Botulinum neurotoxin, produced by the ubiquitous spore forming bacteria Clostridium botulinum, is the sole cause of the neuroparalytic disease botulism. The disease results generally from food poisoning, infrequently from wounds. Infant botulism is not classical food poisoning. The immunologically distinct seven neurotoxin (=NT) types specifically block release of the neurotransmitter acetylcholine from presynapses. The mechanism of this blockage is not known. Definitive diagnosis and the only therapy of botulism depends on a chemically modified form(s) of the NT, i.e. toxoid (immunogen) and the antiserum raised with the immunogen. The NT is now in use as an experimental drug to correct certain muscle conditions, e.g. strabismus (crossed eyes), essential blepharospasm and hemifacial spasm. As part of a long-term on-going project on determination of i) the structure, ii) structure-function relationship and iii) mode of action of the NT, we plan to work in the next three years on the following areas of structure and structure-function relationship: 1. Determine sequence of about 200 amino acid residues from each of types A, B and E NT based on partial enzymatic and chemical cleavage of the separable H and L chains, the two subunits of a dichain NT. 2. Analyze the H chain and its fragments to locate the """"""""binding site"""""""" with which the H chain and its parent NT bind to neuromuscular junctions (NMJ). 3. Determine the structural alterations, covalent and conformational, that accompany 100-500 fold increase in toxicity of a single chain NT following its trypsinization. 4. Conversely, determine the structural alteration(s) that causes loss of toxicity by selectively modifying cationic (Arg, His, Lys) and aromatic (Tyr, Trp) residues that now appear to be important for toxicity. Exact number of residues modified will be related to the % loss of toxic activity and then the """"""""critical"""""""" residue(s) will be located; are they on the H or the L chain? This would delineate the active centers of the NT involved in binding to NMJ and lethality (mouse). 5. The property of ganglioside GT1b to bind with and detoxify NT will be exploited to determine the participating active centers of the NT. 6. Circumscribe the region of the H chain responsible for its ability to form channels in bilayer membranes. 7. Selectively radiolabel the separated H or L chain and combine them to reconstitute highly toxic radiolabeled NT which will unambiguously establish if H or L chain (or both) enters the nerve ending to produce toxicity. Non-toxic NT will be reconstituted from chemically modified L chain and unmodified H chain so that it binds to the NMJ effectively and can act as a specific competitive 'antagonist' for the NT. 8. Initiate crystallization of NT as the first step to determine in the long run the 3-D structure of the NT.
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