The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.     

Molecular basis for tetanus toxin coreceptor interactions

Tetanus toxin (TeNT) elicits spastic paralysis through the cleavage of vesicle-associated membrane protein-2 (VAMP-2) in neurons at the interneuronal junction of the central nervous system. While TeNT retrograde traffics from peripheral nerve endings to the interneuronal junction, there is limited understanding of the neuronal receptors utilized by tetanus toxin for the initial entry into nerve cells. Earlier studies implicated a coreceptor for tetanus toxin entry into neurons: a ganglioside binding pocket and a sialic acid binding pocket and that GT1b bound to each pocket. In this study, a solid phase assay characterized the ganglioside binding specificity and functional properties of both carbohydrate binding pockets of TeNT. The ganglioside binding pocket recognized the ganglioside sugar backbone, Gal-GalNAc, independent of sialic acid-(5) and sialic acid-(7) and GM1a was an optimal substrate for this pocket, while the sialic acid binding pocket recognized sialic acid-(5) and sialic acid-(7) with "b"series of gangliosides preferred relative to "a" series gangliosides. The high-affinity binding of gangliosides to TeNT HCR required functional ganglioside and sialic acid binding pockets, supporting synergistic binding to coreceptors. This analysis provides a model for how tetanus toxin utilizes coreceptors for high-affinity binding to neurons.     

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.     

Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry

Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A–G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.     

Identification of a binding site for ganglioside on the receptor binding domain of tetanus toxin

The carboxyl-terminal region of the tetanus toxin heavy chain (H(C) fragment) binds to di- and trisialylgangliosides on neuronal cell membranes. To determine which amino acids in tetanus toxin are involved in ganglioside binding, homology modeling was performed using recently resolved X-ray crystallographic structures of the tetanus toxin H(C) fragment. On the basis of these analyses, two regions in tetanus toxin that are structurally homologous with the binding domains of other sialic acid and galactose-binding proteins were targeted for mutagenesis. Specific amino acids within these regions were altered using site-directed mutagenesis. The amino acid residue tryptophan 1288 was found to be critical for binding of the H(C) fragment to ganglioside GT1b. Docking of GD1b within this region of the toxin suggested that histidine 1270 and aspartate 1221 were within hydrogen bonding distance of the ganglioside. These two residues were mutagenized and found also to be important for the binding of the tetanus toxin H(C) fragment to ganglioside GT1b. In addition, the H(C) fragments mutagenized at these residues have reduced levels of binding to neurites of differentiated PC-12 cells. These studies indicate that the amino acids tryptophan 1288, histidine 1270, and aspartate 1221 are components of the GT1b binding site on the tetanus toxin H(C) fragment.     

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.     

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Structure of tetanus toxin. II. Toxin binding to ganglioside.

The interaction between tetanus toxin and ganglioside containing 2 N-acetylneuraminic acid residues linked in sequence to one another has been investigated using a new method involving radioactively labeled ganglioside and tetanus toxin adsorbed to Sephadex matrix. Binding between the two components was demonstrated, and it was calculated that in the nanomolar concentration range, tetanus toxin becomes half-saturated at about 5 X 10(-8) M concentration of ganglioside. Removal of the ceramide portion from the ganglioside resulted in the complete loss of binding activity, whereas removal of the terminal N-acetylneuraminic acid residue from the intact ganglioside had no effect. Among the fragments derived from tetanus toxin (Helting, T. B., and Zwisler, O. (1977) J. Biol. Chem. 252, 187-193), only the heavy chain polypeptide exhibited a binding activity of the same order of magnitude as that observed for the native toxin. The light chain polypeptide showed no interaction with ganglioside and among the fragments derived from the toxin by digestion with papain, only Fragment C, at a high protein concentration, displayed marginal binding activity. Using monovalent antibodies directed against specific regions of the tetanus toxin molecule, it was demonstrated that antibodies directed against Fragment C uniquely interfere with the binding process. Anti-light chain serum was ineffective, as well as antitetanus toxoid serum previously absorbed with Fragment C. It is concluded that the binding site for ganglioside is located on the heavy chain portion of tetanus toxin, possibly in or near the region comprised by Fragment C.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphocytes. Saturation binding by monosialylated [3H]-GM1 to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (KD) of 2.2 +/- 1.4 microM and a binding capacity near 2 fmoles/cell. Competitive inhibition of [3H]-GM1 binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM1-congeneric gangliosides. A comparison between the results of these binding studies and ganglioside-induced decrease of CD4 expression demonstrated that every aspect of [3H]-GM1 binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of ganglioside binding to specific sites on CD4+ T-helper lymphocytes.     

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.     

Gangliosides in human, cow and goat milk, and their abilities as to neutralization of cholera toxin and botulinum type A neurotoxin

To elucidate the potential of mammalian milk as to protection of infants from infections, we determined the ganglioside compositions of human, cow and goat milk in relation with cholera toxin and botulinum type A neurotoxin-receptors. Gangliosides accounted for 1 to 2 μmol of lipid-bound sialic acid (LSA) in 100 ml of milk, and GD3 comprised about 69% of LSA in all milk samples. Among the milk samples examined, goat milk was found to contain an amount of gangliosides belonging to the b-pathway representing 15.8% of the total LSA. Accordingly, botulinum neurotoxin bound to GT1b and GQ1b in goat milk, but not to any gangliosides in human or cow milk. On the other hand, GM1, the cholera toxin receptor, was found to be present in all milk samples at concentrations of 0.02% to 0.77% of the total LSA and to be maintained at a relatively constant level in human milk during the postpartum period. Gangliosides from 1 ml of pooled human milk exhibited the ability to attenuate the binding of cholera toxin (30 ng) to GM1 by 93%, and those from 500 μl of goat milk completely inhibited the binding of botulinum type A neurotoxin 1.5 μg to GT1b. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2]. PVP, polyvinylpyrrolidone; LSA, lipid-bound sialic acid.     

Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide.

Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection. In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells. We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide. Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit. The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue. The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13. Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue. The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.     

Interaction Between Tetanus Toxin and Rabbit Kidney: A Comparison with Rat Brain Preparations

125I-Tetanus toxin is bound by basolateral membranes from rabbit kidneys. Fixation is specific, as it is minimally inhibited by the nonbinding (fragment B) moiety of tetanus toxin, whereas the binding moiety (fragment C) is equivalent to the native toxin in inhibiting fixation. Competition is also pronounced with mildly toxoided toxin. Association and dissociation of 125I-toxin are delayed in kidney when compared to brain membranes. The binding sites in kidney membranes are partially sensitive to neuraminidase and resist heating to 56 degrees C, in contrast to those in brain membranes which are very sensitive to both treatments. The binding sites of the two preparations can be discriminated further by variation of the ionic environment. Sodium dodecyl sulfate-disc gel electrophoresis followed by transfer to nitrocellulose, and TLC with consecutive overlay indicate that tetanus toxin exclusively binds to long-chain gangliosides from rat brain. Binding sites in kidney membranes from rabbits and rats can be made visible by the overlay technique. They are apparently heterogeneous and more hydrophobic. We conclude that rabbit kidney contains binding sites for tetanus toxin which resemble gangliosides but differ from the major gangliosides in brain both chemically and with respect to their interaction with tetanus toxin.     

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

Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy-terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three-dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C-terminal beta-trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine-1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar-binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.     

Tyrosine-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurones

Tetanus toxin acts by blocking the release of glycine from inhibitory neurones within the spinal cord. An initial stage in the toxin's action is binding to acceptors on the nerve surface and polysialogangliosides are a component of these acceptor moieties. Using site-directed mutagenesis, we identify tyrosine-1290 of tetanus toxin as a key residue that is involved in ganglioside binding. This residue, which is located at the centre of a shallow pocket on the beta-trefoil domain of the tetanus H(c) fragment, is also shown to play a key role in the functional binding of tetanus toxin to spinal cord neurones leading to the inhibition of neurotransmitter release.     

Neuronal sensitivity to tetanus toxin requires gangliosides.

Tetanus toxin produces spastic paralysis in situ by blocking inhibitory neurotransmitter release in the spinal cord. Although di- and trisialogangliosides bind tetanus toxin, their role as productive toxin receptors remains unclear. We examined toxin binding and action in spinal cord cell cultures grown in the presence of fumonisin B(1), an inhibitor of ganglioside synthesis. Mouse spinal cord neurons grown for 3 weeks in culture in 20 microM fumonisin B(1) develop dendrites, axons, and synaptic terminals similar to untreated neurons, even though thin layer chromatography shows a greater than 90% inhibition of ganglioside synthesis. Absence of tetanus and cholera toxin binding by toxin-horseradish peroxidase conjugates or immunofluorescence further indicates loss of mono- and polysialogangliosides. In contrast to control cultures, tetanus toxin added to fumonisin B(1)-treated cultures does not block potassium-stimulated glycine release, inhibit activity-dependent uptake of FM1-43, or abolish immunoreactivity for vesicle-associated membrane protein, the toxin substrate. Supplementing fumonisin B(1)-treated cultures with mixed brain gangliosides completely restores the ability of tetanus toxin to bind to the neuronal surface and to block neurotransmitter release. These data demonstrate that fumonisin B(1) protects against toxin-induced synaptic blockade and that gangliosides are a necessary component of the receptor mechanism for tetanus toxin.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.     

Tetanus toxin fragment C binds to a protein present in neuronal cell lines and motoneurons

Tetanus Toxin Fragment C Binds to a Protein Present in Neuronal Cell Lines and Motoneurons Tetanus neurotoxin is one of the most powerful protein toxins known, acting in vivo at femtomolar doses. Two main factors determine its high potency: a protease activity restricted to a single intracellular substrate and its absolute neurospecificity. Whereas the enzymatic properties of tetanus toxin have been thoroughly defined, the nature of its neuronal receptor(s) and their involvement in the intracellular trafficking of tetanus toxin are poorly understood. Using binding and crosslinking experiments, we report here on the characterisation of an N-glycosylated 15-kDa interacting protein, which behaves as an integral membrane protein. This putative receptor specifically interacts with the binding domain (fragment C) of tetanus toxin and not with several related botulinum neurotoxins in spinal cord motoneurons and neuronal-like cell lines. Sialic acid-specific lectins antagonise the binding of tetanus toxin to the cell surface and to the 15-kDa protein, supporting the central role of sialic acid residues in the recognition process. Altogether, these results indicate the existence of a neuronal protein receptor for tetanus toxin whose identification is likely to constitute a key step in the analysis of the molecular machinery involved in the toxin internalisation and retrograde transport.