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Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia

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Abstract

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel–nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo’s fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC50 values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC50 value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

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References

  • Barbieri L, Valbonesi P, Bonora E, Gorini P, Bolognesi A, Stirpe F (1997) Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res 25:518–522. doi:10.1093/nar/25.3.518

    Article  CAS  Google Scholar 

  • Barbieri L, Polito L, Bolognesi A, Ciani M, Pelosi E, Farini V, Jha AK, Sharma N, Vivanco JM, Chambery A, Parente A, Stirpe F (2006) Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata. Biochim Biophys Acta 1760:783–792. doi:10.1016/j.bbagen.2006.01.002

    Article  CAS  Google Scholar 

  • Basch E, Gabardi S, Ulbricht C (2003) Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm 60(4):356–359

    Google Scholar 

  • Begam M, Kumar S, Roy S, Campanella JJ, Kapoor HC (2006) Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in Escherichia coli. Phytochemistry 67(22):2441–2449. doi:10.1016/j.phytochem.2006.08.015

    Article  CAS  Google Scholar 

  • Bhaskar A, Deb U, Kumar O, Rao PVL (2008) Abrin induced oxidative stress mediated DNA damage in human leukemic cells and its reversal by N-acetylcysteine. Toxicol In Vitro 22(8):1902–1908. doi:10.1016/j.tiv.2008. 09.013

    Article  CAS  Google Scholar 

  • Bian X, Shen F, Chen Y, Wang B, Deng M, Meng Y (2010) PEGylation of alpha-momorcharin: synthesis and characterization of novel anti-tumor conjugates with therapeutic potential. Biotechnol Lett 32(7):883–890. doi:10.1007/s10529-010-0242-8

    Article  CAS  Google Scholar 

  • Choudhary NL, Yadav OP, Lodha ML (2008) Ribonuclease, deoxyribonuclease, and antiviral activity of Escherichia coli expressed Bougainvillea xbuttiana antiviral protein 1. Biochemistry (Moscow) 73(3):273–277. doi:10.1134/S000629790803005X

    Article  CAS  Google Scholar 

  • Corrado G, Scarpetta M, Alioto D, Maro AD, Polito L, Parente A, Rao R (2008) Inducible antiviral activity and rapid production of the ribosome-inactivating protein I from Phytolacca heterotepala in tobacco. Plant Sci 174:467–474. doi:10.1016/j.plantsci.2008.01.009

    Article  CAS  Google Scholar 

  • De-la-Peña C, Badri DV, Vivanco JM (2008) Novel role for pectin methylesterase in Arabidopsis: a new function showing ribosome-inactivating protein (RIP) activity. Biochim Biophys Acta, Gen Subj 1780(5):773–783. doi:10.1016/j.bbagen.2007.12.013

    Article  Google Scholar 

  • Di MA, Chambery A, Daniele A, Casoria P, Parente A (2007) Isolation and characterization of heterotepalins, type 1 ribosome-inactivating proteins from Phytolacca heterotepala leaves. Phytochemistry 68(6):767–776. doi:10.1016/j.phytochem.2006.12.002

    Article  Google Scholar 

  • Endo Y, Tsurugi K, Franz H (1988) The site of action of the A-chain of mistletoe lectin I on eukaryotic ribosomes: the RNA N-glycosidase activity of the protein. FEBS Lett 231:378–380. doi:10.1016/0014-5793(88)80853-6

    Article  CAS  Google Scholar 

  • Fermani S, Tosi G, Farini V, Polito L, Falini G, Ripamonti A, Barbieri L, Chambery A, Bolognesi A (2009) Structure/function studies on two type 1 ribosome inactivating proteins: bouganin and lychnin. J Struct Biol 168(2):278–287. doi:10.1016/j.jsb.2009.07.010

    Article  CAS  Google Scholar 

  • Girbés T, Ferreras JM, Arias FJ, Stirpe F (2004) Description, distribution, activity and phylogenetic relationship of ribosome-inactivating proteins in plants, fungi and bacteria. Mini Rev Med Chem 4:461–476. doi:10.2174/1389557043403891

    Google Scholar 

  • Ho WKK, Liu SC, Shaw PC, Yeung HW, Ng TB, Chan WY (1991) Cloning of the cDNA of α-momorcharin: a ribosome-inactivating protein. Biochim Biophys Acta 1088:311–314. doi:10.1016/0167-4781(91)90070-3

    Article  CAS  Google Scholar 

  • Hudak KA, Bauman JD, Tumer NE (2002) Pokeweed antiviral protein binds to the cap structure of eukaryotic mRNA and depurinates the mRNA downstream of the cap. RNA 8:1148–1159. doi:10.1017.S1355838202026638

    Article  CAS  Google Scholar 

  • Kovaleva V, Krynytskyy H, Gout I, Gout R (2011) Recombinant expression, affinity purification and functional characterization of Scots pine defensin 1. Appl Microbiol Biotechnol 89:1093–1101. doi:10.1007/s00253-010-2935-2

    Article  CAS  Google Scholar 

  • Morais JKS, Gomes VM, Oliveira JTA, Santos IS, Da Cunha M, Oliveira HD, Oliveira HP, Sousa DOB, Vasconcelos IM (2010) Soybean toxin (SBTX), a protein from soybeans that inhibits the life cycle of plant and human pathogenic fungi. J Agric Food Chem 58:10356–10363. doi:10.1021/jf101688k

    Article  CAS  Google Scholar 

  • Nerurkar P, Ray RB (2010) Bitter melon: antagonist to cancer. Pharm Res 27(6):1049–1053. doi:10.1007/s11095-010-0057-2

    Article  CAS  Google Scholar 

  • Nielsen T, Thrane C, Christophersen C, Anthoni U, Sørensen J (2000) Structure, production characteristics and fungal antagonism of tensin—a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578. J Appl Microbiol 89(6):992–1001. doi:10.1046/j.1365-2672.2000.01201.x

    Article  CAS  Google Scholar 

  • Parente A, Berisio R, Chambery A, Di MA (2010) Type 1 ribosome-inactivating proteins from the ombu tree (Phytolacca dioica L.). In: Lord JM, Hartley MR (eds) Toxic plant proteins. Springer, Heidelberg, pp 79–106

    Chapter  Google Scholar 

  • Park SC, Kim JY, Lee JK, Hwang I, Cheong H, Nah JW, Hahm KS, Park Y (2009) Antifungal mechanism of a novel antifungal protein from pumpkin rinds against various fungal pathogens. J Agric Food Chem 57(19):9299–9304. doi:10.1021/jf902005g

    Article  CAS  Google Scholar 

  • Peumans W, Van Damme E (2010) Evolution of plant ribosome-inactivating proteins. In: Lord JM, Hartley MR (eds) Toxic plant proteins. Springer, Heidelberg, pp 1–26

    Chapter  Google Scholar 

  • Reinbothe S, Reinbothe C, Lehmann J, Becker W, Apel K, Parthier B (1994) JIP60, a methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions. Proc Natl Acad Sci 91:7012–7016

    Article  CAS  Google Scholar 

  • Selitrennikoff CP (2001) Antifungal proteins. Appl Environ Microbiol 67(7):2883–2894. doi:10.1128/AEM.67.7.2883-2894.2001

    Article  CAS  Google Scholar 

  • Sharma N, Park SW, Vepachedu R, Barbieri L, Clani M, Stirpe F, Savary BJ, Vivanco JM (2004) Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity. Plant Physiol 134:171–181. doi:10.1104/pp.103.030205

    Article  CAS  Google Scholar 

  • Stirpe F, Barbieri L, Batelli MG, Soria M, Lappi DA (1992) Ribosome inactivating proteins from plants: present status and future prospects. Nat Biotechnol 10:405–412. doi:10.1038/nbt0492-405

    Article  CAS  Google Scholar 

  • Virgilio M, Lombardi A, Caliandro R, Fabbrini MS (2010) Ribosome-inactivating proteins: from plant defense to tumor attack. Toxins 2(11):2699–2737. doi:10.3390/toxins2112699

    Article  Google Scholar 

  • Vivanco JM, Savary BJ, Flores HE (1999) Characterization of two novel type I ribosome-inactivating proteins from the storage roots of the Andean crop Mirabilis expansa. Plant Physiol 119(4):1447–1456. doi:10.1104/pp.119.4.1447

    Article  CAS  Google Scholar 

  • Walsh TA, Morgan AE, Hey TD (1991) Characterization and molecular cloning of a proenzyme form of a ribosome inactivating protein from maize-novel mechanism of proenzyme activation by proteolytic removal of a 2.8-kilodalton internal peptide segment. J Biol Chem 266:23422–23427

    CAS  Google Scholar 

  • Wong JH, Ng TB, Cheung RCF, Ye XJ, Wang HX, Lam SK, Lin P, Chan YS, Fang EF, Ngai PHK, Xia LX, Ye XY, Jiang Y, Liu F (2010) Proteins with antifungal properties and other medicinal applications from plants and mushrooms. Appl Microbiol Biotechnol 87:1–15. doi:10.1007/s00253-010-2690-4

    Article  Google Scholar 

  • Yang X, Xiao Y, Wang X, Pei Y (2007) Expression of a novel small antimicrobial protein from the seeds of motherwort (Leonurus japonicus) confers disease resistance in tobacco. Appl Environ Microbiol 73(3):939–946. doi:10.1128/AEM.02016-06

    Article  CAS  Google Scholar 

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Acknowledgements

Professor Huimin Yan, Jiaxin Liu, and Hong Deng (Wuhan Institute of Virology, Chinese Academy of Sciences, China) are thanked for their help in preparing the TEM images. We also thank Professor Zhixiong Xie and Doctor Candidate Weikun Tang of Wuhan University for their technical support during the experiments. This work was supported by research grants from the Scientific Research Program of Wuhan Municipality, Hubei Province, People’s Republic of China (200820422170) and the Fundamental Research Fund for the Central Universities (204-274388).

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Authors and Affiliations

  1. State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei Province, People’s Republic of China

    Shuzhen Wang, Yubo Zhang, Ying He, Junjie Yan, Zhihua Wu & Yi Ding

  2. Institute of Hydroecology, Ministry of Water Resources and Chinese Academy of Sciences, 578 Xiongchu Avenue, Wuhan, Hubei Province, People’s Republic of China

    Honggao Liu

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  1. Shuzhen Wang
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  2. Yubo Zhang
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  3. Honggao Liu
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Correspondence to Yi Ding.

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Wang, S., Zhang, Y., Liu, H. et al. Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia . Appl Microbiol Biotechnol 96, 939–950 (2012). https://doi.org/10.1007/s00253-012-3886-6

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