Unexpected carbohydrate cross-binding by Escherichia coli heat-labile enterotoxin. Recognition of human and rabbit target cell glycoconjugates in comparison with cholera toxin

Karlsson, Karl‐Anders; Teneberg, Susann; Ångström, Jonas; Kjellberg, Anders; Hirst, Timothy R.; Bergström, Jakob; Miller‐Podraza, Halina

doi:10.1016/s0968-0896(96)00174-5

Cited by 26 publications

(26 citation statements)

References 13 publications

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“…For clarity, the contribution of the Asn 4 C ␤ H 2 group to the van der Waals surface was omitted but makes the side chain fit even tighter. not bind to human small intestinal glycoproteins on blotting membranes, and hLTB binds only after de-sialylation (38), indicating that certain substitutions on the Gal␤4GlcNAc␤6 (Gal␤3GlcNAc␤3)Gal␤ core obliterates the binding.…”

Section: Discussionmentioning

confidence: 99%

Novel Carbohydrate Binding Site Recognizing Blood Group A and B Determinants in a Hybrid of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin B-subunits

Ångström¹,

Bäckström²,

Berntsson³

et al. 2000

Journal of Biological Chemistry

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The B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) are structurally and functionally related. However, the carbohydrate binding specificities of the two proteins differ. While both CTB and LTB bind to the GM1 ganglioside, LTB also binds to N-acetyllactosamine-terminated glycoconjugates. The structural basis of the differences in carbohydrate recognition has been investigated by a systematic exchange of amino acids between LTB and CTB. Thereby, a CTB/LTB hybrid with a gain-of-function mutation resulting in recognition of blood group A and B determinants was obtained. Glycosphingolipid binding assays showed a specific binding of this hybrid Bsubunit, but not CTB or LTB, to slowly migrating nonacid glycosphingolipids of human and animal small intestinal epithelium. A binding-active glycosphingolipid isolated from cat intestinal epithelium was characterized by mass spectrometry and proton NMR as GalNAc␣3(Fuc␣2)Gal␤4(Fuc␣3)GlcNAc␤3Gal␤4Glc NAc␤3Gal␤4Glc␤1Cer. Comparison with reference glycosphingolipids showed that the minimum binding epitope recognized by the CTB/LTB hybrid was Gal␣3(Fuc␣2)Gal␤4(Fuc␣3)GlcNAc␤. The blood group A and B determinants bind to a novel carbohydrate binding site located at the top of the B-subunit interfaces, distinct from the GM1 binding site, as found by docking and molecular dynamics simulations.Enterotoxins produced by Vibrio cholerae and enterotoxigenic Escherichia coli are causative agents of diarrheal diseases leading to millions of deaths annually (1). Both cholera toxin (CT) 1 and E. coli type I heat-labile enterotoxin (LT) are oligomeric proteins with one A-subunit and five B-subunits (2). The A-subunits have ADP-ribosyltransferase activity, while the B-subunits mediate binding to receptors on the eukaryotic cell surface. LT type I B-subunits originating from porcine and human isolates of enterotoxigenic E. coli (pLTB and hLTB, respectively) share 96% sequence identity with each other, but only approximately 80% with CTB (3). Despite great similarity between the carbohydrate binding sites of the B-subunits, evidenced by recent crystal complexes (4 -6), the carbohydrate binding specificities of CTB and LTB differ. Both B-subunits bind with high affinity to GM1 (7), whereas only LTB interacts with N-acetyllactosamine-terminated glycoconjugates (8 -10). Binding of LTB, but not CTB, to gangliotetraosylceramide and the GD1b ganglioside has also been reported (8, 10, 11). The structural basis of the differences in carbohydrate binding between CTB and hLTB have been investigated by construction of a number of CTB/hLTB hybrids, having CTB amino acids substituted with heterologous amino acids of hLTB (12). By introducing hLTB residues in the 1-25 region and at positions 94 and 95 of CTB, a hybrid B-subunit (designated LCTBH) was created, with N-acetyllactosaminebinding properties almost indistinguishable from hLTB.These studies have now been extended by re-substitution of single amino acids of LCTBH back to the original CTB residues. By re-substituting...

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Section: Discussionmentioning

confidence: 99%

Novel Carbohydrate Binding Site Recognizing Blood Group A and B Determinants in a Hybrid of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin B-subunits

Ångström¹,

Bäckström²,

Berntsson³

et al. 2000

Journal of Biological Chemistry

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“…membranes may be similar or identical. The delipidized membrane preparation contains LT receptors of glycoprotein nature (Holmgren et al, 1982) and could also include non-extractable hydrophilic glycolipids, such as PGCs (Karlsson et al, 1996). In contrast, the LTB residues Asn-94 and Ser-95 had no detectable effect on the binding to the acid GSL preparation containing both GM1 and non-GM1 gangliosides.…”

Section: Discussionmentioning

confidence: 99%

“…Both CT and LT bind with high affinity to ganglioside GM1, Gal␤3GalNac␤4(NeuAc␣3)Gal␤4Glc␤1Cer (Holmgren et al, 1973;1975). In addition, LT also binds significantly to glycoprotein receptors present in the intestines of several species (Griffiths and Critchley, 1991;Holmgren et al, 1982;Shida et al, 1996), to polyglycosylceramides (PGCs), complex glycolipids with carbohydrate chains of more than 30 saccharides (Karlsson et al, 1996), and with low affinity to other glycosphingolipids including GM2, asialo-GM1 and neolactotetraosylceramide (paragloboside) (Fukuta et al, 1988;Teneberg et al, 1994). These non-GM1 receptors have been estimated to account for Ϸ50% or as much as 90% of the total binding activity of LT in human or rabbit intestine, respectively (Holmgren et al, 1982;, and, at least in the rabbit, they are of functional significance, since CTB could not inhibit the fluid secretion induced by LT (Holmgren et al, 1982;Pierce, 1973).…”

Section: Introductionmentioning

confidence: 99%

Structural basis for differential receptor binding of cholera and Escherichia coli heat‐labile toxins: influence of heterologous amino acid substitutions in the cholera B‐subunit

Bäckström

Shahabi

Johansson

et al. 1997

Molecular Microbiology

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SummaryThe closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1-25 region were combined with those in the 75-83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33→Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.

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“…Neolactotetraose (NEO) is a secondary receptor present on the surface of small intestinal epithelial cells (Karlsson et al 1996) that may be competing with GM1, the primary receptor of LTB. The crystal structure of NEO in complex with LTB from porcine isolates (pLTB) shows that NEO binds in the primary binding site of the toxin (Holmner et al 2011).…”

Section: Assignment and Data Depositionmentioning

confidence: 99%

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

et al. 2017

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The major virulence factor of enterotoxigenic Escherichia coli (ETEC) is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1 H, 13 C, 15 N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13 C',

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Unexpected carbohydrate cross-binding by Escherichia coli heat-labile enterotoxin. Recognition of human and rabbit target cell glycoconjugates in comparison with cholera toxin

Cited by 26 publications

References 13 publications

Novel Carbohydrate Binding Site Recognizing Blood Group A and B Determinants in a Hybrid of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin B-subunits

Novel Carbohydrate Binding Site Recognizing Blood Group A and B Determinants in a Hybrid of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin B-subunits

Structural basis for differential receptor binding of cholera and Escherichia coli heat‐labile toxins: influence of heterologous amino acid substitutions in the cholera B‐subunit

1H, 13C, 15N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

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