Papain-Like Proteinases from the Climbing Plant Asclepias Curassavica. Cloning and proteomics analysis

W.D. OBREGÓN; S. TREJO; C. LIGGIERI; S. VAIRO CAVALLI; D. LUFRANO; S. BRONSOMS; F.X. AVILÉS

Bilbao, España

Congreso; XXXI Congreso de la Sociedad Española de Bioquímica y Biología Molecular; 2008

Sociedad Española de Bioquímica y Biología Molecular

Asclepain cI (AcI) and asclepain cII (AcII) two plant cysteine peptidases that share some physical and biochemical features were isolated from latex of Asclepias curassavica, a wild South American Asclepiadaceae. Peptidases were purified to homogeneity by ultracentrifugation and cation exchange chromatography. Purity was confirmed by SDS-PAGE and their molecular weight was characterized by MALDI-TOF mass spectrometry. The SDS-PAGE spots were treated with dithiotreitol and iodoacetamide to block sulfhydril groups and to prevent autolysis and were further digested with trypsin. PMF analysis by MALDI-TOF MS showed the enzymes have some equivalent peptides, revealing close homology among them. By database searching no identification of proteins was possible due to the insufficient information of plant cysteine proteinases consigned. From the total RNA content of a latex sample, the cDNA of both proteases were obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine proteinase conserved domains. Theoretical PMF of partial sequences polypeptide of two cysteine peptidases (187 and 186 aminoacids) obtained by cloning were compared with the PMF of the enzymes previously purified. Subsequently the N-termini of AcI and AcII were characterized by Edman’s sequencing and overlapped with those sequences coming from the cDNA to obtain the full-length sequence of both mature proteinases (212 and 211 residues respectively). Phylogenetic analysis performed with Blast Tool indicates that AcI and AcII belong to the group of papain-like cystein peptidases. Their sequences show a high degree of identity with those of other cysteine plant proteases like caricain, papaya proteinase omega, quimopapain, oryzain, zingipain a and actinidin.