Characterization of papain-like isoenzymes from latex of Asclepias curassavica by molecular biology validated by proteomic approach

WALTER D. OBREGÓN; C. LIGGIERI; SEBASTIÁN A. TREJO; FRANCESC X. AVILÉS; SANDRA E. VAIRO-CAVALLI; NORA PRIOLO

BIOCHIMIE

ELSEVIER

Año: 2009 vol. 91 p. 1457 - 1464

0300-9084

Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders.These pharmacological actions have been attributed to the presence of cysteine proteases in these milkylatices. Asclepias curassavica (Asclepiadaceae), ‘‘scarlet milkweed’’ is a perennial subshrub native to SouthAmerica. In the current paper we report a new approach directed at the selective biochemical andmolecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for theproteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases weredigested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. Noidentification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceaelatex cysteine proteinases available in databases. From total RNA extracted from latex samples,cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceaecysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained bycloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequencesof 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII,characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain thefull-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment andphylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group ofpapain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMFcould be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with verysimilar physicochemical and functional properties, with advantages over other conventional methods(for instance enzyme kinetics) that are time consuming and afford less reliable results.