BIOSYNTHESIS, ASSEMBLY AND SECRETION OF COAGULATION-FACTOR-VIII

Factor VIII is a large complex glycoprotein that is deficient in hemop hilia A. It has a domain organization consisting of A1-A2-B-A3-C1-C2 w here the B domain is a heavily glycosylated region that is dispensable for procoagulant activity. Factor VIII expression is 10- to 20-fold l ower than the homologous coagulation factor V.Factor VIII expression i s limited due to a low level of steady-state messenger RNA in the cyto plasm and inefficient transport of the primary translation product fro m the endoplasmic reticulum to the Golgi apparatus. Within the secreto ry pathway, factor VIII is processed to a heterodimer of the heavy cha in (domains A1-A2-B) in a metal ion association with the light chain ( domains A3-C1-C2). Upon secretion from the cell, von Willebrand factor binds the light chain of factor VIII and stabilizes the factor, preve nting degradation. Protein folding within the mammalian secretory path way is facilitated by molecular chaperones. Within the endoplasmic ret iculum, factor VIII exhibits stable interaction with protein chaperone s identified as the immunoglobulin-binding protein (BiP), calnexin and calreticulin. BiP is a peptide-dependent ATPase that interacts with e xposed hydrophobic surfaces on unfolded proteins or unassembled protei n subunits. A potential BiP binding site within factor VIII has been i dentified. Mutation of a single amino acid residue in the potential Bi P binding site increased the secretion efficiency of factor VIII by th reefold. Interestingly, the proposed BiP binding site is adjacent to a type-1 copper binding site within the A1 domain that is required for interaction between the factor VIII A1 domain and the A3 domain. We pr opose that Cu(I) binds the type-1 copper ion-binding site in the A1 do main and provides the essential requirement for a stable interaction b etween the heavy and light chains. Calnexin and calreticulin are trans membrane and lumenal proteins, respectively localized to the endoplasm ic reticulum, which associate transiently with many soluble and membra ne glycoproteins during folding and subunit assembly. The calnexin and calreticulin interaction with factor VIII occurs primarily through am ino-terminal linked oligosaccharides within the heavily glycosylated f actor VIII B domain and this interaction appears to be required for fa ctor VIII secretion. The findings suggest that factor VIII cycles thro ugh interactions with BiP, calnexin and calreticulin. Although the int eraction with BiP does not appear to be required for factor VIII secre tion, data suggest that the calnexin and/or calreticulin interaction i s required for secretion. The observations suggest a unique requiremen t for carbohydrate processing and calnexin/calreticulin interaction th at may limit the productive secretion of factor VIII and have implicat ions for approaches towards somatic fell gene therapy for hemophilia A .