Binding of Clostridium botulinum Type C and D Neurotoxins to Ganglioside and Phospholipid: NOVEL INSIGHTS INTO THE RECEPTOR FOR CLOSTRIDIAL NEUROTOXINS

Kentaro Tsukamoto,Tomoko Kohda,Masafumi Mukamoto,Kumiko Takeuchi,Hideshi Ihara,Masaki Saito,Shunji Kozaki

doi:10.1074/jbc.m507596200

Abstract

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.

Highlights

Seven types of Clostridium botulinum strains (A through G) are distinguished by differences in the antigenic specificity of their pharmacologically similar neurotoxins
We described how the gene for the mosaic form of Botulinum neurotoxins (BoNTs) is harbored in the isolates related to avian botulism, whereas the authentic BoNT/C may be implicated in the disease affecting mammalians, including cattle, mink, and
Binding of Recombinant HC to Gangliosides and Phospholipids—In the preliminary experiments, we found that BoNT/C and BoNT/D could bind to lipids

Summary

NOVEL INSIGHTS INTO THE RECEPTOR FOR CLOSTRIDIAL NEUROTOXINS*

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. We demonstrated the first time that BoNT/B binds to synaptotagmins I and II in the presence of gangliosides GT1b and GD1a [5,6,7] These proteins are homologous synaptic vesicle membrane proteins thought to function as Ca2ϩ sensors for exocytosis [8]. We provide informative results that BoNT/D elicits toxic action against neural cells in a ganglioside-independent manner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Wild type