Alanine-substituted mutant on Gly373 and Asn375 of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera

doi:10.1016/S2095-3119(18)61933-1

摘要/Abstract

Abstract:

Cry1Ai-h-loop 2 is a mutant of Cry1Ai constructed by exchanging loop 2 from Cry1Ah protein and shows insecticidal activity against Helicoverpa armigera. The toxicity of Cry1Ai-h-loop 2, in contrast to the very low toxicity of Cry1Ai, is closely associated with the eleven residues in the loop 2 region. To characterize the key sites of loop 2 in Cry1Ai-h-loop 2, alanine-substituted mutants were generated. The toxicity of these mutants against H. armigera indicated that dual-mutant on Gly³⁷³and Asn³⁷⁵ caused a significant decrease in toxic activity. ELISA binding and competition binding assays demonstrated that the reduction of toxicity in the mutant of interest was correlated with decreased binding affinity.

Key words: Bacillus thuringiensis , Cry1Ai , Domain II-loop2 , Helicoverpa armigera , binding affinity

. [J]. Journal of Integrative Agriculture, 2019, 18(5): 1064-1071.

LIU Yu-xiao, ZHOU Zi-shan, LIANG Ge-mei, SONG Fu-ping, ZHANG Jie. Alanine-substituted mutant on Gly³⁷³ and Asn³⁷⁵ of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera[J]. Journal of Integrative Agriculture, 2019, 18(5): 1064-1071.

参考文献

Adegawa S, Nakama Y, Endo H, Shinkawa N, Kikuta S, Sato R. 2017. The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor. Biochimica et Biophysica Acta, 1865, 220–231.
Arenas I, Bravo A, Soberón M, Gómez I. 2010. Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin. Journal of Biological Chemistry, 285, 12497–12503.
Atsumi S, Inoue Y, Ishizaka T, Mizuno E, Yoshizawa Y, Kitami M, Sato R. 2008. Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin. The FEBS Journal, 275, 4913–4926.
Bravo A, Gill S S, Soberón M. 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49, 423–435.
Bravo A, Gómez I, Conde J, Muñoz-Garay C, Sánchez J, Miranda R, Zhuang M, Gill S S, Soberón M. 2004. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochimica et Biophysica Acta, 1667, 38–46.
Cantón P E, Zanicthe Reyes E Z, Ruiz de Escudero I, Bravo A, Soberón M. 2011. Binding of Bacillus thuringiensis subsp. israelensis Cry4Ba to Cyt1Aa has an important role in synergism. Peptides, 32, 595–600.
Crickmore N, Baum J, Bravo A, Lereclus D, Narva K, Sampson K, Schnepf E, Sun M, Zeigler D R. 2016. Bacillus thuringiensis toxin nomenclature. [2017-11-12]. http://www.btnomenclature.info/
Gómez I, Sánchez J, Muñoz-Garay C, Matus V, Gill S S, Soberón M, Bravo A. 2014. Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: Two distinct pre-pores are involved in toxicity. Biochemical Journal, 459, 383–396.
Howlader M T, Kagawa Y, Miyakawa A, Yamamoto A, Taniguchi T, Hayakawa T, Sakai H. 2010. Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa. Applied and Environmental Microbiology, 76, 860–865.
Jouzani G S, Valijanian E, Sharafi R. 2017. Bacillus thuringiensis: A successful insecticide with new environmental features and tidings. Applied Microbiology and Biotechnology, 101, 2691–2711.
Juntadech T, Kanintronkul Y, Kanchanawarin C, Katzenmeier G, Angsuthanasombat C. 2014. Importance of polarity of the α4-α5 loop residue-Asn166 in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: Implications for ion permeation and pore opening. Biochimica et Biophysica Acta, 1838, 319–327.
Khaokhiew T, Angsuthanasombat C, Promptmas C. 2009. Correlative effect on the toxicity of three surface-exposed loops in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin. FEMS Microbiology Letters, 300, 139–145.
Lacey L A, Grzywacz D, Shapiro-Ilan D I, Frutos R, Brownbridge M, Goettel M S. 2015. Insect pathogens as biological control agents: Back to the future. Journal of Invertebrate Pathology, 132, 1–41.
Liu Y L, Wang Q Y, Wang F X, Ding X Z, Xia L Q. 2010. Residue 544 in domain III of the Bacillus thuringiensis Cry1Ac toxin is involved in protein structure stability. The Protein Journal, 29, 440–444.
Lu H, Rajamohan F, Dean D H. 1994. Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori. Journal of Bacteriology, 176, 5554–5559.
Lucena W A, Pelegrini P B, Martins-de-Sa D, Fonseca F C, Gomes J E J, de Macedo L L, da Silva M C, Oliveira R S, Grossi-de-Sa M F. 2014. Molecular approaches to improve the insecticidal activity of Bacillus thuringiensis Cry toxins. Toxins, 6, 2393–2423.
Lv Y, Tang Y, Zhang Y L, Xia L Q, Wang F X, Ding X Z, Yi S M, Li W P, Yin J. 2011. The role of β20-β21 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis. Current Microbiology, 62, 665–670.
Pacheco S, Gómez I, Arenas I, Saab-Rincon G, Rodríguez-Almazán C, Gill S S, Bravo A, Soberón M. 2009. Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a “ping pong” binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors. The Journal of Biological Chemistry, 284, 32750–32757.
Palma L, Munoz D, Berry C, Murillo J, Caballero P. 2014. Bacillus thuringiensis toxins: An overview of their biocidal activity. Toxins, 6, 3296–3325.
Pardo-López L, Soberón M, Bravo A. 2013. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiology Reviews, 37, 3–22.
Pérez C, Fernandez L E, Sun J G, Folch J L, Gill S S, Soberón M, Bravo A. 2005. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. Proceedings of the National Academy of Sciences of the United States of America, 102, 18303–18308.
Pigott C R, Ellar D J. 2007. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiology and Molecular Biology Reviews, 71, 255–281.
Rajamohan F, Alzate O, Cotrill J A, Curtiss A, Dean D H. 1996a. Protein engineering of Bacillus thuringiensis δ-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae. Proceedings of the National Academy of Sciences of the United States of America, 93, 14338–14343.
Rajamohan F, Hussain S R, Cotrill J A, Gould F, Dean D H. 1996b. Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb δ-endotoxins suggest loop 3 is involved in initial binding to lepidopteran midguts. The Journal of Biological Chemistry, 271, 25220–25226.
Roh J Y, Nair M S, Liu X S, Dean D H. 2009. Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin. FEMS Microbiology Letters, 295, 156–163.
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean D H. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, 62, 775–806.
Sengupta A, Sarkar A, Priya P, Ghosh Dastidar S, Das S. 2013. New insight to structure-function relationship of GalNAc mediated primary interaction between insecticidal Cry1Ac toxin and HaALP receptor of Helicoverpa armigera. PLoS ONE, 8, e78249.
Shu C L, Liu D M, Zhou Z S, Cai J L, Peng Q, Gao J G, Song F P, Zhang J. 2013. An improved PCR-restriction fragment length polymorphism (RFLP) method for the identification of cry1-type genes. Applied and Environmental Microbiology, 79, 6706–6711.
Tigue N J, Jacoby J, Ellar D J. 2001. The α-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins. Applied and Environmental Microbiology, 67, 5715–5720.
Wang F S, Liu Y Y, Zhang F J, Chai L J, Ruan L F, Peng D H, Sun M. 2012. Improvement of crystal solubility and increasing toxicity against Caenorhabditis elegans by asparagine substitution in block 3 of Bacillus thuringiensis crystal protein Cry5Ba. Applied and Environmental Microbiology, 78, 7197–7204.
Wang F X, Xia L Q, Ding X Z, Zhao X M, Lv Y, Yu Z Q. 2009. N546 in β18-β19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin. Journal of Invertebrate Pathology, 101, 119–123.
Wolfersberger M, Luethy P, Maurer A, Parenti P, Sacchi F V, Giordana B, Hanozet G M. 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comparative Biochemistry and Physiology (Part A: Physiology), 86, 301–308.
Wu K M, Guo Y Y, Lv N. 1999. Geographic variation in susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis insecticidal protein in China. Journal of Economic Entomology, 92, 273–278.
Xue J, Liang G M, Crickmore N, Li H T, He K L, Song F P, Feng X, Huang D F, Zhang J. 2008. Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects. FEMS Microbiology Letters, 280, 95–101.
Zhang X, Candas M, Griko N B, Taussig R, Bulla Jr L A. 2006. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis. Proceedings of the National Academy of Sciences of the United States of America, 103, 9897–902.
Zhou Z S, Lin H Y, Li Y, Shu C L, Song F P, Zhang J. 2014. The minimal active fragment of the Cry1Ai toxin is located between 36I and 605I. Journal of Integrative Agriculture, 13, 1036–1042.
Zhou Z S, Liu Y X, Liang G M, Huang Y P, Bravo A, Soberón M, Song F P, Zhou X P, Zhang J. 2017. Insecticidal specificity of Cry1Ah to Helicoverpa armigera is determined by binding of APN1 via domain II loops 2 and 3. Applied and Environmental Microbiology, 83, e02864–e02880.

Alanine-substituted mutant on Gly³⁷³ and Asn³⁷⁵ of Cry1Ai-h-loop 2 causes reduction in both toxicity and binding against Helicoverpa armigera

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics