Cobrotoxin Research Articles

Cobrotoxin extracted from Naja atra venom relieves arthritis symptoms through anti-inflammation and immunosuppression effects in rat arthritis model

Ethnopharmacological relevanceThe Naja atra (Chinese cobra), primarily distributing in the low or medium altitude areas of southern China and Taiwan, was considered as a medicine in traditional Chinese medicine and used to treat pain, inflammation and arthritis. Aim of the studyTo study the anti-inflammatory and anti-arthritic activities of cobrotoxin (CTX), an active component of the venom from Naja atra. Materials and methodsAdjuvant-induced arthritis (AA) rats were used as the animal model of rheumatoid arthritis. The anti-arthritic effects of CTX were evaluated through the arthritis score, paw edema and histopathology changes of joints. The anti-inflammation effects were assayed by the level of IL-6, TNF-α, IL-1β and the number of inflammatory cells in peripheral blood, as well as the proliferation of fibroblast-like synoviocytes (FLS). The immune level was valued by the proliferation of T cells and the level of CD4 and CD8. ResultsCTX alleviated the disease development of AA rats according to the ameliorating arthritis score, paw edema and histopathology character. At the meanwhile, CTX decreased the levels of IL-6, TNF-α, IL-1β and the numbers of inflammatory cells in peripheral blood. CTX also suppressed the abnormal increasing of CD4+ T cells/ CD8+ T cells ratio, and could significantly inhibit T cell proliferation. Consistent with its effects on inhibiting granuloma's formation, CTX inhibited the proliferation of the cultured FLSs. Further studies on inflammatory signaling in FLSs revealed that CTX could inhibit the NF-κB signaling pathway. ConclusionsCTX has beneficial effects on rheumatoid arthritis by its immune regulation effects and anti-inflammation effects. The inhibition of NF-κB pathway partly contributes to the anti-inflammatory properties of CTX.

Cobrotoxin fromNaja naja atraVenom Ameliorates Adriamycin Nephropathy in Rats

Chronic kidney disease (CKD) becomes a global health problem with high morbidity and mortality. Adriamycin- (ADR-) induced rodent chronic nephropathy is a classic experimental model of human minimal lesion nephrotic syndrome. The present study investigated the effect of cobrotoxin (CTX) on ADR-induced nephropathy. Rats were given 6 mg/kg ADR once through the tail vein to replicate ADR nephropathy model. CTX was administered to rats daily by placing a fast dissolving CTX membrane strip under the tongue starting from 5 days prior to ADR administration until the end of experiment. The results showed that CTX ameliorated the symptoms of ADR nephropathy syndrome with reduced body weight loss, proteinuria, hypoalbuminemia, dyslipidemia, serum electrolyte imbalance, oxidative stress, renal function abnormities, and kidney pathological lesions. Anti-inflammatory cytokine IL-10 expression was elevated after CTX administration in ADR nephropathy model. CTX inhibited the phosphorylation of IκB-α and NF-κB p65 nuclear translocation. Meanwhile, CTX upregulated the protein level of podocyte-specific nephrin and downregulated the level of fibrosis-related TGF-β. These findings suggest that CTX may be a potential drug for chronic kidney diseases.

Activation of M3 muscarinic receptors inhibits T-type Ca 2+ channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca 2+ channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1 μM reversibly inhibited T-currents by ~ 26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca 2+ channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.

Refolding of a small all β-sheet protein proceeds with accumulation of kinetic intermediates

The refolding kinetics of Cobrotoxin (CBTX), a small all β-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen–deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all β-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates.

Cloning, overexpression, and characterization of cobrotoxin

Cobrotoxin (CBTX) is a highly toxic short neurotoxin, isolated from the Taiwan cobra (Naja naja atra) venom. In the present study for the first time we report the cloning and expression of CBTX in high yields (12mg/L) in Escherichia coli. CBTX fused to the IgG-binding domain of protein A (IgG–CBTX) was expressed in the soluble form. The misfolded CBTX portion (of the overexpressed fusion protein) was refolded under optimal redox conditions. The fusion protein (IgG–CBTX) was observed to undergo autocatalytic cleavage to yield CBTX with additional 5 amino acids upstream of its N-terminal end. The far UV and near UV circular dichroism spectra of the recombinant CBTX were identical to those of the toxin isolated from the crude venom source. Recombinant CBTX was isotope labeled (15N and 13C) and all the resonances (1H, 13C, and 15N) in the protein have been unambiguously assigned. 1H–15N HSQC spectrum of recombinant CBTX revealed that the protein is in a biologically active conformation. 1H–15N chemical shift perturbation data showed that recombinant CBTX binds to a peptide derived from the α7 subunit of the Torpedo acetylcholine receptor (AchR) with high affinity. The AchR peptide is found to bind to residues located at the tip of Loop-2 in CBTX. The results of the present study provide an avenue to understand the structural basis for the high toxicity exhibited by CBTX. In addition, complete resonance assignments in CBTX (reported in this study) are expected to trigger intensive research towards the design of new pharmacological agents against certain neural disorders.

Secondary Structure Formation Is the Earliest Structural Event in the Refolding of an All β-Sheet Protein

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight (∼7 kDa) all β-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding.

Structurally Homologous Toxins Isolated from the Taiwan Cobra (Naja naja atra) Differ Significantly in Their Structural Stability

Cardiotoxin and neurotoxin analogues isolated from snake venom sources are highly homologous proteins (>50% homology) with similar three-dimensional structures but exhibit drastically different biological properties. In the present study, we compare the conformational stability of cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX), a neurotoxin analogue, from the Taiwan cobra (Naja naja atra), using circular dichroism spectroscopy and hydrogen–deuterium (H/D) exchange techniques in conjunction with two-dimensional NMR methods. Contrary to expectations, it is found that CTX III and CBTX differ significantly in their structural stabilities. The three-dimensional structure of CBTX is less stable than that of CTX III. The amide protons of residues at the N- and C-terminal ends of the CTX III molecule are strongly protected against H/D exchange, implying that the terminal ends of the molecule are bridged together by significant numbers of hydrogen bonds. However, in CBTX, amide protons at the terminal ends of the molecule do not exhibit an significant protection against H/D exchange. Comparison of the protection factors of the various amide protons in CTX III and CBTX reveals that the extraordinary stability of CTX III stems from the strong network of interactions among the residues at the N- and C-terminal ends and also due to the tight and ordered packing of the nonpolar residues involved in the triple-stranded, anti-parallel, β-sheet segment of the molecule.

The effect of cobrotoxin on cholinergic neurons in the mouse

By using multiple time-point constant-rate infusions of deuterium-labeled phosphorylcholine, appropriate kinetic parameters were obtained for use in the calculation of the turnover rate of acetylcholine (TR ACh) in selected mouse brain regions. After obtaining these data, the relationship between the analgesic agent cobrotoxin (CT) and the activity of central cholinergic neurons was investigated by determination of TR ACh in selected mouse brain regions 3 hours following intracerebroventricular (i.c.v.) injection of CT. There were no obvious changes in the concentrations of ACh and choline (Ch) in the cortex, hippocampus, hypothalamus, midbrain, striatum, or thalamus of the mouse after injection of an analgesic dose of CT (2 μg, i.c.v.). TR ACh in the thalamus and the striatum were significantly increased, as compared to controls. On the other hand, i.c.v. injection of CT was found to significantly reduce TR ACh in the hippocampus and midbrain. These results suggest that the activity of hippocampal and midbrain cholinergic neurons is suppressed by CT, whereas the activity of striatal and thalamic cholinergic neurons is increased by CT at a time when a maximum analgesic response to CT is expressed.

The effect of cholinergic manipulations on the analgesic response to cobrotoxin in mice

Intracerebroventricular (i.c.v.) injection of cobrotoxin (CT), a neurotoxin isolated from the venom of Naja naja atra, produced an antinociceptive response in mice as measured by the tail-flick test. This effect of CT was blocked by systemic administration of atropine, but not by methylatropine or naloxone. Depletion of central acetylcholine (ACh) by hemicholinium-3 (HC-3) blocked the antinociceptive action of cobrotoxin. These results suggest that central cholinergic neurons are important for the mediation of the antinociceptive properties of cobrotoxin.