Rapeseed (Brassica napus) 2-Cys peroxiredoxin. Sequencing of native and recombinant forms and post-translational modifications.

DANIEL CAPORALETTI; MARTIN ARAN; MARÍA ROMINA GIROTTI; ANDREA LLERA; RICARDO WOLOSIUK

Pilar, Buenos Aires

Congreso; First Annual Iberoamerican Congress of Proteomics; 2007

Latina American Human Proteome Organization

Rapeseed (Brassica napus) exhibits a restricted number of entries in databases because the complex tetraploid genome with multiple alleles for each locus makes usually difficult the characterization of DNA. Moreover, low-frequency post-translational modifications of proteins are not included in databases. In this context, tandem mass spectrometry constitutes a useful tool for the identification of not only isolated proteins, either native or recombinant, but also the position and composition of particular modifications. 2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins implicated in cell differentiation, apoptosis and photosynthesis. In this work, we describe the characterization of 2-Cys Prx by Edman sequentiation, Western blot and biochemical analysis. The native form isolated from rapeseed leaves had minor amino acids differences relative to the recombinant protein prepared from the cDNA, due to existence of allelic isoforms. Matching experimental data on MS/MS with the theoretical fragmentation of the modified peptides, we found that 2-Cys Prx could be glutathionylated in one or both conserved cysteines, i.e., Cys53 and Cys175. More interestingly, upon successive reduction, oxidation and incubation with ATP and Mg2+, 2-Cys Prx became phosphorylated. Significantly, the overoxidized forms of Cys175 were phosphorylated while Cys53, the site for sulfenic and sufinic acid formation in mammal counterpart, remains unchanged. Hence, redox (i.e. glutathionylation) and non-redox (phosphorylation) mechanisms converge to conserved cysteine residues for the modulation of 2-Cys Prx functions.