Skip to main content
SpringerLink
Log in

Human Tyrosinase Produced in Insect Cells: A Landmark for the Screening of New Drugs Addressing its Activity

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of l-tyrosine to l-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, l-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Solano, F., Briganti, S., Picardo, M., & Ghanem, G. (2006). Hypopigmenting agents: An updated review on biological, chemical and clinical aspects. Pigment Cell Research, 19, 550–571.

    Article  CAS  Google Scholar 

  2. Gillbro, J., & Olsson, M. (2011). The melanogenesis and mechanisms of skin-lightening agents–existing and new approaches. International Journal of Cosmetic Science, 33, 210–221.

    Article  CAS  Google Scholar 

  3. Matoba, Y., Kumagai, T., Yamamoto, A., Yoshitsu, H., & Sugiyama, M. (2006). Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis. Journal of Biological Chemistry, 281, 8981–8990.

    Article  CAS  Google Scholar 

  4. Sendovski, M., Kanteev, M., Shuster Ben-Yosef, V., Adir, N., & Fishman, A. (2010). Crystallization and preliminary X-ray crystallographic analysis of a bacterial tyrosinase from Bacillus megaterium. Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 66, 1101–1103.

    Article  CAS  Google Scholar 

  5. Ismaya, W. T., Rozeboom, H. J., Weijn, A., Mes, J. J., Fusetti, F., Wichers, H. J., et al. (2011). Crystal structure of Agaricus bisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone. Biochemistry, 50, 5477–5486.

    Article  CAS  Google Scholar 

  6. Fujieda, N., Yabuta, S., Ikeda, T., Oyama, T., Muraki, N., Kurisu, G., et al. (2013). Crystal structures of copper-depleted and copper-bound fungal pro-tyrosinase: insights into endogenous cysteine-dependent copper incorporation. Journal of Biological Chemistry, 288, 22128–22140.

    Article  CAS  Google Scholar 

  7. Takahashi, H., & Parsons, P. G. (1992). Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells. Journal of Investigative Dermatology, 98, 481–487.

    Article  CAS  Google Scholar 

  8. Kong, K.H., (2004). Method for mass-producing of a recombinant human tyrosinase in E. coli. Patent WO 2004048560 A1.

  9. Chen, G., Chen, W., Huang, Y., & Jiang, S. (2012). Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation. Journal of Agriculture and Food Chemistry, 60, 2838–2843.

    Article  CAS  Google Scholar 

  10. Chen, G., Wang, H., & Li, Z., (2005). Human tyrosinase expression carrier and its use. Patent CN 1603417 A.

  11. Dolinska, M. B., Kovaleva, E., Backlund, P., Wingfield, P. T., Brooks, B. P., & Sergeev, Y. V. (2014). Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity. PLoS One, 9, e84494.

    Article  Google Scholar 

  12. Branza-Nichita, N., Negroiu, G., Petrescu, A. J., Garman, E. F., Platt, F. M., Wormald, M. R., et al. (2000). Mutations at critical N-glycosylation sites reduce tyrosinase activity by altering folding and quality control. Journal of Biological Chemistry, 275, 8169–8175.

    Article  CAS  Google Scholar 

  13. Wang, N., & Hebert, D. N. (2006). Tyrosinase maturation through the mammalian secretory pathway: Bringing color to life. Pigment Cell Research, 19, 3–18.

    Article  Google Scholar 

  14. Popescu, C. I., Mares, A., Zdrentu, L., Zitzmann, N., Dwek, R. A., & Petrescu, S. M. (2006). Productive folding of tyrosinase ectodomain is controlled by the transmembrane anchor. Journal of Biological Chemistry, 281, 21682–21689.

    Article  CAS  Google Scholar 

  15. Ando, H., Kondoh, H., Ichihashi, M., & Hearing, V. J. (2007). Approaches to identify inhibitors of melanin biosynthesis via the quality control of tyrosinase. Journal of Investigative Dermatology, 127, 751–761.

    Article  CAS  Google Scholar 

  16. Cioaca, D., Ghenea, S., Spiridon, L. N., Marin, M., Petrescu, A., & Petrescu, S. M. (2011). C-terminus glycans with critical functional role in the maturation of secretory glycoproteins. PLoS One, 6, e19979.

    Article  CAS  Google Scholar 

  17. Bubacco, L., Vijgenboom, E., Gobin, C., Tepper, A. W., Salgado, J., & Canters, G. W. (2000). Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase. Journal of Molecular Catalysis B: Enzymatic, 8, 27–35.

    Article  CAS  Google Scholar 

  18. Greggio, E., Bergantino, E., Carter, D., Ahmad, R., Costin, G., Hearing, V. J., et al. (2005). Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson’s disease. Journal of Neurochemistry, 93, 246–256.

    Article  CAS  Google Scholar 

  19. Espín, J. C., Morales, M., García-Ruiz, P. A., Tudela, J., & García-Cánovas, F. (1997). Improvement of a continuous spectrophotometric method for determining the monophenolase and diphenolase activities of mushroom polyphenol oxidase. Journal of Agriculture and Food Chemistry, 45, 1084–1090.

    Article  Google Scholar 

  20. Espín, J. C., Varón, R., Fenoll, L. G., Gilabert, M., García-Ruíz, P. A., Tudela, J., et al. (2000). Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase. European Journal of Biochemistry, 267, 1270–1279.

    Article  Google Scholar 

  21. Willers, J., Lucchese, A., Mittelman, A., Dummer, R., & Kanduc, D. (2005). Definition of anti-tyrosinase MAb T311 linear determinant by proteome-based similarity analysis. Experimental Dermatology, 14, 543–550.

    Article  CAS  Google Scholar 

  22. Virador, V., Matsunaga, N., Matsunaga, J., Valencia, J., Oldham, R. J., Kameyama, K., et al. (2001). Production of melanocyte-specific antibodies to human melanosomal proteins: Expression patterns in normal human skin and in cutaneous pigmented lesions. Pigment Cell Research, 14, 289–297.

    Article  CAS  Google Scholar 

  23. Tsukamoto, K., Jackson, I. J., Urabe, K., Montague, P. M., & Hearing, V. J. (1992). A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase. EMBO Journal, 11, 519–526.

    CAS  Google Scholar 

  24. Bubacco, L., Salgado, J., Tepper, A. W., Vijgenboom, E., & Canters, G. W. (1999). 1H NMR spectroscopy of the binuclear Cu (II) active site of Streptomyces antibioticus tyrosinase. FEBS Letters, 442, 215–220.

    Article  CAS  Google Scholar 

  25. Vijayan, E., Husain, I., Ramaiah, A., & Madan, N. C. (1982). Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein. Archives of Biochemistry and Biophysics, 217, 738–747.

    Article  CAS  Google Scholar 

  26. Hearing, V. J, Jr. (1987). Mammalian monophenol monooxygenase (tyrosinase): Purification, properties, and reactions catalyzed. Methods in Enzymology, 142, 154–165.

    Article  CAS  Google Scholar 

  27. Wittbjer, A., Dahlback, B., Odh, G., Rosengren, A. M., Rosengren, E., & Rorsman, H. (1989). Isolation of human tyrosinase from cultured melanoma cells. Acta Dermato Venereologica, 69, 125–131.

    CAS  Google Scholar 

  28. Yurkow, E. J., & Laskin, J. D. (1989). Purification of tyrosinase to homogeneity based on its resistance to sodium dodecyl sulfate-proteinase K digestion. Archives of Biochemistry and Biophysics, 275, 122–129.

    Article  CAS  Google Scholar 

  29. Pomerantz, S. H. (1966). The tyrosine hydroxylase activity of mammalian tyrosinase. Journal of Biological Chemistry, 241, 161–168.

    CAS  Google Scholar 

  30. Olivares, C., García-Borrón, J. C., & Solano, F. (2002). Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in mammalian tyrosinase. Implications to the catalytic cycle. Biochemistry, 41, 679–686.

    Article  CAS  Google Scholar 

  31. Jergil, B., Lindbladh, C., Rorsman, H., & Rosengren, E. (1983). Dopa oxidation and tyrosine oxygenation by human melanoma tyrosinase. Acta Dermato Venereologica, 63, 468–475.

    CAS  Google Scholar 

  32. Husain, I., Vijayan, E., Ramaiah, A., Pasricha, J., & Madan, N. (1982). Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C (U)-l-tyrosine incorporation into melanin. Journal of Investigative Dermatology, 78, 243–252.

    Article  CAS  Google Scholar 

  33. Winder, A. J., & Harris, H. (1991). New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. European Journal of Biochemistry, 198, 317–326.

    Article  CAS  Google Scholar 

  34. Eleftherianos, I., & Revenis, C. (2011). Role and importance of phenoloxidase in insect hemostasis. Journal of Innate Immunity, 3, 28–33.

    Article  CAS  Google Scholar 

  35. González-Santoyo, I., & Córdoba-Aguilar, A. (2012). Phenoloxidase: A key component of the insect immune system. Entomologia Experimentalis et Applicata, 142, 1–16.

    Article  Google Scholar 

  36. Simmen, T., Schmidt, A., Hunziker, W., & Beermann, F. (1999). The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal. Journal of Cell Science, 112(Pt 1), 45–53.

    CAS  Google Scholar 

  37. Calvo, P. A., Frank, D. W., Bieler, B. M., Berson, J. F., & Marks, M. S. (1999). A cytoplasmic sequence in human tyrosinase defines a second class of di-leucine-based sorting signals for late endosomal and lysosomal delivery. Journal of Biological Chemistry, 274, 12780–12789.

    Article  CAS  Google Scholar 

  38. Bubacco, L., Spinazze, R., Longa, S. D., & Benfatto, M. (2007). X-ray absorption analysis of the active site of Streptomyces antibioticus tyrosinase upon binding of transition state analogue inhibitors. Archives of Biochemistry and Biophysics, 465, 320–327.

    Article  CAS  Google Scholar 

  39. Bochot, C., Gouron, A., Bubacco, L., Milet, A., Philouze, C., Réglier, M., et al. (2014). Probing kojic acid binding to tyrosinase enzyme: Insights from a model complex and QM/MM calculations. Chemical Communications, 50, 308–310.

    Article  CAS  Google Scholar 

  40. Olivares, C., & Solano, F. (2009). New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins. Pigment cell & melanoma research, 22, 750–760.

    Article  CAS  Google Scholar 

  41. Masamoto, Y., Murata, Y., Baba, K., Shimoishi, Y., Tada, M., & Takahata, K. (2004). Inhibitory effects of esculetin on melanin biosynthesis. Biological and Pharmaceutical Bulletin, 27, 422–425.

    Article  CAS  Google Scholar 

  42. Ryazanova, A. D., Alekseev, A. A., & Slepneva, I. A. (2012). The phenylthiourea is a competitive inhibitor of the enzymatic oxidation of DOPA by phenoloxidase. Journal of Enzyme Inhibition and Medicinal Chemistry, 27, 78–83.

    Article  CAS  Google Scholar 

  43. Goliĉnik, M., & Stojan, J. (2004). Slow-binding inhibition: A theoretical and practical course for students. Biochemistry and Molecular Biology Education, 32, 228–235.

    Article  Google Scholar 

  44. Shi, Y., Chen, Q., Wang, Q., Song, K., & Qiu, L. (2005). Inhibitory effects of cinnamic acid and its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase. Food Chemistry, 92, 707–712.

    Article  CAS  Google Scholar 

  45. Nesterov, A., Zhao, J., Minter, D., Hertel, C., Ma, W., Abeysinghe, P., et al. (2008). 1-(2, 4-Dihydroxyphenyl)-3-(2, 4-dimethoxy-3-methylphenyl) propane, a novel tyrosinase inhibitor with strong depigmenting effects. Chemical & Pharmaceutical Bulletin, 56, 1292–1296.

    Article  CAS  Google Scholar 

  46. Gasparetti, C., Nordlund, E., Jänis, J., Buchert, J., & Kruus, K. (2012). Extracellular tyrosinase from the fungus Trichoderma reeseis hows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1824, 598–607.

    Article  CAS  Google Scholar 

  47. Gouzi, H., Coradin, T., Delicado, E., Ünal, M., & Benmansour, A. (2010). Inhibition kinetics of Agaricus bisporus (JE Lange) Imbach polyphenol oxidase. Open Enzyme Inhibition Journal, 3, 1–7.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. E. Greggio (Department of Biology, University of Padova, Italy) for providing a sample of PMEL (Pigmented Melanoma) cells. This work has been financed by the Assegno di Ricerca (CPDR095797/09) and by the Progetto di Ricerca di Ateneo (CPDA110789/11) from the University of Padova to E.B.

Author information

Authors and Affiliations

  1. Department of Biology, University of Padua, Viale G. Colombo 3, 35121, Padua, Italy

    Stefano Fogal, Marcello Carotti, Laura Giaretta, Federico Lanciai, Leonardo Nogara, Luigi Bubacco & Elisabetta Bergantino

  2. F.I.S.-Fabbrica Italiana Sintetici S.p.A., Viale Milano 26, Montecchio Maggiore, 36075, Vicenza, Italy

    Stefano Fogal

  3. FRI, Food Research and Innovation S.r.l., Via Croce Rossa, 32/2, 35131, Padua, Italy

    Federico Lanciai

Authors
  1. Stefano Fogal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marcello Carotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laura Giaretta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Federico Lanciai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Leonardo Nogara
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Luigi Bubacco
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elisabetta Bergantino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Luigi Bubacco or Elisabetta Bergantino.

Additional information

Stefano Fogal and Marcello Carotti have contributed equally to the work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fogal, S., Carotti, M., Giaretta, L. et al. Human Tyrosinase Produced in Insect Cells: A Landmark for the Screening of New Drugs Addressing its Activity. Mol Biotechnol 57, 45–57 (2015). https://doi.org/10.1007/s12033-014-9800-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-014-9800-y

Keywords

Search

Navigation