Bal Ram Singh

Development of a Fluorescence Internal Quenching Correction Factor to Correct Botulinum Neurotoxin Type A Endopeptidase Kinetics Using SNAPtide

Botulinum neurotoxins (BoNTs), which are highly toxic proteins responsible for botulism, are produced by different strains of Clostridium botulinum. These various strains of bacteria produce seven distinct serotypes, labeled A-G. Once inside cells, the zinc-dependent proteolytic light chain (LC) degrades specific proteins involved in acetylcholine release at neuromuscular junctions causing flaccid paralysis, specifically synaptosomal-associated protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT endopeptidase assays using short substrate homologues have been widely used and developed because of their ease of synthesis, detection limits, and cost. SNAPtide, a 13-amino acid fluorescence resonance energy transfer (FRET) peptide, was used in this study as a SNAP-25 homologue for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage. Signal quenching can become an issue after cleavage since quencher molecules can quench cleaved fluorophore molecules in close proximity, reducing the apparent signal. This reduction in apparent signal provides an inherent error as SNAPtide concentrations are increased. In this study, fluorescence internal quenching (FIQ) correction factors were derived using an unquenched SNAPtide peptide to quantify the signal quenching over a range of SNAPtide concentrations and temperatures. The BoNT/A LC endopeptidase kinetics at the optimally active temperature (37 °C) using SNAPtide were studied and used to demonstrate the FIQ correction factors in this study. The FIQ correction factors developed provide a convenient method to allow for improved accuracy in determining and comparing BoNT/A LC activity and kinetics using SNAPtide over a broad range of concentrations and temperatures.

Type A botulinum neurotoxin complex proteins differentially modulate host response of neuronal cells

Type A Botulinum neurotoxin (BoNT/A), the most potent poison known to mankind, is produced by Clostridium botulinum type A as a complex with neurotoxin-associated proteins (NAPs). Currently BoNT/A in purified and complex forms are both available in therapeutic and cosmetic applications to treat neuromuscular disorders. Whereas Xeomin(®) (incobotulinumtoxin A, Merz Pharmaceuticals, Germany) is free from complexing proteins, Botox(®) (onabotulinumtoxin A, Allergan, USA) contains NAPs, which by themselves have no known role in the intracellular biochemical process involved in the blockade of neurotransmitter release. Since the fate and possible interactions of NAPs with patient tissues after intramuscular injection are not known, it was the aim of this study to evaluate the binding of BoNT/A and/or the respective NAPs to cells derived from neuronal and non-neuronal human tissues, and to further explore neuronal cell responses to different components of BoNT/A. BoNT/A alone, the complete BoNT/A complex, and the NAPs alone, all bind to neuronal SH-SY5Y cells. The BoNT/A complex and NAPs additionally bind to RMS13 skeletal muscle cells, TIB-152 lymphoblasts, Detroit 551 fibroblasts besides the SH-SY5Y cells. However, no binding to these non-neuronal cells was observed with pure BoNT/A. Although BoNT/A, both in its purified and complex forms, bind to SH-SY5Y, the intracellular responses of the SH-SY5Y cells to these BoNT/A components are not clearly understood. Examination of inflammatory cytokine released from SH-SY5Y cells revealed that BoNT/A did not increase the release of inflammatory cytokines, whereas exposure to NAPs significantly increased release of IL-6, and MCP-1, and exposure to BoNT/A complex significantly increased release of IL-6, MCP-1, IL-8, TNF-α, and RANTES vs. control, suggesting that different components of BoNT/A complex induce significantly differential host response in human neuronal cells. Results suggest that host response to different compositions of BoNT/A based therapeutics may play important role in local and systemic symptoms in patients.

The Botulinum Toxin as a Therapeutic Agent: Molecular Structure and Mechanism of Action in Motor and Sensory Systems

Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is the most potent molecule known to mankind. Higher potency of BoNT is attributed to several factors, including structural and functional uniqueness, target specificity, and longevity. Although BoNT is an extremely toxic molecule, it is now increasingly used for the treatment of disorders related to muscle hyperactivity and glandular hyperactivity. Weakening of muscles due to peripheral action of BoNT produces a therapeutic effect. Depending on the target tissue, BoNT can block the cholinergic neuromuscular or cholinergic autonomic innervation of exocrine glands and smooth muscles. In recent observations of the analgesic properties of BoNT, the toxin modifies the sensory feedback loop to the central nervous system. Differential effects of BoNT in excitatory and inhibitory neurons provide a unique therapeutic tool. In this review the authors briefly summarize the structure and mechanism of actions of BoNT on motor and sensory neurons to explain its therapeutic effects and future potential.

Molecular assembly of Clostridium botulinum progenitor M complex of type E

Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. The similarity of the general architecture between the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.

The Botulinum Neurotoxin Complex and the Role of Ancillary Proteins

All seven known serotypes of botulinum neurotoxin (BoNT) are produced in the form of a complex with a group of neurotoxin-associated proteins (NAPs). The BoNT complex is encoded by a gene cluster regulated by its own transcription factor, and the proteins coded by polycistronic messenger ribonucleic acid (mRNA) self-assemble into complexes of 300–900 kDa. Types A, B, C, D, and G complexes contain hemagglutinin (HA), whereas types E and F complexes do not contain HA. Sequence homology among respective BoNTs and NAPs range from 55.3 to 98.5 %, and all the proteins in the BoNT complex belong to a stable class of protein with high longevity inside mammalian cells. A new 250-kDa protein (P-250) with high immunogenicity has been identified in the BoNT/A complex which is not part of the neurotoxin gene cluster. The 33-kDa hemagglutinin (HA-33) is the most abundant NAP. The HA-33 is protease resistant and is highly immunogenic. HA-33 appears to play an important role in the translocation of the neurotoxin across the gut wall, enhancing the endopeptidase activity of BoNT and protection of BoNT against proteases. The role of other NAPs is not as clear, and their role in the biology of the bacteria is not understood at all. BoNT complexes are used as therapeutic product, although a therapeutic product without NAPs appears to retain the properties of the complex-based products. NAPs in therapeutic products may have other subtle long-term effects which need to be investigated.

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.

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.

Evolution of Toxin

It is very intriguing how toxins in certain organisms have evolved. There must have been a drawn out period where evolution ‘tested’ and ‘fine-tuned’ the toxin. To trace back we need to study both animal and bacterial toxins. Animal toxins are very diverse, which makes them important candidates for evolutionary innovation. On the other hand, bacterial toxins always target critical molecules. Toxins typically go after molecules that are either scarce or those involve in signal transductions. Both classes of toxin offer a unique model to study predator-prey relationship, co-evolution, lateral gene transfer, natural selection, and the influence of structure on molecular evolution. Adaptation and counter-adaptation due to evolutionary arms race results in complex traits. Each toxin or poison probably has its own evolutionary “arms race”. To be a strong player in evolutionary “arms race” toxins need structural plasticity. The existence of structural plasticity allows them to evolve as highly specific toxins for their respective targets. Protein toxins are either multi-domain or complex protein, and their larger surface area and subunit flexibility provide structural flexibility needed to survive, adapt and evolve. Several mechanisms and models have been suggested to explain this phenomenon. Point mutations, gene duplication, lateral gene transfer, recombination, and post-translation modification of genes lead to a wide variety of proteins and peptides. Study of these phenomena, relationships and proposed mechanisms can establish evolutionary link of protein toxins to their progenitors.

The botulinum toxin as a therapeutic agent: molecular and pharmacological insights

Botulinum neurotoxins (BoNTs), the most potent toxins known to mankind, are metal- loproteases that act on nerve-muscle junctions to block exocytosis through a very specific and exclusive endopeptidase activity against soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of presynaptic vesicle fusion machinery. This very ability of the toxins to produce flaccid muscle paralysis through chemical denervation has been put to good use, and these potentially lethal toxins have been licensed to treat an ever expanding list of medical disorders and more popularly in the field of esthetic medicine. In most cases, therapeutic BoNT preparations are high-molecular-weight protein complexes consisting of BoNT, complexing proteins, and excipients. There is at least one isolated BoNT, which is free of complexing proteins in the market (Xeomin ® ). Each commercially available BoNT formulation is unique, differing mainly in molecular size and composition of complexing proteins, biological activity, and antigenicity. BoNT serotype A is marketed as Botox ® , Dysport ® , and Xeomin ® , while

Differential endopeptidase activity of different forms of type A botulinum neurotoxin: A unique relationship between the size of the substrate and activity of the enzyme

&NA; Botulinum neurotoxins (BoNTs; serotypes A‐G) are metalloproteases, which cleave and inactivate cellular proteins essential for neurotransmitter release. In bacterial cultures, BoNTs are secreted as a complex of the neurotoxin and a group of neurotoxin associated proteins (NAPs). Under physiological condition (pH 7.4), this complex is believed to be dissociated to separate the neurotoxin from NAPs. BoNT consists of a 50 kDa light (L) chain (LC or catalytic domain) and a 100 kDa heavy (H) chain (or HC) linked through a disulfide bond and other non‐covalent interactions. The cell intoxication involves three major steps; binding, membrane translocation and inhibition of neurotransmitter release. The last step of intoxication, endopeptidase activity, is very unique and specific that can be used for detection of the complex and isolated forms of the toxin. A fluorescent tag‐labeled synthetic peptide (SNAPtide) derived from a segment of SNAP‐25, an intracellular substrate of BoNT/A, is used to detect and assay the endopeptidase activity of BoNT/A. The detection of the signal is based on the change in the fluorescence energy transfer after selective cleavage of the peptide by the BoNT/A. In this report, we demonstrate that SNAPtide as a commonly used substrate widely differ in reaction with BoNT/A complex, BoNT/A, and BoNT/A light chain. These findings have implications for assays used in detection, and in screening potential inhibitors. HighlightsReduction of disulfide bond is required for optimum activity of BoNT/A toxin and BoNT/A Complex.The size of the enzyme and the substrate plays a significant role in the endopeptidase activity.Folding and flexibility between the enzyme and the substrate, impacts its endopeptidase activity.