血小板膜糖蛋白ⅡbL721R和Q860X复合杂合突变导致血小板无力症的分子机制

Molecular mechanisms of Glanzmann thrombasthenia caused by αⅡb L721R and Q860X compound heterozygous mutation

目的探讨血小板膜糖蛋白ⅡbL721R和Q860X复合杂合突变导致血小板无力症的分子机制。方法应用PCR法对先证者αⅡb和β3基因所有外显子及其侧翼序列进行扩增；PCR产物纯化后直接测序，检测其突变基因。突变位点经直接测序证实排除基因多态性。采用PCR定点突变法构建αⅡbL721R和Q860X突变真核表达载体，测序正确后用脂质体将其分别与表达β3亚基的真核表达载体共转染293T和CHO细胞。采用流式细胞术检测转染细胞膜上αⅡbβ3的表达，用Western blot法鉴定αⅡbL721R和Q860X突变体在转染细胞内的总体表达，采用免疫荧光共聚焦显微镜确定αⅡbL721R和Q860X突变体的细胞内定位。结果先证者在αⅡb基因存在T2255G（L721R）和C2671T（QS60X）复合杂合突变。PCDM8ⅡbL721R／PCDM8Ⅲa转染的293T细胞表面αⅡb为野生型的2．1％，β3为野生型的11．0％。PCDM8ⅡbQ860X／PCDM8Ⅲa转染的293T细胞表面αⅡb为野生型的31．9％，133为野生型的18．0％。Western blot法证实突变αⅡbL721R与β3共表达后，可检测到前体αⅡb（pro-αⅡb），但未检测出成熟αⅡb Q860X与β3共表达后，检测到截短型αⅡb蛋白。免疫荧光细胞内共定位研究显示L721R和Q860X突变αⅡbβ3蛋白大部分分布于内质网，仅有少量在高尔基体中。结论αⅡbL721R和Q860X突变不影响αⅡb蛋白的合成和pro-αⅡβ3复合物的形成，但阻碍了pro—αⅡbβ3复合物由内质网向高尔基体的转运，导致细胞内滞留，从而影响了αⅡbβ3在细胞表面的表达，是导致血小板表面缺乏αⅡbβ3复合物、产生血小板无力症的分子机制。

Objective To explore the molecular mechanisms of Glanzmann thrombasthenia caused by αⅡb L721 R and Q860X compound heterozygous mutation. Methods All exons and exon-intron boundaries of αⅡb and β3 gene were amplified by PCR and analyzed by direct DNA sequencing. Gene polymorphisms were excluded by direct DNA sequencing, αⅡb L721R and Q860X mutants expressing vectors were constructed by in vitro site-directed mutagenesis. The expression of αⅡb L721R and Q860X mutants on transfected cell membrane were analyzed by flow cytometry and the whole expression level was confirmed by Western blot. The subcellular localizations of αⅡb L721R and Q860X mutants were determined by immunofluorescent confocal scanning microscopy. Results The αⅡb compound heterozygous mutations, T2255G （ L721 R） and C2671T（Q860X） , were identified in the proband, the former being inherited from the maternal side and the latter the paternal side. The 293T cells cotransfected with mutated αⅡb L721R and wild-type β3 expression plasmids expressed 2.1% of normal amount of αⅡb on the cell surface as shown by FACS, in contrast to 31.9% of normal amount of αⅡb on the cells cotransfected with cDNAs of mutated αⅡb Q860X and wild- type β3 expression plasmids. Western blot of the cell lysates showed no detectable mature αⅡb in cells lysates with L721R mutant. While, truncated αⅡb protein was detected in cell lystes with Q860X mutant. Immunofluoreseence studies demonstrated that both L721R and Q860X mutant pro-αⅡbβ3 complex colocalized in endoplasmic reticulum, but a little in Golgi. Conclusions The L721R and Q860X mutations of αⅡb prevent transport of the pro-αⅡbβ3 complex from the endoplasmic reticulum to the Golgi, hindering its maturation and surface expression. The impaired αⅡbβ3 transport is responsible for the thrombasthenia.