David D, Saenko E L, Santos I M, Johnson D J, Tuddenham E G, McVey J H, Kemball-Cook G
Centro de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal.
Br J Haematol. 2001 Jun;113(3):604-15. doi: 10.1046/j.1365-2141.2001.02399.x.
In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM(-) and CRM(r) variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.
在甲型血友病中，大多数因子VIII（FVIII）错义突变在分子水平上的功能缺陷仍然未知。利用缺失B结构域的FVIII cDNA（FVIIISQ）进行定点诱变，引入了与严重交叉反应物质（CRM）阴性（FVIII-C329S）或轻度/中度CRM降低（FVIII-G1948D）的甲型血友病相关的两种突变。野生型（FVIIISQ-WT）和变异型FVIIISQ蛋白在中国仓鼠卵巢（CHO）细胞中稳定转染后成功表达，并在细胞内、分子和功能水平上进行了部分表征。逆转录聚合酶链反应分析证实，用野生型和两种变异构建体转染的CHO细胞中，转录和mRNA加工均正常。与FVIIISQ-WT相反，对CRM（-）和CRM（r）变异体的免疫荧光分析显示，粗糙内质网内有细胞内FVIII积累，提示转染的CHO细胞存在分泌缺陷。对分泌的FVIIISQ变异蛋白的免疫印迹分析表明，它们与FVIIISQ-WT相似，以未切割的170 kDa多肽、加工后的90 kDa重链和80 kDa轻链的混合群体形式表达。在CHO细胞中表达的缺失B结构域的FVIIISQ变异体的表型分析与患者FVIII活性降低密切相关，此外，表面等离子体共振研究表明，两种错义突变均与凝血酶激活后A2结构域解离速率增加有关。我们得出结论，发现的突变通过活性辅因子FVIIIa的细胞内滞留和稳定性降低导致了甲型血友病的表型。
