The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG
- Author
- Sarah Lebeer, Ingmar JJ Claes, Crina IA Balog, Geert Schoofs, Tine LA Verhoeven, Kris Nys, Ingemar von Ossowski, Willem M de Vos, Hanne LP Tytgat, Patrizia Agostinis, Airi Palva, Els Van Damme (UGent) , André M Deelder, Sigrid CJ De Keersmaecker, Manfred Wuhrer and Jos Vanderleyden
- Organization
- 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.
- Keywords
- glycoprotein, bacterial O-glycosylation, Probiotic, Akt signaling, peptidoglycan hydrolase, FUNCTIONAL-ANALYSIS, GLYCOPROTEINS, BIOSYNTHESIS, MOLECULES, BACTERIA, HOST, PATHOGENS, CELL
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Citation
Please use this url to cite or link to this publication: http://hdl.handle.net/1854/LU-2140368
- MLA
- Lebeer, Sarah, et al. “The Major Secreted Protein Msp1/P75 Is O-Glycosylated in Lactobacillus Rhamnosus GG.” MICROBIAL CELL FACTORIES, vol. 11, 2012, doi:10.1186/1475-2859-11-15.
- APA
- Lebeer, S., Claes, I. J., Balog, C. I., Schoofs, G., Verhoeven, T. L., Nys, K., … Vanderleyden, J. (2012). The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG. MICROBIAL CELL FACTORIES, 11. https://doi.org/10.1186/1475-2859-11-15
- Chicago author-date
- Lebeer, Sarah, Ingmar JJ Claes, Crina IA Balog, Geert Schoofs, Tine LA Verhoeven, Kris Nys, Ingemar von Ossowski, et al. 2012. “The Major Secreted Protein Msp1/P75 Is O-Glycosylated in Lactobacillus Rhamnosus GG.” MICROBIAL CELL FACTORIES 11. https://doi.org/10.1186/1475-2859-11-15.
- Chicago author-date (all authors)
- Lebeer, Sarah, Ingmar JJ Claes, Crina IA Balog, Geert Schoofs, Tine LA Verhoeven, Kris Nys, Ingemar von Ossowski, Willem M de Vos, Hanne LP Tytgat, Patrizia Agostinis, Airi Palva, Els Van Damme, André M Deelder, Sigrid CJ De Keersmaecker, Manfred Wuhrer, and Jos Vanderleyden. 2012. “The Major Secreted Protein Msp1/P75 Is O-Glycosylated in Lactobacillus Rhamnosus GG.” MICROBIAL CELL FACTORIES 11. doi:10.1186/1475-2859-11-15.
- Vancouver
- 1.Lebeer S, Claes IJ, Balog CI, Schoofs G, Verhoeven TL, Nys K, et al. The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG. MICROBIAL CELL FACTORIES. 2012;11.
- IEEE
- [1]S. Lebeer et al., “The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG,” MICROBIAL CELL FACTORIES, vol. 11, 2012.
@article{2140368, 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.}}, articleno = {{15}}, author = {{Lebeer, Sarah and Claes, Ingmar JJ and Balog, Crina IA and Schoofs, Geert and Verhoeven, Tine LA and Nys, Kris and von Ossowski, Ingemar and de Vos, Willem M and Tytgat, Hanne LP and Agostinis, Patrizia and Palva, Airi and Van Damme, Els and Deelder, André M and De Keersmaecker, Sigrid CJ and Wuhrer, Manfred and Vanderleyden, Jos}}, issn = {{1475-2859}}, journal = {{MICROBIAL CELL FACTORIES}}, keywords = {{glycoprotein,bacterial O-glycosylation,Probiotic,Akt signaling,peptidoglycan hydrolase,FUNCTIONAL-ANALYSIS,GLYCOPROTEINS,BIOSYNTHESIS,MOLECULES,BACTERIA,HOST,PATHOGENS,CELL}}, language = {{eng}}, pages = {{14}}, title = {{The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG}}, url = {{http://doi.org/10.1186/1475-2859-11-15}}, volume = {{11}}, year = {{2012}}, }
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