Abstract：
OBJECTIVE: To structurally and functionally characterize three newly identified F9 missense mutations, C268Y, I316F, and G413V, in Chinese hemophilia B patients.
METHODS: FIX mutants were expressed in vitro by transient transfection of Chinese hamster ovary (CHO) cells. One-stage activated partial thromboplastin time (APTT) based assay and enzyme-linked immunosorbent assay (ELISA) were used to measure the coagulation activity and antigen level of FIX in conditioned medium. Western blot analysis was also used to evaluate interference of the mutations with synthesis and secretion of FIX. A structural model of the FIX G413V mutant was constructed and structural disturbance caused by the mutation was determined by molecular dynamics simulations.
RESULTS: Both C268Y and I316F impaired expression of FIX. However, the I316F mutant degraded quickly, whereas the C268Y mutant mostly accumulated intracellularly. The G413V mutant could be synthesized and secreted normally, but procoagulant activity was almost completely lost. This loss is likely mostly due to the impact on the catalytic residue cS195.
CONCLUSIONS: The three FIX mutations identified in Chinese hemophilia B patients either impaired the expression of FIX, as was seen with the I316F and C268Y mutants, or impaired the function of FIX, as was seen with the G413V mutant.
METHODS: FIX mutants were expressed in vitro by transient transfection of Chinese hamster ovary (CHO) cells. One-stage activated partial thromboplastin time (APTT) based assay and enzyme-linked immunosorbent assay (ELISA) were used to measure the coagulation activity and antigen level of FIX in conditioned medium. Western blot analysis was also used to evaluate interference of the mutations with synthesis and secretion of FIX. A structural model of the FIX G413V mutant was constructed and structural disturbance caused by the mutation was determined by molecular dynamics simulations.
RESULTS: Both C268Y and I316F impaired expression of FIX. However, the I316F mutant degraded quickly, whereas the C268Y mutant mostly accumulated intracellularly. The G413V mutant could be synthesized and secreted normally, but procoagulant activity was almost completely lost. This loss is likely mostly due to the impact on the catalytic residue cS195.
CONCLUSIONS: The three FIX mutations identified in Chinese hemophilia B patients either impaired the expression of FIX, as was seen with the I316F and C268Y mutants, or impaired the function of FIX, as was seen with the G413V mutant.
摘要：
目的：为了在结构和功能上表征三个新鉴定的F9错义突变，C268Y,I316F,和G413V，中国血友病B患者。
方法:通过瞬时转染中国仓鼠卵巢(CHO)细胞在体外表达FIX突变体。采用基于一级活化部分凝血活酶时间（APTT）的测定和酶联免疫吸附测定（ELISA）来测量条件培养基中FIX的凝血活性和抗原水平。蛋白质印迹分析也用于评估突变对FIX合成和分泌的干扰。构建了FIXG413V突变体的结构模型，并通过分子动力学模拟确定了突变引起的结构干扰。
结果：C268Y和I316F均损害了FIX的表达。然而,I316F突变体快速降解，而C268Y突变体主要在细胞内积累。G413V突变体可以正常合成和分泌，但是促凝血活性几乎完全丧失。这种损失可能主要是由于对催化残余物cS195的影响。
结论：在中国血友病B患者中发现的三个FIX突变要么损害了FIX的表达，正如I316F和C268Y突变体所看到的那样，或者损害了FIX的功能，正如G413V突变体所见。
方法:通过瞬时转染中国仓鼠卵巢(CHO)细胞在体外表达FIX突变体。采用基于一级活化部分凝血活酶时间（APTT）的测定和酶联免疫吸附测定（ELISA）来测量条件培养基中FIX的凝血活性和抗原水平。蛋白质印迹分析也用于评估突变对FIX合成和分泌的干扰。构建了FIXG413V突变体的结构模型，并通过分子动力学模拟确定了突变引起的结构干扰。
结果：C268Y和I316F均损害了FIX的表达。然而,I316F突变体快速降解，而C268Y突变体主要在细胞内积累。G413V突变体可以正常合成和分泌，但是促凝血活性几乎完全丧失。这种损失可能主要是由于对催化残余物cS195的影响。
结论：在中国血友病B患者中发现的三个FIX突变要么损害了FIX的表达，正如I316F和C268Y突变体所看到的那样，或者损害了FIX的功能，正如G413V突变体所见。
