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 p28 (Smp28) in soluble form to a concentration of more than 6 g/l culture in Saccharomyces cerevisiae was accompanied by a post-translational modification, 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 of on-line reversed-phase HPLC/electrospray mass spectrometry. Comparison with non-modified recombinant Smp28 allowed us to localize the modification to the N-terminal hexapeptide AGEHIK, which had an increased mass of 26 Da. Reversed-phase HPLC of the modified peptide with a shallow acetonitrile gradient revealed the presence of two components of identical mass and amino acid composition. Both peptides were inaccessible to N-terminal Edman sequencing, indicating that a rearrangement of the N-terminal region of recombinant Smp28 had taken place during tryptic digestion leading to two isomeric, N-terminally blocked peptides. Deuterium-exchange mass spectrometry showed that the modified peptides lacked two exchangeable protons, suggesting cyclic modifications implying the N-terminal amino group. Tandem mass spectrometry by means of the nano-electrospray technique and collision-induced dissociation allowed us to identify the modified sites as Ala1, His4 and Lys6 based on a characteristic modified a1 ion of Ala1 (70.0 Da), a modified immonium ion of His4 (136.0 Da) and a modified y1" 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 digestion to two different cyclic peptides.
Roecklin
D
D
Transgène S.A., Strasbourg, France.
Klarskov
K
K
Cavallini
B
B
Sabatié
J
J
Bouchon
B
B
Loew
D
D
Van Dorsselaer
A
A
Bischoff
R
R
D000079:Acetaldehyde / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005982:Glutathione Transferase; D000079:Acetaldehyde