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

Lebeer S, Claes IJ, Balog CI, Schoofs G, Verhoeven TL, Nys K, von Ossowski I, de Vos WM, Tytgat HL, Agostinis P, Palva A, Van Damme EJ, Deelder AM, De Keersmaecker SC, Wuhrer M, Vanderleyden J - Microb. Cell Fact. (2012)

Bottom Line: Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA.Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Microbial and Plant Genetics, K,U,Leuven, Kasteelpark Arenberg 20, box 2460, B-3001 Leuven, Belgium. sarah.lebeer@biw.kuleuven.be

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.

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Identification of Msp1 as a glycoprotein. (A) SDS-PAGE analysis of LGG's secreted proteins present in spent culture supernatant after overnight growth in AOAC medium. The secreted proteins of LGG WT, msp1 mutant, EPS mutant CMPG5351 and CW-PS mutant CMPG5413 are included. Arrows indicate the major secreted proteins of LGG Msp1 and Msp2 confirmed by Edman degradation and MS/MS. The gels are stained with Sypro® Ruby and ProQ Emerald glycoprotein stain. (B) Comparative analysis of the glycosylation of Msp1 in spent culture supernatant of LGG wild-type before and after treatment with TFMS, and after recombinant expression in E. coli. The msp1 mutant CMPG10200 was included as a control. This mutant expresses a C-terminally truncated form of Msp1 and lacks the enzymatic active NlpC/P60 domain [17]. SDS-PAGE gels were compared by ConA lectin blotting and blotting with Msp1 specific antiserum. (C) Comparison of the sensitivity of recombinant non-glycosylated Msp1 purified from E. coli (rec. Msp1) and native glycosylated Msp1 purified from LGG WT (glyc. Msp1) towards pronase E and proteinase K. Protein samples were incubated for 1 h at 37°C with the indicated diluted preparations of proteases (Sigma). SDS-PAGE gels were stained with Sypro® Ruby (Invitrogen). Images were scanned with Typhoon 9400 (GE Healthcare).
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Figure 2: Identification of Msp1 as a glycoprotein. (A) SDS-PAGE analysis of LGG's secreted proteins present in spent culture supernatant after overnight growth in AOAC medium. The secreted proteins of LGG WT, msp1 mutant, EPS mutant CMPG5351 and CW-PS mutant CMPG5413 are included. Arrows indicate the major secreted proteins of LGG Msp1 and Msp2 confirmed by Edman degradation and MS/MS. The gels are stained with Sypro® Ruby and ProQ Emerald glycoprotein stain. (B) Comparative analysis of the glycosylation of Msp1 in spent culture supernatant of LGG wild-type before and after treatment with TFMS, and after recombinant expression in E. coli. The msp1 mutant CMPG10200 was included as a control. This mutant expresses a C-terminally truncated form of Msp1 and lacks the enzymatic active NlpC/P60 domain [17]. SDS-PAGE gels were compared by ConA lectin blotting and blotting with Msp1 specific antiserum. (C) Comparison of the sensitivity of recombinant non-glycosylated Msp1 purified from E. coli (rec. Msp1) and native glycosylated Msp1 purified from LGG WT (glyc. Msp1) towards pronase E and proteinase K. Protein samples were incubated for 1 h at 37°C with the indicated diluted preparations of proteases (Sigma). SDS-PAGE gels were stained with Sypro® Ruby (Invitrogen). Images were scanned with Typhoon 9400 (GE Healthcare).

Mentions: Msp1, described previously as the p75 protein [16], is present in abundant amounts in the spent culture supernatant of LGG. Interestingly, this protein appears to have an aberrant migration pattern when analyzed by SDS-PAGE and is detected with a significantly higher molecular mass (70-75 kDa) (Figure 1A) than the expected mass (46.8 kDa) for a protein with a N-terminal secretion signal (Figure 1B-C). Previously, this protein was named (i.e. p75) based on its size on SDS gels. Because of the apparent inconsistency in molecular mass, we have designated this protein according to its relative abundance as Msp1 (as in the Major secreted protein). While our electrophoretic results are strongly suggestive of post-translational modification, part of the observed discrepancy in molecular mass might also be due to the disproportionately high number of alanine residues (~23% content) in the primary structure of this protein (Figure 1C). Alanine has a low SDS-binding capacity and is known for a high helical propensity causing slow gel migration for proteins with high alanine content [18]. However, we found that a msp1 mutant CMPG10200 [17] (Table 1), expressing a truncated form of Msp1 that lacks a 311-residue C-terminal segment encompassing a large proportion of alanines, still migrates with a larger (~45 kDa) (Figure 2B) than expected size (16.4 kDa) (Figure 1C). Contrastingly, the other major secreted protein of LGG (encoded by LGG_00031 ), here renamed as Msp2 but also known previously as p40 [16], has a predicted molecular mass of 39.7 kDa that corresponds well with its observed migration pattern on SDS gels, suggesting that this protein has not undergone major post-translational modifications (Figure 1A).


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

Lebeer S, Claes IJ, Balog CI, Schoofs G, Verhoeven TL, Nys K, von Ossowski I, de Vos WM, Tytgat HL, Agostinis P, Palva A, Van Damme EJ, Deelder AM, De Keersmaecker SC, Wuhrer M, Vanderleyden J - Microb. Cell Fact. (2012)

Identification of Msp1 as a glycoprotein. (A) SDS-PAGE analysis of LGG's secreted proteins present in spent culture supernatant after overnight growth in AOAC medium. The secreted proteins of LGG WT, msp1 mutant, EPS mutant CMPG5351 and CW-PS mutant CMPG5413 are included. Arrows indicate the major secreted proteins of LGG Msp1 and Msp2 confirmed by Edman degradation and MS/MS. The gels are stained with Sypro® Ruby and ProQ Emerald glycoprotein stain. (B) Comparative analysis of the glycosylation of Msp1 in spent culture supernatant of LGG wild-type before and after treatment with TFMS, and after recombinant expression in E. coli. The msp1 mutant CMPG10200 was included as a control. This mutant expresses a C-terminally truncated form of Msp1 and lacks the enzymatic active NlpC/P60 domain [17]. SDS-PAGE gels were compared by ConA lectin blotting and blotting with Msp1 specific antiserum. (C) Comparison of the sensitivity of recombinant non-glycosylated Msp1 purified from E. coli (rec. Msp1) and native glycosylated Msp1 purified from LGG WT (glyc. Msp1) towards pronase E and proteinase K. Protein samples were incubated for 1 h at 37°C with the indicated diluted preparations of proteases (Sigma). SDS-PAGE gels were stained with Sypro® Ruby (Invitrogen). Images were scanned with Typhoon 9400 (GE Healthcare).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3295695&req=5

Figure 2: Identification of Msp1 as a glycoprotein. (A) SDS-PAGE analysis of LGG's secreted proteins present in spent culture supernatant after overnight growth in AOAC medium. The secreted proteins of LGG WT, msp1 mutant, EPS mutant CMPG5351 and CW-PS mutant CMPG5413 are included. Arrows indicate the major secreted proteins of LGG Msp1 and Msp2 confirmed by Edman degradation and MS/MS. The gels are stained with Sypro® Ruby and ProQ Emerald glycoprotein stain. (B) Comparative analysis of the glycosylation of Msp1 in spent culture supernatant of LGG wild-type before and after treatment with TFMS, and after recombinant expression in E. coli. The msp1 mutant CMPG10200 was included as a control. This mutant expresses a C-terminally truncated form of Msp1 and lacks the enzymatic active NlpC/P60 domain [17]. SDS-PAGE gels were compared by ConA lectin blotting and blotting with Msp1 specific antiserum. (C) Comparison of the sensitivity of recombinant non-glycosylated Msp1 purified from E. coli (rec. Msp1) and native glycosylated Msp1 purified from LGG WT (glyc. Msp1) towards pronase E and proteinase K. Protein samples were incubated for 1 h at 37°C with the indicated diluted preparations of proteases (Sigma). SDS-PAGE gels were stained with Sypro® Ruby (Invitrogen). Images were scanned with Typhoon 9400 (GE Healthcare).
Mentions: Msp1, described previously as the p75 protein [16], is present in abundant amounts in the spent culture supernatant of LGG. Interestingly, this protein appears to have an aberrant migration pattern when analyzed by SDS-PAGE and is detected with a significantly higher molecular mass (70-75 kDa) (Figure 1A) than the expected mass (46.8 kDa) for a protein with a N-terminal secretion signal (Figure 1B-C). Previously, this protein was named (i.e. p75) based on its size on SDS gels. Because of the apparent inconsistency in molecular mass, we have designated this protein according to its relative abundance as Msp1 (as in the Major secreted protein). While our electrophoretic results are strongly suggestive of post-translational modification, part of the observed discrepancy in molecular mass might also be due to the disproportionately high number of alanine residues (~23% content) in the primary structure of this protein (Figure 1C). Alanine has a low SDS-binding capacity and is known for a high helical propensity causing slow gel migration for proteins with high alanine content [18]. However, we found that a msp1 mutant CMPG10200 [17] (Table 1), expressing a truncated form of Msp1 that lacks a 311-residue C-terminal segment encompassing a large proportion of alanines, still migrates with a larger (~45 kDa) (Figure 2B) than expected size (16.4 kDa) (Figure 1C). Contrastingly, the other major secreted protein of LGG (encoded by LGG_00031 ), here renamed as Msp2 but also known previously as p40 [16], has a predicted molecular mass of 39.7 kDa that corresponds well with its observed migration pattern on SDS gels, suggesting that this protein has not undergone major post-translational modifications (Figure 1A).

Bottom Line: Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA.Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre of Microbial and Plant Genetics, K,U,Leuven, Kasteelpark Arenberg 20, box 2460, B-3001 Leuven, Belgium. sarah.lebeer@biw.kuleuven.be

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.

Show MeSH
Related in: MedlinePlus