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 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.
Schatz
Simone M
SM
Baxter BioScience, Biomedical Research Centre, Orth/Donau, Austria.
Zimmermann
Klaus
K
Hasslacher
Meinhard
M
Kerschbaumer
Randolf
R
Dockal
Michael
M
Gritsch
Herbert
H
Turecek
Peter L
PL
Schwarz
Hans P
HP
Dorner
Friedrich
F
Scheiflinger
Friedrich
F
D000409:Alanine / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000493:pharmacokinetics; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000235:genetics; D014841:von Willebrand Factor / Q000378:metabolism
D014841:von Willebrand Factor; D014364:Tryptophan; D005169:Factor VIII; D015951:Factor Xa; D000409:Alanine