Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

Sjoerd van der Post; Durai B Subramani; Malin Bäckström; Malin E V Johansson; Malene B Vester-Christensen; Ulla Mandel; Eric P Bennett; Henrik Clausen; Gunnar Dahlén; Aneta Sroka; Jan Potempa; Gunnar C Hansson

doi:10.1074/jbc.M113.459479

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

Sjoerd van der Post, Durai B Subramani, Malin Bäckström, Malin E V Johansson, Malene B Vester-Christensen, Ulla Mandel, Eric P Bennett, Henrik Clausen, Gunnar Dahlén, Aneta Sroka, Jan Potempa, Gunnar C Hansson

49 Citations (Scopus)

Abstract

The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.

Original language	English
Journal	The Journal of Biological Chemistry
Volume	288
Issue number	20
Pages (from-to)	14636-46
Number of pages	11
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M113.459479
Publication status	Published - 17 May 2013

Access to Document

10.1074/jbc.M113.459479

Cite this

van der Post, S., Subramani, D. B., Bäckström, M., Johansson, M. E. V., Vester-Christensen, M. B., Mandel, U., Bennett, E. P., Clausen, H., Dahlén, G., Sroka, A., Potempa, J., & Hansson, G. C. (2013). Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB). The Journal of Biological Chemistry, 288(20), 14636-46. https://doi.org/10.1074/jbc.M113.459479

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB). / van der Post, Sjoerd; Subramani, Durai B; Bäckström, Malin et al.

In: The Journal of Biological Chemistry, Vol. 288, No. 20, 17.05.2013, p. 14636-46.

Research output: Contribution to journal › Journal article › Research › peer-review

van der Post, S, Subramani, DB, Bäckström, M, Johansson, MEV, Vester-Christensen, MB, Mandel, U , Bennett, EP , Clausen, H, Dahlén, G, Sroka, A, Potempa, J & Hansson, GC 2013, 'Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)', The Journal of Biological Chemistry, vol. 288, no. 20, pp. 14636-46. https://doi.org/10.1074/jbc.M113.459479

@article{09d140663d12442eaea9aa5fb68182c3,

title = "Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)",

abstract = "The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.",

author = "{van der Post}, Sjoerd and Subramani, {Durai B} and Malin B{\"a}ckstr{\"o}m and Johansson, {Malin E V} and Vester-Christensen, {Malene B} and Ulla Mandel and Bennett, {Eric P} and Henrik Clausen and Gunnar Dahl{\'e}n and Aneta Sroka and Jan Potempa and Hansson, {Gunnar C}",

year = "2013",

month = may,

day = "17",

doi = "10.1074/jbc.M113.459479",

language = "English",

volume = "288",

pages = "14636--46",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

number = "20",

}

TY - JOUR

T1 - Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

AU - van der Post, Sjoerd

AU - Subramani, Durai B

AU - Bäckström, Malin

AU - Johansson, Malin E V

AU - Vester-Christensen, Malene B

AU - Mandel, Ulla

AU - Bennett, Eric P

AU - Clausen, Henrik

AU - Dahlén, Gunnar

AU - Sroka, Aneta

AU - Potempa, Jan

AU - Hansson, Gunnar C

PY - 2013/5/17

Y1 - 2013/5/17

N2 - The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.

AB - The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.

U2 - 10.1074/jbc.M113.459479

DO - 10.1074/jbc.M113.459479

M3 - Journal article

C2 - 23546879

SN - 0021-9258

VL - 288

SP - 14636

EP - 14646

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 20

ER -

Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

Abstract

Access to Document

Fingerprint

Cite this