Stress‐related RNase PR‐10c is post‐translationally modified by glutathione in birch
The PR-10c (previously termed as Bet v 1-Sc3) protein of birch belongs to the family of intracellular pathogenesis-related proteins. The high-performance liquid chromatography electrospray ionization ion trap mass spectrometry (HPLC-ESI-MS) analysis of PR-10c-His fusion protein, produced in Escherichia coli, revealed three major peaks and masses. Enzymatic digestions and HPLC-ESI-MS and matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF-MS) analyses of each fraction indicated that PR-10c-His protein is post-translationally modified by carbamylation and S-glutathiolation. Carbamylation was localized into the N-terminal end of PR-10c-His and does not represent a biologically significant modification. The possible nuclease activity of PR-10c was analysed with S-glutathiolated and reduced fractions of PR-10c-His fusion protein. Both forms of PR-10c-His as well as the dimeric form of the protein possess RNase activity which is capable of digesting different RNA substrates. None of the fractions showed activity against single- or double-stranded DNA. The MALDI-TOF-MS analysis of PR-10c polypeptide extracted from zinc-exposed birch roots showed that the protein is post-translationally modified by glutathione (gamma-Glu-Cys-Gly) also in vivo. The S-glutathiolated cysteine residue of PR-10c is not conserved among Bet v 1 homologous proteins and is also unique in the PR-10 family. As far as we know this is the first observation of S-glutathiolation in plants, or any post-translational modification in the PR-10 family of proteins.