Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2007
Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.
Biochemistry, 2013
Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B−synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G−synaptotagmin recognition. These data suggest a reduced length and/or a bend in the Cterminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.
The FASEB Journal, 2013
Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCVreleasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.-Meng, J., Wang, J., Lawrence, G. W., Dolly, J. O. Molecular components required for resting and stimulated endo-cytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons. FASEB J. 27, 3167-3180 (2013). www.fasebj.org Key Words: amphiphysin ⅐ AP2 ⅐ clathrin ⅐ dynamin ⅐ large dense-core vesicles ⅐ small clear synaptic vesicles There is a great need to decipher the uptake processes in different types of neurons for the 7 serotypes of botulinum neurotoxin (BoNT) due to these being the most potent poison known, causing life-threatening botulism by intracellularly inhibiting transmitter release (reviewed in refs. 1, 2). Furthermore, clinical use of innocuous, tiny amounts of the toxin for treating neuronal disorders has kindled interest in the molecular machinery responsible for their trafficking. Type A BoNT (BoNT/A) has proved successful in alleviating the symptoms of Ͼ100 conditions arising from nerve overactivity, e.g., dystonias, spasticity, autonomic hypersecretory disorders, and overactive bladder (reviewed in ref. 3). Also, it can reduce the frequency of chronic migraine and chronic daily headaches (4), although not all types of chronic pain are responsive (5). BoNTs consist of a proteolytic light chain linked via a disulfide and noncovalent bonds to a heavy chain (HC). These neurotoxic proteins bind to intravesicular regions of synaptic vesicle protein 2 (SV2; BoNT/A, /D, /E, and /F) or synaptotagmin (BoNT/B and /G) that become exposed on exocytosis and, thereby, enter neurons by exploiting the recycling of small clear synaptic vesicles (SCSVs; refs. 6-15). Hence, BoNT uptake is accelerated by nerve stimulation (16-18). In comparison, little is known regarding the mode of BoNT entry into nerves that mainly secrete neuropeptides from large dense-core vesicles (LDCVs), such as sensory fibers. Study of the toxins' entry into different neuron types is warranted due to the prospect of yielding insights into differences and commonalities
Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E
Scientific Reports
Botulinum neurotoxins (Bonts) represent a family of bacterial toxins responsible for neuroparalytic disease 'botulism' in human and animals. their potential use as biological weapon led to their classification in category 'A' biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni-NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4 + and CD8 + t cells. increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4 + and CD8 + T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4 + T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E. Clostridium botulinum is Gram-positive anaerobic, spore-forming bacteria that produce botulinum neurotoxins (BoNTs). Botulinum neurotoxins are considered as the most toxic substances known to humankind 1 and the causative agent of botulism 2. BoNTs are also classified by the Centers for Disease Control (CDC) as one of the highest-risk threat agents for bioterrorism ("Class A agents") 3. Botulism is characterized by flaccid paralysis induced by blockade of acetylcholine release at neuromuscular junctions that, if not treated, can be fatal 4. Traditionally, based on their antigenic properties, BoNTs are classified into 7 distinct serotypes (A-G) 5. Recently a new serotype was reported as BoNT-H 6 , it has since been disproven as a novel serotype, collective data now accurately described it as FA chimeric i.e. BoNT/F5A or BoNT/HA rather than a novel new serotype 7,8. All BoNT serotypes act through similar mechanisms on their target neuronal cells. BoNTs are synthesized as an inactive single polypeptide chain of ~ 150 kDa. It is activated after post-translational cleavage in di-chain consisting of a ~ 100 kDa heavy chain (HC) and a ~ 50 kDa light chain (LC) held together via single disulfide bond 9. Structurally, these BoNTs are composed of three functional domains of ~ 50 kDa each; receptor-binding domain (HC C), translocation domain (HC N), and catalytic domain (LC). Intoxication of BoNTs is a multistep process that initiates with the binding of HC C on the presynaptic cell surface followed by internalization of LC mediated by HC N through receptor-mediated endocytosis. Inside the nerve terminal, the LC, which is a zinc-dependent metalloprotease, cleaves one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby blocking synaptic vesicle fusion thus release of acetylcholine 10-12. BoNT serotypes have
Biochemical and Biophysical Research Communications, 2011
Non-toxic derivatives of Botulinum neurotoxin A (BoNT/A) have potential use as neurontargeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.
Biochemical and Biophysical Research Communications, 2011
Among the seven serotypes (A-G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin-proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoN-Ts cellular mode of action, involving these two types of host cells.