Analysis of mutants of tetanus toxin H<sub>C</sub> fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Sinha, Katharine A.; Box, Michael; Lalli, Giovanna; Schiavo, Giampietro; Schneider, Holm; Groves, Michael J.; Siligardi, Giuliano; Fairweather, Neil

doi:10.1046/j.1365-2958.2000.02246.x

Cited by 12 publications

(22 citation statements)

References 22 publications

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“…Asp1147, Asp1214, and Arg1226 play key role in stabilizing any bound receptor in this region. Deletion or mutation of these key residues to Ala or Ser significantly reduces the binding activity of this site 16, 22…”

Section: Resultsmentioning

confidence: 97%

“…Sia1 makes direct hydrogen bonds with the side chains of Asp1147, Asn1216, and Arg1226, and Sia2 is stabilized by interactions with Asp1214, Asn1216, Arg1226, and Tyr1229 (Table II). Recent site‐directed mutagenesis experiments showed that the mutation of key residues Asp1214–Asn1219 resulted in substantially reduced ganglioside binding 22. One of the interactions, the salt bridge observed in TeNT between the guanidino group of Arg1226 and the carboxylate oxygens (O1A and O1B) of sialic acid (Sia2) (Table II) is conserved in GT1b and Sia complexes and is characteristic of TeNT–ganglioside recognition 10, 11.…”

Section: Resultsmentioning

confidence: 99%

“…Mutagenesis studies suggest that the C‐terminal residues 1306–1310 are essential for cell and ganglioside binding, and photoaffinity studies show that the carboxyl‐terminal 34 amino acids of TeNT (residues 1282–1315) are sufficient to support the binding of gangliosides that have the NeuAcα(2,8)NeuAc moiety 3, 17. Recent analysis of mutants of TeNT show that His1271–Asp1282, Asp1214–Asn1219, and the region around Tyr1290 provide key residues for ganglioside binding 16, 22. The X‐ray crystal structures of TeNT with lactose, galactose, GalNAc, sialic acid, and GT1b analog show the presence of four different sites on the surface of TeNT, which are spaced at least 10 Å from each other 10, 11.…”

Section: Introductionmentioning

confidence: 99%

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Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

et al. 2005

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Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

et al. 2005

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“…Other work reported toxin inactivation by genetic methods such as the site-directed mutagenesis of active site residues (Sinha et al, 2000;Nencioni et al, 1991). However, to stabilize the antigen and to obtain the required long-term stability of the antigen, it was in some cases necessary to treat genetically modified vaccine antigen with cross-linking agents (Nencioni et al, 1991;Nencioni et al 1990).…”

Section: Introductionmentioning

confidence: 99%

Unbinding forces of single pertussis toxin–antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

Moreno

Chevalier

Ronzon

et al. 2011

J of Molecular Recognition

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An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed.

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“…1997). The neighborhood of H1293 to the ganglioside binding pocket was confirmed in a mutagenesis study showing reduced in vitro binding of the TeNT H C mutant H1293A to isolated ganglioside GT1b (Sinha et al. 2000).…”

Section: The Mode Of Molecular Cnt‐ganglioside Interactionmentioning

confidence: 82%

Cell entry strategy of clostridial neurotoxins

Binz

Rummel

2009

Journal of Neurochemistry

180

165

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vesicle protein 2; Syt, synaptotagmin; TeNT, tetanus neurotoxin. AbstractTetanus neurotoxin and botulinum neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins' sophisticated access to their site of action.

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Analysis of mutants of tetanus toxin H_C fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Cited by 12 publications

References 22 publications

Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

Unbinding forces of single pertussis toxin–antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

Cell entry strategy of clostridial neurotoxins

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Analysis of mutants of tetanus toxin HC fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Cited by 12 publications

References 22 publications

Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

Common binding site for disialyllactose and tri‐peptide in C‐fragment of tetanus neurotoxin

Unbinding forces of single pertussis toxin–antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

Cell entry strategy of clostridial neurotoxins

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Analysis of mutants of tetanus toxin H_C fragment: ganglioside binding, cell binding and retrograde axonal transport properties