Ganglioside Binding Research Articles

Crystal structure of botulinum neurotoxin subtype A3 cell binding domain in complex with GD1a co‐receptor ganglioside

Botulinum neurotoxins (BoNTs) are one of the most toxic proteins known to humans. Their molecular structure is comprised of three essential domains—a cell binding domain (HC), translocation domain and catalytic domain (light chain) . The HC domain facilitates the highly specific binding of BoNTs to the neuronal membrane via a dual‐receptor complex involving a protein receptor and a ganglioside. Variation in activity/toxicity across subtypes of serotype A has been attributed to changes in protein and ganglioside interactions, and their implications are important in the design of novel BoNT‐based therapeutics. Here, we present the structure of BoNT/A3 cell binding domain (HC/A3) in complex with the ganglioside GD1a at 1.75 Å resolution. The structure revealed that six residues interact with the three outermost monosaccharides of GD1a through several key hydrogen bonding interactions. A detailed comparison of structures of HC/A3 with HC/A1 revealed subtle conformational differences at the ganglioside binding site upon carbohydrate binding.

LTA1 and dmLT enterotoxin-based proteins activate antigen-presenting cells independent of PKA and despite distinct cell entry mechanisms.

Enterotoxin-based proteins are powerful manipulators of mucosal immunity. The A1 domain of heat-labile enterotoxin from E. coli, or LTA1, is a newer adjuvant from this family under investigation for intranasal vaccines. Although LTA1 has been examined in mouse vaccination studies, its ability to directly stimulate immune cells compared to related adjuvant proteins has not been well explored. Here, we perform the first rigorous examination of LTA1’s effect on antigen presenting cells (APC) using a human monocyte cell line THP-1. To better understand LTA1’s stimulatory effects, we compared it to dmLT, or LT-R192G/L211A, a related AB5 adjuvant in clinical trials for oral or parenteral vaccines. LTA1 and dmLT both activated APCs to upregulate MHC-II (HLA-DR), CD86, cytokine secretion (e.g., IL-1β) and inflammasome activation. The effect of LTA1 on surface marker changes (e.g., MHC-II) was highly dose-dependent whereas dmLT exhibited high MHC-II expression regardless of dose. In contrast, cytokine secretion profiles were similar and dose-dependent after both LTA1 and dmLT treatment. Cellular activation by LTA1 was independent of ganglioside binding, as pre-treatment with purified GM1 blocked the effect of dmLT but not LTA1. Unexpectedly, while activation of the inflammasome and cytokine secretion by LTA1 or dmLT was blocked by the protein kinase A inhibitor H89 (similar to previous reports), these responses were not inhibited by a more specific PKA peptide inhibitor or antagonist; thus Indicating that a novel and unknown mechanism is responsible for inflammasome activation and cytokine secretion by LT proteins. Lastly, LTA1 stimulated a similar cytokine profile in primary human monocytes as it did in THP1 cells, including IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, and TNFα. Thus, we report that LTA1 protein programs a dendritic cell-like phenotype in APCs similar to dmLT in a mechanism that is independent of PKA activation and GM1 binding and entry.

An integrated numerical and experimental framework for modeling of CTB and GD1b ganglioside binding kinetics

A mathematical model is developed and validated for a multistep binding process between cholera toxin subunit B (CTB) and GD1b receptors that precedes cholera infection. To study the dynamics of the complex CTB‐GD1b binding mechanisms, cooperative binding effect and GD1b receptor aggregation in the host cell membrane are considered. More reliable parameters for the CTB‐GD1b binding kinetics are estimated by quantitatively calibrating the proposed multistep binding model against the experimental measurements obtained from the novel nanocube‐based biosensor. Specifically, a numerical scheme that includes the sensitivity analysis, parameter estimation and dynamic optimization is implemented for the model calibration. Through this scheme, identifiable model parameters are determined. After those selected parameters are estimated, the calibrated model and the experimental measurements were in reasonable agreement for different CTB and GD1b concentrations, which shows a promising approach for identification of the kinetics of CTB binding to the host cell membrane. © 2018 American Institute of Chemical Engineers AIChE J, 64: 3882–3893, 2018

Detecting Protein-Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs.

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB5). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10days, GM1 binding to CTB5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB5 measured for pre-loaded PDs and PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. Graphical Abstract ᅟ.

Enhancing toxin-based vaccines against botulism

Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HCN) and a receptor binding domain (HCC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHCN/A1 and HCC/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >106-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1W. M-BoNT/A1W vaccination protected against challenge by 106 LD50 Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1W surviving BoNT challenge had dominant antibody responses to the LCHCN domain, but varied antibody responses to HCC. Sera from mice vaccinated with M-BoNT/A1W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.

Human norovirus GII.4(MI001) P dimer binds fucosylated and sialylated carbohydrates.

Human noroviruses (HuNoV), members of the family Caliciviridae, are the major cause of acute viral gastroenteritis worldwide. Successful infection is linked to the ability of the protruding (P) domain of the viral capsid to bind histo-blood group antigens (HBGA). Binding to gangliosides plays a major role for many nonhuman calici- and noroviruses. Increasing evidence points to a broader role of sialylated carbohydrates such as gangliosides in norovirus infection. Here, we compare HBGA and ganglioside binding of a GII.4 HuNoV variant (MI001), previously shown to be infectious in a HuNoV mouse model. Saturation transfer difference nuclear magnetic resonance spectroscopy, native mass spectrometry (MS) and surface plasmon resonance spectroscopy were used to characterize binding epitopes, affinities, stoichiometry and dynamics, focusing on 3'-sialyllactose, the GM3 ganglioside saccharide and B antigen. Binding was observed for 3'-sialyllactose and various HBGAs following a multistep binding process. Intrinsic affinities (Kd) of fucose, 3'-sialyllactose and B antigen were determined for the individual binding steps. Stronger affinities were observed for B antigen over 3'-sialyllactose and fucose, which bound in the mM range. Binding stoichiometry was analyzed by native MS showing the presence of four B antigens or two 3'-sialyllactose in the complex. Epitope mapping of 3'-sialyllactose revealed direct interaction of α2,3-linked sialic acid with the P domain. The ability of HuNoV to engage multiple carbohydrates emphasizes the multivalent nature of norovirus glycan-specificity. Our findings reveal direct binding of a GII.4 HuNoV P dimer to α2,3-linked sialic acid and support a broader role of ganglioside binding in norovirus infection.

Insights into the Mechanisms by Which Clostridial Neurotoxins Discriminate between Gangliosides.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the causative agents of the paralytic diseases botulism and tetanus, respectively. Entry of toxins into neurons is mediated through initial interactions with gangliosides, followed by binding to a protein co-receptor. Herein, we aimed to understand the mechanism through which individual neurotoxins recognize the carbohydrate motif of gangliosides. Using cell-based and in vitro binding assays, in conjunction with structure-driven site-directed mutagenesis, a conserved hydrophobic residue within the BoNTs that contributes to both affinity and specificity toward Sia5-containing gangliosides was identified. We demonstrate that targeted mutations within the Sia5 binding pocket result in the generation of neurotoxins that either bind and enter cells more efficiently (BoNT/A1 and BoNT/B) or display altered ganglioside binding specificity (TeNT). These data support a model in which recognition of Sia5 is largely driven by hydrophobic interactions between the sugar and the Sia5 binding site.

Structural basis of myelin-associated glycoprotein adhesion and signalling.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin–axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified—N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site—that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin–axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.

Ganglioside-Lipid and Ganglioside-Protein Interactions Revealed by Coarse-Grained and Atomistic Molecular Dynamics Simulations.

Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to a ceramide. Gangliosides reside in the outer leaflet of the plasma membrane and play a crucial role in various physiological processes such as cell signal transduction and neuronal differentiation by modulating structures and functions of membrane proteins. Because the detailed behavior of gangliosides and protein-ganglioside interactions are poorly known, we investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin (AQP1) and WALP23 using equilibrium molecular dynamics simulations and potential of mean force calculations at both coarse-grained (CG) and atomistic levels. In atomistic simulations, on the basis of the GROMOS force field, ganglioside aggregation appears to be a result of the balance between hydrogen bond interactions and steric hindrance of the headgroups. GM3 clusters are slightly larger and more ordered than GM1 clusters due to the smaller headgroup of GM3. The different structures of GM1 and GM3 clusters from atomistic simulations are not observed at the CG level based on the Martini model, implying a difference in driving forces for ganglioside interactions in atomistic and CG simulations. For protein-ganglioside interactions, in the atomistic simulations, GM1 lipids bind to specific sites on the AQP1 surface, whereas they are depleted from WALP23. In the CG simulations, the ganglioside binding sites on the AQP1 surface are similar, but ganglioside aggregation and protein-ganglioside interactions are more prevalent than in the atomistic simulations. Using the polarizable Martini water model, results were closer to the atomistic simulations. Although experimental data for validation is lacking, we proposed modified Martini parameters for gangliosides to more closely mimic the sizes and structures of ganglioside clusters observed at the atomistic level.

Anti-ganglioside antibodies are removed from circulation in mice by neuronal endocytosis.

SEE VAN DOORN AND JACOBS DOI101093/BRAIN/AWW078 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE : In axonal forms of Guillain-Barré syndrome, anti-ganglioside antibodies bind gangliosides on nerve surfaces, thereby causing injury through complement activation and immune cell recruitment. Why some nerve regions are more vulnerable than others is unknown. One reason may be that neuronal membranes with high endocytic activity, including nerve terminals involved in neurotransmitter recycling, are able to endocytose anti-ganglioside antibodies from the cell surface so rapidly that antibody-mediated injury is attenuated. Herein we investigated whether endocytic clearance of anti-ganglioside antibodies by nerve terminals might also be of sufficient magnitude to deplete circulating antibody levels. Remarkably, systemically delivered anti-ganglioside antibody in mice was so avidly cleared from the circulation by endocytosis at ganglioside-expressing plasma membranes that it was rapidly rendered undetectable in serum. A major component of the clearance occurred at motor nerve terminals of neuromuscular junctions, from where anti-ganglioside antibody was retrogradely transported to the motor neuron cell body in the spinal cord, recycled to the plasma membrane, and secreted into the surrounding spinal cord. Uptake at the neuromuscular junction represents a major unexpected pathway by which pathogenic anti-ganglioside antibodies, and potentially other ganglioside binding proteins, are cleared from the systemic circulation and also covertly delivered to the central nervous system.

Two Feet on the Membrane: Uptake of Clostridial Neurotoxins.

The extraordinary potency of botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT) is mediated by their high neurospecificity, targeting peripheral cholinergic motoneurons leading to flaccid and spastic paralysis, respectively, and successive respiratory failure. Complex polysialo gangliosides accumulate BoNT and TeNT on the plasma membrane. The ganglioside binding in BoNT/A, B, E, F, G, and TeNT occurs via a conserved ganglioside-binding pocket within the most carboxyl-terminal 25kDa domain HCC, whereas BoNT/C, DC, and D display here two different ganglioside binding sites. This enrichment step facilitates subsequent binding of BoNT/A, B, DC, D, E, F, and G to the intraluminal domains of the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C and synaptotagmin-I/-II, respectively. Whereas an induced α-helical 20-mer Syt peptide binds via side chain interactions to the tip of the HCC domain of BoNT/B, DC and G, the preexisting, quadrilateral β-sheet helix of SV2C-LD4 binds the clinically most relevant serotype BoNT/A mainly through backbone-backbone interactions at the interface of HCC and HCN. In addition, the conserved, complex N559-glycan branch of SV2C establishes extensive interactions with BoNT/A resulting in delayed dissociation providing BoNT/A more time for endocytosis into synaptic vesicles. An analogous interaction occurs between SV2A/B and BoNT/E. Altogether, the nature of BoNT-SV2 recognition clearly differs from BoNT-Syt. Subsequently, the synaptic vesicle is recycled and the bound neurotoxin is endocytosed. Acidification of the vesicle lumen triggers membrane insertion of the translocation domain, pore formation, and finally translocation of the enzymatically active light chain into the neuronal cytosol to halt release of neurotransmitters.

Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides

Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimer's and Parkinson's. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimer's β-amyloid (Aβ1-42) peptide and Parkinson's disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca2+ fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinson's-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimer's and Parkinson's diseases.

The Divergent CD8+ T Cell Adjuvant Properties of LT-IIb and LT-IIc, Two Type II Heat-Labile Enterotoxins, Are Conferred by Their Ganglioside-Binding B Subunits

Poor immune responses elicited by vaccine antigens can be enhanced by the use of appropriate adjuvants. Type II heat-labile enterotoxins (HLT) produced by Escherichia coli are extremely potent adjuvants that augment both humoral and cellular immunity to co-administered antigens. Recent findings demonstrate that LT-IIb and LT-IIc, two type II HLT adjuvants, exhibit potent, yet distinguishable CD8+ T cell adjuvant properties. While LT-IIc elicits a robust and rapid response at one week after administration, LT-IIb engenders a more gradual and slower expansion of antigen-specific CD8+ T cells that correlates with improved immunity. The variations in immune effects elicited by the HLT adjuvants have been generally attributed to their highly divergent B subunits that mediate binding to various gangliosides on cell surfaces. Yet, HLT adjuvants with point mutations in the B subunit that significantly alter ganglioside binding retain similar adjuvant functions. Therefore, the contribution of the B subunits to adjuvanticity remains unclear. To investigate the influence of the B subunits on the enhancement of immune responses by LT-IIb and LT-IIc, chimeric HLT were engineered in which the B subunits of the two adjuvants were exchanged. Comparing the immune potentiating characteristics of both native and chimeric HLT adjuvants, it was found that not all the adjuvant characteristics of the HLT adjuvants were modulated by the respective B subunits. Specifically, the differences in the CD8+ T cell kinetics and protective responses elicited by LT-IIb and LT-IIc did indeed followed their respective B subunits. However, induction of IL-1 from macrophages and the capacity to intoxicate cells in a mouse Y1 adrenal cell bioassay did not correlate with the B subunits. Therefore, it is likely that additional factors other than the B subunits contribute to the effects elicited by the HLT adjuvants.

Ganglioside regulation of AMPA receptor trafficking.

Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10(-4)), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

Deciphering the glycolipid code of Alzheimer's and Parkinson's amyloid proteins allowed the creation of a universal ganglioside-binding peptide.

A broad range of microbial and amyloid proteins interact with cell surface glycolipids which behave as infectivity and/or toxicity cofactors in human pathologies. Here we have deciphered the biochemical code that determines the glycolipid-binding specificity of two major amyloid proteins, Alzheimer's β-amyloid peptide (Aβ) and Parkinson's disease associated protein α-synuclein. We showed that both proteins interact with selected glycolipids through a common loop-shaped motif exhibiting little sequence homology. This 12-residue domain corresponded to fragments 34-45 of α-synuclein and 5-16 of Aβ. By modulating the amino acid sequence of α-synuclein at only two positions in which we introduced a pair of histidine residues found in Aβ, we created a chimeric α-synuclein/Aβ peptide with extended ganglioside-binding properties. This chimeric peptide retained the property of α-synuclein to recognize GM3, and acquired the capacity to recognize GM1 (an Aβ-inherited characteristic). Free histidine (but not tryptophan or asparagine) and Zn2+ (but not Na+) prevented this interaction, confirming the key role of His-13 and His-14 in ganglioside binding. Molecular dynamics studies suggested that the chimeric peptide recognized cholesterol-constrained conformers of GM1, including typical chalice-shaped dimers, that are representative of the condensed cholesterol-ganglioside complexes found in lipid raft domains of the plasma membrane of neural cells. Correspondingly, the peptide had a particular affinity for raft-like membranes containing both GM1 and cholesterol. The chimeric peptide also interacted with several other gangliosides, including major brain gangliosides (GM4, GD1a, GD1b, and GT1b) but not with neutral glycolipids such as GlcCer, LacCer or asialo-GM1. It could inhibit the binding of Aβ1-42 onto neural SH-SY5Y cells and did not induce toxicity in these cells. In conclusion, deciphering the glycolipid code of amyloid proteins allowed us to create a universal ganglioside-binding peptide of only 12-residues with potential therapeutic applications in infectious and neurodegenerative diseases that involve cell surface gangliosides as receptors.

Multiple domains of tetanus toxin direct entry into primary neurons.

Tetanus toxin elicits spastic paralysis by cleaving VAMP-2 to inhibit neurotransmitter release in inhibitory neurons of the central nervous system. As the retrograde transport of tetanus neurotoxin (TeNT) from endosomes has been described, the initial steps that define how TeNT initiates trafficking to the retrograde system are undefined. This study examines TeNT entry into primary cultured cortical neurons by total internal reflection fluorescence (TIRF) microscopy. The initial association of TeNT with the plasma membrane was dependent upon ganglioside binding, but segregated from synaptophysin1 (Syp1), a synaptic vesicle (SV) protein. TeNT entry was unaffected by membrane depolarization and independent of SV cycling, whereas entry of the receptor-binding domain of TeNT (HCR/T) was stimulated by membrane depolarization and inhibited by blocking SV cycling. Measurement of the incidence of colocalization showed that TeNT segregated from Syp1, whereas HCR/T colocalized with Syp1. These studies show that while the HCR defines the initial association of TeNT with the cell membrane, regions outside the HCR define how TeNT enters neurons independent of SV cycling. This provides a basis for the unique entry of botulinum toxin and tetanus toxin into neurons.

Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system

Although Escherichia coli and yeast were commonly used to express recombinant Hc of botulinum neurotoxins, as an alternative, in current study, a 293E expression system was used to express the Hc of botulinum neurotoxin serotype B (BHc) as soluble recombinant protein for experimental vaccine evaluation. Our results demonstrated that the 293E expression system could produce high level of recombinant secreted BHc protein, which was immunorecognized specifically by anti-botulinum neurotoxin serotype B (BoNT/B) sera and showed ganglioside binding activities. The serological response and efficacy of recombinant BHc formulated with aluminum hydroxide adjuvant were evaluated in mice. Immunization with Alhydrogel-formulated BHc subunit vaccine afforded the effective protection against BoNT/B challenge. A frequency- and dose-dependent effect to immunization with BHc subunit vaccine was observed and the ELISA antibody titers correlated well with neutralizing antibody titers and protection. And a solid-phase assay showed that the neutralizing antibodies from the BHc-immunized mice inhibited the binding of BHc to the ganglioside GT1b. Our results also show that the plasmid pABE293SBHc derived of the 293E expression system as DNA vaccine is capable of inducing stronger humoral response and protective efficacy against BoNT/B than the pVAX1SBHc. In summary, immunization with the 293E-expressed BHc protein generates effective immune protection against BoNT/B as E. coli or yeast-expressed BHc, so the efficient expression of botulinum Hc protein for experimental vaccine can be prepared using the 293E expression system.

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.

Sialic Acid Linkage in Glycosphingolipids Is a Molecular Correlate for Trafficking and Delivery of Extracellular Cargo

Gangliosides, glycosphingolipids containing sialic acid moieties, are well known mediators of transmembrane signaling and endocytosis at the plasma membrane. However, little is known about their precise regulatory role at the cell periphery for intracellular sorting of extracellular cargo. Here we inspected published scientific literature for two types of cargoes, namely bacterial toxins and viruses, regarding their usage of gangliosides. We derived a rather simple yet surprisingly consistent framework to classify 20 viruses from 12 different families and five type AB bacterial toxins into two broad categories. We propose that gangliosides with terminally attached sialic acids classify cargo for uptake and trafficking early in the endocytic pathway while gangliosides with internally attached sialic acids associate with uptake and trafficking of cargo late in the endocytic system. Our study provides a testable hypothesis for future investigations into a wide range of trafficking events. It could be utilized as a framework for other intracellular pathogens where lipids are known to be involved in recognition and trafficking. For instance, predictions can be put forward and evaluated based on ganglioside binding patterns and intracellular trafficking routes. Finally, incorporation of our classifier into large scale systems-biology studies could help reveal related molecular determinants in subcellular sorting.

Crystal Structures of Botulinum Neurotoxin DC in Complex with Its Protein Receptors Synaptotagmin I and II

Botulinum neurotoxins (BoNTs) can cause paralysis at exceptionally low concentrations and include seven serotypes (BoNT/A-G). The chimeric BoNT/DC toxin has a receptor binding domain similar to the same region in BoNT/C. However, BoNT/DC does not share protein receptor with BoNT/C. Instead, it shares synaptotagmin (Syt) I and II as receptors with BoNT/B, despite their low sequence similarity. Here, we present the crystal structures of the binding domain of BoNT/DC in complex with the recognition domains of its protein receptors, Syt-I and Syt-II. The structures reveal that BoNT/DC possesses a Syt binding site, distinct from the established Syt-II binding site in BoNT/B. Structure-based mutagenesis further shows that hydrophobic interactions play a key role in Syt binding. The structures suggest that the BoNT/DC ganglioside binding sites are independent of the protein receptor binding site. Our results reveal the remarkable versatility in the receptor recognition of the BoNTs.