血小板膜糖蛋白αⅡ b基因A2334C突变对αⅡ bβ3复合物合成及转运的影响——附一例报告

Effect of glycoproteinαⅡbA2334C mutation on the biosynthesis and transportation of αⅡbβ3 complex

目的 探讨血小板膜糖蛋白αⅡb基因A2334C突变对αⅡbβ3复合物合成的影响。方法 构建αⅡb基因A2334C真核表达载体,测序正确后用脂质体将其与表达人整合素β3亚基的真核表达质粒p3. 1 3a共转染CHO细胞,对转染后细胞用Westernblot法鉴定αⅡb基因A2334C突变体在CHO细胞中的总体表达;用流式细胞仪分析转运至细胞膜表面的αⅡb基因A2334C亚基;为了明确突变亚基的亚细胞定位,构建了αⅡb基因A2334CGFP融合蛋白表达质粒并用激光共聚焦显微镜确定GFP融合蛋白的细胞内定位。结果 Westernblot证明转基因CHO细胞有αⅡbA2334C基因的表达,但是成熟αⅡb的比例较正常对照低;流式细胞仪检测发现在细胞膜上有该亚基的分布,但是只有正常的 25%;进一步的融合蛋白细胞内定位研究显示,αⅡb基因A2334CGFP可以由内质网转运至高尔基体。结论 αⅡb基因A2334C突变没有影响αⅡb的合成及其与β3的结合,但是仅有少部分前体αⅡb能够进一步形成成熟的αⅡb,其膜上的表达仅为正常的 25%。该突变不影响αⅡbβ3由内质网向高尔基体的转运。该突变的致病机制可能是αⅡb的折叠错误使其容易降解导致膜上αⅡbβ3复合物减少及血小板功能减弱。

Objective To study the effect of glycoprotein(GP)αⅡbA2334C mutation on the biosynthesis and expression of αⅡbβ3 complex. Methods The GPαⅡbA2334C eukaryotic expression plasmid pc3.1 2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin β3 expression plasmid pc3.1 3a. The whole expression of αⅡbA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed αⅡbA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy. Results Expression of the mutant protein, αⅡbA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that αⅡbA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus. Conclusions The mutant αⅡbA2334C can be synthesized in CHO cells and form αⅡbβ3 complex. However, only a small fraction of the premature αⅡbA2334C can be transported to Golgi apparatus and transformed to mature αⅡb. The possible pathogenesis of this type Ⅱ thrombasthenia may be that the misfolded αⅡbA2334C is partially degraded in the endoplasmic reticulum causing lower expression of αⅡbβ3 complex on the membrane and resulting in impared function of platelets than normal αⅡb.