The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene
The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene
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DOI：
10.1038/sj.mt.6300268
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发表时间：
2007-10-01
影响因子：
12.4
通讯作者：
Xiao, Weidong
中科院分区：
文献类型：
--
作者：
Chen, Lingxia;Zhu, Fuxiang;Xiao, Weidong
凝血因子VIII（FVIII）作为由重链（HC）和轻链（LC）组成的异源二聚体分泌，其可以独立表达并与生物活性的恢复重新关联。由于腺相关病毒（AAV）载体的大小限制，已经开发了分别递送HC和LC的策略。然而，FVIII HC的分泌效率比LC低10-100倍。在本研究中，我们证明了F309 S突变和增强的B结构域糖基化单独不足以改善FVIII HC分泌，这表明FVIII LC在调节HC分泌中的作用。为了表征FVIII LC的这种作用，我们通过翻译后蛋白反式剪接比较了有和没有LC的FVIII HC分泌。如在体外证明的，LC与HC的连接显著增加HC分泌。通过将FVIII内含肽质粒流体动力学注射到血友病A小鼠中，也在体内证实了这种HC分泌增加。此外，当LC以反式供应时，也可以观察到HC分泌的类似增强，这可能是由于HC和LC在分泌途径中的自发缔合。总之，增强FVIII HC多肽的分泌可能需要FVIII LC多肽以顺式或反式适当结合。这些结果可能有助于设计新策略以改善FVIII基因递送。
Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10-100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.