Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin.
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 (KD values) for TetC binding to a number of related NeuAc-containing carbohydrates (sialyllactose and disialyllactose), as well as six (NeuAc)n oligomers (n = 1-6). KD values were found to range between approximately 10-35 microM. 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.
Conway
Maria C Prieto
MCP
Chemistry and Materials Science Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA.
Whittal
Randy M
RM
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
Baldwin
Michael A
MA
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
Burlingame
A L
AL
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
Balhorn
Rod
R
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Avenue, 94550, Livermore, CA, USA. balnorn2@llnl.gov.
D001665:Binding Sites; D003198:Computer Simulation; D019281:Dimerization; D005732:Gangliosides / Q000737:chemistry*; D008956:Models, Chemical; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013744:Tetanus Toxin / Q000737:chemistry*
D005732:Gangliosides; D010446:Peptide Fragments; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; D019158:N-Acetylneuraminic Acid