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

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of spec ific 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 neurotox in and is thus unable to bind to neurons and effect inhibition of neurotran smitter release, Here we report the chemical conjugation of LHN/A to an alt ernative cell-binding ligand, wheat germ agglutinin (WGA), When applied to a variety of cell lines, including those that are ordinarily resistant to t he effects of neurotoxin, WGA-LHN/A conjugate potently inhibits secretory r esponses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopep tidase-dependent cleavage of the natural botulinum neurotoxin type A substr ate, 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 nonn euronal origin. These observations lead to the conclusion that a clostridia l endopeptidase conjugate that can be used to investigate SNARE-mediated pr ocesses in a variety of cells has been successfully generated.