Skip to main content

Advertisement

SpringerLink
Log in

Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg1648 and Glu1649. By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg1648 FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg1648 FVIII processing site by PCSK6.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

rFVIII:

Recombinant human factor VIII

PACE:

Paired basic amino acid cleaving enzyme

PCSK:

Proprotein convertase subtilisin/kexin

Dhfr:

Dihydrofolate reductase

References

  1. Hockin, M. F., Jones, K. C., Everse, S. J., et al. (2002). A model for the stoichiometric regulation of blood coagulation. Journal of Biological Chemistry, 277, 18322–18333.

    Article  CAS  Google Scholar 

  2. Bolton-Maggs, P. H., & Pasi, K. J. (2003). Haemophilias A and B. Lancet, 361, 1801–1809.

    Article  CAS  Google Scholar 

  3. Mannucci, P. M., & Tuddenham, E. G. (2001). The hemophilias—from royal genes to gene therapy. New England Journal of Medicine, 344, 1773–1779.

    Article  CAS  Google Scholar 

  4. Grillberger, L., Kreil, T. R., Nasr, S., et al. (2009). Emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells. Biotechnology Journal, 4, 186–201.

    Article  CAS  Google Scholar 

  5. Mannucci, P. M. (2008). Back to the future: A recent history of haemophilia treatment. Haemophilia, 14, 10–18.

    Article  Google Scholar 

  6. Pittman, D. D., Tomkinson, K. N., & Kaufman, R. J. (1994). Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells. Journal of Biological Chemistry, 269, 17329–17337.

    CAS  Google Scholar 

  7. Boedeker, B. G. (2001). Production processes of licensed recombinant factor VIII preparations. Seminars in Thrombosis and Hemostasis, 27, 385–394.

    Article  CAS  Google Scholar 

  8. Pipe, S. W., Morris, J. A., Shah, J., et al. (1998). Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin. Journal of Biological Chemistry, 273, 8537–8544.

    Article  CAS  Google Scholar 

  9. Miao, H. Z., Sirachainan, N., Palmer, L., et al. (2004). Bioengineering of coagulation factor VIII for improved secretion. Blood, 103, 3412–3419.

    Article  CAS  Google Scholar 

  10. Pittman, D. D., Alderman, E. M., Tomkinson, K. N., et al. (1993). Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII. Blood, 81, 2925–2935.

    CAS  Google Scholar 

  11. Cerullo, V., Seiler, M. P., Mane, V., et al. (2007). Correction of murine hemophilia A and immunological differences of factor VIII variants delivered by helper-dependent adenoviral vectors. Molecular Therapy, 15, 2080–2087.

    Article  CAS  Google Scholar 

  12. Lind, P., Larsson, K., Spira, J., et al. (1995). Novel forms of B-domain-deleted recombinant factor VIII molecules—Construction and biochemical characterization. European Journal of Biochemistry, 232, 19–27.

    Article  CAS  Google Scholar 

  13. Jankowski, M. A., Patel, H., Rouse, J. C., et al. (2007). Defining ‘full-length’ recombinant factor VIII: A comparative structural analysis. Haemophilia, 13, 30–37.

    Article  CAS  Google Scholar 

  14. Kaufman, R. J., Wasley, L. C., & Dorner, A. J. (1988). Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. Journal of Biological Chemistry, 263, 6352–6362.

    CAS  Google Scholar 

  15. Rehemtulla, A., & Kaufman, R. J. (1992). Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes. Blood, 79, 2349–2355.

    CAS  Google Scholar 

  16. Seidah, N. G., & Chrétien, M. (1999). Proprotein and prohormone convertases: A family of subtilases generating diverse bioactive polypeptides. Brain Research, 848, 45–62.

    Article  CAS  Google Scholar 

  17. Seidah, N. G., & Prat, A. (2012). The biology and therapeutic targeting of the proprotein convertases. Nature Reviews of Drug Discovery, 11, 367–383.

    Article  CAS  Google Scholar 

  18. Peters, R. T., Toby, G., Lu, Q., et al. (2013). Biochemical and functional characterization of a recombinant monomeric factor VIII-Fc fusion protein. Journal of Thrombosis and Haemostasis, 11, 132–141.

    Article  CAS  Google Scholar 

  19. Niwa, H., Yamamura, K., & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193–199.

    Article  CAS  Google Scholar 

  20. Chen, C., Fang, X. D., Zhu, J., et al. (1999). The gene expression of coagulation factor VIII in mammalian cell lines. Thrombosis Research, 95, 105–115.

    Article  CAS  Google Scholar 

  21. Yonemura, H., Sugawara, K., Nakashima, K., et al. (1993). Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains. Protein Engineering, 6, 669–674.

    Article  CAS  Google Scholar 

  22. Tiscornia, G., Singer, O., & Verma, I. M. (2006). Production and purification of lentiviral vectors. Nature Protocols, 1, 241–245.

    Article  CAS  Google Scholar 

  23. Colin, C., Tobaruella, F. S., Correa, R. G., et al. (2010). Cloning and characterization of a novel alternatively spliced transcript of the human CHD7 putative helicase. BMC Research Notes, 3, 252.

    Article  Google Scholar 

  24. Tsuji, A., Ikoma, T., Hashimoto, E., et al. (2002). Development of selectivity of alpha1-antitrypsin variant by mutagenesis in its reactive site loop against proprotein convertase. A crucial role of the P4 arginine in PACE4 inhibition. Protein Engineering, 15, 123–130.

    Article  CAS  Google Scholar 

  25. Urlaub, G., Käs, E., Carothers, A. M., et al. (1983). Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell, 33, 405–412.

    Article  CAS  Google Scholar 

  26. Kaufman, R. J., Wasley, L. C., Spiliotes, A. J., et al. (1985). Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Molecular and Cellular Biology, 5, 1750–1759.

    Article  CAS  Google Scholar 

  27. Garten, W., Hallenberger, S., Ortmann, D., et al. (1994). Processing of viral glycoproteins by the subtilisin-like endoprotease furin and its inhibition by specific peptidylchloroalkylketones. Biochimie, 76, 217–225.

    Article  CAS  Google Scholar 

  28. Pipe, S. W., & Kaufman, R. J. (1997). Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proceedings of the National Academy of Sciences USA, 94, 11851–11856.

    Article  CAS  Google Scholar 

  29. Burton, M., Nakai, H., Colosi, P., et al. (1999). Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein. Proceedings of the National Academy of Sciences USA, 96, 12725–12730.

    Article  CAS  Google Scholar 

  30. VandenDriessche, T., Vanslembrouck, V., Goovaerts, I., et al. (1999). Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proceedings of the National Academy of Sciences USA, 96, 10379–10384.

    Article  CAS  Google Scholar 

  31. Hu, C., & Lipshutz, G. S. (2012). AAV-based neonatal gene therapy for hemophilia A: Long-term correction and avoidance of immune responses in mice. Gene Therapy, 19, 1166–1176.

    Article  CAS  Google Scholar 

  32. Scallan, C. D., Liu, T., Parker, A. E., et al. (2003). Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII. Blood, 102, 3919–3926.

    Article  CAS  Google Scholar 

  33. Moussalli, M., Pipe, S. W., Hauri, H. P., et al. (1999). Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII. Journal of Biological Chemistry, 274, 32539–32542.

    Article  CAS  Google Scholar 

  34. Seidah, N. G., Mayer, G., Zaid, A., et al. (2008). The activation and physiological functions of the proprotein convertases. International Journal of Biochemistry & Cell Biology, 40, 1111–1125.

    Article  CAS  Google Scholar 

  35. Rousselet, E., Benjannet, S., Hamelin, J., et al. (2011). The proprotein convertase PC7: Unique zymogen activation and trafficking pathways. Journal of Biological Chemistry, 286, 2728–2738.

    Article  CAS  Google Scholar 

  36. Tsuji, A., Sakurai, K., Kiyokage, E., et al. (2003). Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix. Biochimica et Biophysica Acta, 1645, 95–104.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are deeply grateful to M.Sc. Marluce da Cunha Mantovani, Zizi de Mendonça, Débora Cristina da Costa, Mariele Santos Moraes, and Thays Rafaelle Viana for excellent technical assistance and to our lab colleagues for discussions and criticisms. This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Banco Nacional de Desenvolvimento Econômico e Social (BNDES), Ministério da Saúde (MS-DECIT), Ministério de Ciência, Tecnologia e Inovação (MCTI), and the University of São Paulo (USP). MAD held a post-doctoral fellowship from the Brazilian National Research Council (CNPq). ESM held a pre-doctoral fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Author information

Authors and Affiliations

  1. Biochemistry Department, Chemistry Institute, University of São Paulo, São Paulo, SP, 05508-000, Brazil

    Marcos A. Demasi, Erika de S. Molina, Fernando H. Lojudice, Angelita Muras & Mari C. Sogayar

  2. NUCEL-Núcleo de Terapia Celular e Molecular, School of Medicine, University of São Paulo, Rua Pangaré, 100 (Cidade Universitária), São Paulo, SP, 05360-130, Brazil

    Marcos A. Demasi, Erika de S. Molina, Christian Bowman-Colin, Fernando H. Lojudice, Angelita Muras & Mari C. Sogayar

Authors
  1. Marcos A. Demasi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erika de S. Molina
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Bowman-Colin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fernando H. Lojudice
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Angelita Muras
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mari C. Sogayar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marcos A. Demasi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Demasi, M.A., de S. Molina, E., Bowman-Colin, C. et al. Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins. Mol Biotechnol 58, 404–414 (2016). https://doi.org/10.1007/s12033-016-9939-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-016-9939-9

Keywords

Search

Navigation