Title :
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 g SV2C-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