A和C结构域糖基化位点对凝血八因子的分泌及活性的影响

Study of the Effects of A and C-Domain Glycosylation Sites on the Secretion and Activity of Recombinant Factor Ⅷ

在血液来源和血液制品安全隐患制约下,基因工程重组凝血八因子已开始替代天然八因子用以治疗血友病等病症,但目前高效表达重组八因子的方法在我国仍有技术瓶颈。该项目利用定点突变技术,通过PCR、质粒抽提、转染以及产物检测这四个步骤来研究重组凝血八因子在A和C结构域添加糖基化位点对八因子表达及活性的影响。结果表明,在重组八因子A结构域Bip结合位点外端增加糖基化位点能够促进八因子的细胞外分泌量,而在Bip结合位点内的突变则不但大大减少了八因子的胞外分泌也使八因子活性几乎全部丧失。与A结构域不同,在八因子C结构域增加糖基化位点的结果表明,无论在蛋白C结合区之内或之外增加糖基化位点不但不能大幅提高八因子的胞外分泌,也使八因子活性基本丧失。这些结果说明只有在A结构域外缘的非Bip结合区进行糖基化改造,才有可能提高八因子分泌并保持其活性。

Because of the safety concerns of blood sources and whole blood products,genetically engineered recombinant factor Ⅷ have begun to replace blood-derived factor Ⅷ for the treatment of hemophilia and related diseases. However,the relatively low expression level of recombinant factor Ⅷ remains a major technology bottleneck to reduce the cost of recombinant factor Ⅷ production. To explore the possible means to increase recombinant factor Ⅷ expression at molecular level,site-directed mutagenesis techniques were used to generate potential glycosylation sites around A and C domains of a B-domain deleted recombinant FⅧ and tested the activity,expression and secretion of various glycosylation constructs. The results showed that generating glycosylation sites at the outer edges of domain A increased secretion of factor Ⅷ while those glycosylation sites generated at the Bip-binding core not only greatly reduced the secretion but also abolished the activity of Factor Ⅷ. Different from A-domain,the glycosylation sites generated in C-domain abolished most factor Ⅷ activity without increasing its secretion regardless whether they were located at the core protein-C binding site or at the edge of C-domain. Taken together,the results suggest that the only glycosylation sites with the potential of increasing factorⅧ secretion are located around the edge of A-domain.