Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

Abstract

Abstract. Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein-lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity.

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

“…Factor VIII contains a validated N-glycosylation site [22] that, in combination with other Nglycans, facilitates binding [23]. Moreover, N-glycosylation of DDR1 at a residue distal to the DSD (N211), that is conserved in DDR2, has been shown to affect receptor activation [24].…”

Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII

“…This may explain the different ratios of the respective oligosaccharides as the B domain contains 17 additional potential Nglycosylation sites. Based on clinical experience, the CHO cell type and the plasma-type N-glycosylation of FVIII result in efficacious and welltolerated products [16]. In common with a BDD rFVIII product expressed in a human cell line [19], but contrasting with the BDDrFVIII investigated here, which is produced in CHO cells, the α1,3-Gal non-human potentially antigenic epitope was not detected in rVIIISingleChain, and this finding may contribute to its safety profile.…”

“…Such modifications may also affect pharmacokinetic characteristics, activity and immunogenicity [16,17]. It is known that the glycosylation pattern of recombinant products strongly depends on the cell type used for expression.…”

“…It is known that the glycosylation pattern of recombinant products strongly depends on the cell type used for expression. Therefore, N-glycosylation should be considered when choosing a production cell [16]. Generally, rVIII-SingleChain had the same N-glycan structures, but in different ratios, to other CHO-cell-derived rFVIII products.…”

Development and Engineering of CSαβ Motif for Biomedical Application

“…Even expressed in eukaryotic cells, glycosylation of proteins is not exactly the same among difference specises (Perego et al, 2010). Unappropriatly modified recombinant proteins might lead to unexpected immune respones, if the proteins are used for medical purpose (Kosloski et al, 2009;Li & d'Anjou, 2009). Protein designing challenges our understanding of the principles underlying protein structure and is also a good method to access our understanding of sequence-structure and structure-function relationship (Nikkhah et al, 2006).…”