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

Abstract

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

Binding activity and immunogenic characterization of recombinant C-terminal quarter and half of the heavy chain of botulinum neurotoxin serotype A

“…28,29 Ganglioside, especially GT1b, is considered a component of the double-receptor system of BoNTs. 24,[30][31][32] The first step in BoNT/A intoxication of neurons starts with binding of C-terminus subdomain of Hc to ganglioside on presynaptic membranes. 17,27 Therefore, our results show that the sera antibody from mice immunized with AHc-C could effectively block AHc or AHc-C binding to ganglioside, which indicates that anti-AHc-C antibodies neutralize or inhibit the first step in BoNT/A intoxication of neurons, i.e., the binding of BoNT/A to ganglioside.…”

“…There into, BoNTs firstly bind cell surface ganglioside and possibly another protein receptor, which mediated solely through the Hc domains. 3,[24][25][26][27] Even, the C-terminal quarter of the heavy chain (Hc-C) could be responsible for the binding of neurotoxin to receptor of the target cells. 5,6 In the current study, we have expressed and purified the C-terminal quarter of the heavy chain of botulinum neurotoxin serotype A (AHc-C, amino acids 1,088 through 1,296) in a fully soluble form by the pTIG-Trx vector which can express Trx together with AHc-C. Our results revealed that the recombinant protein AHc-C was specifically recognized by anti-BoNT/A antibodies.…”

“…Ganglioside is considered a component of the double-receptor system of BoNTs. 3,24 Thus the AHc or AHc-C binding with GT1b was examined to evaluate if the GT1b binding capacity is retained in the recombinant AHc-C purified in this study. Ganglioside binding activities analyzed by ELISA showed recognition of ganglioside by the purified AHc-C as good as AHc (Fig.…”

Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes

“…A polysialoganglioside binding site has been mapped in Hc-C, and is close to the binding site for the luminal domain of the synaptic vesicle (SV) protein synaptotagmin in BoNT/B and /G [12][13][14][15][16]; the Hc-C of BoNT/A and /E contain, in addition to the polysialoganglioside binding site a site of binding for luminal loops of the SV protein SV2 [17][18][19]. (b) This doublereceptor binding mediates the next step which is the endocytosis inside synaptic vesicles [16,20]. The interior of these vesicles becomes acidic owing to the action of a vacuolar-type ATPase proton pump [21].…”

The translocation domain of botulinum neurotoxin A moderates the propensity of the catalytic domain to interact with membranes at acidic pH

“…The three domains are critical for cell intoxication by BoNTs. H C allows recognition of neuronal cells by a double anchorage to a ganglioside and a protein receptor [8][9][10][11]. Following binding to its receptors, BoNT is internalized through clathrin-dynamin mediated endocytosis of recycling neurosecretion vesicles [12].…”