Logan SM, Hui JP, Vinogradov E, Aubry AJ, Melanson JE, Kelly JF, Nothaft H, Soo EC Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches FEBS Journal276(4) (2009)
1014-1023
The structure was elucidated in this paper NCBI PubMed ID:19154343 Publication DOI:10.1111/j.1742-4658.2008.06840.x Journal NLM ID:101229646 Publisher: Blackwell Publishing Correspondence: evelyn.soonrc-cnrc.gc.ca Institutions: NRC-Institute for Biological Sciences, Ottawa, Canada, NRC-Institute for Marine Biosciences, Halifax, Canada
It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.
Logan SM, Hui JP, Vinogradov E, Aubry AJ, Melanson JE, Kelly JF, Nothaft H, Soo EC Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches FEBS Journal276(4) (2009)
1014-1023
The structure was elucidated in this paper NCBI PubMed ID:19154343 Publication DOI:10.1111/j.1742-4658.2008.06840.x Journal NLM ID:101229646 Publisher: Blackwell Publishing Correspondence: evelyn.soonrc-cnrc.gc.ca Institutions: NRC-Institute for Biological Sciences, Ottawa, Canada, NRC-Institute for Marine Biosciences, Halifax, Canada
It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.
Lees-Miller RG, Iwashkiw JA, Scott NE, Seper A, Vinogradov E, Schild S, Feldman MF A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii Molecular Microbiology89(5) (2013)
816-830
The structure was elucidated in this paper NCBI PubMed ID:23782391 Publication DOI:10.1111/mmi.12300 Journal NLM ID:8712028 Publisher: Blackwell Publishing Correspondence: mfeldmanualberta.ca Institutions: Alberta Glycomics Centre, Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada
Multi-drug resistant strains of Acinetobacter baumannii are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general O-glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the O-pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of PglC, the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of A. baumannii 17978 capsular polysaccharide via NMR, demonstrated that the pentasaccharides that decorate the glycoproteins are also the building blocks for capsule biosynthesis. Two linked subunits, but not longer glycan chains, were detected on proteins via MS. The discovery of a bifurcated pathway for O-glycosylation and capsule synthesis not only provides insight into the biology of A. baumannii but also identifies potential novel candidates for intervention against this emerging pathogen.
Gargano AFG, Schouten O, van Schaick G, Roca LS, van den Berg-Verleg JH, Haselberg R, Akeroyd M, Abello N, Somsen GW Profiling of a high mannose-type N-glycosylated lipase using hydrophilic interaction chromatography-mass spectrometry Analytica Chimica Acta1109 (2020)
69-77
NCBI PubMed ID:32252907 Publication DOI:10.1016/j.aca.2020.02.042 Journal NLM ID:0370534 Correspondence: a.garganouva.nl Institutions: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands, Centre for Analytical Sciences Amsterdam, Amsterdam, The Netherlands, Division of BioAnalytical Chemistry, Amsterdam Institute for Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands, van‘t Hoff Institute for Molecular Science, University of Amsterdam, Amsterdam, The Netherlands, DSM Biotechnology Center, Analysis Department, Delft, The Netherlands
Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.
high-resolution mass spectrometry, HILIC, high mannose-type N-glycosylation, intact protein analysis
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]; infection due to Campylobacter jejuni [ICD11: XN4Q5 
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