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N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A

Abstract

Botulinum neurotoxin serotype A1 (BoNT/A1), a licensed drug widely used for medical and cosmetic applications, exerts its action by invading motoneurons. Here we report a 2.0-Å-resolution crystal structure of the BoNT/A1 receptor-binding domain in complex with its neuronal receptor, glycosylated human SV2C. We found that the neuronal tropism of BoNT/A1 requires recognition of both the peptide moiety and an N-linked glycan on SV2. This N-glycan—which is conserved in all SV2 isoforms across vertebrates—is essential for BoNT/A1 binding to neurons and for its potent neurotoxicity. The glycan-binding interface on SV2 is targeted by a human BoNT/A1-neutralizing antibody currently licensed as an antibotulism drug. Our studies reveal a new paradigm of host-pathogen interactions, in which pathogens exploit conserved host post-translational modifications, thereby achieving highly specific receptor binding while also tolerating genetic changes across multiple isoforms of receptors.

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Figure 1: SV2 glycosylation is critical for BoNT/A1 binding and entry into neurons.
Figure 2: Structure of HCA in complex with human gSV2C.
Figure 3: Site-directed-mutagenesis analysis of the SV2-binding site on HCA.
Figure 4: The SV2 glycan binding mode is conserved in BoNT/HA.
Figure 5: The SV2 glycan-binding site on BoNT/A1 is the target of the neutralizing antibody CR1.

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Acknowledgements

This work was partly supported by National Institute of Allergy and Infectious Diseases (NIAID) grants R01AI091823 and R21AI123920 to R.J. and R01AI096169 to M.K.; by National Institute of Neurological Disorders and Stroke (NINDS) grant R01NS080833 to M.D.; and by Bundesministerium für Bildung und Forschung grants FK031A212A to A.R. and FK031A212B to B.G. Dorner (RKI). NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P41 GM103403). The Pilatus 6M detector at the 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). Use of the APS, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. We thank J. Weisemann for cloning HCHA and N. Krez for dissecting the MPN hemidiaphragm tissue. We thank E. Chapman (University of Wisconsin–Madison), E. Johnson (University of Wisconsin–Madison), J. Marks (University of California, San Francisco), and R. Janz (The University of Texas Health Science Center at Houston) for generously providing reagents.

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Contributions

G.Y. and S.M. performed the cloning and mutagenesis. G.Y., K.L., and R.J. carried out the protein expression, purification, characterization, and crystallographic studies. K.P. collected the X-ray diffraction data. S.Z. and M.D. performed all experiments on cultured neurons. A.R. and S.M. generated the full-length BoNT/A1 mutants and performed the MPN assay. D.S., K.B., and M.K. performed the SPR studies. R.J., M.D., and A.R. wrote the manuscript with input from other authors.

Corresponding authors

Correspondence to Andreas Rummel, Min Dong or Rongsheng Jin.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The structure of the HCA–SV2C complex.

(a) The structure of human gSV2C displays a unique pentapeptide-repeat motif, where phenylalanine residues spaced 5 residues apart (except S527) provide important stacking effect to stabilize the structure. These residues are shown in sticks, with the ones that are conserved in all three SV2 isoforms across different species are colored gold. (b) The structures of HCA in complex with the rat bSV2C or human gSV2C are superimposed. The N559 glycan of gSV2C is shown as a transparent sphere model. Residue F563 of human SV2C is replaced by L563 in rat SV2C, which abolishes the cation-π stacking interaction. (c) The protein–protein interface between HCA and the human gSV2C. The plots were generated using LIGPLOT (Laskowski, R.A. et al., J Chem Inf Model 51, 2778-86 2011). BoNT/A and SV2C residues are labeled brown and green, respectively. Hydrogen bonds are indicated by dashed green lines. A similar interaction network is observed in the structure of HCA in complex with the rat bSV2C, except that the cation-π stacking interaction (double arrow) is unique for human SV2C.

Supplementary Figure 2 Expression levels of the deglycosylation mutants of SV2A, 2B, and 2C in neurons.

Hippocampal/cortical neurons cultured from SV2A(-/-)SV2B(-/-) mice were infected with lentiviruses that express either WT SV2A, 2B, and 2C, or indicated deglycosylation mutants. Cell lysates were harvested and subjected to immunoblot analysis. Actin served as a loading control. The lentiviral vector contains two separated synapsin promoters, with one driving expression of SV2 and the other driving expression of GFP. Thus, GFP served as an internal control for viral infection. Immunoblot signals of SV2 were quantified, normalized using GFP signals, and compared between WT and deglycosylation mutants. The same amounts of viruses were used for WT SV2A and SV2A-N573A (panel a), and for WT SV2C and SV2C-N559A (panel c). The deglycosylation mutation has no effect on SV2A and modestly reduced the expression level of SV2C in neurons. However, it severely reduced the expression level of SV2B. As shown in panel b, even with 10-fold more viruses, SV2B-N516A expression was still drastically lower than WT SV2B. The data are presented as mean ± S.D., n = 3.

Supplementary Figure 3 Electron densities of the N559 glycan of SV2C in the gSV2C–HCA complex.

(a) Key glycan-binding residues of HCA and the N559 glycan are shown as stick models. Water molecules facilitating the HCA–glycan association are shown as green spheres. A simulated-annealing omit electron density map contoured at 1.5 σ was overlaid with the final refined model. (b) A different view with a rotation ~90° about a vertical axis.

Supplementary Figure 4 Single-site mutations of HCA and HCHA adopt wild-type-like structures.

The thermal stability of proteins was measured using a fluorescence-based thermal shift assay on a StepOne real-time PCR system (ThermoFisher). Specifically, protein melting was monitored using a hydrophobic dye, SYPRO Orange (Sigma-Aldrich), as the temperature was increased in a linear ramp from 20oC to 95oC. The midpoint of the protein-melting curve (Tm) was determined using the software provided by the instrument manufacturer. The data are presented as mean ± S.D., n = 3. All HCA and HCHA mutants showed Tm values comparable to the wild-type protein, indicating correct protein folding.

Supplementary Figure 5 Characterization of binding between HCA variants and human bSV2C and gSV2C.

(a) Surface plasmon resonance was used to examine the changes of binding affinity between HCA variants and SUMO-bSV2C or gSV2C, respectively. SV2C was covalently immobilized to a CM5 chip as a ligand whereas HCA variants were analytes. Bars from left to right represent the responses when HCA was applied at 10 pM, 1 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, and 200 nM, respectively. RU stands for arbitrary response unit. (b,c) Interactions between HCA variants (preys) and SUMO-bSV2C or gSV2C (baits) were examined by a pull down assay. (d,e) Binding kinetics and affinity between HCA-F953G and immobilized bSV2C (107 RU; panel d) or gSV2C (74 RU; panel e) were determined by injecting 1:3 dilution series ranging from 2,000 nM to 8.23 nM. Values shown represent the mean ±S.D. (n = 2).

Supplementary Figure 6 Binding of glycan-binding-deficient HCA mutants to neurons that express individual SV2 isoforms.

Hippocampal/cortical neurons cultured from SV2A(+/+)SV2B(-/-) mice served as neurons that only express SV2A. Neurons that only express SV2B or SV2C were created by infecting neurons cultured from SV2A(-/-)SV2B(-/-) mice with lentiviruses that express SV2B or SV2C, respectively. Neurons were then exposed to WT or indicated HCA mutant (100 nM), washed, fixed, and subjected to immunostaining analysis. HCA was detected with a monoclonal human anti-BoNT/A antibody (RAZ-1) and SV2 was detected with a mouse monoclonal pan-SV2 antibody. Scale bar, 20 µm.

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Yao, G., Zhang, S., Mahrhold, S. et al. N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat Struct Mol Biol 23, 656–662 (2016). https://doi.org/10.1038/nsmb.3245

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