Clostridial neurotoxins (botulinum and tetanus toxins) are large proteins organized into three functional domains, an amino terminal proteolytic domain, central translocation domain, and a carboxyl terminal receptor binding region. The neurotoxic effects of clostridial neurotoxins results from binding to cell surface receptors, translocation into the neural cell, and the proteolytic cleavage of proteins essential for synaptic vesicle docking/fusion events (SNARE proteins) by the enzymatically active amino terminal domain. The subsequent block of neurotransmitter release at the neuromuscular junction by botulinum toxins or block of inhibitory neurotransmitter release within the central nervous system by tetanus toxin leads to flaccid or spasmodic paralysis of the victim, respectively. The focus of this project has been the development of an in vitro assay for toxin activity and the preparation of a vaccine to the receptor bining C-fragment. Two recombinant toxin light chains (Lc) have been produced for development an in vitro assay for tetanus neurotoxin (TeNT) based on its metalloprotease activity. Full length Lc (Lc-His6, residues 1-457), with a C-terminal histidyl tag, and truncated Lc (GST-sLc, residues 1-427), fused at the N-terminal with glutathione sulfhydryl transferase, have catalytic activity in a qualitative SDS-PAGE assay that uses recombinant substrate. A recombinant substrate, vesicle-associated membrane protein (VAMP) fused with an N-terminal histidyl tag, has been partially purified and found to adsorb to Ni-NTA plates, which will permit development of a solid-phase, quantitative assay. Milligram quantities of TeNT C-fragment (Hc) have been purified by affinity adsorption to Ni-NTA Sepharose. Alternately, a """"""""native"""""""" Hc (i.e., Hc expressed without extraneous epitopes or fusions) has been purified on an affinity column prepared with a Hc-specific monoclonal antibody. Formation of a conjugate with Mening A polysaccharide by reductive amination or with Mening C polysaccharide by cross-linkage of amines is being explored.
