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The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG

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 and Patrizia Agostinis, et al. (2012) MICROBIAL CELL FACTORIES. 11.
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.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
glycoprotein, bacterial O-glycosylation, Probiotic, Akt signaling, peptidoglycan hydrolase, FUNCTIONAL-ANALYSIS, GLYCOPROTEINS, BIOSYNTHESIS, MOLECULES, BACTERIA, HOST, PATHOGENS, CELL
journal title
MICROBIAL CELL FACTORIES
Microb. Cell. Fact.
volume
11
article_number
15
pages
14 pages
Web of Science type
Article
Web of Science id
000301577600001
JCR category
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
JCR impact factor
3.306 (2012)
JCR rank
39/157 (2012)
JCR quartile
1 (2012)
ISSN
1475-2859
DOI
10.1186/1475-2859-11-15
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
2140368
handle
http://hdl.handle.net/1854/LU-2140368
date created
2012-06-12 12:44:14
date last changed
2012-06-13 13:56:51
@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, Andr{\'e} M and De Keersmaecker, Sigrid CJ and Wuhrer, Manfred and Vanderleyden, Jos},
  issn         = {1475-2859},
  journal      = {MICROBIAL CELL FACTORIES},
  keyword      = {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://dx.doi.org/10.1186/1475-2859-11-15},
  volume       = {11},
  year         = {2012},
}

Chicago
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.
APA
Lebeer, Sarah, Claes, I. J., Balog, C. I., Schoofs, G., Verhoeven, T. L., Nys, K., von Ossowski, I., et al. (2012). The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG. MICROBIAL CELL FACTORIES, 11.
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.
MLA
Lebeer, Sarah, Ingmar JJ Claes, Crina IA Balog, et al. “The Major Secreted Protein Msp1/p75 Is O-glycosylated in Lactobacillus Rhamnosus GG.” MICROBIAL CELL FACTORIES 11 (2012): n. pag. Print.