C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor.
Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or H(C)) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C-terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway.
Herreros
J
J
Molecular Neuropathobiology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K. j.herreros@icrf.icnet.uk
Lalli
G
G
Schiavo
G
G
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019281:Dimerization; D008565:Membrane Proteins / Q000378:metabolism*; D020932:Nerve Growth Factor / Q000494:pharmacology; D009466:Neuromuscular Blocking Agents / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013388:Succinimides / Q000494:pharmacology; D013744:Tetanus Toxin / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009466:Neuromuscular Blocking Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013388:Succinimides; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; C028728:tetanus toxin receptor; C011683:bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone; D020932:Nerve Growth Factor; D017868:Cyclic AMP-Dependent Protein Kinases