The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
- Equal contributors
1 Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20, box 2460, B-3001 Leuven, Belgium
2 Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium
3 Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
4 Cell Death Research & Therapy laboratory, Department Molecular and Cell Biology, Faculty of Medicine, K.U.Leuven, Herestraat 49, box 901, B-3000, Belgium
5 Department of Veterinary Biosciences, University of Helsinki, P.O. Box 66, FIN-00014, Helsinki, Finland
6 Department of Molecular Biotechnology, Ghent University, Ghent, Belgium
Microbial Cell Factories 2012, 11:15 doi:10.1186/1475-2859-11-15Published: 1 February 2012
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.
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.
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.