Abstract
Herinase, a new bi-functional fibrinolytic metalloprotease, was purified from a medicinal and edible mushroom Hericium erinaceum. The enzyme was monomeric with a molecular mass of 51 kDa. Analysis of fibrin zymography showed an active band with a similar molecular mass. The N-terminal sequence of herinase VPSSFRTTITDAQLRG was highly distinguished from known fibrinolytic enzymes. Moreover, the enzyme activity was strongly inhibited by EDTA and EGTA, indicating that herinase is a metalloprotease. Herinase exhibited high specificity for the substrate t-PA followed by plasmin. The K m and V max values for H-D-Ile-Pro-Arg-PNA were found to be 4.7 mg and 26.7 U/ml respectively. Similarly, fibrin plate assays revealed that it was able to degrade fibrin clot directly and also able to activate plasminogen. Herinase provoked a rapid degradation of fibrin and fibrinogen α chains and slower degradation of γ chains. It had no activity on the β chains of fibrin and fibrinogen. This result suggests that herinase could possibly contain higher amount of α-fibrinogenase. The activity of herinase was stimulated by metal ions such as Ca2+, Mg2+, and Mn2+, but inhibited by Cu2+, Fe2+, and Zn2+. Herinase exhibited maximum activity at 30 °C and pH 7.0. These results demonstrate that herinase could be a novel fibrinolytic enzyme.
Similar content being viewed by others
References
Weisel, J. W., Stauffacher, C. V., Bullitt, E., & Cohen, C. (1985). A model for fibrinogen: domains and sequence. Science, 230, 1388–1391.
Doolittle, R. F., Yang, Z., & Mochalkin, I. (2001). Crystal structure studies on fibrinogen and fibrin. Annals of the New York Academy of Sciences, 936, 31–43.
Mackman, N. (2008). Triggers, targets and treatments for thrombosis. Nature, 451, 914–918.
Flemmig, M., & Melzig, M. F. (2012). Serine-proteases as plasminogen activators in terms of fibrinolysis. Journal of Pharmacy and Pharmacology, 64, 1025–1039.
Lu, C.L., & Chen, S.N. (2012). Fibrinolytic Enzymes from Medicinal Mushrooms. In: Eshel Faraggi (ed.). Protein Structure, InTech, ISBN: 978-953-51-0555-8, pp.396.
Guillamón, E., García-Lafuente, A., Lozano, M., D'Arrigo, M., Rostagno, M. A., Villares, A., et al. (2010). Edible mushrooms: role in the prevention of cardiovascular diseases. Fitoterapia, 81, 715–723.
Kim, H. C., Choi, B. S., Sapkota, K., Kim, S., Lee, H. J., Yoo, J. C., et al. (2011). Purification and characterization of a novel, highly potent fibrinolytic enzyme from Paecilomyces tenuipes. Process Biochemistry, 46, 1545–1553.
Mizuno, T., Wasa, T., Ito, H., Suzuki, C., & Ukai, N. (1992). Antitumor-active polysaccharides isolated from the fruiting body of Hericium erinaceum, an edible and medicinal mushroom called yamabushitake or houtou. Bioscience, Biotechnology, and Biochemistry, 56, 347–348.
Hazekawa, M., Kataoka, A., Hayakawa, K., Uchimasu, T., Furuta, R., Irie, K., et al. (2010). Neuroprotective effect of repeated treatment with Hericium erinaceum in mice subjected to middle cerebral artery occlusion. Journal of Health Sciences, 56, 296–303.
Son, C. G., Shin, J. W., Cho, J. H., Cho, C. K., Yun, C. H., Chung, W., et al. (2006). Macrophage activation and nitric oxide production by water soluble components of Hericium erinaceum. International Immunopharmacology, 6, 1363–1369.
Yim, M. H., Shin, J. W., Son, J. Y., Oh, S. M., Han, S. H., Cho, J. H., et al. (2007). Soluble components of Hericium erinaceum induce NK cell activation via production of interleukin-12 in mice splenocytes. Acta Pharmacologica Sinica, 28, 901–907.
Xu, H., Wu, P. R., Shen, Z. Y., & Chen, X. D. (2010). Chemical analysis of Hericium erinaceum polysaccharides and effect of the polysaccharides on derma antioxidant enzymes, MMP-1 and TIMP-1 activities. International Journal of Biological Macromolecules, 47, 33–36.
Wang, J. C., Hu, S. H., Su, C. H., & Lee, T. M. (2001). Antitumor and immunoenhancing activities of polysaccharide from culture broth of Hericium spp. The Kaohsiung Journal of Medical Sciences, 17, 461–467.
Kim, S. K., Son, C. G., Yun, C. H., & Han, S. H. (2010). Hericium erinaceum induces maturation of dendritic cells derived from human peripheral blood monocytes. Phytotherapy Research, 24, 14–19.
Kim, Y. S., Jeon, J. H., Im, J., Kang, S. S., Choi, J. N., Ju, H. R., et al. (2011). Induction of intercellular adhesion molecule-1 by water-soluble components of Hericium erinaceum in human monocytes. Journal of Ethnopharmacology, 133, 874–880.
Lee, E. W., Shizuki, K., Hosokawa, S., Suzuki, M., Suganuma, H., Inakuma, T., et al. (2000). Two novel diterpenoids, erinacines H and I from the mycelia of Hericium erinaceum. Bioscience, Biotechnology, and Biochemistry, 64, 2402–2405.
Kenmoku, H., Shimai, T., Toyomasu, T., Kato, N., & Sassa, T. (2002). Erinacine Q, a new erinacine from Hericium erinaceum, and its biosynthetic route to erinacine C in the basidiomycete. Bioscience, Biotechnology, and Biochemistry, 66, 571–575.
Ueda, K., Tsujimori, M., Kodani, S., Chiba, A., Kubo, M., Masuno, K., et al. (2008). An endoplasmic reticulum (ER) stress-suppressive compound and its analogues from the mushroom Hericium erinaceum. Bioorganic & Medicinal Chemistry, 16, 9467–9470.
Xiao, R., Li, Q. W., Perrett, S., & He, R. Q. (2007). Characterisation of the fibrinogenolytic properties of the buccal gland secretion from Lampetra japonica. Biochimie, 89, 383–392.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
Kim, S. H., Choi, N. S., & Lee, W. Y. (1998). Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Analytical Biochemistry, 263, 115–116.
Astrup, T., & Mullertz, S. (1952). The fibrin plate method for estimating fibrinolytic activity. Archives of Biochemistry and Biophysics, 40, 346–351.
Datta, G., Dong, A., Witt, J., & Tu, A. T. (1995). Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus. Archives of Biochemistry and Biophysics, 317, 365–373.
Matsubara, K., Hori, K., Matsuura, Y., & Miyazawa, K. (2000). Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 125, 137–143.
Shen, M. H., Kim, J. S., Sapkota, K., Park, S. E., Choi, B. S., Kim, S., et al. (2007). Purification, characterization and cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia. Journal of Microbiology and Biotechnology, 17, 1271–1283.
Swenson, S., & Markland, F. S., Jr. (2005). Snake venom fibrin(ogen)olytic enzymes. Toxicon, 45, 1021–1039.
Marder, V. J., & Novokhatny, V. (2010). Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential. Journal of Thrombosis and Haemostasis, 8, 433–444.
Frederiks, W. M., & Mook, O. R. (2004). Metabolic mapping of proteinase activity with emphasis on in situ zymography of gelatinases: review and protocols. Journal of Histochemistry and Cytochemistry, 52, 711–722.
Mihara, H., Sumi, H., Yoneta, T., Mizumoto, H., Ikeda, R., Seiki, M., et al. (1991). A novel fibrinolytic enzyme extracted from the earthworm, Lumbricus rubellus. The Japanese Journal of Physiology, 41, 461–472.
Yang, H., Wang, Y., Xiao, Y., Wang, Y., Wu, J., Liu, C., et al. (2011). A bi-functional anti-thrombosis protein containing both direct-acting fibrin(ogen)olytic and plasminogen-activating activities. PLoS One, 6, e17519.
Pinto, A. F., Dobrovolski, R., Veiga, A. B., & Guimarães, J. A. (2004). Lonofibrase, a novel alpha-fibrinogenase from Lonomia obliqua caterpillars. Thrombosis Research, 113, 147–154.
Kim, J. H., & Kim, Y. S. (2001). Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. Bioscience, Biotechnology, and Biochemistry, 65, 356–362.
Lee, S. Y., Kim, J. S., Kim, J. E., Sapkota, K., Shen, M. H., Kim, S., et al. (2005). Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea. Protein Expression and Purification, 43, 10–17.
Park, S. E., Li, M. H., Kim, J. S., Sapkota, K., Kim, J. E., Choi, B. S., et al. (2007). Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia. Bioscience, Biotechnology, and Biochemistry, 71, 2214–2222.
Kim, J. S., Kim, J. E., Choi, B. S., Park, S. E., Sapkota, K., Kim, S., et al. (2008). Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia. Mycological Research, 112, 990–998.
Peng, Y., Yang, X., & Zhang, Y. (2005). Microbial fibrinolytic enzymes: an overview of source, production, properties, and thrombolytic activity in vivo. Applied Microbiology and Biotechnology, 69, 126–132.
Rau, J. C., Beaulieu, L. M., Huntington, J. A., & Church, F. C. (2007). Serpins in thrombosis, hemostasis and fibrinolysis. Journal of Thrombosis and Haemostasis, 5, 102–115.
Gomis-Rüth, F. X., Kress, L. F., Kellermann, J., Mayr, I., Lee, X., Huber, R., et al. (1994). Refined 2.0 A X-ray crystal structure of the snake venom zinc-endopeptidase adamalysin II. Primary and tertiary structure determination, refinement, molecular structure and comparison with astacin, collagenase and thermolysin. Journal of Molecular Biology, 239, 513–544.
Bello, C. A., Hermogenes, A. L., Magalhaes, A., Veiga, S. S., Gremski, L. H., Richardson, M., et al. (2006). Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom. Biochimie, 88, 189–200.
Acknowledgment
This study was supported by research funds from Chosun University (2011).
Conflict of Interest
None
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Choi, BS., Sapkota, K., Choi, JH. et al. Herinase: A Novel Bi-functional Fibrinolytic Protease from the Monkey Head Mushroom, Hericium erinaceum . Appl Biochem Biotechnol 170, 609–622 (2013). https://doi.org/10.1007/s12010-013-0206-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12010-013-0206-2