Testing a Recombinant Form of Tetanus Toxoid as a Carrier Protein for Glycoconjugate Vaccines.

Glycoconjugate vaccines play a major role in the prevention of infectious diseases worldwide, with significant impact on global health, enabling the polysaccharides to induce immunogenicity in infants and immunological memory. Tetanus toxoid (TT), a chemically detoxified bacterial toxin, is among the few carrier proteins used in licensed glycoconjugate vaccines. The recombinant full-length 8MTT was engineered in E. coli with eight individual amino acid mutations to inactivate three toxin functions. Previous studies in mice showed that 8MTT elicits a strong IgG response, confers protection, and can be used as a carrier protein. Here, we compared 8MTT to traditional carrier proteins TT and cross-reactive material 197 (CRM197), using different polysaccharides as models: Group A Streptococcus cell-wall carbohydrate (GAC), Salmonella Typhi Vi, and Neisseria meningitidis serogroups A, C, W, and Y. The persistency of the antibodies induced, the ability of the glycoconjugates to elicit booster response after re-injection at a later time point, the eventual carrier-induced epitopic suppression, and immune interference in multicomponent formulations were also evaluated. Overall, immunogenicity responses obtained with 8MTT glycoconjugates were compared to those obtained with corresponding TT and, in some cases, were higher than those induced by CRM197 glycoconjugates. Our results support the use of 8MTT as a good alternative carrier protein for glycoconjugate vaccines, with advantages in terms of manufacturability compared to TT.

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain.

Botulinum neurotoxin subtype A4 (BoNT/A4) is ~1000-fold less potent than BoNT/A1. This study addresses the basis for low BoNT/A4 potency. Utilizing BoNT/A1-A4 and BoNT/A4-A1 Light Chain-Heavy Chain (LC-HC) chimeras, HC-A4 was responsible for low BoNT/A4 potency. Earlier studies showed BoNT/A1-receptor binding domain (Hcc) bound a ß-strand peptide (556-564) and glycan-N559 within Luminal Domain 4 (LD4) of SV2C, the BoNT/A protein receptor. Relative to BoNT/A1, the Hcc of BoNT/A4 possesses two amino acid variants (D1141 and N1142) within the ß-peptide binding interface and one amino acid variant (R1292) located near the SV2C glycan-N559. Introduction of BoNT/A4 ß-strand peptide variant (D1141 and N1142) into BoNT/A1 reduced toxin potency 30-fold, and additional introduction of the BoNT/A4 glycan-N559 variant (D1141, N1142, and R1292) further reduced toxin potency to approach BoNT/A4. While introduction of BoNT/A1 glycan-N559 variant (G1292) into BoNT/A4 did not alter toxin potency, additional introduction of BoNT/A1 ß-strand peptide variants (G1141, S1142, and G1292) resulted in potency approaching BoNT/A1 potency. Thus, outcomes from these functional and modeling studies indicate that in rodent models, disruption of Hcc -SV2C ß-peptide and -glycan-N559 interactions mediate low BoNT/A4 potency, while in human motor neurons, disruption of Hcc-SV2C ß-peptide alone mediates low BoNT/A4 potency, which link to a species-specific variation at SV2C563.

How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin for humans and is utilized as a therapy for numerous neurologic diseases. BoNT/A comprises a catalytic Light Chain (LC/A) and a Heavy Chain (HC/A) and includes eight subtypes (BoNT/A1-/A8). Previously we showed BoNT/A potency positively correlated with stable localization on the intracellular plasma membrane and identified a low homology domain (amino acids 268-357) responsible for LC/A1 stable co-localization with SNAP-25 on the plasma membrane, while LC/A3 was present in the cytosol of Neuro2A cells. In the present study, steady-state- and live-imaging of a cytosolic LC/A3 derivative (LC/A3V) engineered to contain individual structural elements of the A1 LDH showed that a 59 amino acid region (275-334) termed the MLD was sufficient to direct LC/A3V from the cytosol to the plasma membrane co-localized with SNAP-25. Informatics and experimental validation of the MLD-predicted R1 region (an α-helix, residues 275-300) and R2 region (a loop, α-helix, loop, residues 302-334) both contribute independent steps to the stable co-localization of LC/A1 with SNAP-25 on the plasma membrane of Neuro-2A cells. Understanding how these structural elements contribute to the overall association of LC/A1 on the plasma membrane may identify the molecular basis for the LC contribution of BoNT/A1 to high potency.

Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein.

Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd (Przedpelski et al. mBio, 2020). Here we describe the production of 8MTT in Gor/MetTM E. coli, a strain engineered to have an oxidative cytoplasm, allowing for the expression of soluble, disulfide-bonded proteins. The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.

Multiple Domains of Staphylococcal Superantigen-like Protein 11 (SSL11) Contribute to Neutrophil Inhibition.

Staphylococcus aureus is an opportunistic pathogen producing many immune evasion molecules targeting various components of the host immune defense, including the Staphylococcal superantigen-like protein (SSL 1-14) family. Despite sharing similar structures with the powerful superantigens (SAgs), which cause massive T cell activation, SSLs interfere with a wide range of innate immune defenses. SSLs are divided into two subgroups, SSLs that contain a conserved carbohydrate Sialyl Lewis X [Neu5Acα2-3Galß1-4(Fucα1-3) GlcNAcß, SLeX] binding site and SSLs that lack the SLeX binding site. SSL2-6 and SSL11 possess the SLeX binding site. Our previous studies showed that SSL11 arrests cell motility by inducing cell adhesion in differentiated HL60 (dHL60) cells, while SSL7 did not bind dHL60 cells. SSL7-based chimeras were engineered by exchanging the SSL7 sequence with the corresponding SSL11 sequence and assaying for a gain of SSL11 function, namely, the induction of cell spreading and motility arrest. In addition to the SLeX-binding site, we observed that three beta-strands ß6, ß7, and ß9 and the N-terminal residues, Y16 and Y17, transitioned SSL7 to gain SSL11 activities. These studies define the structure-function properties of SSL11 that may allow SSL11 to inhibit S. aureus clearance by the host innate immune system, allowing S. aureus to maintain a carrier state in humans, an understudied aspect of S. aureus pathogenesis.

Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1-A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.

A Novel High-Potency Tetanus Vaccine.

Chemically inactivated tetanus toxoid (CITT) is clinically effective and widely used. However, CITT is a crude nonmalleable vaccine that contains hundreds of Clostridium tetani proteins, and the active component is present in variable and sometimes minor percentages of vaccine mass. Recombinant production of a genetically inactivated tetanus vaccine offers an opportunity to replace and improve the current tetanus vaccine. Previous studies showed the feasibility of engineering full-length tetanus toxin (TT) in Escherichia coli In the present study, full-length TT was engineered with eight individual amino acid mutations (8MTT) to inactivate catalysis, translocation, and host receptor-binding functions, retaining 99.4% amino acid identity to native tetanus toxin. 8MTT purified as a 150-kDa single-chain protein, which trypsin nicked to a 100-kDa heavy chain and 50-kDa light chain. The 8MTT was not toxic for outbred mice and was >50 million-fold less toxic than native TT. Relative to CITT, 8MTT vaccination elicited a strong immune response and showed good vaccine potency against TT challenge. The strength of the immune response to both vaccines varied among individual outbred mice. These data support 8MTT as a candidate vaccine against tetanus and a malleable candidate conjugate vaccine platform to enhance the immune response to polysaccharides and other macromolecular molecules to facilitate a rapid response to emerging microbial pathogens.IMPORTANCE Chemical inactivation is a clinically effective mechanism to detoxify protein toxins to produce vaccines against microbial infections and to serve as a platform for production of conjugate polysaccharide vaccines. This method is widely used for the production of protein toxin vaccines, including tetanus toxoid. However, chemical modification alters the protein structure with unknown effects on antigenicity. Here, a recombinant full-length tetanus toxin (TT) is engineered with 8 mutations (8MTT) that inactivate three toxin functions: catalysis, translocation, and receptor binding. 8MTT is nontoxic and elicits a potent immune response in outbred mice. 8MTT also represents a malleable platform for the production of conjugate vaccines, which can facilitate a rapid vaccine response against emerging microbial pathogens.

Tetanus Toxin cis-Loop Contributes to Light-Chain Translocation.

The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving soluble N-ethylmaleimide-sensitive factor attachment protein receptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, ß-lactamase (ßlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop (767DKE769) connecting α15 and α16 (cis-loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis-loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis-loop in LC translocation, independently of pore formation.IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, ß-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (ßlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K768 in LC translocation. K768 was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Resolving the Molecular Steps in Clostridial Neurotoxin Light Chain Translocation.

The clostridial neurotoxins (CNTs), botulinum toxin and tetanus toxin, are the most toxic proteins for humans. Neurotoxicity is based upon the specificity of the CNTs for neural host receptors and substrates. CNTs are organized into three domains, a Light Chain (LC) that is a metalloprotease and a Heavy Chain (HC) that has two domains, an N-terminal LC translocation domain (HCN) and a C-terminal receptor binding domain (HCC). While catalysis and receptor binding functions of the CNTs have been developed, our understanding of LC translocation is limited. This is due to the intrinsic complexity of the translocation process and limited tools to assess the step-by-step events in LC translocation. Recently, we developed a novel, cell-based TT-reporter to measure LC translocation as the translocation of a ß-lactamase reporter across a vesicle membrane in neurons. Using this approach, we identified a role for a cis-Loop, located within the HCN, in LC translocation. In this commentary, we describe our current understanding of how CNTs mediate LC translocation and place the role of the cis-Loop in the LC translocation process relative to other independent functions that have been implicated in LC translocation. Understanding the basis for LC translocation will enhance the use of CNTs in vaccine development and as human therapies.

Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action.

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen, which causes superficial to lethal clinical infections. Neutrophils are the most abundant leukocytes in the blood and are the first defense mechanism against S. aureus infections. Here we show Staphylococcal Superantigen-Like protein 11 (SSL11) from MRSA USA300_FPR3757 mediated differentiated human neutrophil-like cells (dHL60) motility arrest by inducing cell adhesion and "locking" cells in adhesion stage, without inducing oxidative burst. Pre-incubation of SSL11 with the glycan Sialyl Lewis X blocked SSL11 function and de-glycosylation of dHL60 cells by PNGase F abolished SSL11 binding, suggesting that SSL11 functions via interacting with glycans. This is the first description of a bacterial toxin inhibiting neutrophil motility by inducing adhesion and "locking" cells in an adhesion stage. Therefore, this study might provide a new target against S. aureus infections.

Detection of ADP-Ribosylating Bacterial Toxins.

Many bacterial toxins catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to a host protein. Greater than 35 bacterial ADP-ribosyltransferase toxins (bARTTs) have been identified. ADP-ribosylation of host proteins may be specific or promiscuous. Despite this diversity, bARTTs share a common reaction mechanism, three-dimensional active site structure, and a conserved active site glutamic acid. Here, we describe how to measure the ADP-ribosylation of host proteins as purified proteins or within a cell lysate.

Light Chain Diversity among the Botulinum Neurotoxins.

Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A⁻G). The Centers for Disease Control classifies BoNTs as 'Category A' select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies.

Protein Toxins That Utilize Gangliosides as Host Receptors.

Subsets of protein toxins utilize gangliosides as host receptors. Gangliosides are preferred receptors due to their extracellular localization on the eukaryotic cell and due to their essential nature in host physiology. Glycosphingolipids, including gangliosides, are mediators of signal transduction within and between eukaryotic cells. Protein toxins possess AB structure-function organization, where the A domain encodes a catalytic function for the posttranslational modification of a host macromolecule, including proteins and nucleic acids, and a B domain, which encodes host receptor recognition, including proteins and glycosphingolipids, alone or in combination. Protein toxins use similar strategies to bind glycans by pockets and loops, generally employing hydrogen bonding and aromatic stacking to stabilize interactions with sugars. In some cases, glycan binding facilitates uptake, while in other cases, cross-linking or a second receptor is necessary to stimulate entry. The affinity that protein toxins have for host glycans is necessary for tissue targeting, but not always sufficient to cause disease. In addition to affinity for binding the glycan, the lipid moiety also plays an important role in productive uptake and tissue tropism. Upon endocytosis, the protein toxin must escape to another intracellular compartment or into cytosol to modify a host substrate, modulating host signaling, often resulting in cytotoxic or apoptotic events in the cell, and a unique morbidity for the organism. The study of protein toxins that utilize gangliosides as host receptors has illuminated numerous eukaryotic cellular processes, identified the basis for developing interventions to prevent disease through vaccines and control bacterial diseases through therapies. In addition, subsets of these protein toxins have been utilized as therapeutic agents to treat numerous human inflictions.

The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC. There are seven immunological BoNT serotypes (A to G); each serotype includes genetic variants, termed subtypes. Only two subtypes, BoNT/A1 and BoNT/B1, are currently used as therapeutics. BoNT serotype A (BoNT/A) subtypes A2 to A8 show distinct potency, duration of action, and pathology relative to BoNT/A1. Specifically, BoNT/A3 possesses shorter duration of action and elicits distinct symptoms in mice at high toxin doses. In this report, we analyzed the roles of LC and HC of BoNT/A3 for duration of action, neuronal cell entry, and mouse pathology by using clostridium-derived recombinant hybrid BoNTs consisting of reciprocal LC and HC (BoNTA1/A3 and BoNTA3/A1). Hybrid toxins were processed in their expression host to a dichain BoNT consisting of LC and HC linked via a disulfide bond. The LC and HC defined BoNT potency in mice and BoNT toxicity for cultured neuronal cells, while the LC defined the duration of BoNT action in cell and mouse models. Protein alignment identified a previously unrecognized region within the LC subtype A3 (LC/A3) relative to the other LC serotype A (LC/A) subtypes (low primary acid homology [LPH]) that correlated to intracellular LC localization. This study shows the utility of recombinant hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.IMPORTANCE Botulinum neurotoxins are the most potent protein toxins for humans and potential bioterrorism threats, but they are also widely used as pharmaceuticals. Within the large family of BoNTs, only two subtypes are currently used as pharmaceuticals, with a large number of BoNT subtypes remaining as untapped potential sources for unique pharmaceuticals. Here, two recombinant hybrid toxins were engineered, consisting of domains from two BoNT subtypes that possess distinct duration of action and activity in human neurons and mice. We define the functional domains responsible for BoNT action and demonstrate creation of functional hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.

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.

Protein Structure Facilitates High-Resolution Immunological Mapping.

Select agents (SA) pose unique challenges for licensing vaccines and therapies. In the case of toxin-mediated diseases, HHS assigns guidelines for SA use, oversees vaccine and therapy development, and approves animal models and approaches to identify mechanisms for toxin neutralization. In this commentary, we discuss next-generation vaccines and therapies against ricin toxin and botulinum toxin, which are regulated SA toxins that utilize structure-based approaches for countermeasures to guide rapid response to future biothreats.

When Escherichia coli doesn't fit the mold: A pertussis-like toxin with altered specificity.

Bacterial toxins introduce protein modifications such as ADP-ribosylation to manipulate host cell signaling and physiology. Several general mechanisms for toxin function have been established, but the extent to which previously uncharacterized toxins utilize these mechanisms is unknown. A study of an Escherichia coli pertussis-like toxin demonstrates that this protein acts on a known toxin substrate but displays distinct and dual chemoselectivity, suggesting this E. coli pertussis-like toxin may serve as a unique tool to study G-protein signaling in eukaryotic cells.

Vaccines against Botulism.

Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin.

Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons.

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans but also are common therapies for neurological diseases. BoNTs are dichain toxins, comprising an N-terminal catalytic domain (LC) disulfide bond linked to a C-terminal heavy chain (HC) which includes a translocation domain (HN) and a receptor binding domain (HC). Recently, the BoNT serotype A (BoNT/A) subtypes A1 and A2 were reported to possess similar potencies but different rates of cellular intoxication and pathology in a mouse model of botulism. The current study measured HCA1 and HCA2 entry into rat primary neurons and cultured Neuro2A cells. We found that there were two sequential steps during the association of BoNT/A with neurons. The initial step was ganglioside dependent, while the subsequent step involved association with synaptic vesicles. HCA1 and HCA2 entered the same population of synaptic vesicles and entered cells at similar rates. The primary difference was that HCA2 had a higher degree of receptor occupancy for cells and neurons than HcA1. Thus, HCA2 and HCA1 share receptors and entry pathway but differ in their affinity for receptor. The initial interaction of HCA1 and HCA2 with neurons may contribute to the unique pathologies of BoNT/A1 and BoNT/A2 in mouse models.

Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E were insoluble, prohibiting studies towards understanding the mechanisms of toxin action and substrate recognition. In this work, the molecular basis of insolubility of the recombinant LCs of two representative subtypes of BoNT/E, E1(Beluga) and E3 (Alaska), was determined. Hydrophobicity profile and structural modeling predicted a C-terminal candidate region responsible for the insolubility of LC/Es. Deletion of C-terminal 19 residues of LC/E(1-400) resulted in enhanced solubility, from 2 to ∼50% for LC/EAlaska and from 16 to ∼95% for LC/EBeluga. In addition, resides 230-236 were found to contribute to a different solubility level of LC/EAlaska when compared to LC/EBeluga. Substituting residues (230)TCI(232) in LC/EAlaska to the corresponding residues of (230)KYT(232) in LC/EBeluga enhanced the solubility of LC/EAlaska to a level approaching that of LC/EBeluga. Among these LC/Es and their derivatives, LC/EBeluga 1-400 was the most soluble and stable protein. Each LC/E derivative possessed similar catalytic activity, suggesting that the C-terminal region of LC/Es contributed to protein solubility, but not catalytic activity. In conclusion, this study generated a soluble and stable recombinant LC/E and provided insight into the structural components that govern the solubility and stability of the LCs of other BoNT serotypes and Tetanus toxin.