Hostname: page-component-cd9895bd7-hc48f Total loading time: 0 Render date: 2024-12-22T18:07:52.463Z Has data issue: false hasContentIssue false
  • English
  • Français

Functional expression of the Spodoptera exigua chitinase to examine the virtually screened inhibitor candidates

Published online by Cambridge University Press:  22 May 2019

L. Zhang ,
Z. Guan Open the ORCID record for Z. Guan [Opens in a new window] ,
Z. Pan ,
H. Ge ,
D. Zhou ,
J. Xu  and
W. Zhang
L. Zhang
Affiliation:
State Key Laboratory of Biocontrol and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Z. Guan
Affiliation:
State Key Laboratory of Biocontrol and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
Z. Pan
Affiliation:
State Key Laboratory of Biocontrol and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
H. Ge
Affiliation:
Medical College, Qingdao University, No. 38 Dengzhou Road, Qingdao 266021, China
D. Zhou
Affiliation:
Guangdong Provincial Key Laboratory of New Technology in Rice Breeding, Rice Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
J. Xu
Affiliation:
Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
W. Zhang*
Affiliation:
State Key Laboratory of Biocontrol and School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China
*
*Author for correspondence Phone: + 86 20 39332963 Fax: +86 20 39943515 E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Chitinase is responsible for insect chitin hydrolyzation, which is a key process in insect molting and pupation. However, little is known about the chitinase of Spodoptera exigua (SeChi). In this study, based on the SeChi gene (ADI24346) identified in our laboratory, we constructed the recombinant baculovirus P-Chi for the expression of recombinant SeChi (rSeChi) in Hi5 cells. The rSeChi was purified by chelate affinity chromatography, and the purified protein showed activity comparable with that of a commercial SgChi, suggesting that we harvested active SeChi for the first time. The purified protein was subsequently tested for enzymatic properties and revealed to exhibit its highest activity at pH 8 and 40 C. Using homology modeling and molecular docking techniques, the three-dimensional model of SeChi was constructed and screened for inhibitors. In two rounds of screening, twenty compounds were selected. With the purified rSeChi, we tested each of the twenty compounds for inhibitor activity against rSeChi, and seven compounds showed obvious activity. This study provided new information for the chitinase of beet armyworm and for chitinase inhibitor development.

Keywords

Spodoptera exiguachitinasebaculovirus expression systemhomology modelinginhibitor screening
Type
Research Paper
Information
Bulletin of Entomological Research , Volume 109 , Issue 6 , December 2019 , pp. 741 - 751
Check for updates
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Footnotes

†

The first two authors contributed equally to this work

References

Andersen, S.O. (2003) Biochemistry of Insect Cuticle. Annual Review of Entomology 24, 29–59.Google Scholar
Arakane, Y. & Muthukrishnan, S. (2010) Insect chitinase and chitinase-like proteins. Cellular and Molecular Life Sciences 67, 201–216.Google Scholar
Arakane, Y., Zhu, Q., Matsumiya, M., Muthukrishnan, S. & Kramer, K.J. (2003) Properties of catalytic, linker and chitin-binding domains of insect chitinase. Insect Biochemistry & Molecular Biology 33, 631–648.Google Scholar
Arora, N., Ahmad, T., Rajagopal, R. & Bhatnagar, R.K. (2003) A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1. Biochemical & Biophysical Research Communications 307, 620–625.Google Scholar
Bernard, A., Payton, M. & Radford, K.R. (2001) Protein expression in the baculovirus system. Current protocols in Neuroscience Chapter 4, Unit 4 19.Google Scholar
Carlini, C.R. & Grossi-De-Sá, M.F. (2002) Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon 40, 1515–1539.Google Scholar
Chen, L., Zhou, Y., Qu, M., Zhao, Y. & Yang, Q. (2014) Fully deacetylated chitooligosaccharides act as efficient glycoside hydrolase family 18 chitinase inhibitors. Journal of Biological Chemistry 289, 17932.Google Scholar
Chen, L., Liu, T., Duan, Y., Lu, X. & Yang, Q. (2017) Microbial secondary metabolite, phlegmacin B1, as a novel inhibitor of insect chitinolytic enzymes. Journal of Agricultural and Food Chemistry 65, 3851–3857.Google Scholar
Fan, Y., Zhang, Y., Yang, X., Pei, X., Guo, S. & Pei, Y. (2007) Expression of a Beauveria bassiana chitinase (Bbchit1) in Escherichia coli and Pichia pastoris. Protein Expression & Purification 56, 93–99.Google Scholar
Fan, X.J., Mi, Y.X., Ren, H., Zhang, C., Li, Y. & Xian, X.X. (2015) Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth lithocolletis ringoniella. Biochemistry 80, 242–250.Google Scholar
Fan, X.J., Yang, C., Zhang, C., Ren, H. & Zhang, J.D. (2018) Cloning, site-directed mutagenesis, and functional analysis of active residues in lymantria dispar chitinase. Applied Biochemistry and Biotechnology 184, 12–24.Google Scholar
Fitches, E., Wilkinson, H., Bell, H., Bown, D.P., Gatehouse, J.A. & Edwards, J.P. (2004) Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase. Insect Biochemistry and Molecular Biology 34, 1037–1050.Google Scholar
Fukamizo, T. (2000) Chitinolytic enzymes: catalysis, substrate binding, and their application. Current Protein and Peptide Science 1, 105–124.Google Scholar
Gu, Q., Xu, J. & Gu, L. (2010) Selecting diversified compounds to build a tangible library for biological and biochemical assays. Molecules 15, 5031–5044.Google Scholar
Hao, C.J., Chai, B.F., Wei, W., Yi, S. & Liang, A.H. (2005) Polyclonal antibody against Manduca sexta chitinase and detection of chitinase expressed in transgenic cotton. Biotechnology Letters 27, 97–102.Google Scholar
Henrissat, B. & Bairoch, A. (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal 293(Pt 3), 781.Google Scholar
Hirose, T., Maita, N., Gouda, H., Koseki, J., Yamamoto, T., Sugawara, A., Nakano, H., Hirono, S., Shiomi, K. & Watanabe, T. (2013) Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor. Proceedings of the National Academy of Sciences of the United States of America 110, 15892.Google Scholar
Hu, S.B., Liu, P., Ding, X.Z., Yan, L., Sun, Y.J., Zhang, Y.M., Li, W.P. & Xia, L.Q. (2009) Efficient constitutive expression of chitinase in the mother cell of Bacillus thuringiensis and its potential to enhance the toxicity of Cry1Ac protoxin. Applied Microbiology & Biotechnology 82, 1157.Google Scholar
Kim, J.S., Choi, J.Y., Roh, J.Y., Lee, H.Y., Jang, S.S. & Je, Y.H. (2007) Production of recombinant polyhedra containing Cry1Ac fusion protein in insect cell lines. Journal of Microbiology and Biotechnology 17, 739–744.Google Scholar
Kitts, P.A. and Possee, R.D. (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810–817.Google Scholar
Kramer, K.J. & Muthukrishnan, S. (1997) Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochemistry & Molecular Biology 27, 887.Google Scholar
Lehane, M.J. (1997) Peritrophic matrix structure and function. Annual Review of Entomology 42, 525.Google Scholar
Lertcanawanichakul, M., Wiwat, C., Bhumiratana, A. & Dean, D.H. (2004) Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. Aizawai toward Lymantria dispar larvae. Current Microbiology 48, 175–181.Google Scholar
Liang, T.W., Chen, Y.Y., Pan, P.S. & Wang, S.L. (2014) Purification of chitinase/chitosanase from Bacillus cereus and discovery of an enzyme inhibitor. International Journal of Biological Macromolecules 63, 8.Google Scholar
Liu, T., Chen, L., Zhou, Y., Jiang, X., Duan, Y. & Yang, Q. (2017) Structure, catalysis and inhibition of OfChi-h, the Lepidoptera-exclusive insect chitinase. Journal of Biological Chemistry 292, 2080–2088.Google Scholar
Luckow, V.A., Lee, S.C., Barry, G.F. & Olins, P.O. (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. Journal of Virology 67, 4566.Google Scholar
Lv, M. (2007) Study on chitinase inhibitors. Doctoral dissertation. Zhejiang University of Technology.Google Scholar
Maccari, G., Deodato, D., Fiorucci, D., Orofino, F., Truglio, G.I., Pasero, C., Martini, R., De, L.F., Docquier, J.D. & Botta, M. (2017) Design and synthesis of a novel inhibitor of T. Viride chitinase through an in silico target fishing protocol. Bioorganic & Medicinal Chemistry Letters 27, 3332–3336.Google Scholar
Merzendorfer, H. & Zimoch, L. (2003) Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. Journal of Experimental Biology 206, 4393–4412.Google Scholar
Müller, M. (1992) Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes. Experientia 48, 118–129.Google Scholar
Paek, A., Park, H.Y. & Jeong, S.E. (2012) Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, mamestra brassicae. Molecules & Cells 33, 439.Google Scholar
Reynolds, S.E. & Samuels, R.I. (1996) Physiology and biochemistry of insect moulting fluid. Advances in Insect Physiology 26, 157–232.Google Scholar
Rinaudo, M. (2007) Chitin and chitosan – properties and applications. Cheminform 38, 603–632.Google Scholar
Sakuda, S., Isogai, A., Matsumoto, S. & Suzuki, A. (1987) Search for microbial insect growth regulators. II. Allosamidin, a novel insect chitinase inhibitor. Journal of Antibiotics 40, 296.Google Scholar
Saville, G.P., Thomas, C.J., Possee, R.D. & King, L.A. (2002) Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif. Journal of General Virology 83, 685–694.Google Scholar
Schrempf, H. (2001) Recognition and degradation of chitin by streptomycetes. Antonie van Leeuwenhoek 79, 285–289.Google Scholar
Shen, Z. & Jacobs-Lorena, M. (1998) A type I peritrophic matrix protein from the malaria vector Anopheles gambiae binds to chitin. Cloning, expression, and characterization. Journal of Biological Chemistry 273, 17665.Google Scholar
Shinoda, T., Kobayashi, J., Matsui, M. & Chinzei, Y. (2001) Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virus-encoded chitinase gene. Insect Biochemistry & Molecular Biology 31, 521–532.Google Scholar
Shoichet, B.K. (2011) Virtual screening of chemical libraries. Nature 432, 862.Google Scholar
Somers, P.J., Yao, R.C., Doolin, L.E., Mcgowan, M.J., Fukuda, D.S. & Mynderse, J.S. (1987) Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516. Journal of Antibiotics 40, 1751–1756.Google Scholar
Wu, W., Lin, T., Pan, L., Yu, M., Li, Z., Pang, Y. & Yang, K. (2006) Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K Gene. Journal of Virology 80, 11475–11485.Google Scholar
Wu, Q., Liu, T. & Yang, Q. (2013) Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis. Insect Science 20, 147–157.Google Scholar
Xie, J., Zhou, Z., Zhang, X., Lan, X. & Hu, H. (2006) Preliminary screening of chitinase inhibitor producing strains. Chinese Journal of Antibiotics 31, 39–41.Google Scholar
Yan, J., Cheng, Q., Narashimhan, C.B., Li, S. & Aksoy, S. (2002) Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochemistry & Molecular Biology 32, 979–989.Google Scholar
Zhang, D., Chen, J., Yao, Q., Pan, Z., Chen, J. & Zhang, W. (2012) Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference. Archives of Insect Biochemistry & Physiology 79, 220–234.Google Scholar
Zheng, Y., Zheng, S., Cheng, X., Ladd, T., Lingohr, E.J., Krell, P.J., Arif, B.M., Retnakaran, A. & Feng, Q. (2002) A molt-associated chitinase cDNA from the spruce budworm, Choristoneura fumiferana. Insect Biochemistry and Molecular Biology 32, 1813–1823.Google Scholar
Zhu, Q., Arakane, Y., Banerjee, D., Beeman, R.W., Kramer, K.J. & Muthukrishnan, S. (2008) Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochemistry & Molecular Biology 38, 452–466.Google Scholar
Supplementary material: File

Zhang et al. supplementary material

Zhang et al. supplementary material Figure S1

Download Zhang et al. supplementary material(File)
File 328.1 KB