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首页> 外文期刊>Platelets >AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3.
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AlphaIIbG236E causes Glanzmann thrombasthenia by impairing association with beta3.

机译：AlphaIIbG236E通过削弱与beta3的结合而导致Glanzmann血虚症。

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Glanzmann thrombasthenia (GT) is a recessively inherited bleeding disorder caused by the quantitative or qualitative deficiency of the platelet fibrinogen receptor, integrin alphaIIbbeta3. The N-terminal domain of the alphaIIb subunit is folded in a beta-propeller that plays the role of binding fibrinogen and associating with the ligand-binding region of beta3. Analysing the mutations of Italian GT patients we found that a patient had a alphaIIb G236E missense substitution that substitutes a glycine from the highly conserved PhiPhiGPhi motif of blade 4 of the beta-propeller. To verify experimentally the effect of the substitution of glycine 236 human embryonic kidney (HEK) cells were transfected with normal or mutated alphaIIb in conjunction with normal beta3. Using flow cytometry analysis we found the percentage of HEK cells transfected with alphaIIbG236Ebeta3 that reacted with anti alphaIIbbeta3 was very low. In HEK cells transfected with either alphaIIbbeta3 or alphaIIbG236Ebeta3 and lysed, when immunoblotting was done in non-reducing conditions a band reacting with an antibody against alphaIIb was present in both lysates, although less intense in cells transfected with alphaIIbG236Ebeta3. In reducing condition alphaIIb from cells transfected with alphaIIbbeta3 was nearly all mature, while in cells transfected with alphaIIbG236Ebeta3 the ratio pro-alphaIIb: alphaIIb was 1 : 1, with signs of degradation of the mutated protein. Cell lysates were then immunoprecipitated with antibodies against alphaIIb and immunoblotted with an antibody reacting with beta3. While in immunoblots from cells transfected with alphaIIbbeta3 a band corresponding to beta3 was strongly detectable, in immunoblots originating from cells transfected with alphaIIbG236Ebeta3 no band at the same level of normal beta3 was detected. Immunofluorescence studies showed accumulation of alphaIIbG236Ebeta3 in the endoplasmic reticulum and minimal transport to the Golgi. In conclusion we demonstrated that the alphaIIbG236E mutation causes GT by impairing the association with beta3 during biogenesis of the receptor.
机译：格兰兹曼性血小板减少症（GT）是一种隐性遗传性出血性疾病，由血小板纤维蛋白原受体整联蛋白alphaIIbbeta3的定量或定性缺乏引起。 alphaIIb亚基的N末端结构域在一个β-螺旋桨中折叠，该螺旋桨起着结合血纤蛋白原的作用，并与β3的配体结合区相关联。分析意大利GT患者的突变后，我们发现患者具有alphaIIb G236E错义替代，该替代取代了β-螺旋桨叶片4的高度保守的PhiPhiGPhi基序中的甘氨酸。为了实验验证甘氨酸替代的效果，将正常或突变的alphaIIb与正常beta3一起转染了236个人胚胎肾（HEK）细胞。使用流式细胞仪分析，我们发现被alphaIIbG236Ebeta3转染的HEK细胞与抗alphaIIbbeta3反应的百分比非常低。在用alphaIIbbeta3或alphaIIbG236Ebeta3转染并裂解的HEK细胞中，在非还原条件下进行免疫印迹时，两种裂解物中均存在与针对alphaIIb的抗体反应的条带，尽管在用alphaIIbG236Ebeta3转染的细胞中强度较低。在还原条件下，用αIIbβ3转染的细胞中的αIIb几乎全部成熟，而在用αIIbG236Eβ3转染的细胞中，前αIIb∶αIIb之比为1∶1，具有突变蛋白降解的迹象。然后将细胞裂解物用针对alphaIIb的抗体进行免疫沉淀，并使用与beta3反应的抗体进行免疫印迹。在用alphaIIbbeta3转染的细胞进行的免疫印迹中，可以强烈检测到对应于beta3的条带，而在用alphaIIbG236Ebeta3转染的细胞产生的免疫印迹中，未检测到相同水平的正常beta3的条带。免疫荧光研究表明，αIIbG236Ebeta3在内质网中积累，并且向高尔基体的转运极少。总之，我们证明了alphaIIbG236E突变通过在受体的生物发生过程中削弱与beta3的结合而导致GT。

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