Abstract
We describe the characterization of IETI, the first trypsin inhibitor purified from Inga edulis, a tree widely distributed in Brazil. Two-step chromatography was used to purify IETI, a protein composed of a single peptide chain of 19,685.10 Da. Amino-terminal sequencing revealed that IETI shows homology with the Kunitz family, as substantiated by its physical–chemical features, such as its thermal (up to 70 °C) and wide-range pH stability (from 2 to 10), and the value of its dissociation constant (6.2 nM). IETI contains a single reactive site for trypsin, maintained by a disulfide bridge; in the presence of DTT, its inhibitory activity was reduced in a time- and concentration-dependent manner. IETI presented activity against Candida ssp., including C. buinensis and C. tropicalis. IETI inhibitory activity triggered yeast membrane permeability, affecting cell viability, thus providing support for the use of IETI in further studies for the control of fungal infections.
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References
Abad-Zapatero C et al (1996) Structure of a secreted aspartic protease from C. albicans complexed with a potent inhibitor: implications for the design of antifungal agents. Protein Sci 5:640–652
Azarkan M, Martinez-Rodriguez S, Buts L, Baeyens-Volant D, Garcia-Pino A (2011) The plasticity of the β-trefoil fold constitutes an evolutionary platform for protease inhibition. J Biol Chem 286:43726–43734
Bahar AA, Ren D (2013) Antimicrobial peptides. Pharmaceuticals 6:1543–1575
Bhattacharyya A, Babu CR (2009) Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features. Phytochemistry 70:703–712. https://doi.org/10.1016/j.phytochem.2009.04.001
Bijina B et al (2011) Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative. Saudi J Biol Scie 18:273–281. https://doi.org/10.1016/j.sjbs.2011.04.002
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Broekaert WF, Terras FR, Cammue BP, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–59
Carmona-Gutierrez D, Eisenberg T, Büttner S, Meisinger C, Kroemer G, Madeo F (2010) Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 17:763–773
Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124:803–814. https://doi.org/10.1016/j.cell.2006.02.008
da Silva Bezerra C et al (2016) Exploiting the biological roles of the trypsin inhibitor from Inga vera seeds: a multifunctional Kunitz inhibitor. Process Biochem 51:792–803
Di Ciero L et al (1998) The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds. J Protein Chem 17:827–834
Heckman DS, Geiser DM, Eidell BR, Stauffer RL, Kardos NL, Hedges SB (2001) Molecular evidence for the early colonization of land by fungi and plants. Science 293:1129–1133
Huang Y, Huang J, Chen Y (2010) Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 1:143–152
Kim J-Y, Park S-C, Kim M-H, Lim H-T, Park Y, Hahm K-S (2005) Antimicrobial activity studies on a trypsin–chymotrypsin protease inhibitor obtained from potato. Biochem Biophys Res Commun 330:921–927. https://doi.org/10.1016/j.bbrc.2005.03.057
Kobayashi CCBA, Fernandes OdFL, Miranda KC, de Sousa ED, Silva MdRR (2004) Candiduria in hospital patients: a study prospective. Mycopathologia 158:49–52
Kraszewska J, Beckett MC, James TC, Bond U (2016) Comparative analysis of the antimicrobial activities of plant defensin-like and ultrashort peptides against food-spoiling bacteria. Appl Environ Microbiol 82:4288–4298
Kumar A, Zarychanski R, Pisipati A, Kumar A, Kethireddy S, Bow EJ (2018) Fungicidal versus fungistatic therapy of invasive Candida infection in non-neutropenic adults: a meta-analysis. Mycology. https://doi.org/10.1080/21501203.2017.1421592
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Li Z et al (2014) Reconstructed mung bean trypsin inhibitor targeting cell surface GRP78 induces apoptosis and inhibits tumor growth in colorectal cancer. Int J Biochem Cell Biol 47:68–75. https://doi.org/10.1016/j.biocel.2013.11.022
Lingaraju MH, Gowda LR (2008) A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge. Biochim Biophys Acta (BBA) Proteins Proteom 1784:850–855. https://doi.org/10.1016/j.bbapap.2008.02.013
Macedo MLR, Garcia VA, Freire MGM, Richardson M (2007) Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd. Phytochemistry 68:1104–1111. https://doi.org/10.1016/j.phytochem.2007.01.024
Madeo F et al (1999) Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145:757–767
Melo FR et al (2002) Inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking. Proteins Struct Funct Bioinform 48:311–319
Migliolo L, de Oliveira AS, Santos EA, Franco OL, de Sales MP (2010) Structural and mechanistic insights into a novel non-competitive Kunitz trypsin inhibitor from Adenanthera pavonina L. seeds with double activity toward serine- and cysteine-proteinases. J Mol Graph Model 29:148–156. https://doi.org/10.1016/j.jmgm.2010.05.006
Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217
Oard S, Karki B (2006) Mechanism of β-purothionin antimicrobial peptide inhibition by metal ions: molecular dynamics simulation study. Biophys Chem 121:30–43
Oliva MLV, Silva MCC, Sallai RC, Brito MV, Sampaio MU (2010) A novel subclassification for Kunitz proteinase inhibitors from leguminous seeds. Biochimie 92:1667–1673. https://doi.org/10.1016/j.biochi.2010.03.021
Oliveira CFR et al (2012) Purification and biochemical properties of a Kunitz-type trypsin inhibitor from Entada acaciifolia (Benth.) seeds. Process Biochem 47:929–935. https://doi.org/10.1016/j.procbio.2012.02.022
Onesti S, Brick P, Blow DM (1991) Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds. J Mol Biol 217:153–176. https://doi.org/10.1016/0022-2836(91)90618-g
Page KR, Chaisson R, Sande M (2008) CHAPTER 34—cryptococcosis and other fungal infections (histoplasmosis and coccidioidomycosis) in HIV-infected patients. In: Global HIV/AIDS medicine. W.B. Saunders, Edinburgh, pp 375–391
Ramalho SR et al (2018) A novel peptidase Kunitz inhibitor from Platypodium elegans seeds is active against Spodoptera frugiperda larvae. J Agric Food Chem 66:1349-1358
Ramos VdS et al (2012) Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens. Comp Biochem Physiol C Toxicol Pharmacol 156:148–158. https://doi.org/10.1016/j.cbpc.2012.07.007
Rawlings ND, Barret JA, Bateman A (2012) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 40:D343–D350
Richardson M, Campos FAP, Xavier-Filho J, Macedo MLR, Maia GMC, Yarwood A (1986) The amino acid sequence and reactive (inhibitory) site of the major trypsin isoinhibitor (DE5) isolated from seeds of the Brazilian Carolina tree (Adenanthera pavonina L.). Biochim Biophys Acta (BBA) Protein Struct Mol Enzymol 872:134–140. https://doi.org/10.1016/0167-4838(86)90156-1
Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73–85
Sanglard D, Kuchler K, Ischer F, Pagani J, Monod M, Bille J (1995) Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39:2378–2386
Srinivasan A, Giri AP, Harsulkar AM, Gatehouse JA, Gupta VS (2005) A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts anti-metabolic effect on podborer (Helicoverpa armigera) larvae. Plant Mol Biol 57:359–374
Sumikawa JT, Nakahata AM, Fritz H, Mentele R, Sampaio MU, Oliva MLV (2006) A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge. Planta Med 72:393–397
Tang H et al (2014) An improved genome release (version Mt4. 0) for the model legume Medicago truncatula. BMC Genom 15:312
Taveira GB, Carvalho AO, Rodrigues R, Trindade FG, Da Cunha M, Gomes VM (2016) Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species. BMC Microbiol 16:12
Terada S, Fujimura S, Kino S, Kimoto E (1994) Purification and characterization of three proteinase inhibitors from Canavalia lineata seeds. Biosci Biotechnol Biochem 58:371–375
Thery T, Arendt EK (2018) Antifungal activity of synthetic cowpea defensin Cp-thionin II and its application in dough. Food Microbiol 73:111–121
Thevissen K, Terras FR, Broekaert WF (1999a) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458
Thevissen K, Terras FRG, Broekaert WF (1999b) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl Environ Microbiol 65:5451–5458
Vriens K, Cammue B, Thevissen K (2014) Antifungal plant defensins: mechanisms of action and production. Molecules 19:12280–12303
Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD (2016) Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol 7:2173. https://doi.org/10.3389/fmicb.2016.02173
Wu Z et al (2003) Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3. Proc Natl Acad Sci 100:8880–8885
Zaragoza O, Rodrigues ML, De Jesus M, Frases S, Dadachova E, Casadevall A (2009) The capsule of the fungal pathogen Cryptococcus neoformans. Adv Appl Microbiol 68:133–216. https://doi.org/10.1016/S0065-2164(09)01204-0
Zottich U et al (2013) An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi. Biochim Biophys Acta (BBA) Gen Subj 1830:3509–3516
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This work was supported by CNPq (407127/2013-5), CAPES and FINEP.
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Communicated by Yusuf Akhter.
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Dib, H.X., de Oliveira, D.G.L., de Oliveira, C.F.R. et al. Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp.. Arch Microbiol 201, 223–233 (2019). https://doi.org/10.1007/s00203-018-1598-8
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DOI: https://doi.org/10.1007/s00203-018-1598-8