Expression Studies of Recombinant FVIII Proteins Exhibiting Mutations in the B-Domain
K. Klempau, H. Singer, C. Klein, H.-H. Brackmann, P. Hanfland, T. Tonn, R. Schwaab and J. Oldenburg
Introduction
The phenotype of hemophilia A is due to the deficiency or absence of coagulation fac- tor VIII (FVIII) caused by a great number of heterogeneous mutations within the large FVIII gene, including various point mutations, inversions and deletions or in- sertions. Missense mutations have been of special interest because they point to func- tional important regions of the FVIII molecule. Notably, missense mutations are sig- nificantly underrepresented in the middle part of the B-domain, indicating that this part of the FVIII molecule may be less important for its function. Furthermore, the successful treatment with a B-domain deleted recombinant FVIII-concentrate, shows that the B-domain might be dispensable for a functional FVIII protein. However, in some hemophilia A patients a missense mutation was the only sequence variation found within the FVIII gene, even by sequencing the complete coding region (Fig. 1).
Materials and Methods
Missense mutations within the B-domain were identifying in hemophilia A patients by mutation screening and sequencing of FVIII gene. Using oligonucleotide site directed mutagenesis, we constructed FVIII mutants with missense mutations with- in the B-domain to study the effect of these mutations. To investigate the causality of such missense mutations expression studies were performed in Chinese hamster ovary cells (CHO), transfected with vector constructs encoding the FVIII protein variants (Fig. 2).
Results and Discussion
In the present study five mutated rFVIII-proteins Pro928Arg, Val993Leu, Asp1241Glu, Arg1310Gly and Asn1441Lys were expressed in CHO cells and charac- terized for FVIII activity (FVIII:C), FVIII antigen (FVIII:Ag)-level and the specific activity (FVIII:C[%]/FVIII:Ag[%]). The results were compared to the expression of wildtype FVIII protein. The protein variant Arg1241Glu has also been reported in the normal population thus representing a polymorphism. Four of the five mis- sense mutations (Pro928Arg, Val993Leu, Asp1241Glu, Arg1310Gly) showed signifi-
I. Scharrer/W. Schramm (Ed.)
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cantly expression of functional FVIII protein thus probably not being causative for a hemophilic phenotype. However, there were some differences in the specific activity and expression/secretion levels, which may point to a FVIII:C modulating effect. The Arg1310Gly variant showed secretion and specific activity (~1) very similar to nor- mal FVIII expression. The rFVIII-proteins Asp1241Glu and Pro928Arg exhibited a higher specific activity (~1,7-2), but secretion- and expression levels were only 50%
of the wild type protein. The rFVIII-protein Val993Leu showed nearly the same spe- cific activity (~0,9) as normal rFVIII, but slightly higher expression and secretion levels compared to wildtype FVIII. The expression studies of the Asn1441Lys variant gave inconclusive results and need further evaluation (Table 1).
Expression Studies of Recombinant FVIII Proteins Exhibiting Mutations 345
740 825 911 997 1083 1169 1255 1341 1427 1513 1599 1685
1685
782 868 954 1040 1126 1212 1298 1384 1470 1556 1642
928 993
1241
1310
1441
