Mutation of the surface-exposed amino acid Trp to Ala in the FVIII C2 domain results in defective secretion of the otherwise functional protein

The C2 domain of factor VIII (FVIII) is important for FVIII-phospholipid (PL) and FVIII-von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain-PL interface. Within loop four, the solvent-exposed...

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Bibliographic Details
Published in:British journal of haematology 2004-06, Vol.125 (5), p.629-637
Main Authors: Schatz, Simone M, Zimmermann, Klaus, Hasslacher, Meinhard, Kerschbaumer, Randolf, Dockal, Michael, Gritsch, Herbert, Turecek, Peter L, Schwarz, Hans P, Dorner, Friedrich, Scheiflinger, Friedrich
Format: Article
Language:English
Subjects:
Alanine - genetics
Blotting, Western
Cell Line
Electrophoresis, Polyacrylamide Gel
Factor VIII - chemistry
Factor VIII - genetics
Factor VIII - pharmacokinetics
Factor Xa - metabolism
Humans
Mutation - genetics
Protein Binding
Protein Structure, Tertiary
Tryptophan - genetics
von Willebrand Factor - metabolism
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Summary:The C2 domain of factor VIII (FVIII) is important for FVIII-phospholipid (PL) and FVIII-von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain-PL interface. Within loop four, the solvent-exposed amino acid, Trp(2313), is believed to contribute to FVIII-PL binding. To analyse this interaction, the amino-acid exchange Trp(2313) to Ala (W2313A) was introduced into the C2 domain of B-domain-deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl-l-serine vesicles showed, however, a 28-fold higher K(D) for W2313A, indicating the important role of Trp(2313) in the FVIII-PL interaction. In conclusion, the C2-domain-surface-exposed residue Trp(2313), is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl-l-serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo.
ISSN:0007-1048
DOI:10.1111/j.1365-2141.2004.04959.x