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J. Biol. Chem., Vol. 280, Issue 42, 35164-35171, October 21, 2005

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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¶, and Shunji Kozaki1

From the Department of Veterinary Science, Graduate School of Life and Environmental Sciences and the Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan and the ^¶Department of Oncology and Pharmacodynamics, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose, Tokyo 204-8588, Japan

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.

Received for publication, July 13, 2005 , and in revised form, August 15, 2005.

^* This work was supported in part by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB200358 and AB200360.

¹ To whom correspondence should be addressed. Tel.: 81-72-254-9504; Fax: 81-72-254-9499; E-mail: kozaki{at}center.osakafu-u.ac.jp.

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