The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.
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.
Chen
Lingxia
L
Department of Pediatrics, University of Pennsylvania Medical Center and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Zhu
Fuxiang
F
Li
Juan
J
Lu
Hui
H
Jiang
Haiyan
H
Sarkar
Rita
R
Arruda
Valder R
VR
Wang
Jinhui
J
Zhao
Jennifer
J
Pierce
Glenn F
GF
Ding
Qiulan
Q
Wang
Xuefeng
X
Wang
Hongli
H
Pipe
Steven W
SW
Liu
Xiang-Qin
XQ
Xiao
Xiao
X
Camire
Rodney M
RM
Xiao
Weidong
W
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010957:Plasmids; D011499:Protein Processing, Post-Translational
D005169:Factor VIII