Critical analysis in the advancement of cell-based assays for botulinum neurotoxin.

The study on botulinum neurotoxins (BoNTs) has rapidly evolved for their structure and functions as opposed to them being poisons or cures. Since their discoveries, the scientific community has come a long way in understanding BoNTs' structure and biological activity. Given its current application as a tool for understanding neurocellular activity and as a drug against over 800 neurological disorders, relevant and sensitive assays have become critical for biochemical, physiological, and pharmacological studies. The natural entry of the toxin being ingestion, it has also become important to examine its mechanism while crossing the epithelial cell barrier. Several techniques and methodologies have been developed, for its entry, pharmacokinetics, and biological activity for identification, and drug efficacy both in vivo and in vitro conditions. However, each of them presents its own challenges. The cell-based assay is a platform that exceeds the sensitivity of mouse bioassay while encompassing all the steps of intoxication including cell binding, transcytosis, endocytosis, translocation and proteolytic activity. In this article we review in detail both the neuronal and nonneuronal based cellular interaction of BoNT involving its transportation, and interaction with the targeted cells, and intracellular activities.

Clostridial Neurotoxins: Structure, Function and Implications to Other Bacterial Toxins.

Gram-positive bacteria are ancient organisms. Many bacteria, including Gram-positive bacteria, produce toxins to manipulate the host, leading to various diseases. While the targets of Gram-positive bacterial toxins are diverse, many of those toxins use a similar mechanism to invade host cells and exert their functions. Clostridial neurotoxins produced by Clostridial tetani and Clostridial botulinum provide a classical example to illustrate the structure-function relationship of bacterial toxins. Here, we critically review the recent progress of the structure-function relationship of clostridial neurotoxins, including the diversity of the clostridial neurotoxins, the mode of actions, and the flexible structures required for the activation of toxins. The mechanism clostridial neurotoxins use for triggering their activity is shared with many other Gram-positive bacterial toxins, especially molten globule-type structures. This review also summarizes the implications of the molten globule-type flexible structures to other Gram-positive bacterial toxins. Understanding these highly dynamic flexible structures in solution and their role in the function of bacterial toxins not only fills in the missing link of the high-resolution structures from X-ray crystallography but also provides vital information for better designing antidotes against those toxins.

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design.

BACKGROUND: A natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A). METHODS: In this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC. RESULTS: Studies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site. CONCLUSION: The NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT. GENERAL SIGNIFICANCE: Insights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.

Evolutionary Features in the Structure and Function of Bacterial Toxins.

Toxins can function both as a harmful and therapeutic molecule, depending on their concentrations. The diversity in their function allows us to ask some very pertinent questions related to their origin and roles: (a) What makes them such effective molecules? (b) Are there evolutionary features encoded within the structures of the toxins for their function? (c) Is structural hierarchy in the toxins important for maintaining their structure and function? (d) Do protein dynamics play a role in the function of toxins? and (e) Do the evolutionary connections to these unique features and functions provide the fundamental points in driving evolution? In light of the growing evidence in structural biology, it would be appropriate to suggest that protein dynamics and flexibility play a much bigger role in the function of the toxin than the structure itself. Discovery of IDPs (intrinsically disorder proteins), multifunctionality, and the concept of native aggregation are shaking the paradigm of the requirement of a fixed three-dimensional structure for the protein's function. Growing evidence supporting the above concepts allow us to redesign the structure-function aspects of the protein molecules. An evolutionary model is necessary and needs to be developed to study these important aspects. The criteria for a well-defined model would be: (a) diversity in structure and function, (b) unique functionality, and (c) must belong to a family to define the evolutionary relationships. All these characteristics are largely fulfilled by bacterial toxins. Bacterial toxins are diverse and widely distributed in all three forms of life (Bacteria, Archaea and Eukaryotes). Some of the unique characteristics include structural folding, sequence and functional combination of domains, targeting a cellular process to execute their function, and most importantly their flexibility and dynamics. In this work, we summarize certain unique aspects of bacterial toxins, including role of structure in defining toxin function, uniqueness in their enzymatic function, and interaction with their substrates and other proteins. Finally, we have discussed the evolutionary aspects of toxins in detail, which will help us rethink the current evolutionary theories. A careful study, and appropriate interpretations, will provide answers to several questions related to the structure-function relationship of proteins, in general. Additionally, this will also allow us to refine the current evolution theories.

Natural Compounds and Their Analogues as Potent Antidotes against the Most Poisonous Bacterial Toxin.

Botulinum neurotoxins (BoNTs), the most poisonous proteins known to humankind, are a family of seven (serotype A to G) immunologically distinct proteins synthesized primarily by different strains of the anaerobic bacterium Clostridium botulinum Being the causative agents of botulism, the toxins block neurotransmitter release by specifically cleaving one of the three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, thereby inducing flaccid paralysis. The development of countermeasures and therapeutics against BoNTs is a high-priority research area for public health because of their extreme toxicity and potential for use as biowarfare agents. Extensive research has focused on designing antagonists that block the catalytic activity of BoNTs. In this study, we screened 300 small natural compounds and their analogues extracted from Indian plants for their activity against BoNT serotype A (BoNT/A) as well as its light chain (LCA) using biochemical and cellular assays. One natural compound, a nitrophenyl psoralen (NPP), was identified to be a specific inhibitor of LCA with an in vitro 50% inhibitory concentration (IC50) value of 4.74 ± 0.03 µM. NPP was able to rescue endogenous synaptosome-associated protein 25 (SNAP-25) from cleavage by BoNT/A in human neuroblastoma cells with an IC50 of 12.2 ± 1.7 µM, as well as to prolong the time to the blocking of neutrally elicited twitch tensions in isolated mouse phrenic nerve-hemidiaphragm preparations.IMPORTANCE The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins, involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small-molecule antidotes against BoNT with the potential to reverse the paralysis syndrome of botulism. In principle, small-molecule inhibitors (SMIs) can gain entry into BoNT-intoxicated cells if they have a suitable octanol-water partition coefficient (log P) value and other favorable characteristics (P. Leeson, Nature 481:455-456, 2012, https://doi.org/10.1038/481455a). Several efforts have been made in the past to develop SMIs, but inhibitors effective under in vitro conditions have not in general been effective in vivo or in cellular models (L. M. Eubanks, M. S. Hixon, W. Jin, S. Hong, et al., Proc Natl Acad Sci U S A 104:2602-2607, 2007, https://doi.org/10.1073/pnas.0611213104). The difference between the in vitro and cellular efficacy presumably results from difficulties experienced by the compounds in crossing the cell membrane, in conjunction with poor bioavailability and high cytotoxicity. The screened nitrophenyl psoralen (NPP) effectively antagonized BoNT/A in both in vitro and ex vivo assays. Importantly, NPP inhibited the BoNT/A light chain but not other general zinc endopeptidases, such as thermolysin, suggesting high selectivity for its target. Small-molecule (nonpeptidic) inhibitors have better oral bioavailability, better stability, and better tissue and cell permeation than antitoxins or peptide inhibitors.

A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin.

Botulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results suggest that the C-terminus of LCA plays a critical role in introducing a flexible structure, which is essential for its biological function. This is the first report of such a massive structural role of the C-terminus of a protein being critical for maintaining a functional state.

A Novel Surface Plasmon Resonance Biosensor for the Rapid Detection of Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) are Category A agents on the NIAID (National Institute of Allergy and Infectious Diseases) priority pathogen list owing to their extreme toxicity and the relative ease of production. These deadly toxins, in minute quantities (estimated human i.v. lethal dose LD50 of 1-2 ng/kg body weight), cause fatal flaccid paralysis by blocking neurotransmitter release. The current gold standard detection method, the mouse-bioassay, often takes days to confirm botulism. Furthermore, there are no effective antidotes known to reverse the symptoms of botulism, and as a result, patients with severe botulism often require meticulous care during the prolonged paralytic illness. To combat potential bio-terrorism incidents of botulinum neurotoxins, their rapid detection is paramount. Surface plasmon resonance (SPR) is a very sensitive technique to examine bio-molecular interactions. The label-free, real-time analysis, with high sensitivity and low sample consumption makes this technology particularly suitable for detection of the toxin. In this study, we demonstrated the feasibility in an assay with a newly designed SPR instrument for the rapid detection of botulinum neurotoxins. The LOD (limit of detection) of the Newton Photonics (NP) SPR based assay is 6.76 pg/mL for Botulinum Neurotoxin type A Light Chain (BoNT/A LC). We established that the detection sensitivity of the system is comparable to the traditional mouse LD50 bioassay in BoNT/A using this SPR technology.

Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.

In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A.

PURPOSE: A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT. METHODS: We determined lethality (LD50) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0 µg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model. RESULTS: drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin. CONCLUSIONS: Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.

Structural and functional analysis of botulinum neurotoxin subunits for pH-dependent membrane channel formation and translocation.

The structure-function relationship of Botulinum Neurotoxin (BoNT) proteins is greatly influenced by pH. While the low pH of endosome favors membrane interaction of the heavy chain (HC) for the formation of a membrane channel and translocation of the light chain (LC), the catalytic activity of the LC requires a neutral pH for cleavage of the soluble NSF attachment protein receptor (SNARE) complex in the cytosol. In this study, we monitored secondary structural characteristics of LC, HC and holotoxin at individual pHs 4.5 and 7.2 and at the transition pH4.5 to 7.2 to identify the structural signatures underlying their function. The HC showed higher thermal stability at pH4.5 with a melting temperature (Tm) of 60.4°C. The structural analysis of HC in the presence of liposomes showed no difference in ellipticity with that of HC at pH7.2 at 208 and 222 nm but a 25.2% decrease in ellipticity at 208 nm at acidic pH, indicating low pH-induced structural changes that might facilitate interaction with the membrane. Further, HC showed 18% release of K+ ions from liposomes at pH4.5 as against 6% at neutral pH, reinforcing its role in membrane channel formation. LC on the other hand, showed maximum ellipticity at pH7.2, a condition that is relevant to its endopeptidase activity in the cytosol of the neurons. Also, the similarity in the structures at pH7.2 and transition pH4.5 to 7.2 suggested that the flexibility acquired by the protein at low pH was reversible upon exposure to neutral pH for cleavage of SNARE proteins.

Resolution of sub-nanosecond motions in botulinum neurotoxin endopeptidase: An evidence of internal flexibility.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind, which act on the peripheral nervous system leading to flaccid paralysis. Although co-crystal structure of BoNT/A light chain (LC) reveals some unique features of the biological function of this molecule, structural characteristics in solution reveal its dynamic features, not available through the published crystal structures. In this study, we have examined internal flexibility of this molecule by measuring rotational correlation time as a function of viscosity, using frequency domain fluorescence anisotropy decay technique. Fluorescence anisotropy decay of BoNT/A LC resolved sub-nanosecond local motion (faster component), interpreted as internal flexibility of the molecule was affected significantly with viscosity. Both local and global movements were affected by viscosity, which indicates the accessibility of protein core and flexibility of overall structure. In conclusion, this work demonstrates the presence of flexibility in the internal peptide segments, which appears to play a significant role in BoNT/A LC biological function.

Role of neurotoxin associated proteins in the low pH induced structural changes in the botulinum neurotoxin complex.

Botulinum Neurotoxin (BoNT) produced by the bacterium Clostridium botulinum as a complex with NAPs causes botulism. It has been known that the NAPs protect the toxin from both extremes of pHs and proteases of the GI tract. In an attempt to emulate the physiological conditions encountered by the toxin, we examined BoNT/A, BoNT/A complex, and NAPs under different pH conditions and monitored their structural characteristics by far-UV CD and thermal denaturation analysis. BoNT/A complex showed the maximum CD signal with a mean residue weight ellipticity of -1.8 × 10(5)° cm(2)/dmol at 222 nm at both acidic and neutral pHs. Thermal denaturation analysis revealed NAPs to be the most stable amongst the three protein samples examined. Interestingly and quite uniquely, at pH 2.5, there was an increase in CD signal for BoNT complex as a function of temperature, which correlated with the NAPs profile, indicating a shielding effect of NAPs on BoNT complex at low pH. Calculation of the weighted mean of the ellipticities at the Tm for thermal unfolding of toxin and NAPs at neutral and acidic pHs showed variation with that of BoNT complex, suggesting structural reorganization in BoNT complex upon the association of NAPs and BoNT. In conclusion, this study reveals the structural behavior of BoNT complex and NAPs with pH changes substantially, which could be quite relevant for BoNT survival under extreme pH conditions in vivo.

Current strategies for designing antidotes against botulinum neurotoxins.

INTRODUCTION: Botulinum neurotoxins (BoNTs) are proteins responsible for the deadly paralytic disease botulism. Extreme toxicity, ease of production and lack of antidotes against BoNT makes it a category A biothreat agent, according to the United States Center of Disease Control and Prevention. The only available therapy for BoNT is an equine antitoxin antibody or/and a protracted respiratory support system. Even then, antibody treatment can only prevent further exposure of the toxin and cannot rescue already intoxicated neurons. AREAS COVERED: In this article, the authors provide a summary of the current status of inhibitors and antitoxins used against BoNTs. In particular, the authors focus on new strategies used in the development of novel therapeutics. They also outline the major steps involved in BoNT's mechanism of action and identify specific inhibitors for each step. EXPERT OPINION: Several previous efforts have resulted in less than satisfactory results that are due, in part, to a lack of sustained effort in addition to a poor understanding of the unique structural features of the toxin. BoNT is a double-edged sword with both toxic effects and therapeutic benefits, excluding vaccination as a preventative measure. The long lasting intracellular endopeptidase activity, which causes an extended period of muscle paralysis, necessitates the need to identify effective inhibitor(s) against BoNT, and this could ultimately lead to new therapeutic options.

Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin.

Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37°C. LCE, on the other hand, was although optimally active at 37°C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics.

Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.

Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I1 [Formula: see text] I2 [Formula: see text] U, with two intermediate states I1 and I2. BoNT/A loses almost all its secondary structure in 3.75 M urea (I1), yet it displays a native-like secondary structure in 5 M urea (I2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin's mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.

RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin.

A surface plasmon resonance based RNA aptasensor for rapid detection of natively folded type A botulinum neurotoxin is reported. Using detoxified recombinant type A botulinum neurotoxin as the surrogate, the aptasensor detects active toxin within 90 min. The detection limit of the aptasensor in phosphate buffered saline, carrot juice, and fat free milk is 5.8 ng/ml, 20.3 ng/ml and 23.4 ng/ml, respectively, while that in 5-fold diluted human serum is 22.5 ng/ml. Recovery of toxin from disparate sample matrices are within 91-116%. Most significant is the ability of this aptasensor to effectively differentiate the natively folded toxin from denatured, inactive toxin, which is important for homeland security surveillance and threat assessment. The aptasensor is stable for more than 30 days and over 400 injections/regeneration cycles. Such an aptasensor holds great promise for rapid detection of active botulinum neurotoxin for field surveillance due to its robustness, stability and reusability.

Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.

Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.

Comparative immunochemical characteristics of botulinum neurotoxin type A and its associated proteins.

Clostridium botulinum strains secrete their neurotoxins (BoNT) along with a group of neurotoxin-associated proteins (NAPs) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the G.I. tract. A major component of NAPs is Hn-33, a 33 kDa protein, which is also protease resistant and strongly protects BoNT. The complex form of BoNT/A is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic purposes. Immune response against this formulation could hinder its long-term use; therefore, it is important to characterize the immunological properties of the associated proteins. This study aims to understand the immunological reactivity of BoNT/A complex, BoNT, NAPs, and Hn-33 through a series of competitive enzyme-linked immunosorbent assays (ELISA). The results indicated that BoNT/A complex competed 6 times more with complex antibodies compared to the neurotoxin confirming that the higher immunogenicity of BoNT/A complex was indeed a result of the associated proteins with the neurotoxin complex. While the nearly identical immuno-reactivity of BoNT/A complex and Hn-33 with Hn-33 antibodies indicated that the reactivity was due to the higher immunogenicity not the abundance of Hn-33 in the complex. Both the ELISA and immuno-blot results implied that Hn-33 is primarily responsible for eliciting the antibody response in BoNT/A complex.

Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.