title: Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells.
abstract: Coagulation factors V and VIII are homologous glycosylated plasma proteins that provide essential functions for hemostasis. Factor V is secreted as a single chain polypeptide, whereas factor VIII is processed intracellularly to yield a metal-ion-associated heterodimer that is stabilized through interaction with von Willebrand factor. In transfected mammalian cells, factor V is more efficiently secreted than factor VIII. To provide insight into the different secretion efficiencies, we compared the post-translational processing requirements for factor V and factor VIII expressed in mammalian cells. In contrast to factor VIII, factor V was not detected in association with the immunoglobulin-binding protein (BiP), a chaperonin protein of the endoplasmic reticulum (ER). Depletion of intracellular ATP levels by treatment of cells with low concentrations of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), protonophore that uncouples oxidative phosphorylation, inhibited secretion of factor VIII but had no effect on the secretion of factor V. Inhibition of N-linked oligosaccharide addition by treatment with tunicamycin prevented secretion of both factor V and factor VIII, whereas treatment with an inhibitor of complex oligosaccharide addition, deoxymannojirimycin, did not affect secretion, although the specific activities of both factor V and factor VIII were slightly increased. Thus, complex oligosaccharide addition was not required for secretion or functional activity of either factor V or factor VIII. Depletion of intralumenal calcium with the ionophore A23187 did not affect secretion of either factor V or factor VIII. In the presence of A23187, the secreted factor V was fully functional, whereas the factor VIII heavy and light chains were not associated and the secreted molecule was inactive. In addition, A23187 treatment inhibited addition of serine/threonine (O)-linked oligosaccharides to factor V and factor VIII. The differences between factor V and factor VIII were further evaluated by characterization of a single chain mutant factor VIII. The single chain factor VIII was secreted with an efficiency similar to wild-type factor VIII and also required von Willebrand factor for stabilization. In addition, the activity of single chain factor VIII was also inhibited by A23187 treatment, suggesting a unique metal-ion requirement within the secretory pathway for functional factor VIII folding. The differences identified in BiP association, ATP requirements, and metal-ion dependence for effective functional secretion of these two molecules may underlie mechanisms accounting for their different secretion efficiencies.
authors.affiliation: Genetics Institute, Cambridge, Massachusetts 02140.
authors.forename: D D
authors.initials: DD
authors.lastname: Pittman
authors.forename: K N
authors.initials: KN
authors.lastname: Tomkinson
authors.forename: R J
authors.initials: RJ
authors.lastname: Kaufman
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002413:Cations, Divalent; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D000091342:Endoplasmic Reticulum Chaperone BiP; D004789:Enzyme Activation; D005165:Factor V / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism*; D006031:Glycosylation; D006360:Heat-Shock Proteins; D006801:Humans; D066298:In Vitro Techniques; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D002413:Cations, Divalent; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005165:Factor V; D005169:Factor VIII; D013917:Thrombin