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Open AccessArticle

Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography

1
Department of Chemistry, University of Oslo, Postbox 1033 Blindern, 0315 Oslo, Norway
2
Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
3
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana; Slovenia
*
Author to whom correspondence should be addressed.
Current address: Department of Pharmacy, University of Oslo, Postbox 1068 Blindern, 0316 Oslo, Norway.
Current address: Lek d.d., Kolodvorska 27, 1234 Mengeš, Slovenia.
Int. J. Mol. Sci. 2019, 20(3), 703; https://doi.org/10.3390/ijms20030703
Received: 28 January 2019 / Accepted: 31 January 2019 / Published: 6 February 2019
Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of mortality in children under five years of age and is a great burden on developing countries. The major virulence factor of the bacterium is the heat-labile enterotoxin (LT), a close homologue of the cholera toxin. The toxins bind to carbohydrate receptors in the gastrointestinal tract, leading to toxin uptake and, ultimately, to severe diarrhea. Previously, LT from human- and porcine-infecting ETEC (hLT and pLT, respectively) were shown to have different carbohydrate-binding specificities, in particular with respect to N-acetyllactosamine-terminating glycosphingolipids. Here, we probed 11 single-residue variants of the heat-labile enterotoxin with surface plasmon resonance spectroscopy and compared the data to the parent toxins. In addition we present a 1.45 Å crystal structure of pLTB in complex with branched lacto-N-neohexaose (Galβ4GlcNAcβ6[Galβ4GlcNAcβ3]Galβ4Glc). The largest difference in binding specificity is caused by mutation of residue 94, which links the primary and secondary binding sites of the toxins. Residue 95 (and to a smaller extent also residues 7 and 18) also contribute, whereas residue 4 shows no effect on monovalent binding of the ligand and may rather be important for multivalent binding and avidity. View Full-Text
Keywords: bacterial toxin; cholera toxin; Escherichia coli heat-labile enterotoxin; lectin; N-acetyllactosamine binding; neutral glycosphingolipids; protein–carbohydrate interactions; surface plasmon resonance spectroscopy; X-ray crystal structure bacterial toxin; cholera toxin; Escherichia coli heat-labile enterotoxin; lectin; N-acetyllactosamine binding; neutral glycosphingolipids; protein–carbohydrate interactions; surface plasmon resonance spectroscopy; X-ray crystal structure
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MDPI and ACS Style

Heggelund, J.E.; Heim, J.B.; Bajc, G.; Hodnik, V.; Anderluh, G.; Krengel, U. Specificity of Escherichia coli Heat-Labile Enterotoxin Investigated by Single-Site Mutagenesis and Crystallography. Int. J. Mol. Sci. 2019, 20, 703.

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