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 LH(N)/A) that retains catalytic activity can be prepared by proteolysis. The LH(N)/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 LH(N)/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-LH(N)/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.
Chaddock
J A
JA
Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom. john.chaddock@camr.org.uk
Purkiss
J R
JR
Friis
L M
LM
Broadbridge
J D
JD
Duggan
M J
MJ
Fooks
S J
SJ
Shone
C C
CC
Quinn
C P
CP
Foster
K A
KA
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002460:Cell Line; D003014:Clostridium botulinum / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D014316:Tritium; D014909:Wheat Germ Agglutinins / Q000302:isolation & purification / Q000378:metabolism
D007328:Insulin; D018377:Neurotransmitter Agents; D014909:Wheat Germ Agglutinins; D014316:Tritium; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; D005998:Glycine