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Taxonomic group: bacteria / Proteobacteria (Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11: XN6P4

]

The structure was elucidated in this paper
NCBI PubMed ID: 23000215
Publication DOI: 10.1016/j.carres.2012.05.015
Journal NLM ID: 0043535
Publisher: Elsevier
Correspondence: S. Drouillard sophie.drouillard

cermav.cnrs.fr
Institutions: Centre de recherche sur Macromolecules Vegetales (CERMAV-CNRS), BP 53, F-38041 Grenoble Cedex 9, France

The α-Gal epitope is a carbohydrate structure, Galα-3Galβ-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galβ-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galβ-4GlcNAcβ-3Galβ-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for β-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for β-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galβ-4GlcNAcβ-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific β-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.

Escherichia coli, Oligosaccharides, fermentation, Metabolic engineering, α-Gal epitope, a-Gal epitope

Structure type: oligomer
Location inside paper: p.89, fig.6, (13)
Contained glycoepitopes: IEDB_115013,IEDB_130645,IEDB_130646,IEDB_130649,IEDB_130655,IEDB_130697,IEDB_135813,IEDB_135815,IEDB_136044,IEDB_136906,IEDB_137340,IEDB_137472,IEDB_137776,IEDB_140108,IEDB_140110,IEDB_140122,IEDB_141496,IEDB_141794,IEDB_141807,IEDB_142487,IEDB_142488,IEDB_144998,IEDB_146664,IEDB_146694,IEDB_149558,IEDB_150939,IEDB_151528,IEDB_151531,IEDB_158549,IEDB_158550,IEDB_190606,IEDB_418918,IEDB_689191,IEDB_918314,IEDB_983931,SB_145,SB_165,SB_166,SB_173,SB_187,SB_192,SB_195,SB_30,SB_40,SB_6,SB_7,SB_87,SB_88

Methods: 13C NMR, 1H NMR, TLC, ESI-MS, chemical methods, genetic methods
Enzymes that release or process the structure: JHP1032 (β-3GlcNAcT), β-1,4-galactosyltransferase (recombinant β-4GalT), α-1,3-galactosyltransferase (bovine α-3GalT)
Synthetic data: enzymatic
Comments, role: genetically engineered strain Escherichia coli

Related record ID(s): 29178, 29490, 29491, 29492, 29493, 29494, 29495, 29496, 29497, 29498, 29499, 29500, 29501, 29502, 29503, 29504, 29505, 29506, 29507, 29508, 29509, 29510, 29511, 29512, 29514
NCBI Taxonomy refs (TaxIDs): 562
Reference(s) to other database(s): GTC:G38300KM
Show glycosyltransferases

NMR conditions: in D2O at 353 K [as TSV]

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,4,3,4,3,4,3,4,3,4,3	aDGalp	96.68	?	?	?	72.15	62.25
4,3,4,3,4,3,4,3,4,3,4	bDGalp	104.09	?	79.02	66.32	?	62.12
4,3,4,3,4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.26	?	61.52
4,3,4,3,4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.5
4,3,4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.5
4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,2	Ac	175.84	23.43
4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.52
4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,2	Ac	175.84	23.43
4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.52
4	bDGalp	104.09	?	83.19	?	?	62.07
	aDGlcp	93.05	?	?	80.10	?	61.66


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,4,3,4,3,4,3,4,3,4,3	aDGalp	5.18	3.92	?	4.05	4.23	?
4,3,4,3,4,3,4,3,4,3,4	bDGalp	4.58	3.72	?	4.22	?	?
4,3,4,3,4,3,4,3,4,3,2	Ac	-	2.06
4,3,4,3,4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,4,3,4,3,2	Ac	-	2.06
4,3,4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,4,3,2	Ac	-	2.06
4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,2	Ac	-	2.06
4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,2	Ac	-	2.06
4,3	bDGlcpN	4.73	3.80	?	?	?	?
4	bDGalp	4.47	3.66	?	4.18	?	?
	aDGlcp	5.25	3.61	?	?	?	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,4,3,4,3,4,3,4,3,4,3	aDGalp	96.68/5.18	?/3.92	?/?	?/4.05	72.15/4.23	62.25/?
4,3,4,3,4,3,4,3,4,3,4	bDGalp	104.09/4.58	?/3.72	79.02/?	66.32/4.22	?/?	62.12/?
4,3,4,3,4,3,4,3,4,3,2	Ac		23.43/2.06
4,3,4,3,4,3,4,3,4,3	bDGlcpN	103.49/4.73	56.61/3.80	?/?	80.26/?	?/?	61.52/?
4,3,4,3,4,3,4,3,4	bDGalp	104.09/4.50	?/3.66	83.19/?	?/4.18	?/?	62.07/?
4,3,4,3,4,3,4,3,2	Ac		23.43/2.06
4,3,4,3,4,3,4,3	bDGlcpN	103.49/4.73	56.61/3.80	?/?	80.10/?	?/?	61.5/?
4,3,4,3,4,3,4	bDGalp	104.09/4.50	?/3.66	83.19/?	?/4.18	?/?	62.07/?
4,3,4,3,4,3,2	Ac		23.43/2.06
4,3,4,3,4,3	bDGlcpN	103.49/4.73	56.61/3.80	?/?	80.10/?	?/?	61.5/?
4,3,4,3,4	bDGalp	104.09/4.50	?/3.66	83.19/?	?/4.18	?/?	62.07/?
4,3,4,3,2	Ac		23.43/2.06
4,3,4,3	bDGlcpN	103.49/4.73	56.61/3.80	?/?	80.10/?	?/?	61.52/?
4,3,4	bDGalp	104.09/4.50	?/3.66	83.19/?	?/4.18	?/?	62.07/?
4,3,2	Ac		23.43/2.06
4,3	bDGlcpN	103.49/4.73	56.61/3.80	?/?	80.10/?	?/?	61.52/?
4	bDGalp	104.09/4.47	?/3.66	83.19/?	?/4.18	?/?	62.07/?
	aDGlcp	93.05/5.25	?/3.61	?/?	80.10/?	?/?	61.66/?

¹H NMR data:

Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,4,3,4,3,4,3,4,3,4,3	aDGalp	5.18	3.92	?	4.05	4.23	?
4,3,4,3,4,3,4,3,4,3,4	bDGalp	4.58	3.72	?	4.22	?	?
4,3,4,3,4,3,4,3,4,3,2	Ac		2.06
4,3,4,3,4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,4,3,4,3,2	Ac		2.06
4,3,4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,4,3,2	Ac		2.06
4,3,4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,4,3,2	Ac		2.06
4,3,4,3	bDGlcpN	4.73	3.80	?	?	?	?
4,3,4	bDGalp	4.50	3.66	?	4.18	?	?
4,3,2	Ac		2.06
4,3	bDGlcpN	4.73	3.80	?	?	?	?
4	bDGalp	4.47	3.66	?	4.18	?	?
	aDGlcp	5.25	3.61	?	?	?	?

¹³C NMR data:

Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,4,3,4,3,4,3,4,3,4,3	aDGalp	96.68	?	?	?	72.15	62.25
4,3,4,3,4,3,4,3,4,3,4	bDGalp	104.09	?	79.02	66.32	?	62.12
4,3,4,3,4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.26	?	61.52
4,3,4,3,4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.5
4,3,4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,4,3,2	Ac	175.84	23.43
4,3,4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.5
4,3,4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,4,3,2	Ac	175.84	23.43
4,3,4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.52
4,3,4	bDGalp	104.09	?	83.19	?	?	62.07
4,3,2	Ac	175.84	23.43
4,3	bDGlcpN	103.49	56.61	?	80.10	?	61.52
4	bDGalp	104.09	?	83.19	?	?	62.07
	aDGlcp	93.05	?	?	80.10	?	61.66

The spectrum also has 33 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

CC(=O)N[C@H]1[C@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](CO)O[C@H](O)[C@H](O)[C@H]3O)[C@@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@@H]3O[C@H](CO)[C@@H](O[C@@H]4O[C@H](CO)[C@H](O)[C@H](O[C@@H]5O[C@H](CO)[C@@H](O[C@@H]6O[C@H](CO)[C@H](O)[C@H](O[C@@H]7O[C@H](CO)[C@@H](O[C@@H]8O[C@H](CO)[C@H](O)[C@H](O[C@@H]9O[C@H](CO)[C@@H](O[C@@H]%10O[C@H](CO)[C@H](O)[C@H](O[C@H]%11O[C@H](CO)[C@H](O)[C@H](O)[C@H]%11O)[C@H]%10O)[C@H](O)[C@H]9NC(C)=O)[C@H]8O)[C@H](O)[C@H]7NC(C)=O)[C@H]6O)[C@H](O)[C@H]5NC(C)=O)[C@H]4O)[C@H](O)[C@H]3NC(C)=O)[C@H]2O)[C@@H]1O

SVG MW SMILES 3D mol Ionize