Lactobacillus plantarum WCFS1 O-linked protein glycosylation: An extended spectrum of target proteins and modification sites detected by mass spectrometry

Abstract

It has recently been shown that the major autolysin Acm2 from Lactobacillus plantarum WCFS1 undergoes intracellu-lar O-GlcNAcylation [Fredriksen L, Mathiesen G, Moen A, Bron PA, Kleerebezem M, Eijsink VG, Egge-Jacobsen W. 2012. The major autolysin Acm2 from Lactobacillus plan-tarum undergoes cytoplasmic O-glycosylation. J Bacteriol. 194(2):325-333]. To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsYand the DNA trans-locase FtsK1. Among the other, glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for N-acetyl-hexosamine (HexNAc) were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK). © The Author 2013.