Inhibition of vesicular secretion in both neuronal and nonneuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from Clostridium botulinum (termed LHN/A) that retains catalytic activity can be prepared by proteolysis, The LHN/A, however, lacks the putative native binding domain (H-C) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release, Here we report the chemical conjugation of LHN/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA), When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LHN/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate, These data confirm that the function of the H-C domain of C. botulinum neurotoxin type A is limited to binding to cell surface moieties, The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.