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PMID:26717484
| Citation |
Ferrara, TF, Schneider, VK, Kishi, LT, Carmona, AK, Alves, MF, Belasque-Júnior, J, Rosa, JC, Hunter, WB, Henrique-Silva, F and Soares-Costa, A (2015) Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing. PLoS ONE 10:e0145132 |
|---|---|
| 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. |
| Links |
PubMed PMC4696824 Online version:10.1371/journal.pone.0145132 |
| Keywords |
Amino Acid Sequence; Animals; Biocatalysis/drug effects; Cathepsin B/chemistry; Cathepsin B/genetics; Cathepsin B/isolation & purification; Cathepsin B/metabolism; Citrus/parasitology; Gene Expression Regulation, Enzymologic/drug effects; Hemiptera/drug effects; Hemiptera/enzymology; Hemiptera/genetics; Hemiptera/growth & development; Larva/genetics; Molecular Sequence Data; Peptides/metabolism; Plant Diseases/parasitology; Plant Diseases/prevention & control; Protease Inhibitors/pharmacology; Real-Time Polymerase Chain Reaction; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Sequence Alignment; Sequence Analysis, Protein; Tandem Mass Spectrometry; Trypsin/metabolism |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0004197: cysteine-type endopeptidase activity |
ECO:0000247: |
UniProtKB:A9JSG7 UniProtKB:A9JSI1 UniProtKB:Q8MNY7 UniProtKB:R4WRE6
|
F |
In the paper, the gene sequence of Diaphorina citri is compared in a sequence analysis with Nilaparvata lugens, Aphis citricidus, Riportus pedestris, and Acyrthosiphon pisum. Figure 1 shows this sequence alignment. The other bacteria in the alignment display similar cathepsin B-like cysteine peptidase activity from hemipterans. The dashed boxes in Figure 1 display a probable occlusion loop characteristic of cathepsin B-like cysteine peptidases. |
complete | |||
Notes
See also
References
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