MUTAGENESIS OF A POTENTIAL IMMUNOGLOBULIN-BINDING PROTEIN-BINDING SITE ENHANCES SECRETION OF COAGULATION-FACTOR-VIII

Coagulation factor VIII (FVIII) and factor V are homologous glycoprote ins that have a domain structure of A1-A2-B-A3-C1-C2, FVIII is a heter odimer of the heavy chain (domains A1-A2-B) and the light chain (domai ns A3-C1-C2) in a metal ion-dependent association between the Al-and A 3 domains. Previous studies identified a 110-amino acid region within the FVIII Al-domain that inhibits its secretion and contains multiple short peptide sequences that have potential to bind immunoglobulin-bin ding protein (BiP), FVIII secretion requires high levels of intracellu lar ATP, consistent with an ATP-dependent release from BiP, Site-direc ted mutagenesis was used to elucidate the importance of the potential BiP binding sites in FVIII secretion, Mutation of Phe at position 309 to Ser or Ala enhanced the secretion of functional FVIII and reduced i ts ATP dependence. The F309S FVIII had a specific activity, thrombin a ctivation profile, and heat inactivation properties similar to those o f wild-type FVIII, However, F309S FVIII displayed increased sensitivit y to EDTA-mediated inactivation that is known to occur through metal i on chelation-induced dissociation of the heavy and light chains of FVI II. The results support that Phe(309) is important in high affinity he avy and light chain interaction, and this correlates with a high affin ity BiP-binding site, Introduction of the F309S mutation into other se cretion defective FVIII mu mutants rescued their secretion, demonstrat ing the ability of the this mutation to improve secretion of mutant FV III proteins retained in the cell.