A novel mass spectrometric strategy "BEMAP" reveals Extensive O-linked protein glycosylation in Enterotoxigenic Escherichia coli.

Anders Boysen,Martin R Larsen,Giuseppe Palmisano,Jakob Møller-Jensen,Thøger Jensen Krogh,Iain G Duggin

doi:10.1038/srep32016

Abstract

The attachment of sugars to proteins via side-chain oxygen atoms (O-linked glycosylation) is seen in all three domains of life. However, a lack of widely-applicable analytical tools has restricted the study of this process, particularly in bacteria. In E. coli, only four O-linked glycoproteins have previously been characterized. Here we present a glycoproteomics technique, termed BEMAP, which is based on the beta-elimination of O-linked glycans followed by Michael-addition of a phosphonic acid derivative, and subsequent titanium dioxide enrichment. This strategy allows site-specific mass-spectrometric identification of proteins with O-linked glycan modifications in a complex biological sample. Using BEMAP we identified cell surface-associated and membrane vesicle glycoproteins from Enterotoxigenic E. coli (ETEC) and non-pathogenic E. coli K-12. We identified 618 glycosylated Serine and Threonine residues mapping to 140 proteins in ETEC, including several known virulence factors, and 34 in E. coli K-12. The two strains had 32 glycoproteins in common. Remarkably, the majority of the ETEC glycoproteins were conserved in both strains but nevertheless were only glycosylated in the pathogen. Therefore, bacterial O-linked glycosylation is much more extensive than previously thought, and is especially important to the pathogen.

Highlights

Enterotoxigenic Escherichia coli (ETEC) is the major source of E. coli mediated diarrhoea in humans and livestock[1]
Our method, termed BEMAP, represents a refinement of a previous β-elimination/Michael addition experimental strategy originally described for phosphorylated peptides and O-GlcNAcylated peptides[26]
The novelty of BEMAP lies in the use of 2-Aminoethyl phosphonic acid (AEP) for nucleophilic peptide tagging

Summary

Results and Discussion

BEMAP enables selective and efficient enrichment of O-linked glycopeptides. The intimate coupling between bacterial protein glycosylation and pathophysiology found in several species[6] prompted us to identify novel glycosylated proteins in ETEC strain H10407. The outer membrane protein fractions of H10407 and MG1655 were isolated using exactly the same outer membrane- and TiO2 enrichment protocol as for phosphopeptide purification and subjected to BEMAP analysis for site specific identification of O-linked glycosylation sites This identified a total of 547 glycosylated residues (Fig. 2A) which could be assigned to 127 proteins (Fig. 2B). Out of the 93 glycoproteins identified in the ETEC sample only seven proteins were uniquely expressed in the pathogen (Fig. 2B), implying that ETEC O-glycosylates its protein to a much higher extent than to the non-pathogen, just as tyrosine phosphorylation has been proposed to be in EHEC34 This indicates that post-translational modifications (PTMs) could play a role in the pathophysiology of E. coli. The combined list revealed that 84 H10407 FliC Ser/Thr residues out of 100 possible were modified This extent of modification surpasses any reported number of O-linked glycosylation sites on a single protein in bacteria[6,44]. It remains to be determined which glycosyltransferase recognises the individual motifs

Conclusion

Author Contributions

Additional Information