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Taxonomic group: bacteria / Firmicutes (Phylum: Firmicutes)
Host organism: Homo sapiens
Associated disease: diarrhea [ICD11: ME05.1

, ICD11: SA55

]; infection due to Clostridium difficile [ICD11: XN0SE

]

The structure was elucidated in this paper
Publication DOI: 10.1074/jbc.M116.749481
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: a.dell

imperial.ac.uk
Institutions: BioPharmaSpec, Suite 3.1 Lido Medical Centre, St. Saviours Road, Jersey JE2 7LA, United Kingdom, Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa, Ontario K1A 0R6, Canada, Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, United Kingdom, Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

Glycosylation of flagellins is a well recognized property of many bacterial species. In this study, we describe the structural characterization of novel flagellar glycans from a number of hypervirulent strains of C. difficile We used mass spectrometry (nano-LC-MS and MS/MS analysis) to identify a number of putative glycopeptides that carried a variety of glycoform substitutions, each of which was linked through an initial N-acetylhexosamine residue to Ser or Thr. Detailed analysis of a LLDGSSTEIR glycopeptide released by tryptic digestion, which carried two variant structures, revealed that the glycopeptide contained, in addition to carbohydrate moieties, a novel structural entity. A variety of electrospray-MS strategies using Q-TOF technology were used to define this entity, including positive and negative ion collisionally activated decomposition MS/MS, which produced unique fragmentation patterns, and high resolution accurate mass measurement to allow derivation of atomic compositions, leading to the suggestion of a taurine-containing peptidylamido-glycan structure. Finally, NMR analysis of flagellin glycopeptides provided complementary information. The glycan portion of the modification was assigned as α-Fuc3N-(1→3)-α-Rha-(1→2)-α-Rha3OMe-(1→3)-β-GlcNAc-(1→)Ser , and the novel capping moiety was shown to be comprised of taurine, alanine, and glycine. This is the first report of a novel O-linked sulfonated peptidylamido-glycan moiety decorating a flagellin protein.

bacteria, modification, glycosylation, gram-positive bacteria, Flagellin, Clostridium difficile, nuclear magnetic resonance (NMR), mass spectrometry (MS), sulfonated

Structure type: oligomer
Location inside paper: p.25444, fig.5, structure 3, table 3
Trivial name: flagellin glycopeptide
Compound class: glycopeptide
Contained glycoepitopes: IEDB_133754,IEDB_135813,IEDB_135849,IEDB_136105,IEDB_137340,IEDB_137477,IEDB_1394181,IEDB_141807,IEDB_151531,IEDB_225177,IEDB_885823

Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, ESI-MS, composition analysis, CID-MS/MS, GPC, enzymatic digestion, LC-MS/MS, GF
Comments, role: glycan fraction was obtained by digestion flagellin protein with proteinase K. Substitution position in Thr was assumed

Related record ID(s): 11524, 11860
NCBI Taxonomy refs (TaxIDs): 645463
Show glycosyltransferases

NMR conditions: in D2O at 298 K [as TSV]

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2,3	Me	57.2-57.7
3,3,2	a?Rhap	103.2	67.0	80.5	72.2	70.1	18.2
3,3,3	Me	57.2-57.7
3,3	a?Rhap	100.9	76.5	80.4	72.2	70.3	17.6
3,2	Ac	175.8	23.7
3	b?GlcpN	100.2	56.8	83.0	69.7	77.0	62.0
	x?Thr	?	59.3	75.9	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2,3	Me	3.44-3.45
3,3,2	a?Rhap	4.89	4.27	3.45	3.46	3.75	1.29
3,3,3	Me	3.44-3.45
3,3	a?Rhap	5.09	3.99	3.56	3.51	4.01	1.25
3,2	Ac	-	2.11
3	b?GlcpN	4.62	3.79	3.60	3.50	3.45	3.75-3.92
	x?Thr	-	4.53	4.32	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2,3	Me	57.2-57.7/3.44-3.45
3,3,2	a?Rhap	103.2/4.89	67.0/4.27	80.5/3.45	72.2/3.46	70.1/3.75	18.2/1.29
3,3,3	Me	57.2-57.7/3.44-3.45
3,3	a?Rhap	100.9/5.09	76.5/3.99	80.4/3.56	72.2/3.51	70.3/4.01	17.6/1.25
3,2	Ac		23.7/2.11
3	b?GlcpN	100.2/4.62	56.8/3.79	83.0/3.60	69.7/3.50	77.0/3.45	62.0/3.75-3.92
	x?Thr		59.3/4.53	75.9/4.32	?/?

¹H NMR data:

Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2,3	Me	3.44 3.45
3,3,2	a?Rhap	4.89	4.27	3.45	3.46	3.75	1.29
3,3,3	Me	3.44 3.45
3,3	a?Rhap	5.09	3.99	3.56	3.51	4.01	1.25
3,2	Ac		2.11
3	b?GlcpN	4.62	3.79	3.60	3.50	3.45	3.75 3.92
	x?Thr		4.53	4.32	?

¹³C NMR data:

Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2,3	Me	57.2 57.7
3,3,2	a?Rhap	103.2	67.0	80.5	72.2	70.1	18.2
3,3,3	Me	57.2 57.7
3,3	a?Rhap	100.9	76.5	80.4	72.2	70.3	17.6
3,2	Ac	175.8	23.7
3	b?GlcpN	100.2	56.8	83.0	69.7	77.0	62.0
	x?Thr	?	59.3	75.9	?

The spectrum also has 2 signals at unknown positions (not plotted).

There are 16 chemically distinct structures. Please, select:

CO[C@@H]1[C@H](O)[C@@H](O[C@H]2[C@@H](OC)[C@H](O)[C@@H](C)O[C@@H]2O[C@H]2[C@H](O)[C@@H](CO)O[C@@H](O[C@@H](C)[C@@H](N)C(=O)O)[C@@H]2NC(C)=O)O[C@H](C)[C@H]1O

SVG MW SMILES 3D mol Ionize