Identification and characterization of a recombinant cysteine peptidase (AsCathL) from leaf-cutting ant Atta sexdens Linnaeus, 1758 (Hymenoptera, Formicidae)

Autor(es)

Santos Correa, Katia Celina ; Moreira, Ariele Cristina ; Ibrahim, Amr Galal Abd El-Raheem ; Ramos de Jesus, Hugo Cesar ; Micocci, Kelli Cristina ; Crizostomo Kock, Flavio Vinicius ; Bueno, Odair C. ; Venancio, Tiago ; Henrique-Silva, Flavio ; Souza, Dulce Helena F.

• Tipo de documento: Artigo Científico
• Fonte: Protein Expression and Purification; v. 201, p. 10-pg., 2023-01-01.

Resumo

Cysteine peptidases are involved in physiological processes of insect development and have been considered as potential targets for the development of insect control strategies. In this study, we obtained a recombinant cysteine cathepsin L (AsCathL) from leaf-cutting ant (Atta sexdens), a species from the order Hymenoptera who causes enormous damage to crops, natural forests and reforested areas. RT-qPCR showed AsCathL expression throughout insect development and in all body parts of the adult insect analysed, suggesting its role as a lysosomal cathepsin. AsCathL encodes a protein of 320 amino acid residues consisting of a pro-peptide and the mature with amino acids sequence over 67% similarity with lysosomal cathepsin L of species from Lepidoptera and Diptera. Phylogenetic tree revealed that AsCathL is very similar to predicted cathepsins found in other ants. Recombinant AsCathL was expressed in insoluble form by Escherichia coli Arctic Express (DE3) RIL, purified under denaturing conditions and refolded. The enzyme showed hydrolytic activity in vitro towards synthetic substrate Z-Phe-Arg-AMC at acidic pH. Synthetic inhibitor E-64 acted against peptidase activity and a study regarding the interaction between E-64 and AsCathL using nuclear magnetic resonance (NMR) revealed that 83.18% from all E-64 molecules are irreversibly bound to AsCathL. In addition, the proteolytic activity of AsCathL was strongly inhibited by recombinant sugarcane cystatins with K-i ranging from 0.6 nM to 2.95 nM. To the best of our knowledge this is the first report characterizing a cysteine peptidase from leaf-cutting ants, which may contribute to future studies of ants' cathepsins. (AU)

• Processo FAPESP: 17/15455-4 - Contribuição aos estudos integrados para o controle de formigas cortadeiras - busca de novos inseticidas e fungicidas - nanoencapsulamento
• Beneficiário: Hugo César Ramos de Jesus
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 15/21517-7 - Caracterização funcional de uma catepsina recombinante da formiga cortadeira Atta sexdens
• Beneficiário: Katia Celina Santos Correa
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 14/12169-2 - Estudos de vias metabólicas importantes no controle de formigas cortadeiras
• Beneficiário: Dulce Helena Ferreira de Souza
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 18/16040-5 - Controle Biorracional de Insetos Pragas
• Beneficiário: Flavio Vinicius Crizostomo Kock
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 18/06297-9 - Produção e caracterização de proteínas quiméricas contendo domínio catalítico quitinase e módulos para ligação em quitina
• Beneficiário: Dulce Helena Ferreira de Souza
• Modalidade de apoio: Auxílio à Pesquisa - Regular