Recombinant expression and characterization of a cysteine peptidase from Xanthomonas citri subsp citri

Xanthomonas citri subsp citri (Xac) is the bacterium responsible for citrus canker disease in citrus plants. the aim of this study was to describe the recombinant expression, purification, and characterization of a cysteine peptidase from Xac strain 306, which is a candidate for involvement in the pathogenicity of this bacterium. the gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein in P. pastoris. the same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin substrate with a catalytic efficiency (k(cat)/K-m) of 47 mu M-1.s(-1). the purified (HIS)CPXAC displayed maximal catalytic activity at pH 5.5 and 30 degrees C. the recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K-i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence in a secreted protein from Xanthomonas spp.Fundo de Defesa da CitriculturaFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP, BrazilUniv Fed Sao Carlos, Lab Bioquim & Biol Mol Microrganismos, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFundo Def Citricultura, Araraquara, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilFAPESP: 98/14138-2FAPESP: 05/59833-5Web of Scienc

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambdaex = 320 nm and lambdaem = 420 nm) at 37ºC, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 µM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 µM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 µM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de MedicinaUniversidade de São Paulo Faculdade de Medicina Instituto do CoraçãoUniversidade de São Paulo Escola de Educação Física e Esporte Laboratório de Bioquímica da Atividade MotoraUNIFESP, EPM, Depto. de BiofísicaUNIFESP, EPM, Depto. de MedicinaSciEL

Intramolecularly quenched fluorogenic substrate for angiotensin I-converting enzyme

UNIFESP, Dept Biophys, Sao Paulo, BrazilUNIFESP, Dept Med, Div Nephrol, Sao Paulo, BrazilUNIFESP, Dept Biophys, Sao Paulo, BrazilUNIFESP, Dept Med, Div Nephrol, Sao Paulo, BrazilWeb of Scienc

Phenolic compounds in raw and cooked rice (Oryza sativa L.) and their inhibitory effect on the activity of angiotensin I-converting enzyme

Whole rice has been widely studied due to the abundance of bioactive compounds in its pericarp. Some of the beneficial effects of these compounds on human health have been attributed to their antioxidant and other biological activities, such as enzyme inhibition. in this work, we evaluated the contents of total, soluble and insoluble phenolic compounds of 6 red and 10 non-pigmented genotypes of whole rice as well as their inhibitory effect on the activity of angiotensin I-converting enzyme (ACE). the effects of cooking on phenolics and their inhibitory activities were also investigated. Red genotypes showed high content of phenolics, mainly soluble compounds, at an average of 409.7 mg ferulic acid eq./100 g, whereas overall lower average levels (99.4 mg ferulic acid eq./100 g) at an approximate soluble/insoluble compound ratio of 1:1 were observed in non-pigmented rice. Pigmented rice displayed a greater inhibitory effect on ACE than non-pigmented rice. in fact, a significant correlation between the content of soluble phenolics and ACE inhibition was observed (r = 0.8985, p < 0.05). in addition to significantly reducing the levels of total phenolics and ACE inhibition, cooking altered the soluble/insoluble compound ratio, especially among red rice genotypes. (C) 2011 Elsevier B.V. All rights reserved.Univ São Paulo, Dept Food & Expt Nutr, Fac Pharmaceut Sci, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

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

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

Alignment of <i>DCcathB</i> with similar cathepsin B-like cysteine peptidases from hemipterans.

<p><i>Diaphorina citri</i> (genbank accession number: 110456454), <i>Nilaparvata lugens</i> (genbank accession number: 22535408), <i>Aphis citricidus</i> (genbank accession number: 161343879), <i>Riptortus pedestris</i> (genbank accession number: 501291537), <i>Acyrthosiphon pisum</i> (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The <i>DCcathB</i> 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 <i>DCcathB</i> 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.</p

<i>DCcathB</i> expression analysis by RT-qPCR in <i>D</i>. <i>citri</i> egg, nymph and adult (A) and head, gut and remaining tissues (B).

<p>The quantification calibrator in developmental phases was egg and for the tissues was gut. Error bars were calculated according [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145132#pone.0145132.ref060" target="_blank">60</a>]. The difference was significant when the p-value was lower than 0.05.</p

<i>DCcathB</i> expression.

<p>A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_<i>DCcathB</i> 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 <i>DCcathB</i>. 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.</p