Binding of Clostridium botulinum type C and D neurotoxins to ganglioside and phospholipid. Novel insights into the receptor for clostridial neurotoxins.
Clostridium botulinum neurotoxins (BoNTs) act on nerve endings to block acetylcholine release. Their potency is due to their enzymatic activity and selective high affinity binding to neurons. Although there are many pieces of data available on the receptor for BoNT, little attempt has been made to characterize the receptors for BoNT/C and BoNT/D. For this purpose, we prepared the recombinant carboxyl-terminal domain of the heavy chain (H(C)) and then examined its binding capability to rat brain synaptosomes treated with enzymes and heating. Synaptosomes treated with proteinase K or heating retained binding capability to both H(C)/C and H(C)/D, suggesting that a proteinaceous substance does not constitute the receptor component. We next performed a thin layer chromatography overlay assay of H(C) with a lipid extract of synaptosomes. Under physiological or higher ionic strengths, H(C)/C bound to gangliosides GD1b and GT1b. These data are in accord with results showing that neuraminidase and endoglycoceramidase treatment decreased H(C)/C binding to synaptosomes. On the other hand, H(C)/D interacted with phosphatidylethanolamine but not with any ganglioside. Using cerebellar granule cells obtained from GM3 synthase knock-out mice, we found that BoNT/C did not elicit a toxic effect but that BoNT/D still inhibited glutamate release to the same extent as in granule cells from wild type mice. These observations suggested that BoNT/C recognized GD1b and GT1b as functional receptors, whereas BoNT/D induced toxicity in a ganglioside-independent manner, possibly through binding to phosphatidylethanolamine. Our results provide novel insights into the receptor for clostridial neurotoxin.
Tsukamoto
Kentaro
K
Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan.
Kohda
Tomoko
T
Mukamoto
Masafumi
M
Takeuchi
Kumiko
K
Ihara
Hideshi
H
Saito
Masaki
M
Kozaki
Shunji
S
D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry*; D001921:Brain / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003014:Clostridium botulinum / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K / Q000494:pharmacology; D005089:Exocytosis; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D007477:Ions; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry*; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012799:Sialyltransferases / Q000737:chemistry; D013574:Synaptosomes / Q000378:metabolism
D005732:Gangliosides; D007477:Ions; D009498:Neurotoxins; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D011994:Recombinant Proteins; C096325:botulinum toxin type D; C483858:phosphatidylethanolamine; D004247:DNA; D012799:Sialyltransferases; C021343:haematoside synthetase; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase; D019286:Endopeptidase K; D001905:Botulinum Toxins; C096324:botulinum toxin type C