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Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

Ferrara TF, Schneider VK, Kishi LT, Carmona AK, Alves MF, Belasque-Júnior J, Rosa JC, Hunter WB, Henrique-Silva F, Soares-Costa A - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

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.

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CID-MS/MS spectra of tryptic peptides from DCCathB.Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from D. citri ESTs.
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pone.0145132.g003: CID-MS/MS spectra of tryptic peptides from DCCathB.Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from D. citri ESTs.

Mentions: For the verification of the DCcathB amino acid sequence, the purified protein was subjected to sequencing by mass spectrometry. The MALDI TOF/TOF data obtained by CID-MS/MS provided the amino acid sequences of two peptides: QNCYNPSYESTYR (m/z 1625.07 ± 0.41 at residue position 249 to 261) (Fig 3A) and SGVYQHNFGDSIGLHAVR (m/z 1957.32 ± 0.38 at residue position 303 to 320) (Fig 3B). These tryptic peptides correspond to 8.3% sequence coverage and a total MASCOT score of 185. The results obtained by mass spectrometry identified two peptides present in the amino acid sequence of the predicted protein obtained in the USDA database of the D. citri transcriptome (lcl/Sequence 1 ORF: 50.1174 Frame +2 Cathepsin B) as well in the sequence generated by the sequencing of the pPICZαC_DCcathB expression plasmid. The results obtained by mass spectrometry confirm the identity of the purified protein as a cathepsin B-like cysteine peptidase.


Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

Ferrara TF, Schneider VK, Kishi LT, Carmona AK, Alves MF, Belasque-Júnior J, Rosa JC, Hunter WB, Henrique-Silva F, Soares-Costa A - PLoS ONE (2015)

CID-MS/MS spectra of tryptic peptides from DCCathB.Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from D. citri ESTs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696824&req=5

pone.0145132.g003: CID-MS/MS spectra of tryptic peptides from DCCathB.Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from D. citri ESTs.
Mentions: For the verification of the DCcathB amino acid sequence, the purified protein was subjected to sequencing by mass spectrometry. The MALDI TOF/TOF data obtained by CID-MS/MS provided the amino acid sequences of two peptides: QNCYNPSYESTYR (m/z 1625.07 ± 0.41 at residue position 249 to 261) (Fig 3A) and SGVYQHNFGDSIGLHAVR (m/z 1957.32 ± 0.38 at residue position 303 to 320) (Fig 3B). These tryptic peptides correspond to 8.3% sequence coverage and a total MASCOT score of 185. The results obtained by mass spectrometry identified two peptides present in the amino acid sequence of the predicted protein obtained in the USDA database of the D. citri transcriptome (lcl/Sequence 1 ORF: 50.1174 Frame +2 Cathepsin B) as well in the sequence generated by the sequencing of the pPICZαC_DCcathB expression plasmid. The results obtained by mass spectrometry confirm the identity of the purified protein as a cathepsin B-like cysteine peptidase.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil.

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.

Show MeSH