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Site-specific O-glycosylation on the MUC2 mucin protein inhibits cleavage by the Porphyromonas gingivalis secreted cysteine protease (RgpB).

Artikel i vetenskaplig tidskrift
Författare 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
Publicerad i The Journal of biological chemistry
Volym 288
Nummer/häfte 20
Sidor 14636-46
ISSN 1083-351X
Publiceringsår 2013
Publicerad vid Institutionen för odontologi
Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
Sidor 14636-46
Språk en
Länkar dx.doi.org/10.1074/jbc.M113.459479
Ämnesord Adhesins, Bacterial, metabolism, Amino Acid Sequence, Animals, CHO Cells, Chromatography, Colitis, microbiology, Colon, metabolism, Cricetinae, Cysteine Endopeptidases, metabolism, Epithelium, metabolism, Glycosylation, Humans, Mass Spectrometry, Molecular Sequence Data, Mucin-2, metabolism, Porphyromonas gingivalis, enzymology
Ämneskategorier Medicinska grundvetenskaper

Sammanfattning

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.

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