血小板膜糖蛋白αⅡbA477P(A446P)突变对αⅡbβ3复合物表达的影响(英文)  

Effect of αⅡb Ala477Pro (A446P) mutation of the platelet glycoprotein on the expression of αⅡbβ3 complex

作　　者：袁小瑜[1] 陈方平[1] 夏昆[2] 付敢[1] 蹇在伏[1] 解勤之[1] 王光平[1] 

机构地区：[1]中南大学湘雅医院血液科,长沙410008 [2]中南大学国家医学遗传学重点实验室,长沙410078

出　　处：《中南大学学报（医学版）》2009年第8期712-717,共6页Journal of Central South University ：Medical Science

摘　　要：目的:探讨血小板膜糖蛋白αⅡbA477P(A446P)突变对αⅡbβ3蛋白表达的影响。方法:用重叠延伸PCR法对正常的αⅡb-pcDNA3.1+质粒载体进行体外定点诱变,构建了αⅡbA477P突变质粒载体;经体外真核细胞COS-7细胞转染试验后,从转录水平、蛋白质水平和膜表面复合物水平检测突变体在真核细胞内的表达。结果:突变的αⅡbA477P(A446P)基因在真核细胞内有转录,Western印迹显示细胞内有αⅡb蛋白的表达,但突变αⅡbA477P(A446P)β3复合物在细胞膜表面的表达量仅为正常αⅡbβ3复合物的12.95%。结论:αⅡbA477P(A446P)显著降低了αⅡbβ3复合物在细胞膜表面的表达,其机制可能是该位点的突变干扰了αⅡb和β3正常结合或者影响了αⅡb蛋白的正常构型。Objective To explore the effect of glycoprotein（GP） αⅡb Ala477Pro（A446P） mutation on the expression of integrin αⅡbβ3.Methods The αⅡbA477P（A446P） eukaryotic expression plasmid αⅡbA477P（A446P）-pcDNA3.1＋ was constructed by site-directed mutagenesis.Cos-7 cells were transfected with the mutational plasmid or the normal plasmid αⅡb-pcDNA3.1＋ with β3-pcDNA3.1＋.The expression of αⅡbA477P（A446P）was tested with the RT-PCR,Western blot,and flow cytometry.Results RT-PCR revealed the transcriptional script of αⅡbA477P（A446P）.Western blot showed the expression of αⅡbA477P（A446P） protein.Flow cytometry demonstrated that the expression of αⅡbA477P（A446P）β3 on the membrane was only 12.95% of the normal αⅡbβ3 complex.Conclusion αⅡbA477P（A446P） mutation distinctly reduces the expression of αⅡbβ3 complex on the membrane.This mutation may interfere the formation of αⅡbβ3 complex or impair the proper conformation of αⅡb subunit.

关 键 词：血小板无力症 整联蛋白αⅡbβ3 β-螺旋 突变 表达 

分 类 号：R55[医药卫生—血液循环系统疾病]