title: Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.
abstract: Coagulation factor VIII (FVIII) and factor V are homologous glycoproteins that have a domain structure of A1-A2-B-A3-C1-C2. FVIII is a heterodimer of the heavy chain (domains A1-A2-B) and the light chain (domains A3-C1-C2) in a metal ion-dependent association between the A1- and A3-domains. Previous studies identified a 110-amino acid region within the FVIII A1-domain that inhibits its secretion and contains multiple short peptide sequences that have potential to bind immunoglobulin-binding protein (BiP). FVIII secretion requires high levels of intracellular ATP, consistent with an ATP-dependent release from BiP. Site-directed 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 its ATP dependence. The F309S FVIII had a specific activity, thrombin activation profile, and heat inactivation properties similar to those of wild-type FVIII. However, F309S FVIII displayed increased sensitivity to EDTA-mediated inactivation that is known to occur through metal ion chelation-induced dissociation of the heavy and light chains of FVIII. The results support that Phe309 is important in high affinity heavy and light chain interaction, and this correlates with a high affinity BiP-binding site. Introduction of the F309S mutation into other secretion defective FVIII mutants rescued their secretion, demonstrating the ability of the this mutation to improve secretion of mutant FVIII proteins retained in the cell.
authors.affiliation: Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
authors.forename: M
authors.initials: M
authors.lastname: Swaroop
authors.forename: M
authors.initials: M
authors.lastname: Moussalli
authors.forename: S W
authors.initials: SW
authors.lastname: Pipe
authors.forename: R J
authors.initials: RJ
authors.lastname: Kaufman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D000091342:Endoplasmic Reticulum Chaperone BiP; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
chemical_list: D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D005169:Factor VIII; D004492:Edetic Acid