Liu H C, Shen M C, Eng H L, Wang C H, Lin T M
Institute of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan; Department of Medical Research, E-DA Hospital/I-SHOU University, Kaohsiung, Taiwan.
Haemophilia. 2014 Jul;20(4):e318-26. doi: 10.1111/hae.12476. Epub 2014 Jun 3.
Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.
先天性因子V（FV）缺乏症是一种罕见的遗传性疾病。我们确定了FV蛋白A1结构域中的一个错义突变Asp68His与严重FV缺乏症相关的机制。我们通过使用特异性分泌和降解途径抑制剂进行体外表达研究来表征突变型FV-Asp68His蛋白，并通过免疫荧光染色分析突变蛋白的细胞内转运。Asp68His突变导致条件培养基中FV蛋白水平极低，但FV特异性活性正常。FV野生型（wt）和FV-Asp68His mRNA之间相似的mRNA降解率表明，Asp68His突变在转录水平上不影响FV表达。使用一种特异性分泌途径抑制剂布雷菲德菌素A来证明，与FV-wt蛋白相比，FV-Asp68His突变蛋白转运到细胞外的效率较低。此外，我们表明Asp68His突变通过MG132介导的蛋白酶体降解途径导致细胞内降解增加。在转染的细胞裂解物中，FV-wt蛋白有多种翻译后修饰形式，但FV-Asp68His蛋白未完全糖基化。我们进一步观察到，FV-Asp68His蛋白仅在内质网中被回收，未转运至高尔基体，导致分泌受损。这些结果强烈表明，Asp68His突变可能导致FV蛋白细胞内运输缺陷、降解增强和分泌受损。
