遗传性白内障小鼠晶状体的比较蛋白质组分析

Comparative proteomic analysis of lens in congenital inherited cataract mice

目的:比较研究先天性遗传性白内障的晶状体蛋白质表达谱的改变。方法:采用自发的Crygs基因突变先天性隐性遗传白内障小鼠模型,分别提取白内障与正常小鼠晶状体总蛋白,进行固相pH梯度(IPG)等电聚焦双向电泳,R-250染色,使用PDQuest7.30图像分析软件分析电泳图像。选择部分差异蛋白点胶上酶解,应用MALDI-TOF/TOF仪器进行串联质谱(MS/MS)鉴定及分析。结果:上样量为882μg时,白内障和正常小鼠分别检测到(417±53)个蛋白点(n=3)和(370±41)个蛋白点(n=3)。上样量为190μg时,白内障和正常小鼠分别检测到(60±7)个蛋白点和(57±5)个蛋白点(n=3)。对γS、BFSP/filensin、γF、βA1、βB1、βB2和αB等7种差异蛋白进行了鉴定。突变小鼠晶状体正常γS、念珠状纤维结构蛋白(BFSP/filensin)缺失,γF表达下调(<4倍),异常βA1、βB1、βB2和αB表达上调(>4倍),分子量比正常小,提示βA1、βB1、βB2和αB可能发生裂解。结论:蛋白质的双向电泳和质谱分析有助于基因突变后下游蛋白的功能分析。Crygs基因突变导致γS晶状体蛋白的异常,并引起细胞骨架蛋白(BFSP/filensin)和其它晶状体蛋白(γF、βA1、βB1、βB2和αB等)继发改变,从而间接诱发白内障。

AIM： To separate total lens proteins of congenital inherited cataract in mice and to observe the alteration of proteins after gene mutation. METHODS： We studied the mice with a spontaneous mutation of crystallin gamma S （Crygs） transmitted as a recessive trait. Total proteins were extracted and separated using immobilized pH gradient （ IPG）, two - dimensional electrophoresis （2 - DE） and colloidal Coomassie brilliant blue （CBB） staining. The image analysis was carried out using PDQuest 7.30 software package. Several significantly differential proteins in gel were identi- fied by matrix assisted laser adsorption/ionizationtime of flight- tandem mass spectrometry （MALDI- TOF- MS/MS）. RESULTS： As the 882 μg sample was added, we detected （417 ＋ 53 ） spots and （370 ±41 ）spots in cataract and normal lens, respectively. As the 190 μg sample was loaded, we detected （60 ±7） spots and （57±5） spots in cataract and norreal lens, respectively. Seven kinds of differential proteins were identified, including BFSP/filensin, γS, γF, βA1, βB1, βB2 and orB. In crystalline lens of mutant mice, γS and beaded -filament structure protein （BFSP/filensin） were not detected. γF was downexpressed （ 〈4 fold） while βA1, βB1, βB2 and αB were overexpressed （ 〉4 fold） in mutant cataract. The latter proteins were less MW than normal, suggesting that they were possibly truncated. CONCLUSION： 2 - DE and mass spectrometry can help to assess and analyze the function of proteins as a novel approach. The mutant Crygs gene can lead to the abnormity of ＂/S crystallin, which can induce the changes of skeletonal protein （BFSP/filensin） and other crystallins γF, βA1, βB1, βB2 and αB） , and then evoke cataract secondarily.