Mashayekhi Amir, Shahbazi Shirin, Omrani Mirdavood
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University Tehran, Iran.
Department of Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Biotechnol. 2018 Apr 18;16(1):e1813. doi: 10.21859/ijb.1813. eCollection 2018 Apr.
Coagulation Factor VII is a vitamin K-dependent serine protease which has a pivotal role in the initiation of the coagulation cascade. The congenital Factor VII deficiency is a recessive hemorrhagic disorder that occurs due to mutations of gene. In the present study C91S (p.C91S) substitution was detected in a patient with FVII deficiency. This mutation has not been characterized by a functional study.
In this study, we aimed to evaluate the impact of C91S substitution on factor VII expression and function.
The complete cDNA was isolated from HepG2 cell line and inserted into the pcDNA3.1 mammalian expression vector. The desired mutation was generated by the site-directed mutagenesis and the wild-type and mutated constructs were transfected into CHO-K1 cells. The protein activity and antigen level (antigen concentration) were validated in the culture medium and cell lysate of the transiently transformed cells. An immunocytochemistry procedure was also performed to evaluate the intracellular localization of the mutated and the wild-type FVII, as well.
The present study has demonstrated that C91S antigen expression was increased in the transfected CHO-K1 cells compared to the wild-type (WT) protein. Despite an increased protein secretion, the factor VII coagulant activity was diminished following C91S substitution when it was assessed by a standard one-stage analysis. In addition, the immunocytochemistry procedure revealed that there was no difference in the intracellular localization of the C91S mutated FVII compared to the WT protein.
Our results present that C91S mutation has an effect on the coagulation activity, secretion, biosynthesis, and probably folding of the FVII leading to the FVII deficiency.
凝血因子VII是一种维生素K依赖的丝氨酸蛋白酶，在凝血级联反应的启动中起关键作用。先天性因子VII缺乏症是一种隐性出血性疾病，由基因突引起。在本研究中，在一名因子VII缺乏症患者中检测到C91S（p.C91S）替代。该突变尚未通过功能研究进行表征。
在本研究中，我们旨在评估C91S替代对因子VII表达和功能的影响。
从HepG2细胞系中分离出完整的cDNA，并将其插入pcDNA3.1哺乳动物表达载体中。通过定点诱变产生所需的突变，并将野生型和突变型构建体转染到CHO-K1细胞中。在瞬时转化细胞的培养基和细胞裂解物中验证蛋白质活性和抗原水平（抗原浓度）。还进行了免疫细胞化学程序以评估突变型和野生型FVII的细胞内定位。
本研究表明，与野生型（WT）蛋白相比，转染的CHO-K1细胞中C91S抗原表达增加。尽管蛋白质分泌增加，但通过标准的一步分析法评估时，C91S替代后因子VII凝血活性降低。此外，免疫细胞化学程序显示，与WT蛋白相比，C91S突变的FVII在细胞内定位上没有差异。
我们的结果表明，C91S突变对FVII的凝血活性、分泌、生物合成以及可能的折叠有影响，导致FVII缺乏。
