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Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice

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Abstract

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe’s disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean β-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation−phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA−/−) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.

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Abbreviations

IP:

intraperitoneal

AMD:

acid maltase deficiency

GSDII:

glycogen storage disease type II

exon 6 neo:

exon 6 neomycin resistent

rhGAA:

recombinant GAA

tobrhGAA:

recombinant GAA produced in tobacco seeds

GAA:

acid maltase

ERT:

enzyme replacement therapy

References

  1. Fischer, R., Schillberg, S., Hellwig, S., Twyman, R. M., & Drossard, J. (2012). GMP issues for recombinant plant-derived pharmaceutical proteins. Biotechnology Advances, 30, 434–439.

    Article  CAS  Google Scholar 

  2. Lico, C., Santi, L., Twyman, R. M., Pezzotti, M., & Avesani, L. (2012). The use of plants for the production of therapeutic human peptides. Plant Cell Reports, 31, 439–451.

    Article  CAS  Google Scholar 

  3. Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., & Fischer, R. (2003). Molecular farming in plants: host systems and expression technology. Trends in Biotechnology, 21, 570–578.

    Article  CAS  Google Scholar 

  4. Twyman, R. M., Ramessar, K., Quemada, H., Capell, T., & Christou, P. (2009). Plant biotechnology: the importance of being accurate. Trends in Biotechnology, 27, 609–612.

    Article  CAS  Google Scholar 

  5. Kusnadi, A. R., Evangelista, R. L., Hood, E. E., Howard, J. A., & Nikolov, Z. L. (1998). Processing of transgenic corn seed and its effect on the recovery of recombinant beta-glucuronidase. Biotechnology and Bioengineering, 60, 44–52.

    Article  CAS  Google Scholar 

  6. Reggi, S., Marchetti, S., Patti, T., De Amicis, F., Cariati, R., Bembi, B., & Fogher, C. (2005). Recombinant human acid β-glucosidase stored in tobacco seed is stable, active and taken up by human fibroblasts. Plant Molecular Biology, 57, 101–113.

    Article  CAS  Google Scholar 

  7. Stoeger, E., Vaquero, C., Torres, E., Sack, M., Nicholson, L., Drossard, J., Williams, S., Keen, D., Perrin, Y., Christou, P., & Fischer, R. (2000). Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies. Plant Molecular Biology, 42, 583–590.

    Article  Google Scholar 

  8. Kermode, A. R. (2006). Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biology. Canadian Journal of Botany, 84, 679–694.

    Article  CAS  Google Scholar 

  9. Kermode, A. R. (2012). Seed Expression Systems for Molecular Farming. In A. Wang & S. Ma (Eds.), Molecular farming in plants: recent advances and future prospects (pp. 89–123). New York: Springer.

    Chapter  Google Scholar 

  10. Lau, O. S., & Sun, S. S. M. (2009). Plant seeds as bioreactors for recombinant protein production. Biotechnology Advances, 27, 1015–1022.

    Article  CAS  Google Scholar 

  11. Boothe, J., Nykiforuk, C., Shen, Y., Zaplachinski, S., Szarka, S., Kuhlman, P., Murray, E., Morck, D., & Moloney, M. M. (2010). Seed-based expression system for plant molecular farming. Plant Biotechnology Journal, 8, 588–606.

    Article  CAS  Google Scholar 

  12. Stoger, E., & Ma, J. K. (2005). Sowing the seeds of success: Pharmaceutical proteins from plants. Current Opinion in Biotechnology, 16, 167–173.3.

    Article  CAS  Google Scholar 

  13. Gomord, V., Fischette, A. C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson, C., Michaud, D., & Faye, L. (2010). Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnology Journal, 8, 564–587.

    Article  CAS  Google Scholar 

  14. Saint-Jore-Dupas, C., Faye, L., & Gomord, V. (2007). From planta to pharma with glycosylation in the toolbox. Trends in Biotechnology, 25, 317–323.

    Article  CAS  Google Scholar 

  15. Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fischette-Lainé, A. C., Gomord, V., & Faye, L. (1998). N-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant N-glycans. Plant Molecular Biology, 38, 31–48.

    Article  CAS  Google Scholar 

  16. Gomord, V., & Faye, L. (2004). Posttranslational modification of therapeutic proteins in plants. Current Opinion in Plant Biology, 7, 171–181.

    Article  CAS  Google Scholar 

  17. Kermode, A. R. (1996). Mechanisms of intracellular protein transport and targeting. Critical Reviews in Plant Sciences, 15, 285–423.

    Article  CAS  Google Scholar 

  18. He, X., Galpin, J. D., Tropak, M. B., Mahuran, D., Haselhorst, T., von Itzstein, M., Kolarich, D., Packer, N. H., Miao, Y., Jiang, L., Grabowski, G. A., Clarke, L. A., & Kermode, A. R. (2012). Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants. Glycobiology, 22, 492–503.

    Article  CAS  Google Scholar 

  19. Hers, H. G. (1963). Alpha-glucosidase deficiency in generalized glycogen storage disease (Pompe's Disease). Biochemical Journal, 86, 11–16.

    CAS  Google Scholar 

  20. Kornfeld, S. (1986). Trafficking of lysosomal enzymes in normal and disease states. The Journal of Clinical Investigation, 77, 1–6.

    Article  CAS  Google Scholar 

  21. Rosenfeld, M. G., Kreibich, G., Popov, D., Kato, K., & Sabatini, D. D. (1982). Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution. The Journal of Cell Biology, 93, 135–141.

    Article  CAS  Google Scholar 

  22. Oude Elferink, R.P.J. (1985) Biosynthesis, transport and processing of lysosomal alpha glucosidase. PhD Thesis, University of Amsterdam.

  23. Slonim, A., Bulone, L., Ritz, S., Goldberg, T., Chen, A., & Martiniuk, F. (2000). Identification of two subtypes of infantile acid maltase deficiency: Evaluation of twenty-two patients and review of the literature. Journal of Pediatrics, 137, 283–285.

    Article  CAS  Google Scholar 

  24. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Roger, S. D., & Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science, 227, 1229–1231.

    Article  CAS  Google Scholar 

  25. Doyle, J. J., & Doyle, J. L. (1997). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19, 11–15.

    Google Scholar 

  26. Martiniuk, F., Honig, J., & Hirschhorn, R. (1984). Further studies of the structure of human placental acid alpha-glucosidase. Archives of Biochemistry and Biophysics, 231, 454–460.

    Article  CAS  Google Scholar 

  27. Martiniuk, F., & Hirschhorn, R. (1981). Characterization of neutral isozymes of human alpha-glucosidase. Biochimica et Biophysica Acta, 658, 248–261.

    Article  CAS  Google Scholar 

  28. Martiniuk, F., Chen, A., Donnabella, V., Arvanitopoulos, E., Slonim, A., Raben, N., Plotz, P., & Rom, W. N. (2000). Correction of glycogen storage disease type II by enzyme replacement with a recombinant human acid maltase produced by over-expression in a CHO DHFRneg cell line. Biochem Biophys Res Comm., 276, 917–923.

    Article  CAS  Google Scholar 

  29. Raben, N., Nagaraju, K., Lee, E., Kessler, P., Byrne, B., Lee, L., LaMaurex, M., King, J., Sauer, B., & Plotz, P. (1998). Targeted disruption of the acid alpha glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. Journal of Biological Chemistry, 273, 19086–19092.

    Article  CAS  Google Scholar 

  30. Bijvoet, A. G., Kroos, M. A., Pieper, F. R., Van der Vliet, M., De Boer, H. A., Van der Ploeg, A. T., Verbeet, M. P., & Reuser, A. J. (1998). Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice. Human Molecular Genetics, 1815–24.

  31. Maga, J. A., Zhou, J., Kambampati, R., Peng, S., Wang, X., Bohnsack, R. N., Thomm, A., Golata, S., Tom, P., Dahms, N. M., Byrne, B. J., & Lebowitz, J. H. (2013). Glycosylation-independent lysosomal targeting of acid α-glucosidase enhances muscle glycogen clearance in Pompe mice. Journal of Biological Chemistry, 288, 1428–1438.

    Article  CAS  Google Scholar 

  32. Van der Ploeg, A. T., Bolhuis, P. A., Wolterman, R. A., Visser, J. W., Loonen, M. C., Busch, H. F., & Reuser, A. J. (1988). Prospect for enzyme therapy in glycogenosis II variants: a study on cultured muscle cells. Journal of Neurology, 235, 392–396.

    Article  Google Scholar 

  33. Van der Ploeg, A. T., Kroos, M. A., Willemsen, R., Brons, N. H., & Reuser, A. J. (1991). Intravenous administration of phosphorylated acid alpha-glucosidase leads to uptake of enzyme in heart and skeletal muscle of mice. The Journal of Clinical Investigation, 87, 513–518.

    Article  Google Scholar 

  34. Van den Hout, J., Kamphoven, J., Winkel, L. P., Arts, W. F., De Klerk, J. B., Loonen, M. C., Vulto, A. G., Cromme-Dijkhuis, A., Weisglas-Kuperus, N., Hop, W., Van Hirtum, H., Van Diggelen, O. P., Boer, M., Kroos, M. A., Van Doorn, P. A., Van der Voort, E., Sibbles, B., Van Corven, E. J., Brakenhoff, J. P., Van Hove, J., Smeitink, J. A., de Jong, G., Reuser, A. J., & Van der Ploeg, A. T. (2004). Long-term intravenous treatment of Pompe disease with recombinant human α-glucosidase from milk. Pediatrics, 113, 448–457.

    Article  Google Scholar 

  35. Kikuchi, T., Yang, H. W., Pennybacher, M., Ichihara, N., Mizutani, M., van Hove, J. L. K., & Chen, Y. T. (1998). Clinical and metabolic correction of Pompe disease by enzyme therapy in acid maltase-deficient quail. Journal of Clinical Laboratory, 101, 827–833.

    CAS  Google Scholar 

  36. Xu, X., Gan, Q., Clough, R. C., Pappu, K. M., Howard, J. A., Baez, J. A., & Wang, K. (2011). Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase. BMC Biotechnology, 11, 69–80.

    Article  CAS  Google Scholar 

  37. De Marchis, F., Balducci, C., Pompa, A., Riise Stensland, H. M., Guaragno, M., Pagiotti, R., Menghini, A. R., Persichetti, E., Beccari, T., & Bellucci, M. (2011). Human α-mannosidase produced in transgenic tobacco plants is processed in human α-mannosidosis cell lines. Plant Biotechnology Journal, 9, 1061–1073.

    Article  Google Scholar 

  38. Nozoye, T., Takaiwa, F., Tsuji, N., Yamakawa, T., Arakawa, T., Hayashi, Y., & Matsumoto, Y. (2009). Production of Ascaris suum As14 protein and its fusion protein with cholera toxin B subunit in rice seeds. Journal of Veterinary Medical Science, 71, 995–1000.

    Article  CAS  Google Scholar 

  39. Oszvald, M., Kang, T. J., Tomoskozi, S., Jenes, B., Kim, T. G., Cha, Y. S., Tamas, L., & Yang, M. S. (2008). Expression of cholera toxin B subunit in transgenic rice endosperm. Molecular Biotechnology, 40, 261–268.

    Article  CAS  Google Scholar 

  40. Blais, D. R., & Altosaar, I. (2006). Human CD14 expressed in seeds of transgenic tobacco displays similar proteolytic resistance and bioactivity with its mammalian-produced counterpart. Transgenic Research, 15, 151–164.

    Article  CAS  Google Scholar 

  41. Samyn-Petit, B., Wajda Dubos, J. P., Chirat, F., Coddeville, B., Demaizieres, G., Farrer, S., Slomianny, M. C., Theisen, M., & Delannoy, P. (2003). Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants. European Journal of Biochemistry, 270, 3235–3242.

    Article  CAS  Google Scholar 

  42. Woodard, S. L., Mayor, J. M., Bailey, M. R., Barker, D. K., Love, R. T., Lane, J. R., Delaney, D. E., McComas-Wagner, J. M., Mallubhotla, H. D., Hood, E. E., Dangott, L. J., Tichy, S. E., & Howard, J. A. (2003). Maize (Zea mays)-derived bovine trypsin: characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnology and Applied Biochemistry, 38, 123–130.

    Article  CAS  Google Scholar 

  43. Martiniuk, F., Tzall, S., & Chen, A. (1992). Recombinant human acid alpha-glucosidase generated in bacteria: antigenic, but enzymatically inactive. DNA and Cell Biology, 11, 701–706.

    Article  CAS  Google Scholar 

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Acknowledgements

This research was supported in part by a grant UL1 TR000038 from the National Center for Advancing Translational Sciences, National Institutes of Health.

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Authors and Affiliations

  1. Department of Medicine, Division of Pulmonary and Critical Care Medicine, New York University School of Medicine, New York, NY, 10016, USA

    Frank Martiniuk & William N. Rom

  2. iProDynamic Therapeutics, Inc, New York, NY, 10128, USA

    Frank Martiniuk

  3. Plantechno Srl, Via Staffolo 60, 26041, Casalmaggiore, Italy

    Serena Reggi & Corrado Fogher

  4. Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY, USA

    Kam-Meng Tchou-Wong

  5. Università Cattolica S. Cuore, Via E. Parmense 84, 29100, Piacenza, Italy

    Matteo Busconi

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  1. Frank Martiniuk
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  2. Serena Reggi
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Correspondence to Frank Martiniuk.

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Martiniuk, F., Reggi, S., Tchou-Wong, KM. et al. Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice. Appl Biochem Biotechnol 171, 916–926 (2013). https://doi.org/10.1007/s12010-013-0367-z

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