title: O-glycosylation as a novel control mechanism of peptidoglycan hydrolase activity.
abstract: Acm2, the major autolysin of Lactobacillus plantarum, is a tripartite protein. Its catalytic domain is surrounded by an O-glycosylated N-terminal region rich in Ala, Ser, and Thr (AST domain), which is of low complexity and unknown function, and a C-terminal region composed of five SH3b peptidoglycan (PG) binding domains. Here, we investigate the contribution of these two accessory domains and of O-glycosylation to Acm2 functionality. We demonstrate that Acm2 is an N-acetylglucosaminidase and identify the pattern of O-glycosylation (21 mono-N-acetylglucosamines) of its AST domain. The O-glycosylation process is species-specific as Acm2 purified from Lactococcus lactis is not glycosylated. We therefore explored the functional role of O-glycosylation by purifying different truncated versions of Acm2 that were either glycosylated or non-glycosylated. We show that SH3b domains are able to bind PG and are responsible for Acm2 targeting to the septum of dividing cells, whereas the AST domain and its O-glycosylation are not involved in this process. Notably, our data reveal that the lack of O-glycosylation of the AST domain significantly increases Acm2 enzymatic activity, whereas removal of SH3b PG binding domains dramatically reduces this activity. Based on this antagonistic role, we propose a model in which access of the Acm2 catalytic domain to its substrate may be hindered by the AST domain where O-glycosylation changes its conformation and/or mediates interdomain interactions. To the best of our knowledge, this is the first time that O-glycosylation is shown to control the activity of a bacterial enzyme.
authors.affiliation: Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rolain
authors.forename: Elvis
authors.initials: E
authors.lastname: Bernard
authors.forename: Audrey
authors.initials: A
authors.lastname: Beaussart
authors.forename: Hervé
authors.initials: H
authors.lastname: Degand
authors.forename: Pascal
authors.initials: P
authors.lastname: Courtin
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Egge-Jacobsen
authors.forename: Peter A
authors.initials: PA
authors.lastname: Bron
authors.forename: Pierre
authors.initials: P
authors.lastname: Morsomme
authors.forename: Michiel
authors.initials: M
authors.lastname: Kleerebezem
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chapot-Chartier
authors.forename: Yves F
authors.initials: YF
authors.lastname: Dufrêne
authors.forename: Pascal
authors.initials: P
authors.lastname: Hols
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D006031:Glycosylation; D048191:Lactiplantibacillus plantarum / Q000201:enzymology / Q000378:metabolism; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000118:Acetylglucosaminidase; D009238:N-Acetylmuramoyl-L-alanine Amidase
keywords: Autolysin; Bacterial SH3b Domain; Cell Surface Enzymes; Cell Wall; Glycoprotein; Glycoside Hydrolases; Lactobacillus; N-Acetylglucosaminidase; O-Glycosylation; Peptidoglycan