title: The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation.
abstract: The major autolysin Acm2 from the probiotic strain Lactobacillus plantarum WCFS1 contains high proportions of alanine, serine, and threonine in its N-terminal so-called AST domain. It has been suggested that this extracellular protein might be glycosylated, but this has not been experimentally verified. We used high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the possible occurrence of glycans on peptides generated from lactobacillary surface proteins by protease treatment. This approach yielded five glycopeptides in various glycoforms, all derived from the AST domain of Acm2. All five glycopeptides contained the hydroxy-amino acids serine and threonine, suggesting that Acm2 is O-glycosylated. By using lectin blotting with succinylated wheat germ agglutinin, and by comparing the wild-type strain with an Acm2-negative derivative (NZ3557), we found that the attached N-acetylhexosamines are most likely N-acetylglucosamines (GlcNAc). NZ3557 was further used as a genetic background to express an Acm2 variant lacking its secretion signal, resulting in intracellular expression of Acm2. We show that this intracellular version of Acm2 is also glycosylated, indicating that the GlcNAc modification is an intracellular process.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Aas, Norway.
authors.forename: Lasse
authors.initials: L
authors.lastname: Fredriksen
authors.forename: Geir
authors.initials: G
authors.lastname: Mathiesen
authors.forename: Anders
authors.initials: A
authors.lastname: Moen
authors.forename: Peter A
authors.initials: PA
authors.lastname: Bron
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
authors.forename: Vincent G H
authors.initials: VG
authors.lastname: Eijsink
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004269:DNA, Bacterial; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006020:Glycopeptides / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D048191:Lactiplantibacillus plantarum / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006020:Glycopeptides; D009238:N-Acetylmuramoyl-L-alanine Amidase
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