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.
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: van der Post
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden.
authors.forename: Durai B
authors.initials: DB
authors.lastname: Subramani
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Bäckström
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden.
authors.forename: Malin E V
authors.initials: MEV
authors.lastname: Johansson
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and Odontology, University of Copenhagen, DK 2200 Copenhagen, Denmark.
authors.forename: Malene B
authors.initials: MB
authors.lastname: Vester-Christensen
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and Odontology, University of Copenhagen, DK 2200 Copenhagen, Denmark.
authors.forename: Ulla
authors.initials: U
authors.lastname: Mandel
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and Odontology, University of Copenhagen, DK 2200 Copenhagen, Denmark.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Bennett
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine and Odontology, University of Copenhagen, DK 2200 Copenhagen, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.affiliation: Department of Oral Microbiology, University of Gothenburg, 405 30 Gothenburg, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dahlén
authors.affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Sroka
authors.affiliation: Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; Oral Health and Systemic Diseases Group, University of Louisville School of Dentistry, Louisville, Kentucky 40202.
authors.forename: Jan
authors.initials: J
authors.lastname: Potempa
authors.affiliation: Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden. Electronic address: gunnar.hansson@medkem.gu.se.
authors.forename: Gunnar C
authors.initials: GC
authors.lastname: Hansson
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002845:Chromatography; D003092:Colitis / Q000382:microbiology; D003106:Colon / Q000378:metabolism; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D000080867:Gingipain Cysteine Endopeptidases; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D055262:Mucin-2 / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000201:enzymology*
chemical_list: D018829:Adhesins, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D055262:Mucin-2; D003546:Cysteine Endopeptidases
keywords: Bacterial Pathogenesis; Glycoprotein Biosynthesis; Glycosyltransferases; Mucins; Mucus
references.citation: J Biol Chem. 1994 Jan 7;269(1):406-11
references.pmid: 8276827
references.citation: J Proteome Res. 2009 May;8(5):2418-25
references.pmid: 19290618
references.citation: Biochem J. 2003 Jun 1;372(Pt 2):335-45
references.pmid: 12597771
references.citation: Glycobiology. 2012 Jun;22(6):736-56
references.pmid: 22183981
references.citation: Glycobiology. 1999 Jan;9(1):43-52
references.pmid: 9884405
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5645-50
references.pmid: 22451922
references.citation: Genome Biol. 2012 Jun 14;13(6):R42
references.pmid: 22698087
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 15;108 Suppl 1:4659-65
references.pmid: 20615996
references.citation: In Vitro Cell Dev Biol. 1987 Jun;23(6):436-42
references.pmid: 3597283
references.citation: J Biol Chem. 1993 Sep 5;268(25):18771-81
references.pmid: 8360170
references.citation: Anal Biochem. 1999 Mar 15;268(2):305-17
references.pmid: 10075821
references.citation: Am J Med Genet A. 2010 Apr;152A(4):896-903
references.pmid: 20358599
references.citation: Biochem J. 2011 May 15;436(1):61-70
references.pmid: 21338337
references.citation: Nat Biotechnol. 2011 Jul 31;29(8):735-41
references.pmid: 21804562
references.citation: Benef Microbes. 2010 Nov;1(4):343-50
references.pmid: 21831773
references.citation: Mol Cell Proteomics. 2010 Feb;9(2):415-26
references.pmid: 19875382
references.citation: Periodontol 2000. 2010 Oct;54(1):15-44
references.pmid: 20712631
references.citation: Curr Protein Pept Sci. 2003 Dec;4(6):397-407
references.pmid: 14683426
references.citation: PLoS Pathog. 2010 May 13;6(5):e1000902
references.pmid: 20485566
references.citation: FEMS Microbiol Lett. 2007 May;270(1):75-81
references.pmid: 17439635
references.citation: Glycobiology. 2001 Oct;11(10):821-9
references.pmid: 11588158
references.citation: J Biol Chem. 1998 Aug 21;273(34):21648-57
references.pmid: 9705298
references.citation: J Periodontal Res. 1999 Nov;34(8):464-72
references.pmid: 10697803
references.citation: J Biol Chem. 2010 Nov 19;285(47):36293-303
references.pmid: 20837471
references.citation: J Clin Invest. 2011 Apr;121(4):1657-66
references.pmid: 21383503
references.citation: J Biochem. 1998 Feb;123(2):305-12
references.pmid: 9538207
references.citation: J Biol Chem. 1997 Sep 19;272(38):23503-14
references.pmid: 9295285
references.citation: J Biol Chem. 1998 Jul 24;273(30):18857-63
references.pmid: 9668061
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 30;105(39):15064-9
references.pmid: 18806221
references.citation: Science. 2003 Mar 28;299(5615):2074-6
references.pmid: 12663928
references.citation: Proc Natl Acad Sci U S A. 2009 Aug 4;106(31):12921-5
references.pmid: 19617566
references.citation: Nature. 2010 Mar 4;464(7285):59-65
references.pmid: 20203603
references.citation: J Biol Chem. 2003 Apr 18;278(16):13944-51
references.pmid: 12582180
references.citation: Cell Mol Life Sci. 2011 Nov;68(22):3635-41
references.pmid: 21947475
references.citation: Science. 2005 Mar 25;307(5717):1915-20
references.pmid: 15790844
references.citation: J Biol Chem. 2006 Jul 7;281(27):18370-7
references.pmid: 16638743
references.citation: J Biol Chem. 2002 Dec 6;277(49):47248-56
references.pmid: 12374796
references.citation: Nat Methods. 2009 May;6(5):359-62
references.pmid: 19377485
references.citation: Nature. 2011 May 12;473(7346):174-80
references.pmid: 21508958
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9298-303
references.pmid: 16754877
references.citation: J Biol Chem. 2011 Nov 18;286(46):40122-32
references.pmid: 21937429