Analysis of mutants of tetanus toxin H<sub>C</sub> fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Sinha, Katharine A.; Box, Michael; Lalli, Giovanna; Schiavo, Giampietro; Schneider, Holm; Groves, Michael J.; Siligardi, Giuliano; Fairweather, Neil

doi:10.1046/j.1365-2958.2000.02091.x

Cited by 67 publications

(45 citation statements)

References 38 publications

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“…The requirement for gangliosides as a component of the neuronal receptor(s) for TeNT is well established (9,10,29). Although numerous structural and biochemical studies have described the interactions of TeNT with gangliosides in vitro, few studies have addressed ganglioside function in vivo.…”

Section: Discussionmentioning

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: Discussionmentioning

confidence: 99%

“…Only three sialic acids (of six sugar residues of GT2) are visible in the structure, and two of them make direct interactions with HCR/T. (29).…”

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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 recombinant H C domain from tetanus neurotoxin (TeNT) used as a negative control for binding was prepared and purified as previously described (31).…”

Section: Methodsmentioning

confidence: 99%

Binding of Clostridium difficile Surface Layer Proteins to Gastrointestinal Tissues

et al. 2002

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Clostridium difficile is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients. While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified. Previous studies have shown binding of C. difficile whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized. In this paper, we have investigated tissue binding of C. difficile surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the slpA gene. The adherence of C. difficile to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence. Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative. A similar pattern was observed in the mouse. By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP. By Western blotting analysis, we have identified two potential ligands of the C. difficile SLPs, one of which may be specific to the gut. By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin. These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by C. difficile and in the subsequent inflammatory reaction.

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“…The HC production in the BL21 E. coli strain and purification was performed as previously described. 11 The obtained HC fragment was additionally covalently linked to a bi-functional poly(ethylene glycol) (PEG) spacer. Briefly, a bi-functional 5 kDa PEG (JenKem Technology USA, Plano, TX, USA) bearing an N-hydroxysuccinimide and a maleimide end group was used as indicated by the manufacturer, at a 2.5 PEG/HC protein molar ratio.…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

Lopes

Oliveira

Estevão

et al. 2016

IJN

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A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies.

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Analysis of mutants of tetanus toxin H_C fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Cited by 67 publications

References 38 publications

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Binding of Clostridium difficile Surface Layer Proteins to Gastrointestinal Tissues

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

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Analysis of mutants of tetanus toxin HC fragment: ganglioside binding, cell binding and retrograde axonal transport properties

Cited by 67 publications

References 38 publications

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Binding of Clostridium difficile Surface Layer Proteins to Gastrointestinal Tissues

In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

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Analysis of mutants of tetanus toxin H_C fragment: ganglioside binding, cell binding and retrograde axonal transport properties