title: Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.
abstract: Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
authors.forename: Taíse Fernanda da Silva
authors.initials: TF
authors.lastname: Ferrara
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
authors.forename: Vanessa Karine
authors.initials: VK
authors.lastname: Schneider
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
authors.forename: Luciano Takeshi
authors.initials: LT
authors.lastname: Kishi
authors.affiliation: Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Adriana Karaoglanovic
authors.initials: AK
authors.lastname: Carmona
authors.affiliation: Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil.
authors.forename: Marcio Fernando Madureira
authors.initials: MF
authors.lastname: Alves
authors.affiliation: Department of Phytopathology and Nematology, University of São Paulo, Piracicaba, São Paulo, SP, Brazil.
authors.forename: Jose
authors.initials: J
authors.lastname: Belasque-Júnior
authors.affiliation: USDA, ARS, 2001 South Rock Road, Fort Pierce, Florida, United States of America.
authors.forename: José César
authors.initials: JC
authors.lastname: Rosa
authors.affiliation: Protein Chemistry Center and Department of Molecular and Cellular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Wayne Brian
authors.initials: WB
authors.lastname: Hunter
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
authors.forename: Flávio
authors.initials: F
authors.lastname: Henrique-Silva
authors.affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.
authors.forename: Andrea
authors.initials: A
authors.lastname: Soares-Costa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002957:Citrus / Q000469:parasitology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006430:Hemiptera / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D007814:Larva / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010935:Plant Diseases / Q000469:parasitology* / Q000517:prevention & control*; D011480:Protease Inhibitors / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D010455:Peptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014357:Trypsin; D002401:Cathepsin B