【作者】 徐佳；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学（专业学位）， 2013， 博士

【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。更多还原

【Abstract】 βB2-Crystallin is one of major protein constituents of the human lens. Its high solubility and stability are critical for lens clarity and refraction. Many congenital cataract-linked mutations in βB2-crystallin gene (CRYBB2) have been reported. However, researches focused on the pathopoiesis mechanism of these mutations are deficient, thus there is a missing link between the influence of these mutations on the protein structure/function and the onset of cataract.In this research, we studied the mechanism underlying several mutations including the A2V mutation at the N-terminus and four tryptophan (Trp) related mutations (S31W, R145W, W59C, W151C). We cloned βB2-crystallin coding sequence from human lens cDNA library, inserted it in the expression plasmid pET28a, and PCR-based site-directed mutagenesis was performed to construct the mutant plasmids. Then the wild type βB2-crystallin and the mutants were overexpressed in E. coli and purified. The effect of mutations on βB2-crystallin structure and stability was investigated via biophysical methods such as circular dichroism, fluorescence and size-exclusion chromatography. The cell level study was conducted by overexpressing the WT and mutated βB2-crystallin gene in HeLa cells with expression plasmid pEGFP-C3or pcDNA3.1-flag to characterize the subcellular localization of target proteins, and analyze the effect of the mutant proteins on cell apoptosis.The results showed that the A2V mutation did not affect the secondary and tertiary structures, but retard the tetramerization of βB2-crystallin at high protein concentrations. The mutation also slightly decreased the thermal stability and promoted the thermal aggregation of βB2-crystallin. For the Trp related mutations, The W59C and W151C mutations affected the tertiary structure of βB2-crystallin by increasing the hydrophobic exposure and severely decrease the solubility of the mutants both in vitro and in the cells. The S31W and R145W mutations mainly influenced the stability of βB2-crystallin by promoting thermal aggregation. All four Trp mutations reduced the stability of βB2-crystallin against denaturation induced by chemical denaturants. When exposed under UV radiation, the mutants except R145W were more prone to aggregate than the wild type protein.更多还原