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Title: Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

Abstract

Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using {alpha}(1-3,4)-fucosidase, endo-{beta}-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides thatmore » contain {alpha}2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1005928
Resource Type:
Journal Article
Journal Name:
Biochemistry-US
Additional Journal Information:
Journal Volume: 48; Journal Issue: (24) ; 06, 2009; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
ENGLISH
Subject:
36 MATERIALS SCIENCE; ACETYLCHOLINE; AFFINITY; CARBOHYDRATES; CHROMATOGRAPHY; CRYSTAL STRUCTURE; GALACTOSE; GANGLIOSIDES; GLYCOPROTEINS; MOTORS; MUTATIONS; NERVE CELLS; OLIGOSACCHARIDES; POLYPEPTIDES; PROTEINS; SACCHARIDES; SIALIC ACID; SPECIFICITY; SULFATES

Citation Formats

Fu, Zhuji, Chen, Chen, Barbieri, Joseph T, Kim, Jung-Ja P, Baldwin, Michael R, and MCW). Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F. United States: N. p., 2010. Web. doi:10.1021/bi9002138.
Fu, Zhuji, Chen, Chen, Barbieri, Joseph T, Kim, Jung-Ja P, Baldwin, Michael R, & MCW). Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F. United States. doi:10.1021/bi9002138.
Fu, Zhuji, Chen, Chen, Barbieri, Joseph T, Kim, Jung-Ja P, Baldwin, Michael R, and MCW). Mon . "Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F". United States. doi:10.1021/bi9002138.
@article{osti_1005928,
title = {Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F},
author = {Fu, Zhuji and Chen, Chen and Barbieri, Joseph T and Kim, Jung-Ja P and Baldwin, Michael R and MCW)},
abstractNote = {Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using {alpha}(1-3,4)-fucosidase, endo-{beta}-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain {alpha}2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.},
doi = {10.1021/bi9002138},
journal = {Biochemistry-US},
issn = {0006-2960},
number = (24) ; 06, 2009,
volume = 48,
place = {United States},
year = {2010},
month = {2}
}