Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface
| Autor: | Fatbardha Varfaj, Jennifer P. DeAngelis, Hironao Wakabayashi, Philip J. Fay |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Protein subunit
Immunology Blotting Western Biochemistry Hemostasis Thrombosis and Vascular Biology Cofactor Factor IXa Cell Line Protein structure Thrombin Cricetinae medicine Animals Humans Point Mutation Amino Acids Polyacrylamide gel electrophoresis Thermostability chemistry.chemical_classification Factor VIII biology Chemistry Temperature Cell Biology Hematology Amino acid Protein Structure Tertiary Amino Acid Substitution biology.protein Biophysics Thermodynamics Biological Assay Electrophoresis Polyacrylamide Gel Mutant Proteins medicine.drug |
| Zdroj: | Blood. 112(7) |
| ISSN: | 1528-0020 |
| Popis: | Factor VIII consists of a heavy chain (A1A2B domains) and light chain (A3C1C2 domains), whereas the contiguous A1A2 domains are separate subunits in the cofactor, factor VIIIa. The intrinsic instability of the cofactor results from weak affinity interactions of the A2 subunit within factor VIIIa. The charged residues Glu272, Asp519, Glu665, and Glu1984 appear buried at the interface of the A2 domain with either the A1 or A3 domain, and thus may impact protein stability. To determine the effects of these residues on procofactor/cofactor stability, these residues were individually replaced with either Ala or Val, and stable BHK cell lines expressing the B-domainless proteins were prepared. Specific activity and thrombin generation parameters for 7 of the 8 variants were more than 80% the wild-type value. Factor VIII activity at 52°C to 60°C and the decay of factor VIIIa activity after thrombin activation were monitored. Six of the 7 variants showing wild-type-like activity demonstrated enhanced stability, with the Glu1984Val variant showing a 2-fold increase in thermostability and an approximately 4- to 8-fold increase in stability of factor VIIIa. These results indicate that replacement of buried charged residues is an effective alternative to covalent modification in increasing factor VIII (VIIIa) stability. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|