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.
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Abbreviations
- aPTT:
-
Activated partial thromboplastin time
- BPS:
-
Brain phosphatidylserine
- BSA:
-
Bovine serum albumin
- CHO:
-
Chinese hamster ovarian cell line
- DEA:
-
Diethanolamine buffer
- Degly FVIII:
-
Deglycosylated recombinant human factor VIII
- DMPC:
-
Dimyristoylphosphatidylcholine
- ELISA:
-
Enzyme-linked immunosorbent assay
- Endo:
-
Endoglycosidase
- FVIII:
-
Factor VIII
- PB:
-
Phosphate buffer
- PBA:
-
Phosphate buffer with 1% bovine serum albumin
- PBT:
-
Tween 20 containing phosphate buffer
- p-NPP:
-
p-nitrophenylphosphate
- PS:
-
Phospatidylserine
- rFVIII:
-
Recombinant human factor VIII
- SEC:
-
Size exclusion chromatography
- SDS-PAGE:
-
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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Acknowledgements
The authors thank the Pharmaceutical Sciences Instrumentation Facility, University at Buffalo (UB), for the use of the Circular Dichroism and the Fluorescence spectrophotometers. We thank the Hemophilia Center of Western New York for providing rFVIII. We express gratitude to Dr. Robert Straubinger (UB) for suggestions and review of this manuscript. This work was supported by NHLBI, National Institute of Health grant R01 HL-70227 to SVB.
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Matthew P. Kosloski and Razvan D. Miclea equally contributed to the manuscript.
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Kosloski, M.P., Miclea, R.D. & Balu-Iyer, S.V. Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII. AAPS J 11, 424–431 (2009). https://doi.org/10.1208/s12248-009-9119-y
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DOI: https://doi.org/10.1208/s12248-009-9119-y