Limits...
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.

Show MeSH

Related in: MedlinePlus

DCcathB expression.A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_DCcathB supplemented with 0.75% methanol. M: molecular weight BenchMark™ Protein Ladder (Invitrogen). Induction Times: 1–0 h; 2–24 h; 3–48 h; 4–72 h; 5–96 h; 6–120 h; 7–144 h. B–Purification of DCcathB. M: molecular weight marker (Invitrogen). 1—Eluate. 2—Wash buffer without imidazole. 3—Protein eluted in 10 mM imidazole. 4—Protein eluted in 10 mM imidazole. 5—Protein eluted in 25 mM imidazole. 6—Protein eluted in 25 mM imidazole.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696824&req=5

pone.0145132.g002: DCcathB expression.A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_DCcathB supplemented with 0.75% methanol. M: molecular weight BenchMark™ Protein Ladder (Invitrogen). Induction Times: 1–0 h; 2–24 h; 3–48 h; 4–72 h; 5–96 h; 6–120 h; 7–144 h. B–Purification of DCcathB. M: molecular weight marker (Invitrogen). 1—Eluate. 2—Wash buffer without imidazole. 3—Protein eluted in 10 mM imidazole. 4—Protein eluted in 10 mM imidazole. 5—Protein eluted in 25 mM imidazole. 6—Protein eluted in 25 mM imidazole.

Mentions: The SDS-PAGE 12% analysis revealed the expression of the recombinant protein at 24 hours of induction (Fig 2A) and the purified DCcathB as a single and intact band of approximately 50 kDa corresponding to the protein eluted in imidazole fractions of 10 and 25 mM (Fig 2B). The yield of purified DCcathB protein was 2 mg/L of P. pastoris cell culture.


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)

DCcathB expression.A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_DCcathB supplemented with 0.75% methanol. M: molecular weight BenchMark™ Protein Ladder (Invitrogen). Induction Times: 1–0 h; 2–24 h; 3–48 h; 4–72 h; 5–96 h; 6–120 h; 7–144 h. B–Purification of DCcathB. M: molecular weight marker (Invitrogen). 1—Eluate. 2—Wash buffer without imidazole. 3—Protein eluted in 10 mM imidazole. 4—Protein eluted in 10 mM imidazole. 5—Protein eluted in 25 mM imidazole. 6—Protein eluted in 25 mM imidazole.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145132.g002: DCcathB expression.A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_DCcathB supplemented with 0.75% methanol. M: molecular weight BenchMark™ Protein Ladder (Invitrogen). Induction Times: 1–0 h; 2–24 h; 3–48 h; 4–72 h; 5–96 h; 6–120 h; 7–144 h. B–Purification of DCcathB. M: molecular weight marker (Invitrogen). 1—Eluate. 2—Wash buffer without imidazole. 3—Protein eluted in 10 mM imidazole. 4—Protein eluted in 10 mM imidazole. 5—Protein eluted in 25 mM imidazole. 6—Protein eluted in 25 mM imidazole.
Mentions: The SDS-PAGE 12% analysis revealed the expression of the recombinant protein at 24 hours of induction (Fig 2A) and the purified DCcathB as a single and intact band of approximately 50 kDa corresponding to the protein eluted in imidazole fractions of 10 and 25 mM (Fig 2B). The yield of purified DCcathB protein was 2 mg/L of P. pastoris cell culture.

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