Characterization of the proteolytic system present in Vasconcellea quercifolia latex.
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.
Laboratorio de Investigación de Proteínas Vegetales, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, C.C. 711, B1900AVW, La Plata, Argentina.
María José
MJ
Torres
Sebastián Alejandro
SA
Trejo
Walter David
WD
Obregón
Francesc Xavier
FX
Avilés
Laura María Isabel
LM
López
Claudia Luisa
CL
Natalucci
D000595:Amino Acid Sequence; D001483:Base Sequence; D031241:Caricaceae / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007840:Latex / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010802:Phylogeny; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis
D007840:Latex; D010940:Plant Proteins; D057056:Cysteine Proteases; D010447:Peptide Hydrolases