title: Selection against glycosylation sites in potential target proteins of the general HMWC N-glycosyltransferase in Haemophilus influenzae.
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 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.
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia; Institute for Glycomics, Griffith University, Gold Coast Campus, Gold Coast, QLD 4222, Australia.
authors.forename: Jayde A
authors.initials: JA
authors.lastname: Gawthorne
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Nikki Y
authors.initials: NY
authors.lastname: Tan
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Ulla-Maja
authors.initials: UM
authors.lastname: Bailey
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Davis
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Linette W
authors.initials: LW
authors.lastname: Wong
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Ranjitha
authors.initials: R
authors.lastname: Naidu
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Fox
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Gold Coast, QLD 4222, Australia.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
authors.affiliation: Australian Infectious Disease Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia. Electronic address: b.schulz@uq.edu.au.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Schulz
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D006192:Haemophilus Infections / Q000382:microbiology*; D006193:Haemophilus influenzae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D008969:Molecular Sequence Data; D040901:Proteomics
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D006023:Glycoproteins; D005964:Glucosyltransferases; C410788:N-glycosyltransferase, Nocardia aerocolonigenes
keywords: Glycobiology; Leloir pathway; Mass spectrometry; Metabolic engineering; Protein evolution; Proteomics