Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry

Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A–G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.     

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.      

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.     

Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.     

Unique ganglioside recognition strategies for clostridial neurotoxins

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E … H … SXWY … G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

Molecular basis for tetanus toxin coreceptor interactions

Tetanus toxin (TeNT) elicits spastic paralysis through the cleavage of vesicle-associated membrane protein-2 (VAMP-2) in neurons at the interneuronal junction of the central nervous system. While TeNT retrograde traffics from peripheral nerve endings to the interneuronal junction, there is limited understanding of the neuronal receptors utilized by tetanus toxin for the initial entry into nerve cells. Earlier studies implicated a coreceptor for tetanus toxin entry into neurons: a ganglioside binding pocket and a sialic acid binding pocket and that GT1b bound to each pocket. In this study, a solid phase assay characterized the ganglioside binding specificity and functional properties of both carbohydrate binding pockets of TeNT. The ganglioside binding pocket recognized the ganglioside sugar backbone, Gal-GalNAc, independent of sialic acid-(5) and sialic acid-(7) and GM1a was an optimal substrate for this pocket, while the sialic acid binding pocket recognized sialic acid-(5) and sialic acid-(7) with "b"series of gangliosides preferred relative to "a" series gangliosides. The high-affinity binding of gangliosides to TeNT HCR required functional ganglioside and sialic acid binding pockets, supporting synergistic binding to coreceptors. This analysis provides a model for how tetanus toxin utilizes coreceptors for high-affinity binding to neurons.     

Dynamic properties of the colicin E1 ion channel

Abstract The mechanism of channel formation and action of channel-forming colicins is a paradigm for the study of dynamic aspects of membrane-protein interactions. The following experimental results concerning interaction of the colicin E1 channel domain with target membranes, in vitro and in vivo, are discussed: (1) the nature of the translocation-competent state of the channel-forming domain; (2) unfolding of the colicin channel peptide during in vitro binding and anchoring of the channel to liposome membranes at acidic pH; (3) reversal of channel peptide binding to liposomes by an alkaline-directed pH shift; (4) voltage-driven translocation and gating of the ion channel, discussed in the context of a four-helix model for a monomeric channel; (5) rescue of colicin-treated cells by high levels of external K⁺; (6) trypsin rescue of cells depolarized by the colicin ion channel; and (7) interaction of the channel domain with its immunity protein.     

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.     

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ∼150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the “W” pocket) and the sialic acid-binding site (the “R” pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT)³ produced by the spore-forming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicle-associated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ∼150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (∼50 kDa) and a C-terminal heavy chain domain (∼100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptor-binding domain (HCR/T, ∼50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of α-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5–9). Current models implicate a dual receptor mechanism for the binding of the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10). Complex gangliosides are sialic acid-containing glycosphingolipids that are located on the outer leaflet of cell membranes and contain a common “core” (GA1) consisting of Gal(β1–3)GalNAc(β1–4)Gal(β1–4)Glc(β1–1)Cer to which one or more N-acetylneuraminic acids (sialic acids) are bound, yielding “a” and “b” series gangliosides (11, 12). Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site and a sialic acid-binding site (13). Previous studies showed that Trp¹²⁸⁹ is the key residue for the lactose-binding site, and Arg¹²²⁶ is the key residue for the sialic acid-binding site (14). In this study, we denote the lactose-binding site as the “W” pocket and the sialic acid-binding site as the “R” pocket. Binz and co-workers (14) showed that functional R and W binding sites were required for TeNT toxicity (7). These biochemical and cellular studies were supported by a co-crystal structure of HCR/T bound to a GT1b-β anomer analog, which showed that the W and R carbohydrate-binding pockets were located at different regions of TeNT (7). We recently reported that the W pocket binds gangliosides via the GA1 core structure, whereas the R pocket binds gangliosides via di- or tri-sialic acid moieties (15) where simultaneous binding of TeNT to two gangliosides was synergistic (see Fig. 1a). In the current study, gangliosides were identified that bound exclusively to either the W pocket or R pocket, which allowed the characterization of the role of ganglioside binding to the W and R pockets as dual receptors for TeNT entry into neurons.Open in a separate windowFIGURE 1.Interaction of the HCR domain of TeNT with its putative cellular receptor. a, HCR/T has two ganglioside-binding sites. The W pocket binds to the terminal GalNAc-Gal of the ganglioside (illustrated by GM1a). The R pocket binds to the di-sialic acid of the ganglioside (illustrated by GD3). b, alternating lanes of molecular mass marker proteins and cortical neuron lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained for protein with Ponceau S (bottom panel), and then the membrane strips were incubated with 10 nm of the indicated HCR/T (HCR/T wild type (wt), HCR/T (R+, W−), HCR/T (R−, W+), or HCR/T (R−, W−)) followed by HRP-conjugated α-FLAG antibody. The bands were visualized with SuperSignal; exposed film is shown (upper panel). The asterisk denotes the position of purified gangliosides resolved under identical conditions. Migration of the molecular mass marker proteins is indicated (kDa) in the left-most lane in the upper panel.     

Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.     

The colicin Ia receptor,Cir, is also the translocator for colicin Ia

Colicin Ia, a channel‐forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor‐binding domain bound to Cir, a 22‐stranded plugged β‐barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no ‘second receptor’ has ever been identified for TonB‐dependent colicins, such as Ia. We show that if the receptor‐binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild‐type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N‐terminal translocation (T) domain and its channel‐forming C‐terminal domain. Deletion of colicin Ia's receptor‐binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor‐binding domain, Cir also binds weakly to its translocation domain.     

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria.

Mitochondrial precursor proteins made in the cytosol bind to a hetero-oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This 'trans' domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N-terminal targeting sequence of a translocation-arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration-driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3- to 8-fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursor's basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.     

Uptake across the cell envelope and insertion into the inner membrane of ion channel-forming colicins in E coli.

Pore-forming colicins exert their lethal effect on E coli through formation of a voltage-dependent channel in the inner (cytoplasmic-membrane) thus destroying the energy potential of sensitive cells. Their mode of action appears to involve 3 steps: i) binding to a specific receptor located in the outer membrane; ii) translocation across this membrane; iii) insertion into the inner membrane. Colicin A has been used as a prototype of pore-forming colicins. In this review, the 3 functional domains of colicin A respectively involved in receptor binding, translocation and pore formation, are defined. The components of sensitive cells implicated in colicin uptake and their interactions with the various colicin A domains are described. The 3-dimensional structure of the pore-forming domain of colicin A has been determined recently. This structure suggests a model of insertion into the cytoplasmic membrane which is supported by model membrane studies. The role of the membrane potential in channel functioning is also discussed.     

How botulinum and tetanus neurotoxins block neurotransmitter release

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.     

Botulinum Neurotoxin Light Chain Refolds at Endosomal pH for its Translocation

Botulinum neurotoxins (BoNTs), the most poisonous member of class A biothreat agent, cause neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. In its mechanism of action, the catalytic domain (light chain (LC) of BoNT) is transported to the cytosol by the heavy chain (HC) in order to reach its proteolytic substrates. The BoNT HC forms a membrane channel under acidic conditions encountered in endosomes to serve as a passageway for LC to enter into cytosol. We demonstrate here that BoNT/A LC undergoes unique structural changes under the low pH conditions, and adopts a molten globule state, exposing substantial number of hydrophobic groups. The flexibility of the molten globular structure combined with retention of the secondary structure and exposure of specific residues of LC for interaction with the HC, allows its translocation through the narrow endosomal membrane channel.     

The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD₅₀ of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a “dual receptor” mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C.     

Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells

The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.