Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium 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 ∼150-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 spore-forming 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 vesicle-associated 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 ∼150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (∼50 kDa) and a C-terminal heavy chain domain (∼100 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 receptor-binding domain (HCR/T, ∼50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of α-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 the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10). 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(β1–3)GalNAc(β1–4)Gal(β1–4)Glc(β1–1)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 Trp¹²⁸⁹ is the key residue for the lactose-binding site, and Arg¹²²⁶ is the key residue for the sialic acid-binding site (14). In this study, we denote the lactose-binding site as the “W” pocket and the sialic acid-binding site as the “R” pocket. Binz and co-workers (14) showed that functional R and W binding sites were required for TeNT toxicity (7). These biochemical and cellular studies were supported by a co-crystal structure of HCR/T bound to a GT1b-β anomer analog, which showed that the W and R carbohydrate-binding pockets were located at different regions of TeNT (7). We recently reported that the W pocket binds gangliosides via the GA1 core structure, whereas the R pocket binds gangliosides via di- or tri-sialic acid moieties (15) where simultaneous binding of TeNT to two gangliosides was synergistic (see Fig. 1a). In the current study, gangliosides were identified that bound exclusively to either the W pocket or R pocket, which allowed the characterization of the role of ganglioside binding to the W and R pockets as dual receptors for TeNT entry into neurons.Open in a separate windowFIGURE 1.Interaction of the HCR domain of TeNT with its putative cellular receptor. a, HCR/T has two ganglioside-binding sites. The W pocket binds to the terminal GalNAc-Gal of the ganglioside (illustrated by GM1a). The R pocket binds to the di-sialic acid of the ganglioside (illustrated by GD3). b, alternating lanes of molecular mass marker proteins and cortical neuron lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained for protein with Ponceau S (bottom panel), and then the membrane strips were incubated with 10 nm of the indicated HCR/T (HCR/T wild type (wt), HCR/T (R+, W−), HCR/T (R−, W+), or HCR/T (R−, W−)) followed by HRP-conjugated α-FLAG antibody. The bands were visualized with SuperSignal; exposed film is shown (upper panel). The asterisk denotes the position of purified gangliosides resolved under identical conditions. Migration of the molecular mass marker proteins is indicated (kDa) in the left-most lane in the upper panel.