The Secretion Efficiency of Factor VIII Can Be Regulated by the Size and Oligosaccharide Content of the B Domain. (2005)

Abstract Factor VIII (FVIII) shares an identical domain structure (A1-A2-B-A3-C1-C2) with factor V (FV), another coagulation cofactor. FVIII and FV share ~40% amino acid identity within their A and C domains. The B domains of both cofactors are encoded by unusually large single exons and share no amino acid homology to each other or any identified protein, although they have conserved a high content of asparagine (N)-linked oligosaccharides. The B domain of FVIII is dispensable for functional activity and is extensively glycosylated (18 asparagine (N)-linked glycosylation attachment sites). B domain-deleted (BDD)-FVIII generates higher concentrations of mRNA compared to full-length FVIII and therefore increased primary translation product but exhibits a reduced rate of secretion and increased intracellular retention. Bioengineering of the factor VIII (FVIII) B domain effectively improves the trafficking of FVIII within the secretion pathway. We have used a series of B domain variants to map key regions of the B domain that regulate mRNA levels and secretion efficiency. Each construct has increased B domain size (ranging from 29 to 774 amino acids (aa) and increased number of N-linked oligosaccharides (from 1 through 18). BDD-FVIII, FVIII wild-type (WT) and FVIII B domain variants were transfected into COS and CHO cell lines and the media harvested for analysis of FVIII activity and antigen. The addition of even a few N-linked oligosaccharides within a short B domain spacer (optimal at 226 aa and 6 N-linked oligosaccharides, designated 226aa/N6) improves secretion over BDD-FVIII approximately 5 to 10-fold. With additional increase in B domain size and oligosaccharide content, secretion efficiency declined in a stepwise fashion to similar levels observed for FVIII WT. Chimeras were generated in which the B domain of FVIII was replaced with the full B domain of FV or the first 159 aa of the FV B domain with consensus sites for 5 N-linked oligosaccharides. The two FVIII/FV chimeras were expressed 7-fold and 6-fold higher respectively than BDD-FVIII. An alternative construct was prepared in which the human FVIII B domain was replaced with the FVIII B domain from puffer fish (fugu rubripes). The fugu FVIII B domain is 224 aa and shares only 6% sequence identity to the human FVIII B domain, yet has a high density of N-linked oligosaccharides (11). This construct was secreted as efficiently as the human 226aa/N6 human construct. Finally, the FVIII B domain was replaced with a 56 aa sequence from the completely unrelated human lysosome membrane glycoprotein, LAMP-1. The LAMP-1 sequence contained 5 consensus sites for N-linked oligosaccharides. The FVIII/LAMP-1 chimera was secreted 4-fold higher than BDD-FVIII. Thus the secretion efficiency of FVIII can be regulated by the size and oligosaccharide content of the B domain. The results suggest that it is primarily the presence of N-linked oligosaccharides that improves secretion efficiency, though sequences unique to the full-length FVIII B domain may compromise expression regardless of oligosaccharide content possibly through reduced mRNA accumulation. FVIII that can be bioengineered for improved secretion will be an alternative for gene therapy strategies as well as recombinant FVIII production in manufacturing or transgenic strategies.