Thermostable serine protease inhibitor from Death cap (Amanita phalloides)

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Phichaya Khamai
Ketpaillin Chimong
Kritchai Pooncharoen
Widsanusan Chartarrayawadee

Abstract

Background: Protease inhibitor plays an important role in many biological processes in an organism, its selective binding toward protease potentially tuning down some specific biological processes such as enzymatic catalysis regulation, protein signaling as well as protein clearance in order to accomplish homeostasis. Several protein-based protease inhibitors have been isolated and identified, the majority are directed toward serine protease.


Objectives: This study aimed to find a potential protease inhibitor from the local Northern Thailand Death cap (Amanita phalloides) together with biochemical characterization of its general properties.


Materials and methods: Death cap extract collected from Phayao Province, Thailand was initially performed trypsin inhibitory activity assay using BSA and alzoalbumin as substrates. Detection of its inhibition activity was assessed by SDS-PAGE and spectrophotometry. Additionally, molecular size was observed by filtering the extract through 3 kDa molecular cut off membrane. Finally, hydrophobic property was verified by passing the filtrate through phenyl sepharose column chromatography.


Results: Death cap contained serine protease inhibitory activity. Molecular size of inhibitor was suspected to be less than 3 kDa. Hydrophobic property of this inhibitor was observed. Interestingly, its inhibitory property retained after heat inactivation at 100 ᵒC for 10 min.


Conclusion: A novel heat-tolerant inhibitor from the water extract of Death cap was characterized to be a small peptide with hydrophobic property, which could be used as a new peptide protease inhibitor targeting to serine protease that benefit for agricultural and medical field.

Article Details

How to Cite
Khamai, P., Chimong, K., Pooncharoen, K., & Chartarrayawadee, W. (2019). Thermostable serine protease inhibitor from Death cap (Amanita phalloides). Journal of Associated Medical Sciences, 52(3), 158–162. Retrieved from https://he01.tci-thaijo.org/index.php/bulletinAMS/article/view/155211
Section
Research Articles

References

[1] Sabotič J, Kos J. Microbial and fungal protease inhibitors - Current and potential applications. Appl Microbiol Biotechnol 2012;93:1351–75.

[2] Odani S, Tominaga K, Kondou S, Hori H, Koide T, Hara S, et al. The inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes. Eur J Biochem 1999;262:915–23.

[3] Ferry N., Jouanin L., Ceci L. R., Mulligan E. A., Emami K., Gatehouse J. A. GAMR. Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus. Mol Ecol 2005,Jan14(1);337–49.

[4] Shamsi TN, Parveen R, Fatima S. Characterization, biomedical and agricultural applications of protease inhibitors: A review. Int J Biol Macromol 2016;91:1120–33.

[5] Habib, H. and Fazili KM. Plant Protease Inhibitors: A Defense Strategy in Plants. Biotechnol Mol Biol Rev 2007;2:68–85.

[6] Beverly Waxler SFR. Aprotinin: A Serine Protease Inhibitor with Therapeutic Actions: Its Interaction with ACE Inhibitors. Curr Pharm Des 2003;9:777–87.

[7] Cleynen I, Jüni P, Bekkering GE, Nüesch E, Mendes CT, Schmied S, et al. Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: A systematic review. PLoS One 2011;6.

[8] Avanzo P, Sabotič J, Anžlovar S, Popovič T, Leonardi A, Pain RH, et al. Trypsin-specific inhibitors from the basidiomycete Clitocybe nebularis with regulatory and defensive functions. Microbiology 2009;155:3971–81.
[9] Li P, Deng W, Li T. The molecular diversity of toxin gene families in lethal Amanita mushrooms. Toxicon 2014;83:59–68.

[10] Tang S, Zhou Q, He Z, Luo T, Zhang P, Cai Q, et al. Cyclopeptide toxins of lethal amanitas: Compositions, distribution and phylogenetic implication. Toxicon 2016;120:78–88.

[11] Labia DMR. “alpha-Amanitin: a possible suicide substrate-like toxin involving the sulphoxide moiety of the bridged cyclopeptide”. Drug Metab Drug Interact 1988;6:265–274.

[12] Randall RJ, Lewis A. Protein measurement with the Folin Phenol Reagent. The Journal of Biological chemistry 1951;193:265–75.

[13] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5.

[14] Tomarelli RM, Charney J HM. The use of azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity. J Lab Clin Med. J Lab Clin Med 1949;34:428–433.

[15] Santi L, Maggioli C, Mastroroberto M, Tufoni M, Napoli L, Caraceni P. Acute Liver Failure Caused by Amanita phalloides Poisoning. Int J Hepatol 2012;2012:1–6.

[16] Stasyk T, Lutsik-Kordovsky M, Wernstedt C, Antonyuk V, Klyuchivska O, Souchelnytskyi S, et al. A new highly toxic protein isolated from the death cap Amanita phalloides is an l-amino acid oxidase. FEBS J 2010;277:1260–9.

[17] Broussard, C.N. Aggarwal, A., Lacey, S.R., Gramlich, T., Henderson, J.M. and Younossi ZM. Mushroom poisoning—from diarrhea to liver transplantation. Am J Gastroenterol 2001;96:3195–8.

[18] DohmaeN.TakioK.TsumurayaY.HashimotoY. The Complete Amino Acid Sequences of Two Serine Proteinase Inhibitors from the Fruiting Bodies of a Basidiomycete, Pleurotus ostreatusNo Title. Arch Biochem Biophys 1995;316:498–506.

[19] Ali PPM, Sapna K, Mol KRR, Bhat SG, Chandrasekaran M, Elyas KK. Trypsin inhibitor from edible mushroom Pleurotus floridanus active against proteases of microbial origin. Appl Biochem Biotechnol 2014;173:167–78.