Identification by peptide mass fingerprinting of a cysteine protease cloned from Bromelia hieronymi fruits

BRUNO, MARIELA A.; TREJO, SEBASTIÁN A.; AVILÉS, FRANCESC X.; CAFFINI, NÉSTOR O.; LÓPEZ, LAURA M.

Salta

Congreso; Latin American Protein Society Meeting-SAB 2010-Workshop CeBEM; 2010

Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3´RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for the polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M-1 cm-1). Bh-CP1 sequence shows a high percentage of identity with those of other cystein plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159 and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modelling results suggest the enzyme belongs to the á+â class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the á domain, while the â domain is stabilized by another disulfide bridge (Cys153-Cys203). Peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely, hieronymain I, II and III) were performed and compared with the theoretical PMF of Bh-CP1. The experimental and theoretical mass values matches were selected with a mass tolerance of 0.045 Da. No mass peptide coincidences were found between hieonymain I and Bh-CP1 and only two mass peptides of hieronymain III matched with those of Bh-CP1; however, when hieronymain II and Bh-CP1 peptide masses were confronted, eight matches afforded (84.9% intensity coverage and 29.6% sequence coverage). This proteomic approach provides additional information on structural characterization of hieronymain I, II and III and affords strong evidence that Bh-CP1 would correspond to hieronymain II.