ADDITION OF ACETALDEHYDE TO THE N-TERMINUS OF A RECOMBINANT SCHISTOSOMA-MANSONI GLUTATHIONE-S-TRANSFERASE UPON HIGH-LEVEL EXPRESSION IN SACCHAROMYCES-CEREVISIAE

Intracellular expression of recombinant Schistosoma mansoni protein p2 8 (Smp28) in soluble form to a concentration of more than 6 g/l cultur e in Saccharomyces cerevisiae was accompanied by a posttranslational m odification, which occurred during the late stage of the culture. The modified protein, which had a reduced isoelectric point, was isolated by anion-exchange HPLC and characterized by tryptic mapping by means o f on-line reversed-phase HPLC/electrospray mass spectrometry. Comparis on with non-modified recombinant Smp28 allowed us to localize the modi fication to the N-terminal hexapeptide AGEHIK, which had an increased mass of 26 Da. Reversed phase HPLC of the modified peptide with a shal low acetonitrile gradient revealed the presence of two components of i dentical mass and amino acid composition. Both peptides were inaccessi ble to N-terminal Edman sequencing, indicating that a rearrangement of the N-terminal region of recombinant Smp28 had taken place during try ptic digestion leading to two isomeric, N-terminally blocked peptides. Deuterium-exchange mass spectrometry showed that the modified peptide s lacked two exchangeable protons, suggesting cyclic modifications imp lying the N-terminal amino group. Tandem mass spectrometry by means of the nano-electrospray technique and collision-induced dissociation al lowed us to identify the modified sites as Ala1, His4 and Lys6 based o n a characteristic modified a(1) ion of Ala1 (70.0 Da), a modified imm onium ion of His4 (136.0 Da) and a modified y(1) '' ion (173.2 Da) of Lys6. Combination of all the above results led to the conclusion that recombinant Smp28 was initially modified at its N-terminus by addition of acetaldehyde to form an aldimine which rearranged during tryptic d igestion to two different cyclic peptides.