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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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Alignment of DCcathB with similar cathepsin B-like cysteine peptidases from hemipterans.Diaphorina citri (genbank accession number: 110456454), Nilaparvata lugens (genbank accession number: 22535408), Aphis citricidus (genbank accession number: 161343879), Riptortus pedestris (genbank accession number: 501291537), Acyrthosiphon pisum (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The DCcathB predicted peptide signal of seventeen residues is underlined in black. The probable occlusion loop characteristic of cathepsin B-like cysteine peptidases is represented by the dashed box. The potential cleavage site between the propeptide (residues 32 and 69) and mature DCcathB is indicated by an arrow. The predicted conserved catalytic triad C-H-N is indicated by asterisks. Alignment was generated using the Multalign program with default parameters.
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pone.0145132.g001: Alignment of DCcathB with similar cathepsin B-like cysteine peptidases from hemipterans.Diaphorina citri (genbank accession number: 110456454), Nilaparvata lugens (genbank accession number: 22535408), Aphis citricidus (genbank accession number: 161343879), Riptortus pedestris (genbank accession number: 501291537), Acyrthosiphon pisum (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The DCcathB predicted peptide signal of seventeen residues is underlined in black. The probable occlusion loop characteristic of cathepsin B-like cysteine peptidases is represented by the dashed box. The potential cleavage site between the propeptide (residues 32 and 69) and mature DCcathB is indicated by an arrow. The predicted conserved catalytic triad C-H-N is indicated by asterisks. Alignment was generated using the Multalign program with default parameters.

Mentions: The features of DCcathB were displays in Fig 1. The alignment shows the presence of the region corresponding to the occlusion loop, which is characteristic of cathepsin B-like enzymes [62]. The cleavage site between the pro-peptide and mature protein is predicted to occur in the sequence PL^DESD…, resulting in D70 as the N-terminal amino acid of the mature enzyme. The sequence of pro-peptide region contains a unique N-glycosylation site at N40 and the sequence correspondent to the mature protein contains three N-glycosylation sites at N102, N235 and N253. The papain cysteine peptidase family has a pro-region that performs functions such as transport to endosomal or lysosomal compartments for the correct folding of the active mature protein and pro-enzyme activity inhibition [49].


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)

Alignment of DCcathB with similar cathepsin B-like cysteine peptidases from hemipterans.Diaphorina citri (genbank accession number: 110456454), Nilaparvata lugens (genbank accession number: 22535408), Aphis citricidus (genbank accession number: 161343879), Riptortus pedestris (genbank accession number: 501291537), Acyrthosiphon pisum (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The DCcathB predicted peptide signal of seventeen residues is underlined in black. The probable occlusion loop characteristic of cathepsin B-like cysteine peptidases is represented by the dashed box. The potential cleavage site between the propeptide (residues 32 and 69) and mature DCcathB is indicated by an arrow. The predicted conserved catalytic triad C-H-N is indicated by asterisks. Alignment was generated using the Multalign program with default parameters.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145132.g001: Alignment of DCcathB with similar cathepsin B-like cysteine peptidases from hemipterans.Diaphorina citri (genbank accession number: 110456454), Nilaparvata lugens (genbank accession number: 22535408), Aphis citricidus (genbank accession number: 161343879), Riptortus pedestris (genbank accession number: 501291537), Acyrthosiphon pisum (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The DCcathB predicted peptide signal of seventeen residues is underlined in black. The probable occlusion loop characteristic of cathepsin B-like cysteine peptidases is represented by the dashed box. The potential cleavage site between the propeptide (residues 32 and 69) and mature DCcathB is indicated by an arrow. The predicted conserved catalytic triad C-H-N is indicated by asterisks. Alignment was generated using the Multalign program with default parameters.
Mentions: The features of DCcathB were displays in Fig 1. The alignment shows the presence of the region corresponding to the occlusion loop, which is characteristic of cathepsin B-like enzymes [62]. The cleavage site between the pro-peptide and mature protein is predicted to occur in the sequence PL^DESD…, resulting in D70 as the N-terminal amino acid of the mature enzyme. The sequence of pro-peptide region contains a unique N-glycosylation site at N40 and the sequence correspondent to the mature protein contains three N-glycosylation sites at N102, N235 and N253. The papain cysteine peptidase family has a pro-region that performs functions such as transport to endosomal or lysosomal compartments for the correct folding of the active mature protein and pro-enzyme activity inhibition [49].

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
Related in: MedlinePlus