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Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis.

ABSTRACT: Among bacterial species demonstrated to have protein O-glycosylation systems, that of Bacteroides fragilis and related species is unique in that extracytoplasmic proteins are glycosylated at serine or threonine residues within the specific three-amino acid motif D(S/T)(A/I/L/M/T/V). This feature allows for computational analysis of the proteome to identify candidate glycoproteins. With the criteria of a signal peptidase I or II cleavage site or a predicted transmembrane-spanning region and the presence of at least one glycosylation motif, we identified 1021 candidate glycoproteins of B. fragilis. In addition to the eight glycoproteins identified previously, we confirmed that another 12 candidate glycoproteins are in fact glycosylated. These included four glycoproteins that are predicted to localize to the inner membrane, a compartment not previously shown to include glycosylated proteins. In addition, we show that four proteins involved in cell division and chromosomal segregation, two of which are encoded by candidate essential genes, are glycosylated. To date, we have not identified any extracytoplasmic proteins containing a glycosylation motif that are not glycosylated. Therefore, based on the list of 1021 candidate glycoproteins, it is likely that hundreds of proteins, comprising more than half of the extracytoplasmic proteins of B. fragilis, are glycosylated. Site-directed mutagenesis of several glycoproteins demonstrated that all are glycosylated at the identified glycosylation motif. By engineering glycosylation motifs into a naturally unglycosylated protein, we are able to bring about site-specific glycosylation at the engineered sites, suggesting that this glycosylation system may have applications for glycoengineering.

SUBMITTER: Fletcher CM

PROVIDER: S-EPMC3030326 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis.

Fletcher C Mark CM Coyne Michael J MJ Comstock Laurie E LE

The Journal of biological chemistry 20101129 5

Among bacterial species demonstrated to have protein O-glycosylation systems, that of Bacteroides fragilis and related species is unique in that extracytoplasmic proteins are glycosylated at serine or threonine residues within the specific three-amino acid motif D(S/T)(A/I/L/M/T/V). This feature allows for computational analysis of the proteome to identify candidate glycoproteins. With the criteria of a signal peptidase I or II cleavage site or a predicted transmembrane-spanning region and the p ...[more]

PMID: 21115495

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Project description:Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.

Dataset Information

Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis.

Publications

Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis.

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