Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin

Leong, Jenny; Vinal, A C; Dallas, Walter S.

doi:10.1128/iai.48.1.73-77.1985

Cited by 77 publications

(32 citation statements)

References 41 publications

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“…Human infectious strain 240-3 also has Arg 13 (Tsuji et al ., 1987). However, Leong et al (1985) found this codon to be CAC (His) in E. coli strain H74-114, which is infectious for humans and was isolated in the United States. This matches the residue His 13…”

Section: Structural Basis Of Binding Specifcitymentioning

confidence: 99%

“…found at this position in CT and hence in the present structure. Leong et al (1985) proposed that the LT variants are evolutionarily related, with a common ancestral sequence most similar to that of strain H10407. This strain-specific sequence variability at residue 13 may be crucial to analysis of the reported differences in saccharide binding specificity between CT and LT.…”

Section: "mentioning

confidence: 99%

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Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide

et al. 1994

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Cholera toxin (CT) is an AB, hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM, on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GMI pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GMI to cholera toxin thus resembles a 2-fingered grip: the Gal(P1-3)CalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTBs:GMl pentasaccharide complex described here provides a detailed view of a pr0tein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in OM,-mediated signal transduction.Keywords: cholera toxin; crystal structure; ganglioside GM1; sugar binding specificity Cholera is a severe disease that can lead to death within a few hours. The major clinical symptoms are caused by a toxin released after adhesion of the noninvasive Vibrio cholerue bacteria to the proximal small intestine of the host. Cholera toxin (CT) acts intracellularly to catalyze ADP-ribosylation of residue Arg 187 in the a subunit of the trimeric protein G,. The modified G,, loses its GTPase activity and remains constitutively in its GTP-bound state (Cassel & Pfeuffer, 1978), which in turn causes a continuous stimulation of adenylate cyclase. The resulting elevated levels of cyclic AMP lead to massive loss of fluids, the characteristic pathology of enterotoxigenic disease.Cholera toxin is an AB, hexamer consisting of 5 identical B subunits and a single A subunit. It is structurally and functionally related to a larger group of bacterial enterotoxins that includes the closely related Escherichia coli heat-labile enterotoxin (LT) as well as pertussis toxin, diphtheria toxin, shigella toxin, and Pseudomonas aeruginosa exotoxin A. In this class of tox- ins, the biological functions of target cell recognition and enzymatic activity are separated into distinct domains. In the case of CT and LT, cell recognition and binding are carried out by the ...

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Section: Structural Basis Of Binding Specifcitymentioning

confidence: 99%

Section: "mentioning

confidence: 99%

Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide

et al. 1994

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“…The two forms of the heat-labile enterotoxin, pLT and hLT, differ slightly in primary sequence, with the sequence of the humanderived toxin being marginally more similar to that of cholera toxin (Leong et al, 1985). In particular, the human-derived (hLTB) and porcine-derived (pLTB) B-subunits sequences differ at 4 amino acid positions, of 103 (Domenighini et al, 1995).…”

Section: Comparison Of Ltb Derived From Human and Porcine Strains Of mentioning

confidence: 99%

Structural studies of receptor binding by cholera toxin mutants

et al. 1997

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The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain.We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33 -+ Arg complexed with the GMI oligosaccharide, as well as the 2.2-A refinement of a Gly 33 -+ Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33 -+ Arg CTB mutant are occupied by bound GMl oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GMI oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33 -+ Arg mutant complexes.The protein conformation observed for the binding-deficient Gly 33 -+ Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33 -+ Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.

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“…Several variants of CT-B have been identified that differ in two or three residues (reviewed in Finkelstein ef ai, 1987). LTp-l and LTh-l differ in terms of their B subunits at four or five positions (Tsuji ef a/., 1987;Leong et al, 1986), and differ from CT-B at approximately 25% of the positions. All of these enterotoxins bind to GM1, but the type I LTs also bind to several glycoprotein receptors to which CT does not bind (Griffiths ef ai, 1986).…”

Section: Introductionmentioning

confidence: 99%

Analysis of structure and function of the B subunit of cholera toxin by the use of site‐directed mutagenesis

Jobling

Holmes

1991

Molecular Microbiology

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Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.

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Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin

Cited by 77 publications

References 41 publications

Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide

Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide

Structural studies of receptor binding by cholera toxin mutants

Analysis of structure and function of the B subunit of cholera toxin by the use of site‐directed mutagenesis

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Nucleotide sequence comparison between heat-labile toxin B-subunit cistrons from Escherichia coli of human and porcine origin

Cited by 77 publications

References 41 publications

Crystal structure of cholera toxin B‐pentamer bound to receptor GM1 pentasaccharide

Crystal structure of cholera toxin B‐pentamer bound to receptor GM1 pentasaccharide

Structural studies of receptor binding by cholera toxin mutants

Analysis of structure and function of the B subunit of cholera toxin by the use of site‐directed mutagenesis

Contact Info

Product

Resources

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Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide

Crystal structure of cholera toxin B‐pentamer bound to receptor G_M1 pentasaccharide