| Gloydius intermedius is the most toxic species among six pit-vipers in China, which subordinates to the Genus Gloydius, Crotalinae of Viperidae, and distributes in northwest and north China. Snake venom phospholipase (SvPLA2) is the most toxic components of snake venom, and also possess a variety of toxicological and pharmacological activities besides enzymatic activity, which may have potentials in application. However, the structure and function relationship, molecular mechanism of envenomation of the PLA2from Gloydius intermedius remain to be elucidated, for this reason, in the present study, the main neurotoxin was isolated and the neuro-muscular effects were explored.Methods and procedures:1. Purification of Gintexin from the crude venom(1)Size exclusion chromatographyPLA2s were separated by using size exclusion chromatography (Sephacryl-200HR) from Gloydius intermedius crude venom. After elution with50mmol/L ammonium bicarbonate buffer (pH8.0), at a flow rate of0.3mL/min. The fractions were collected. The purity was analyzed by12%SDS-PAGE. For each peak, the the phospholipase A2activity and acute toxicity on mice were detected.(2)Purification of P3fraction by reversed phase-high performance liquid chromatography and Mass spectrometry analysisP3fraction was further separated by reverse phase high performance liquid chromatography(RP-HPLC), and the fractions with PLA2activity were identified by Mass spectrometry. The obtained amino acid sequences were corresponded to the amino acid sequencec deduced from the PLA2clones of the Gloydius intermedius venom gland cDNA library, then subjected to bioinformatics analysis.2. Blocking effects of Gintexin on neuromuscular junctions(1)Neuromuscular blocking effectsRat phrenic nerve-diaphragm and the chicken biventer nerve-muscle preparations were prepared. These preparations were fixed in the bath, with a pertension of0.5g and nerve stimulation (6v0.9Hz0.2ms) to balance at least for15min.40mM KCl or55 μM ACh were added to the bath to test the function of the preparations, then different concentrations of Gintexin(2μg/mL、10μg/mL、50μg/mL) were added. The twitch tension was recorded. After the contractions were completely blocked,40mM KCl or55μM ACh were added, and the integrity of the muscle and the acetylcholine receptors on postsynaptic membrane were evaluated.(2) Determine the acting sites by immunohistochemistryThe neuromuscular preparations blocked from the above mentioned experiments were taken and fixed in10%formaldehyde solution for24h, and followed by the conventional gradient alcohol dehydration, transparent processing with xylene, paraffin embedding, finally subjected to serial section(3μm thickness), the blank sections were obtained. Then dewaxing, inactivating endogenous HRP, antigen repair, serum blocking were performed for immunohistochemistry assay. First antibodies(rabbit anti PLA2and SNAP25diluted in1:1000and1:100with PBS buffer respectively) were added and kept at4℃overnight. Then second antibody labelled with HRP was used and developed with DAB as substrate. The nuclei were stained with hematoxylin and mounted. At last, they were observed under a photomicrosope.3. Muscle damage(1) Histochemic analysisThe blank section of the above mentioned NMJ preparations were taken for hematoxylin-eosin (HE) staining and Masson staining, so as to evaluate the muscle and connective tissue damage.(2)The determination of creatine kinaseKunming Mice(body weight18-20g) were divided into different groups. The crude venom(8μg in50μL saline solution) and Gintexin(1.5μg in50μL saline solution) were injected into the right gastrocnemius muscle of mice, with same volume of saline solution as control. At different time points (1h,3h,6h,9h,12h and24h), blood samples were picked in the eyes, centrifuged at10000rpm for10min and the supernatants were takek to determine the blood creatine kinase activity by dynamic analysis method.Results(1)By using size exclusion chromatography, five fractions were obtained (P1-5) from Gloydius intermedius crude venom. P3showed both the PLA2enzyme activity and lethal toxicity on mice.After separation with RP-HPLC, we found that P3fraction contained several types of PLA2isoformes. Among them, Gintexin-A, Gintexin-B and Gin-E6b were the most abundant, with sparse amount of Gin-E6a. Gintexin-A and Gintexin-B accounted for at leat65%. The molecular weights of these four PLA2isoforms by mass spectrometry were9847Da,14177Da,13662Da and14074Da, respectively. The partial amino acid sequences of the four PLA2were searched and compared with the full amino acid sequences deduced from the cDNA of the four PLA2from the venom gland cDNA library. After the amino acid sequence multiple alignment, like rattlesnake Crotoxin toxins, Gintexin-A and Gintexin-B shared high homology with Crotoxin A and Crotoxin B of Crotalus durissus, and thus formed heterodimer. So in the following experiments, we could use P3fraction as Gintexin to detect the toxicological effects.(3) The primary assay showed that the blocking effect of Gintexin on the chicken biventer NMJ preparation was more sensitive than that on rat phrenic nerve-diaphragm preparation. When in vitro tested on chicken biventer NMJ preparations at different final concentration(2μg/mL, lOμg/mL and50μg/mL), the blocking times were49±0.8min(n=3),40±0.7min(n=3) and30±0.8min(n=3), respectively, but these blocking had no effects on the acetylcholine receptors of the postsynaptic membrane.(4) Immunohistochemical experiments showed that the action sites of Gintexin were not on the nerve fibers, but on the neuromuscular junctions. Based on these observation, Gintexin acted on the synapses of NMJ. Combined with the above mentioned in vitro experiment results, Gintexin was a new presynaptic neurotoxin.(5) By histochemical HE and Masson staining, we could observed that the muscle was damaged, and the damage effect of Gintexin was weaker than that of the crude venom. On both occasion, the extracellular connective tissues were not affected.(6)The CK activity assay results:3hours after crude venom injection, the CK level increased to the highest level(7563.33±639.24U/L), while for the Gintexin groups, the CK level reached high levels of4126±559.11U/L and4220.67±586.75U/L three and six hours after the injection, respectively.Conclusion:A presynaptic neurotoxic PLA2with slight myotoxicity has been successfully separated from the venom of Gloydius intermedius by using size exclusion chromatography and reversed phase-high performance liquid chromatography (RP-HPLC), designated as Gintexin. GintexinA and Gintexin B may form heterodimer through non covalent bond. This work may therefore establish the basic foundation for the further investigation of PLA2molecular evolution and molecular mechanism of envenomation. |
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