Only the complex N559-glycan in the synaptic vesicle glycoprotein 2C mediates high affinity binding to botulinum neurotoxin serotype A1

Abstract

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 gSV2CLD-Fc 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/A more 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.

Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology

“…BoNT/A1 and BoNT/E1 bind different segments of the fourth lumenal loop of SV2, a multispanning integral protein of synaptic vesicles of unknown function (Dong et al, 2006; Mahrhold et al, 2006; Binz and Rummel, 2009; Benoit et al, 2014). Three isoforms of SV2 (A, B, C) are expressed at motor nerve terminals, but SV2C appears to be the one binding BoNT/A1 more efficiently than SV2A or B via its glycosylated fourth luminal domain (Mahrhold et al, 2006; Benoit et al, 2014; Mahrhold et al, 2016), whereas BoNT/E1 binds isoforms A and B, but not C (Dong et al, 2008). …”

“…Of the five putative N-glycosylation sites of SV2C, only the Asn-559 is involved; this N-glycan is highly complex with a tetra antennary structure, which is likely to interact with an extensive area of HC/A1 (Mahrhold et al, 2016). The protein-protein HC/A-SV2C contacts involve mostly the backbones of the two proteins, through the pairing of two solvent-exposed β -strands, one from each partner (Benoit et al, 2014).…”

Puzzling Out Synaptic Vesicle 2 Family Members Functions

“…SV2C exhibits the highest affinity for BoNT/A (Dong et al, 2006). More precisely, one glycosylated asparagine present in the center of the interface between SV2 and the toxin (residue 573 in SV2A, 516 in SV2B and 559 in SV2C) seems to be implicated in BoNT/A’s mode of entry (Mahrhold et al, 2016; Yao et al, 2016). Interaction studies of SV2C with BoNT/A suggest an efficient and rapid binding/unbinding of the pair mediated by the backbone of SV2C rather than a side-chain interaction, as Syt1 and Syt2 do for BoNT/B and G. Interestingly, the binding of SV2C to BoNT/A is facilitated at pH 5, close to SV conditions, whereas the binding of Syt2 with BoNT/B is pH-independent (Weisemann et al, 2016).…”

Characterization of clostridium botulinum neurotoxin serotype A (BoNT/A) and fibroblast growth factor receptor interactions using a novel receptor dimerization assay

“…The specific binding site on H C /A for SV2 has been identified as an exposed beta-strand loop in the center of the binding domain [19, 20, 22, 79]. The observation that a variant of H C /A (T1145A;T1146A [20]) with mutations in residues important for SV2 binding shows reduced ability to dimerize FGFR3c suggests that these residues, directly or indirectly, affect binding to FGFRs.…”