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The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity. [ABSTRACT FROM AUTHOR]

The extraordinary potency of botulinum neurotoxins (BoNTs) is mediated by their high neurospecificity, targeting peripheral cholinergic motoneurons leading to flaccid paralysis and successive respiratory failure. Complex polysialo gangliosides accumulate BoNTs on the plasma membrane and facilitate subsequent binding to synaptic vesicle membrane proteins which results in toxin endocytosis. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 (SV2) isoforms A-C mediates uptake of the clinically most relevant serotype BoNT/A1. SV2C-LD4 exhibits the strongest protein-protein interaction and comprises five putative N-glycosylation sites (PNG sites). Here, we expressed human SV2C-LD4 fused to human IgG-Fc in prokaryotic and eukaryotic expression systems to analyse the effect of N-glycosylation of SV2C on the interaction with BoNT/A1. Mass spectrometric analysis of gSV2CLDFc demonstrates glycosylation of N534, N559 and N565, the latter two residing at the BoNT/A interface. Mutational analysis demonstrates that only the N559-glycan, but not N565-glycan increases affinity of BoNT/A for human gSV2C-LD4. The N559- glycan was characterised as a complex core-fucosylated type with a heterogeneity ranging up to tetra-antennary structure with bisecting N-acetylglucosamine which can establish extensive interactions with BoNT/A. The mutant gSV2CLD-Fc N559A displayed a 50-fold increased dissociation rate kd resulting in an overall 12-fold decreased binding affinity in surface plasmon resonance (SPR) experiments. The delayed dissociation might provide BoNT/Amore time for endocytosis into synaptic vesicles. In conclusion, we show the importance of the complex N559- glycan of SV2C-LD4, adding a third anchor point beside a ganglioside and the SV2C-LD4 peptide, for BoNT/A neuronal cell surface binding and uptake. [ABSTRACT FROM AUTHOR]