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  • Selection against glycosylation sites in potential target proteins of the general HMWC N-glycosyltransferase in Haemophilus influenzae

    Author(s)
    Gawthorne, Jayde A
    Tan, Nikki Y
    Bailey, Ulla-Maja
    Davis, Margaret R
    Wong, Linette W
    Naidu, Ranjitha
    Fox, Kate L
    Jennings, Michael P
    Schulz, Benjamin L
    Griffith University Author(s)
    Jennings, Michael P.
    Year published
    2014
    Metadata
    Show full item record
    Abstract
    The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase ...
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    The HMWABC system of non-typeable Haemophilus influenzae (NTHi) encodes the HMWA adhesin glycoprotein, which is glycosylated by the HMWC glycosyltransferase. HMWC is a cytoplasmic N-glycosyltransferase, homologues of which are widespread in the Pasteurellaceae. We developed an assay for nonbiased detection of glycoproteins in NTHi based on metabolic engineering of the Leloir pathway and growth in media containing radiolabelled monosaccharides. The only glycoprotein identified in NTHi by this assay was HMWA. However, glycoproteomic analyses ex vivo in Escherichia coli showed that HMWC of NTHi was a general glycosyltransferase capable of glycosylating selected asparagines in proteins other than its HMWA substrate, including Asn78 in E. coli 30S ribosomal protein S5. The equivalent residue in S5 homologues in H. influenzae or other sequenced Pasteurellaceae genomes is not asparagine, and these organisms also showed significantly fewer than expected potential sites of glycosylation in general. Expression of active HMWC in E. coli resulted in growth inhibition compared with expression of inactive enzyme, consistent with glycosylation by HMWC detrimentally affecting the function of some E. coli proteins. Together, this supports the presence of a selective pressure in the Pasteurellaceae against glycosylation sites that would be modified by the general N-glycosyltransferase activity of HMWC.
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    Journal Title
    Biochemical and Biophysical Research Communications
    Volume
    445
    Issue
    3
    DOI
    https://doi.org/10.1016/j.bbrc.2014.02.044
    Subject
    Medicinal and biomolecular chemistry
    Biochemistry and cell biology
    Bacteriology
    Infectious agents
    Medical biochemistry and metabolomics
    Publication URI
    http://hdl.handle.net/10072/67069
    Collection
    • Journal articles

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