Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

Abstract

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to obtain an estimate of the dissociation constants (K D values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc) n oligomers (n ϭ 1-6). K D values were found to range between ϳ10 -35 M. The strength of the interactions between the C fragment and (NeuAc) n are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. These results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer) and that NeuAc may play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria, and viruses to facilitate their entrance into cells. (J Am Soc Mass Spectrom 2006, 17, 967-976)

The HC Fragment of Tetanus Toxin forms Stable, Concentration-dependent Dimers via an Intermolecular Disulphide Bond

“…Confirming our results, Prieto Conway et al recently demonstrated dimerisation of TeNT H C (polydirpersed only) using ESI-ToF mass spectrometry. 21 Mutagenesis experiments demonstrated that Cys869 is essential for oligomerisation, as H-C Cys869Ala was predominantly monomeric as shown by SDS-PAGE and SEC. In contrast, substitution of Cys1301 or Cys1077 for alanine had no effect on dimerisation.…”

Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway

“…GT1b is a trisialo sphingolipid with a branched carbohydrate structure containing a single N -acetylneuraminic acid (NeuAc) on one arm and a NeuAc dimer on the other (Figure 3). The strongest and most specific ganglioside association with tetanus toxin is with GT1b because the targeting domain of the toxin contains two binding sites that can accommodate NeuAc residues separated by a distance of approximately 25 Å [43]. Studies based on atomic-force microscopy/total internal-reflection fluorescence microscopy (TMAFM/TIRFM) reinforce this result, suggesting that the membrane activity of fragment C is dependent on both the ganglioside concentration in the membrane and the pH of the medium [44].…”