title: The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG.
abstract: BACKGROUND
  Although the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes.
  
  
  RESULTS
  Here, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O-glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein.
  
  
  CONCLUSIONS
  In this study we have provided the first evidence of protein O-glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.
authors.affiliation: Centre of Microbial and Plant Genetics, K,U,Leuven, Kasteelpark Arenberg 20, box 2460, B-3001 Leuven, Belgium. sarah.lebeer@biw.kuleuven.be
authors.forename: Sarah
authors.initials: S
authors.lastname: Lebeer
authors.forename: Ingmar J J
authors.initials: IJ
authors.lastname: Claes
authors.forename: Crina I A
authors.initials: CI
authors.lastname: Balog
authors.forename: Geert
authors.initials: G
authors.lastname: Schoofs
authors.forename: Tine L A
authors.initials: TL
authors.lastname: Verhoeven
authors.forename: Kris
authors.initials: K
authors.lastname: Nys
authors.forename: Ingemar
authors.initials: I
authors.lastname: von Ossowski
authors.forename: Willem M
authors.initials: WM
authors.lastname: de Vos
authors.forename: Hanne L P
authors.initials: HL
authors.lastname: Tytgat
authors.forename: Patrizia
authors.initials: P
authors.lastname: Agostinis
authors.forename: Airi
authors.initials: A
authors.lastname: Palva
authors.forename: Els J M
authors.initials: EJ
authors.lastname: Van Damme
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
authors.forename: Sigrid C J
authors.initials: SC
authors.lastname: De Keersmaecker
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
mesh_terms: D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D004926:Escherichia coli / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D006801:Humans; D007780:Lacticaseibacillus casei / Q000378:metabolism; D052201:Lacticaseibacillus rhamnosus / Q000378:metabolism*; D013058:Mass Spectrometry; D020066:Merozoite Surface Protein 1 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D006020:Glycopeptides; D020066:Merozoite Surface Protein 1; D011994:Recombinant Proteins; D051057:Proto-Oncogene Proteins c-akt