The effects of novel mutations in A1 domain of human coagulation factor VIII on its secretion level in cultured mammalian cells

Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one of the main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known as BiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of the porcine FVIII and analysis of the hFVIII A110 region, that inhibits its secretion, function of three novel B-domain deleted hFVIII mutant including; two single-mutants (Leu299Phe and Phe309Thr) and a double-mutant (Tyr323His/Lys325Arg) were examined in three mammalian cell lines (HEK-293T, COS, CHO) for the hFVIII secretion efficiency. The double-mutant construct displayed the highest hFVIII expression level, about seven-fold as much the base-line. The double-mutant hFVIII was biologically active and its inactivation patterns by EDTA and heat was similar to that of the non-mutant hFVIII. Semi-quantitative RT-PCR results showed the highest mRNA level for the double-mutant hFVIII. Both of the mutated residues in the double-mutant are located in a hydrophobic heptamer (320MEAYVKV326) that seems to be involved in Bip-binding activity. None of the L299F and F309T hFVIII mutants exhibited improved secretion. This result has provided convincing evidence for the increasing effect of the double-mutant on the hFVIII secretion and transcription efficiencies.