasing doses during UV-A-exposure. Compared to the deamidation of asparagines (Asn) 78 residue to aspartic acid (Asp) or both Asn78 and Asn146 residues to Asp, the deamidation of Asn146 residue to Asp resulted in a greater loss of chaperone activity. The UV-A-induced loss of chaperone activity due to structural changes was studied. The ANS-binding data suggested that the αB-Asn146Asp mutant protein had a relatively compact structure and an increase in surface hydrophobic patches compared to WT and two other deamidated proteins. Similarly, UV-A-exposure altered the Trp microenvironment in the deamidated mutant proteins compared to the WT αB-crystallin. Far-UV CD spectral analyses showed almost no changes among WT and deamidated species on UV-A-exposure except that the αB-Asn146Asp mutant protein showed maximum changes in the random coil structure relative to WT αB-crystallin and two other deamidated proteins. The UV-A-exposure also resulted in the aggregation of WT and the three deamidated mutant proteins with species of greater mass compared to the non-UV-A exposed species. Among the four spots recovered after two-dimensional (2D)-gel electrophoresis from WT and the three deamidated species, the Met and Trp residues of αB-Asn146Asp mutant showed maximum oxidation after UV-A exposure, which might account for its greater loss in chaperone activity compared to WT αB-crystallin and two other deamidated species. Conclusions After UV-A-exposure, the deamidated αB-Asn146Asp mutant protein showed a complete loss of chaperone activity compared to WT αB and αB-Asn78Asp and αB-Asn78/146Asp deamidated species. Apparently, this loss of chaperone activity was due to oxidative changes leading to its greater structural alteration compared to other αB-species.