B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways

Summary

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60–80%) and insoluble fractions (for 20–40%). The incubation of the expressing cells with tunicamycin (5 μg/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH₄Cl, leupeptin, clasto-Lactacystin β-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Keywords

Degradation - factor VIII - furin - lysosome

• References

• 1 Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost 1999; 82: 165-74.
• 2 Do H, Healey JF, Waller EK. et al. Expression of factor VIII by murine liver sinusoidal endothelial cells. J Biol Chem 1999; 274: 19587-92.
• 3 Hollestelle MJ, Thinnes T, Crain K. et al. Tissue distribution of factor VIII gene expression in vivo --a closer look. Thromb Haemost 2001; 86: 855-61.
• 4 Wion KL, Kelly D, Summerfield JA. et al. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 1985; 317: 726-9.
• 5 Kaufman RJ, Wasley LC, Davies MV. et al. Effects of von Willebrand Factor coexpression on the synthesis and secretion of factor VIII in Chinese Hamster Ovary cells. Mol Cell Biol 1989; 9: 1233-42.
• 6 Pittman DD, Alderman EM, Tomkinson KN. et al. Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood 1993; 81: 2925-35.
• 7 Lind P, Larsson K, Spira J. et al. Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization. Eur J Biochem 1995; 232: 19-27.
• 8 Becker S, Simpson JC, Pepperkok R. et al. Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells. Thromb Haemost 2004; 92: 23-35.
• 9 Kaufman S, Pipe SW, Tagliavacca L. et al. Biosynthesis, assembly and secretion of coagulation factor VIII. Blood Coagul Fibrinolysis 1997; 8 (Suppl. 02) Suppl S3-14.
• 10 Tagliavacca L, Wang Q, Kaufman RJ. ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion. Biochemistry 2000; 39: 1973-81.
• 11 Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science 2001; 291: 2364-9.
• 12 Pipe SW, Morris JA, Shah J. et al. Differential interaction of coagulation factor VIII and factor Vwith protein chaperones calnexin and calreticulin. J Biol Chem 1998; 273: 8567-44.
• 13 Dorner AJ, Bole DG, Kaufman RJ. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol 1987; 105: 2665-74.
• 14 Gething MJ. Role and regulation of the ER chaperone BiP. Semin Cell Dev Biol 1999; 10: 465-72.
• 15 Hampton RY. ER stress response: getting the UPR hand on misfolded proteins. Curr Biol 2000; 10: R518-R21.
• 16 Moussalli M, Pipe SW, Hauri HP. et al. Mannosedependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII. J Biol Chem 1999; 274: 32539-42.
• 17 Zhang B, Cunningham MA, Nichols WC. et al. Bleeding due to disruption of a cargo-specific ER-to- Golgi transport complex. Nat Genet 2003; 34: 220-5.
• 18 Cunningham MA, Pipe SW, Zhang B. et al. LMAN1 is a molecular chaperone for the secretion of coagulation factor VIII. J Thromb Haemost 2003; 1: 2360-7.
• 19 Vehar GA, Keyt B, Eaton D. et al. Structure of Human Factor VIII. Nature 1984; 312: 337-42.
• 20 Miao HZ, Sirachainan N, Palmer L. et al. Bioengineering of coagulation factor VIII for improved secretion. Blood 2004; 103: 3412-9.
• 21 Eaton DL, Wood I W, Eaton D. et al. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25: 8343-7.
• 22 Toole JJ, Pittman DD, Orr EC. et al. Alarge region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. Proc Natl Acad Sci U SA 1986; 83: 5939-4.
• 23 Pittman DD, Marquette KA, Kaufman RJ. Role of the B-domain for factor VIII and factor V expression and function. Blood 1994; 84: 4214-25.
• 24 Sandberg H, Almstedt A, Brandt J. et al. Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ Thromb Haemost 2001; 85: 93-100.
• 25 Chao H, Mao L, Bruce AT. et al. Sustained expression of human factor VIII in mice using a parvovirus- based vector. Blood 2000; 95: 1594-9.
• 26 Connelly S, Gardner JM, Lyons RM. et al. Sustained expression of therapeutic levels of human factor VIII in mice. Blood 1996; 87: 4671-7.
• 27 Gnatenko DV, Saenko EL, Jesty J. et al. Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy. Br J Haematol 1999; 104: 27-36.
• 28 Vanden Driessche T, Vanslembrouck V, Goovaerts I. et al. Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc Natl Acad Sci USA 1999; 96: 10379-84.
• 29 Plantier JL, Rodriguez MH, Enjolras N. et al. A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII. Thromb Haemost 2001; 86: 596-603.
• 30 Marquette KA, Pittman DD, Kaufman RJ. A 110-amino acid regionwithin the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells. J Biol Chem 1995; 270: 10297-303.
• 31 Roelse JC, De Laaf RT, Timmermans SM. et al. Intracellular accumulation of factor VIII induced by missense mutations Arg593-->Cys and Asn618-->Ser explains cross-reacting material-reduced haemophilia A. Br J Haematol 2000; 108: 241-6.
• 32 Kaufman RJ. Post-transcriptional modifications required for coagulation factor secretion and function. Thromb Haemost 1998; 79: 1068-79.
• 33 Denault JB, Leduc R. Furin/PACE/SPC1: a convertase involved in exocytic and endocytic processing of precursor proteins. FEBS Lett 1996; 379: 113-6.
• 34 Steiner DF. The proprotein convertases. Curr Opin Chem Biol 1998; 2: 31-9.
• 35 Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 2000; 10: 524-30.
• 36 Wetzel R. Mutations and off-pathway aggregation of proteins. Trends Biotech 1994; 12: 193-8.
• 37 Kim J, Klionsky DJ. Autophagy, cytoplasm-tovacuole targeting pathway, and pexophagy in yeast and mammalian cells. Ann Rev Biochem 2000; 69: 303-42.
• 38 Schubert U, Anton LC, Gibbs J. et al. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 2000; 13: 770-4.
• 39 Garcia-Mata R, Bebok Z, Sorscher EJ. et al. Characterizationand dynamics of aggresome formationbya cytosolic GFP-chimera. J Cell Biol 1999; 146: 1239-54.
• 40 Johnston JA, Ward CL, Kopito RR. Aggresomes: a cellular response to misfolded proteins. J Cell Biol 1998; 143: 1883-98.
• 41 Ortel TL, Moore KD, Ezban M. et al. Effect of heterologous factor Vheavy chain sequences on the secretion of recombinant human factor VIII. Thromb Haemost 1996; 75: 36-44.
• 42 Parodi AJ. Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation. Biochem J 2000; 348 Pt 1 1-13.
• 43 Roth J. Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem Rev 2002; 102: 285-303.
• 44 Kitzmuller C, Caprini A, Moore SE. et al. Processing of N-linked glycans during endoplasmic-reticulum- associated degradation of a short-lived variant of ribophorin I. Biochem J 2003; 376: 687-96.
• 45 Kornfeld S, Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol 1989; 5: 483-525.
• 46 Mellman I. Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 1996; 12: 575-625.
• 47 Baba MO, sumi M, Scott SV. et al. Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J Cell Biol 1997; 139: 1687-95.
• 48 Cabral CM, Liu Y, Sifers RN. Dissecting glycoprotein quality control in the secretory pathway. Trends Biochem Sci 2001; 26: 619-24.
• 49 Noda T, Farquhar MG. A non-autophagic pathway for diversion of ER secretory proteins to lysosomes. J Cell Biol 1992; 119: 85-97.
• 50 Kaufman RJ. Biological regulation of factor VIII activity. Annu Rev Med 1992; 43: 325-39.
• 51 Chen C, Fang XD, Zhu J. et al. The gene expression of coagulation factor VIII in mammalian cell lines. Thromb Res 1999; 95: 105-15.
• 52 Doering CB, Healey JF, Parker ET. et al. High level expression of recombinant porcine coagulation factor VIII. J Biol Chem 2002; 277: 38345-9.
• 53 Haack A, Schmitt C, Poller W. et al. Analysis of expression kinetics and activity of a new B-domain truncated and full-length FVIII protein in three different cell lines. Ann Hematol. 1999; 78: 111-6.
• 54 Herlitschka SE, Schlokat U, Falkner FG. et al. High expression of a B-domain deleted factor VIII gene in a human hepatic cell line. J Biotechnol 1998; 61: 165-73.
• 55 Pittman DD, Tomkinson KN, Kaufman RJ. Posttranslational requirements for functional factor V and factor VIII secretion in mammalian cells. J Biol Chem 1994; 269: 17329-37.