Binding of Clostridium botulinum Type C and D Neurotoxins to Ganglioside and Phospholipid

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.

Subunit Vaccine against the Seven Serotypes of Botulism

“…Montecucco and colleagues (24) proposed that BoNTs bind neurons through coreceptor interactions. Recent studies have implicated gangliosides as the coreceptor for the BoNTs, except for BoNT/D, which appears to utilize another class of small molecules as the coreceptor (35). To address the molecular basis for the neutralizing capacity of HCR immunization, a solid-phase assay was developed.…”

Unique Biological Activity of Botulinum D/C Mosaic Neurotoxin in Murine Species

“…injection into SD rats (female, 8 weeks old, approximately 200 g; Japan SLC, Inc., Shizuoka, Japan), and ddY and C57BL/6J mice (male, 4 weeks old, approximately 20 g; Japan SLC, Inc.) to obtain the mean 50% lethal dose (LD 50 ) by the Reed and Muench calculation (29). The toxicity of BoNT/DC to GM3 synthasedeficient (knockout) mice (42) was assayed by the intravenous injection method (21). The Ethics Committee of the Graduate School of Life and Environmental Sciences, Osaka Prefecture University, approved all animal tests.…”

“…Cerebellar granule neurons from 8-day-old C57BL/6J mice and SD rats were prepared according to a previously reported method (42). The cells were plated on polyethylenimine-coated 4-well culture dishes (Nalge Nunc, Rochester, NY) at a density of 2.6 ϫ 10 5 cells/cm 2 in HK-MEM (minimal essential medium containing 25 mM K ϩ , 10 mM glucose, 30 nM Na 2 SeO 4 , 80 g/ml gentamicin, pH 7.4) with 5% horse serum and 5% fetal calf serum and maintained overnight at 37°C in 5% CO 2 .…”

“…August 2012 Volume 80 Number 8 iai.asm.org 2887 periments were performed as described previously (42). The Glu content was determined using a high-performance liquid chromatography (HPLC)-electron capture detector (HTEC-500; Eicom, Kyoto, Japan), which consists of a GU-GEL matrix separation column, a Glu oxidaseimmobilized column, and a platinum electrode for electrochemical detection.…”

“…Synaptic vesicle glycoprotein SV2C and synaptotagmin I or II are considered to be the protein receptors for BoNT/A and B, respectively (8,23). Meanwhile, for BoNT/C, gangliosides GD1b and GT1b have been identified as binding components (42). Although BoNT/D lacks the SXWY ganglioside-binding motif that is present in other BoNT serotypes, it has recently been reported that the toxin binds to b-series gangliosides (14).…”