The Structures of the HC Fragment of Tetanus Toxin with Carbohydrate Subunit Complexes Provide Insight into Ganglioside Binding

Emsley, Paul; Fotinou, Constantina; Black, Isobel; Fairweather, Neil F.; Charles, Ian G.; Watts, Colin; Hewitt, Eric W.; Isaacs, Neil W.

doi:10.1074/jbc.275.12.8889

Cited by 137 publications

(142 citation statements)

References 34 publications

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“…interactions are consistent with previous co-crystal structures showing the di-sialic acid moiety of the ganglioside GD3 oligosaccharide bound to the R pocket (28) or lactose binding to the W pocket (13). The demonstration that TeNT can simultaneously bind two oligosaccharides within the carbohydratebinding pockets is further supported by mass spectroscopy data showing that TeNT simultaneously bound two molecules of GT1b and that mutation within either carbohydrate-binding pocket reduced TeNT toxicity (14).…”

Section: Discussionsupporting

confidence: 77%

“…Complex gangliosides are sialic acid-containing glycosphingolipids that are located on the outer leaflet of cell membranes and contain a common "core" (GA1) consisting of Gal(␤1-3)GalNAc(␤1-4)Gal(␤1-4)Glc(␤1-1)Cer to which one or more N-acetylneuraminic acids (sialic acids) are bound, yielding "a" and "b" series gangliosides (11,12). Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site and a sialic acid-binding site (13). Previous studies showed that Trp 1289 is the key residue for the lactose-binding site, and Arg 1226 is the key residue for the sialic acid-binding site (14).…”

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confidence: 99%

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Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Chen

Kim

et al. 2009

Journal of Biological Chemistry

119

134

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Tetanus neurotoxin (TeNT) is an exotoxin produced byClostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ϳ150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT) 3 produced by the sporeforming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicleassociated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ϳ150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (ϳ50 kDa) and a C-terminal heavy chain domain (ϳ100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptorbinding domain (HCR/T, ϳ50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of ␣-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5-9). Current models implicate a dual receptor mechanism for the binding of t...

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Section: Discussionsupporting

confidence: 77%

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confidence: 99%

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Chen

Kim

et al. 2009

Journal of Biological Chemistry

119

134

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“…The Hc-C subdomain contains a modified β-trefoil folding motif present in several other proteins that possess recognition and binding functions, such as interleukin-1, fibroblast growth factor and the Kunitz-type trypsin inhibitors. However, the Hc-C sequence is poorly conserved among clostridial NTs (Umland et al, 1997;Lacy et al, 1998;Lacy and Stevens, 1999;Emsley et al, 2000). The critical importance of the last 34 residues from Hc-C, in particular His 1293 of TeNT, for binding the oligosaccharide portion of polysialogangliosides was demonstrated by photoaffinity labelling (Shapiro et al, 1997).…”

Section: Transcytotic Pathway and Gangliosides: The Case Of Clostridimentioning

confidence: 99%

Bacterial protein toxins and lipids: role in toxin targeting and activity

Geny

Popoff

2006

Biology of the Cell

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All bacterial toxins, which globally are hydrophilic proteins, interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Intracellular active toxins follow various trafficking pathways, the sorting of which is greatly dependent on the nature of the receptor, notably lipidic receptor or receptor embedded into a distinct environment such as lipid microdomains. Numerous other toxins act locally on cell membrane. Indeed, phospholipase activity is a common mechanism shared by several membrane‐damaging toxins. In addition, many toxins active intracellularly or on cell membrane modulate host cell phospholipid pathways. Unusually, a few bacterial toxins require a lipid post‐translational modification to be active. Thereby, lipids are obligate partners of bacterial toxins.

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“…However, a second carbohydrate binding site, the sialic acid binding site, was described only for TeNT (26,27). The sialic acid binding site in TeNT could be used to interact with the sialic acid-containing glycoprotein receptor (21,24).…”

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confidence: 99%

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Rummel

Eichner

Weil

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

171

216

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Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.synaptotagmin ͉ tetanus B otulinum neurotoxins (BoNTs) (serotypes A-G) are the causative agents of the disease botulism. The neurotoxins are produced as Ϸ150-kDa single-chain proteins in Clostridium botulinum and subsequently cleaved by proteases, yielding an Ϸ100-kDa heavy chain (HC) and an Ϸ50-kDa light chain (LC). These chains remain connected via a single disulfide bridge, noncovalent interactions, and a HC-derived peptide loop wrapped around the LC. The LCs act as zinc metallopeptidases, which solely hydrolyze one of three SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins: vesicle associated membrane protein/synaptobrevin, synaptosomal-associated protein of 25 kDa, or syntaxin. Together, these substrate molecules constitute the core of the vesicular fusion machinery. Thus, cleavage of one of these proteins inhibits the release of neurotransmitters from synaptic vesicles into the synaptic cleft. The HCs mediate the neurospecific binding, uptake by receptor-mediated endocytosis, and transport of the LC across the endosomal membrane into the cytosol, where the LCs encounter their substrates. The Ϸ50-kDa

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The Structures of the HC Fragment of Tetanus Toxin with Carbohydrate Subunit Complexes Provide Insight into Ganglioside Binding

Cited by 137 publications

References 34 publications

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Bacterial protein toxins and lipids: role in toxin targeting and activity

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

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