한국응용곤충학회지 (Korean journal of applied entomology)

  • 제53권4호
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  • Pages.331-338
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  • 2014
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  • 1225-0171(pISSN)
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  • 2287-545X(eISSN)
한국응용곤충학회 (Korean Society of Applied Entomology)
권호 표지
DOI QR코드

DOI QR Code

바이러스 유래 시스타틴 재조합 단백질의 곤충 면역 및 발육 억제효과

Inhibitory Effects of a Recombinant Viral Cystatin Protein on Insect Immune and Development

  • 김영태 (안동대학교 생명자원과학과) ;
  • 엄성현 (안동대학교 생명자원과학과) ;
  • 박지영 (안동대학교 생명자원과학과) ;
  • 김용균 (안동대학교 생명자원과학과)
  • Kim, Yeongtae (Department of Bioresource Sciences, Andong National University) ;
  • Eom, Seonghyun (Department of Bioresource Sciences, Andong National University) ;
  • Park, Jiyeong (Department of Bioresource Sciences, Andong National University) ;
  • Kim, Yonggyun (Department of Bioresource Sciences, Andong National University)
  • 투고 : 2014.07.18
  • 심사 : 2014.09.18
  • 발행 : 2014.12.01
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초록

시스타틴(cystatin: CST)은 C1A류 시스테인 단백질분해효소에 대한 경쟁적 가역억제자로서 동식물류에서 파파인과 같은 캐셉신을 억제대상으로 작용하게 된다. 바이러스 유래 CST (CpBV-CST1)이 폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus)에서 동정되었다. 기존 연구는 이 유전자의 과발현이 배추좀나방(Plutella xylostella) 유충의 면역 및 발육을 교란한다는 것을 보여 주었다. 본 연구는 이 유전자의 단백질 기능을 분석하기 위해 세균발현시스템을 이용하여 재조합단백질(rCpBV-CST1)을 형성하여 단백질분해효소에 대한 활성억제효과를 결정하고, 곤충의 면역과 발육에 대한 생리적 억제효과를 분석했다. 이 유전자 번역부위는 138 개 아미노산으로 약 15 kDa 크기의 단백질로 추정되었다. CpBV-CST1이 먼저 pGEX 발현벡터에 재조합되고, BL21 STAR (DE3) competent cells에 형질전환된 후 0.5 mM IPTG로 4 시간동안 과발현되었다. 분리된 재조합단백질은 파파인에 대한 뚜렷한 억제효과를 나타냈다. 이 재조합단백질은 파밤나방(Spodoptera exigua)에 대해서 혈구소낭형성의 세포성 면역반응을 억제하고, 경구로 처리할 때 배추좀나방의 유충발육을 처리 농도에 비례하여 제한시켰다. 이상의 결과는 CpBV-CST1이 해충 밀도 억제에 응용될 수 있음을 제시하고 있다.

Cystatins (CSTs) are reversible and competitive inhibitors of C1A cysteine proteases, corresponding to papain-like cathepsins in plants and animals. A viral CST (CpBV-CST1) was identified from a polydnavirus, Cotesia plutellae bracovirus (CpBV). Our previous study indicated that a transient expression of CpBV-CST1 interfered with immune response and development of Plutella xylostella larvae. To directly demonstrate the protein function, this study produced a recombinant CpBV-CST1 protein (rCpBV-CST1) using bacterial expression system to determine its inhibitory activity against cysteine protease and to assess its physiological alteration in insect immune and development. The open reading frame of CpBV-CST1 encodes a polypeptide of 138 amino acids (${\approx}15kDa$). rCpBV-cystatin protein in BL21 STAR (DE3) competent cells containing a recombinant pGEX4T-3:CpBV-CST1 was over-expressed by 0.5 mM IPTG for 4 h. In biological activity assay, the purified rCpBV-CST1 showed a significant inhibition against papain activity. It inhibited a cellular immune response of hemocyte nodule formation in the beet armyworm, Spodoptera exigua. Moreover, its oral administration retarded larval development of the diamondback moth, Plutella xylostella in a dose-dependent manner. These results suggest that CpBV-CST1 may be applied to control insect pest populations.

키워드

참고문헌

  1. Abrahamson, M., Alvarez-Fernandez, M., Nathanson, C.M., 2003. Cystatins. Biochem. Soc. Symp. 70, 179-199.
  2. Agarwala, K.L., Kawabata, S., Hirata, M., Miyagi, M., Tsunasawa, S., Iwanaga, S., 1996. A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning, and tissue localization. J. Biochem. 119, 85-94. https://doi.org/10.1093/oxfordjournals.jbchem.a021220
  3. Attardo, G.M., Strickler-Dinglasan, P., Perkin, S.A.H., Caler, E., Bonaldo, M.F., Soareo, M.B., El-Sayeed, N., Aksoy, S., 2006. Analysis of fat body transcriptome from the adult tsetse fly, Glossina morsitans. Insect Mol. Biol. 15, 411-424. https://doi.org/10.1111/j.1365-2583.2006.00649.x
  4. Brix, K., Dunkhorst, A., Mayer, K., Jordans, S., 2008. Cysteine cathepsins: cellular roadmap to different functions. Biochimie 90, 194-207. https://doi.org/10.1016/j.biochi.2007.07.024
  5. Chen, Y., Gao, F., Ye, X., Wei, S., Shi, M., Zheng, H., Chen, X., 2011. Deep sequencing of Cotesia vestalis bracovirus reveals the complexity of a polydnavirus genome. Virology 414, 42-50. https://doi.org/10.1016/j.virol.2011.03.009
  6. Cho, W.-L., Tsao, S.-M Hays, A.R., Walter, R., Chen, J.-S., Snigirevskaya, E.S., Raikhel, A.S., 1999. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J. Biol. Chem. 274, 13311-13321. https://doi.org/10.1074/jbc.274.19.13311
  7. De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide analysis of the Drosophila immune response by using oligo nucleotide microarray. Proc. Natl. Acad. Sci. USA 98, 12590-12595. https://doi.org/10.1073/pnas.221458698
  8. Desjardins, C., Gundersen-Rindal, D., Hostetler, J., Tallon, L.J., Fadrosh, D.W., Fuester, R.W., Pedroni, M.J., Haas, B.J., Schatz, M.C., Jones, K.M., Crabtree, J., Forberger, H., Nene, V., 2008. Comparative genomics of mutualistic viruses of Glyptapanteles parasitic wasps. Genome Biol. 9, R138. https://doi.org/10.1186/gb-2008-9-9-r138
  9. Dickinson, D.P., 2002. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease. Crit. Rev. Oral Biol. Med. 13, 238-275. https://doi.org/10.1177/154411130201300304
  10. Dubin, G., 2005. Proteinaceous cysteine protease inhibitors. Cell Mol. Life Sci. 62, 653-669. https://doi.org/10.1007/s00018-004-4445-9
  11. Espagne, E., Douris, V., Lalmanach, G., Provost, B., Cattolico, L., Lesobre, J., Kurata, S., Iatrou, K., Drezen, J.M., Huguet, E., 2005. A virus essential for insect host-parasite interactions encodes cystatins. J. Virol. 79, 9765-9776. https://doi.org/10.1128/JVI.79.15.9765-9776.2005
  12. Goh, H.G., Lee, S.G., Lee, B.P., Chois, G.M., Kim, J.H., 1990. Simple mass-rearing of beet armyworm, Spodoptera exigua. Kor. J. Appl. Entomol. 29, 180-183.
  13. Haunerland, N.H., Shirk, P.D., 1995. Regional and functional differentiation in the insect fat body. Annu. Rev. Entomol. 40, 121-145. https://doi.org/10.1146/annurev.en.40.010195.001005
  14. Hegedus, D., O'Grady, M., Chamankhah, M., Baldwin, D., Gleddie, S., Braun, L., Erlandson, M., 2002. Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifers root maggots (Delia radicum). Insect Biochem. Mol. Biol. 32, 1585-1596. https://doi.org/10.1016/S0965-1748(02)00099-1
  15. Homma, K., Kurata, S., Natori, S., 1994. Purification, characterization, and cDNA cloning of procathepsin L from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly) and its involvement in the differentiation of imaginal disc. J. Biol. Chem. 269, 15258-15264.
  16. Kim, Y., 2006. Polydnavirus and its novel application to insect pest control. Kor. J. Appl. Entomol. 45, 241-259.
  17. Kim, Y., Hepat, R., Kim, Y., 2013. A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus. J. Asia Pac. Entomol. 16, 449-455. https://doi.org/10.1016/j.aspen.2013.06.006
  18. Koo, Y.D., Ahn, J.E., Salzman, R.A., Moon, J., Chi, Y.H., Yun, D.J., Lee, S.Y., Koiwa, H., Zhu-salzman, K., 2008. Functional expression of an insect cathespin B-like counter-defence protein. Insect Mol. Biol. 17, 235-245. https://doi.org/10.1111/j.1365-2583.2008.00799.x
  19. Kurata, S., Saito, H. Natori, S., 1992. The 29-kDa hemocyte proteinase dissociates fat body at metamorphosis of Sarcophaga. Dev. Biol. 153, 115-121. https://doi.org/10.1016/0012-1606(92)90096-Y
  20. Lavine, M.D., Strand, M.R., 2002. Insect hemocytes and their role in cellular immune responses. Insect Biochem. Mol. Biol. 32, 1295-1309. https://doi.org/10.1016/S0965-1748(02)00092-9
  21. Lecaille, F., Kaleta, J., Bromme, D., 2002. Human and parasitic papain- like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem. Rev. 102, 4459-4488. https://doi.org/10.1021/cr0101656
  22. Levy, F., Rabel, D., Charlet, M., Bulet, P., Hoffmann, J.A., Ehret-Sabatier, L., 2004. Peptidomic and proteomic analysis of the systemic immune response of Drosophila. Biochimie 86, 607-616. https://doi.org/10.1016/j.biochi.2004.07.007
  23. Li, H., Tang, H., Sivakumar, S., Philip, J., Harrison, R.L., Gatehouse, J.A., Bonning, B., 2008. Insecticidal activity of a basement membrane-degrading protease against Heliothis virescens (Fabricius) and Acyrthosiphon pisum (Harris). J. Insect Physiol. 54, 777-789. https://doi.org/10.1016/j.jinsphys.2008.02.008
  24. Liu, J., Shi, G.P., Zhang, W.Q., Zhang, G.R., Xy, W.H., 2006. Cathepsin L function in insect moulting: moleculoar cloning and functional analysis in cotton bollworm, Helicoverpa armigera. Insect Mol. Biol. 15, 823-834. https://doi.org/10.1111/j.1365-2583.2006.00686.x
  25. Nusawardani, T., Ruberson, J.R., Obrycki, J.J., Bonning, B., 2005. Effects of a protease-expressing recombinant baculovirus insecticide on the parasitoid Cotesia marginiventris (Cresson). Biol. Control 35, 46-54. https://doi.org/10.1016/j.biocontrol.2005.06.006
  26. Olsson, S.L., Ek, B., Bjork, I., 1999. The affinity and kinetics of inhibition of cyteine proteinases by intact recombinant bovine cystatin C. Biochim. Biophys. Acta 1432, 73-81. https://doi.org/10.1016/S0167-4838(99)00090-4
  27. Ratcliffe, N.A., Rowley, A.F., 1979. Role of hemocytes in defence against biological agents, in: Gupta, A.P. (Ed.) Insect hemocytes: development, forms, functions and techniques. Cambridge University Press, Cambridge, UK, pp. 331-414.
  28. Rawlings, N.D., Barrett, A.J., 1990. Evolution of proteins of the cystatin superfamily. J. Mol. Evol. 30, 60-71. https://doi.org/10.1007/BF02102453
  29. Rawlings, N.D., Tolle, D.P., Barrett, A.J., 2004. Evolutionary families of peptidase inhibitors. Biochem. J. 378, 705-716. https://doi.org/10.1042/BJ20031825
  30. Saito, H., Kurata, S., Natori, S., 1992. Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances. Eur. J. Biochem. 209, 939-944. https://doi.org/10.1111/j.1432-1033.1992.tb17366.x
  31. SAS Institute, Inc., 1989. SAS/STAT User's Guide, Release 6.03 ed. SAS Institute, Cary, NC.
  32. Seo, S.Y., Jeon, M.Y., Chun, W.S., Lee, S.H., Seo, J.A., Yi, Y.G., Hong, Y.P., Kim, Y., 2011. Structure-activity analysis of benzylideneacetone for effective control of plant pests. Kor. J. Appl. Entomol. 50, 107-113. https://doi.org/10.5656/KSAE.2011.04.0.15
  33. Serbielle, C., Moreau, S., Veillard, F., Voldoire, E., Bézier, A., Mannucci, M.A., Volkoff, A.N., Drezen, J.M., Lalmanach, G., Huguet, E., 2009. Identification of parasite-responsive cysteine proteases in Manduca sexta. Biol. Chem. 390, 493-502.
  34. Shindo, T., Van Der Hoorn, R.A., 2008. Papain-like cysteine protease: key players at molecular battlefields employed by both plants and their invaders. Mol. Plant Pathol. 9, 119-125.
  35. Song, K.H., Jung, M.K., Eum, J.H., Hwang, I.C., Han, S.S., 2008. Proteomic analysis of parasitized Plutella xylostella larvae plasma. J. Insect Physiol. 54, 1271-1280. https://doi.org/10.1016/j.jinsphys.2008.06.010
  36. Tang, H., Li, H., Meng Lei, S., Harrison, R.L., Bonning, B.C., 2007. Tissue specificity of a baculovirus-expressed, basement smembrane-degrading protease in larvae of Heliothis virescens. Tissue Cell 39, 431-443. https://doi.org/10.1016/j.tice.2007.08.003
  37. Turk, B., Turk, V., Turk, D., 1997. Structural and functional aspect of papain-like cysteine proteinases and their protein inhibitors. Biol. Chem. Hopp. Seyler. 378, 141-150.
  38. Turk, V., Bode, W., 1991. The cystatins: protein inhibitors of cysteine proteinases. Fed. Eur. Biol. Soc. Lett. 285, 213-234. https://doi.org/10.1016/0014-5793(91)80804-C
  39. Uchida, K., Ohmori, D., Ueno, T., Nishizuka, M., Eshita, Y., Fukunaga, A., Kominami, E., 2001. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens. Dev. Biol. 237, 68-78. https://doi.org/10.1006/dbio.2001.0357
  40. Yamamoto, Y., Watabe, S., Kageyama, T., Takahashi, S., 1999. Purification and characterization of Bombyx cysteine proteinase specific inhibitors from the hemolymph of Bombyx mori. Arch. Insect Biochem. Physiol. 41, 119-129.
  41. Zhang,Y., Lu, Y.X., Liu, J., Feng, Q.L., Xu, W.H., 2013. A regulatory pathway, ecdysone-transcription factor Relish-cathepsin L, is involved in insect fat body dissociation. PLOS Genetics 9, e1003273. https://doi.org/10.1371/journal.pgen.1003273
  42. Zhou, J., Ueda, M., Umemiya, R., Battsetseg, B., Boldbaatar, D., Xuan, X., Fujisaka, K., 2006. A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. Insect Biochem. Mol. Biol. 36, 527-535. https://doi.org/10.1016/j.ibmb.2006.03.003

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  2. Construction of a Transgenic Tobacco Expressing a Polydnaviral Cystatin vol.54, pp.1, 2015, https://doi.org/10.5656/KSAE.2015.01.1.055