Biochemical and PMF MALDI-TOF Analyses of Two Novel Papain-Like Plant Proteinases

Obregón, Walter David; Liggieri, Constanza Silvina; Morcelle, Susana R.; Trejo, Sebastián A.; Avilés, Francesc X.; Priolo, Nora

doi:10.2174/092986609789353736

Cited by 10 publications

(19 citation statements)

References 38 publications

(53 reference statements)

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“…Using MASCOT search engine it was not possible to identify this enzyme; therefore, it may be considered a novel proteinase. Protein identification and differentiation by PMF has been adopted in our group as an excellent tool to differentiate, in a fast and unequivocal way, proteases with very similar physicochemical and functional properties (Obregón et al 2009a;Torres et al 2010). The robustness of the method even allowed the differentiation of the homologue isoenzymes of the latex of A. curassavica (Obregón et al 2009a).…”

Section: Resultsmentioning

confidence: 99%

“…PCR was made using specific primers that recognize highly conserved sequences of latex peptidases of the family Apocynaceae (NTapo 1 5 0 -GTTGAATTGCCAGAT TCTGTAGATTGG-3 0 , NTapo 2 5 0 -CCAGATTCTGTAG ATTGGCGG-3 0 (Obregón et al 2009a) in combination with R 0 . Nt 1-8 (5 0 -yTkCCdGATTCCGATGTTTGGmG-3 0 ), effective for the isolation of cDNAs of other cysteine proteases from latex ) was also used combined with R 0 .…”

Section: N-terminal Sequencementioning

confidence: 99%

“…Araujiain aII as well as asclepain cI, asclepain cII (Obregón et al 2009a), asclepain f ), philibertain gA and philibertain gB (Sequeiros et al 2005), all enzymes from latex of Apocynaceae family, presented Fig. 8 Distance map of papain-like cysteine peptidases.…”

Section: Peptidase Cloning and Sequence Analysismentioning

confidence: 99%

“…Latices from Asclepiadoideae subfamily have been used in folk medicine as antiparasitic agents against gastrointestinal nematodes and in wound healing in addition to other ailments. The pharmacological actions of these milky latices have been attributed to the presence of cysteine peptidases (Stepek et al 2004;Obregón et al 2009a). Shivaprasad et al (2009) have shown that the PLCPs present in Asclepias curassavica latex exhibited strong procoagulant action and were found to be specific in their action (thrombin like).…”

Section: Introductionmentioning

confidence: 99%

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Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex

Obregón

Lufrano

Liggieri

et al. 2011

Planta

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Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.

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Section: Resultsmentioning

confidence: 99%

Section: N-terminal Sequencementioning

confidence: 99%

Section: Peptidase Cloning and Sequence Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex

Obregón

Lufrano

Liggieri

et al. 2011

Planta

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“…In this sense, protein identification using mass spectrometry would be the most appropriate choice, and is typically accomplished by either PMF or amino acid sequencing of the peptide using tandem mass spectrometry (MS=MS). Both techniques consist on high-throughput approaches, a very important advantage when compared with other methods, such as western blotting or kinetic determinations in the case of enzymes (Damodaran et al 2007;Obregó n et al 2009). …”

Section: Introductionmentioning

confidence: 99%

Papain Purification Insights: Monitoring by Electrophoretic Approaches and MALDI-TOF Peptide Mass Fingerprint Analyses

et al. 2011

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Papain was purified from dried Carica papaya latex by fractioned salt precipitation in presence of sodium tetrathionate to preserve enzymatic activity. Purification was followed by different electrophoretic methods. Identification of the purified product was afforded by submitting the peptides obtained by tryptic digestion of papain to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis. Comparison of the peptide masses analyzed by peptide mass fingerprinting (PMF) MALDI-TOF and those obtained by theoretical tryptic digestion, revealed the presence of some peptides belonging the other three endopeptidases contained in papaya latex (very similar to papain in molecular weight and pI) in the purified fraction of papain. The PMF by MALDI-TOF could be applied as a method to follow papain purification.

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Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Torres

Trejo

Obregón

et al. 2012

Planta

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Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia ("oak leaved papaya") latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman's degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.

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Biochemical and PMF MALDI-TOF Analyses of Two Novel Papain-Like Plant Proteinases

Cited by 10 publications

References 38 publications

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex

Papain Purification Insights: Monitoring by Electrophoretic Approaches and MALDI-TOF Peptide Mass Fingerprint Analyses

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

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