先天性白内障相关的βB2晶状体蛋白突变的致病机制研究

发布时间：2018-07-10 14:12

本文选题：βB2晶状体蛋白 + 先天性白内障　；参考：《浙江大学》2013年博士论文

【摘要】：βB2晶状体蛋白是人眼晶状体中重要的结构蛋白,与维持晶状体的透明性与屈光性密切相关,其潜在的生理功能尚不明确。目前国内外针对βB2晶状体蛋白基因突变导致先天性白内障的报道已有不少,但是针对突变具体的致病机制研究仍很缺乏。为了更好地阐明白内障的发生机理,从现有的与βB2晶状体蛋白相关的基因突变中,选取数个有意义的关键位点(位于蛋白N末端的突变A2V,以及四个与色氨酸相关的突变S31W、R145W、W59C、W151C),研究这些突变对pB2晶状体蛋白结构、稳定性、细胞内分布等方面的影响。通过PCR从人眼cDNA文库中克隆βB2晶状体蛋白的完整编码序列,构建野生型pB2晶状体蛋白和包含各突变位点的质粒,原核表达质粒采用pET28a,真核表达质粒采用pEGFP-C3和pcDNA3.1-flag;原核表达获取目标蛋白并纯化,采用生物化学和生物物理学研究手段,明确突变前后蛋白结构和理化性质方面的改变；并转染HeLa细胞,使野生型和突变型蛋白分别在细胞中过表达,观察蛋白在细胞内定位以及对细胞凋亡的影响。我们发现,A2V突变没有改变pB2晶状体蛋白的二、三级结构,却影响了蛋白的四级构象,破坏了蛋白同源四聚体的形成；降低了蛋白的热稳定性,使蛋白在高温下更容易聚集；不影响对抗化学变性剂和紫外辐射的稳定性,却使蛋白在复性过程更易发生聚集。W59C和W151C突变使βB2的疏水暴露增加,直接导致突变后蛋白的溶解度显著降低；S31W和R145W这两个突变则导致了蛋白热稳定性的明显下降；另外我们证实了色氨酸相关的突变影响了βB2晶状体蛋白对于紫外辐照的稳定性,除R145W外,其余三个突变体的稳定性较野生型下降,R145W却比野生型更能抵抗紫外能量的损伤；将四个突变体在HeLa细胞中过表达后,W59C和W151C在胞内出现了明显的聚集现象,这可能与它们的的溶解度有关,与蛋白水平的结果一致,说明溶解度下降很可能是这两个突变在出生即表现为白内障的原因。以上结果表明,βB2晶状体蛋白的N末端的突变会对自身的寡聚化造成影响,位于疏水核心的色氨酸残基突变会显著影响溶解度和稳定性,而结构域表面亲水的氨基酸突变为色氨酸后则会降低蛋白稳定性,促进蛋白聚集。据此认为,βB2晶状体蛋白基因突变导致的先天性白内障,其发病机制是多样的。
[Abstract]:尾 B2 crystallin is an important structural protein in human lens, which is closely related to the transparency and refraction of the lens, and its potential physiological function is not clear. There have been many reports at home and abroad about the mutation of 尾 B2 crystallin gene leading to congenital cataract, but there is still a lack of research on the specific pathogenetic mechanism of the mutation. In order to better elucidate the mechanism of cataract, from the existing gene mutations related to 尾 B2 crystallin, Several significant key sites (A _ 2V at the N-terminal protein and four tryptophan related mutations S31WN R145WN W59C W151C) were selected to study the effects of these mutations on the structure, stability and intracellular distribution of pB2 crystallin. The complete coding sequence of 尾 B2 crystallin was cloned from the human eye cDNA library by PCR, and the wild type pB2 crystallin and plasmids containing various mutation sites were constructed. The prokaryotic expression plasmid was pET28a, the eukaryotic expression plasmid was pEGFP-C3 and pcDNA3.1-flag. the target protein was obtained and purified by prokaryotic expression, and the changes of protein structure and physicochemical properties before and after mutation were determined by biochemical and biophysical methods. The wild-type and mutant proteins were overexpressed in HeLa cells respectively. The localization of proteins in the cells and the effect on apoptosis were observed. We found that the mutation of A2V did not change the secondary and tertiary structure of pB2 crystallin, but affected the conformation of the protein, destroyed the formation of homologous tetramer, reduced the thermal stability of the protein, and made the protein gather more easily at high temperature. The stability of anti-chemical denaturant and ultraviolet radiation was not affected, but the aggregation of protein. W59C and W151C mutation increased the hydrophobic exposure of 尾 B2 during renaturation, which directly reduced the solubility of protein. Two mutations, S31W and R145W, resulted in a significant decrease in the thermal stability of the protein. In addition, we confirmed that tryptophan related mutations affected the stability of 尾 B2 crystallin to UV irradiation, except for R145W. The stability of the other three mutants was more stable than that of wild type R145W, but they were more resistant to the damage of ultraviolet energy than wild type. After the four mutants were overexpressed in HeLa cells, W59C and W151C showed obvious aggregation in the cell. This may be related to their solubility and consistent with the results of protein levels, suggesting that the decline in solubility may be the cause of the cataract at birth. These results indicate that the N-terminal mutation of 尾 B2 crystallin will affect the oligomerization of 尾 B2, and the tryptophan residue mutation at the hydrophobic core will significantly affect the solubility and stability of 尾 B2 crystallin. When the hydrophilic amino acids on the surface of domain mutated to tryptophan, the stability of protein was decreased and protein aggregation was promoted. It is concluded that the pathogenesis of congenital cataract caused by 尾 B2 crystallin gene mutation is diverse.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R776.1

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