Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Abstract

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...

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

“…Initially, the HCR binds to ganglioside co-receptors present in the neuronal plasma membrane (Step 1) (10,24). This interaction presumably promotes partitioning into the bilayer by tethering the HCT close to the membrane, such that its orientation relative to the bilayer is optimal for subsequent channel formation.…”

Unique Ganglioside Recognition Strategies for Clostridial Neurotoxins

“…A Unique Asparagine Residue Stabilizes HCR/T R1226L ⅐ Ganglioside Complexes-Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site defined by the conserved GBM and a second sialic acid-binding site defined by a critical arginine residue (Arg-1226) (32,35,39). Therefore, to directly compare binding mediated through the conserved GBM, it is necessary to use a mutant form of the TeNT HCR (HCR/T R1226L ) in which the sialic acid-binding site is inactivated (32,39).…”

“…Most notable, however, is that BoNTs bind only to gangliosides that have an ␣2,3-linked N-acetylneuraminic acid residue (denoted Sia5) attached to Gal4 of the oligosaccharide core (10,33,34), whereas the corresponding ganglioside-binding pocket on TeNT can also bind to GM1a, a ganglioside lacking the Sia5 sugar residue (supplemental Fig. S1) (32,35). Together, these observations suggest that ganglioside-binding affinity and specificity are encoded by residues located outside of the conserved motif.…”

Enhancing the Protective Immune Response against Botulism

“…1B). Since W is conserved within the GBPs of BoNT serotypes A, B, E, F, G and tetanus toxin (17,20,(44)(45)(46)(47), we propose that this mutation can be introduced into each HCR serotype to generate a potentially less reactive but more potent vaccine. In addition, since BoNT/C and BoNT/D possess comparable aromatic residues within another ganglioside binding loop within the HCR (48,49), we propose that the respective W and F may be modified to A to reduce potential reactivity and enhance vaccine potency.…”

“…For example, BoNT/ A(W1266L) had 0.7% of the toxicity of wild-type BoNT/A in a paralytic half-time assay (18). Mutations in the analogous tryptophan in the lactose binding pocket (GBP equivalent) of tetanus toxin also reduced ganglioside binding affinity (19,20).…”