Dataset Information

Effects of Mlx-8, a phospholipase A₂ from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

ABSTRACT:

Background

Here, we described the presence of a neurotoxin with phospholipase A₂ activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs).

Methods

The purification, molecular mass determination, partial amino acid sequencing, phospholipase A₂ activity determination, inhibition of the binding of the selective muscarinic ligand [³H]QNB and inhibition of the total [³H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined.

Results

Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A₂ enzymatic activity. The pK_i values were determined for Mlx-8 toxin and the M₁ selective muscarinic antagonist pirenzepine in hippocampus membranes via [³H]QNB competition binding assays. The pK_i values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [³H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A₂. This suggests that the inhibition of the phospholipase A₂ activity of the venom did not alter its ability to bind to displace [³H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [³H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus.

Conclusion

The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A₂ characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [³H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [³H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

SUBMITTER: Dos Santos RTF

PROVIDER: S-EPMC6986814 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Effects of Mlx-8, a phospholipase A<sub>2</sub> from Brazilian coralsnake <i>Micrurus lemniscatus</i> venom, on muscarinic acetylcholine receptors in rat hippocampus.

Dos Santos Roberta Tancredi Francesco RTF Silva Marcelo Florencio Passos MFP Porto Rafael Marques RM Lebrun Ivo I Gonçalves Luís Roberto de Camargo LRC Batista Isabel de Fátima Correia IFC Sandoval Maria Regina Lopes MRL Abdalla Fernando Maurício Francis FMF

The journal of venomous animals and toxins including tropical diseases 20200127

<h4>Background</h4>Here, we described the presence of a neurotoxin with phospholipase A<sub>2</sub> activity isolated from <i>Micrurus lemniscatus</i> venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs).<h4>Methods</h4>The purification, molecular mass determination, partial amino acid sequencing, phospholipase A<sub>2</sub> activity determination, inhibition of the binding of the selective muscarinic ligand [<sup>3</sup>H]QNB and inhibition of the total [<sup>3</sup>H]ino ...[more]

PMID: 32063920

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Project description:BackgroundEndogenous phospholipases A2 (PLA2) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of β-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The β-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated.Methodsβ-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM medium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry.ResultsProteomic analysis revealed fragments on β-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. β-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes.ConclusionThese findings indicate that β-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.

Project description:Venom gland transcriptomes and proteomes of six Micrurus taxa (M. corallinus, M. lemniscatus carvalhoi, M. lemniscatus lemniscatus, M. paraensis, M. spixii spixii, and M. surinamensis) were investigated, providing the most comprehensive, quantitative data on Micrurus venom composition to date, and more than tripling the number of Micrurus venom protein sequences previously available. The six venomes differ dramatically. All are dominated by 2-6 toxin classes that account for 91-99% of the toxin transcripts. The M. s. spixii venome is compositionally the simplest. In it, three-finger toxins (3FTxs) and phospholipases A₂ (PLA₂s) comprise >99% of the toxin transcripts, which include only four additional toxin families at levels ≥0.1%. Micrurus l. lemniscatus venom is the most complex, with at least 17 toxin families. However, in each venome, multiple structural subclasses of 3FTXs and PLA₂s are present. These almost certainly differ in pharmacology as well. All venoms also contain phospholipase B and vascular endothelial growth factors. Minor components (0.1-2.0%) are found in all venoms except that of M. s. spixii. Other toxin families are present in all six venoms at trace levels (<0.005%). Minor and trace venom components differ in each venom. Numerous novel toxin chemistries include 3FTxs with previously unknown 8- and 10-cysteine arrangements, resulting in new 3D structures and target specificities. 9-cysteine toxins raise the possibility of covalent, homodimeric 3FTxs or heterodimeric toxins with unknown pharmacologies. Probable muscarinic sequences may be reptile-specific homologs that promote hypotension via vascular mAChRs. The first complete sequences are presented for 3FTxs putatively responsible for liberating glutamate from rat brain synaptosomes. Micrurus C-type lectin-like proteins may have 6-9 cysteine residues and may be monomers, or homo- or heterodimers of unknown pharmacology. Novel KSPIs, 3× longer than any seen previously, appear to have arisen in three species by gene duplication and fusion. Four species have transcripts homologous to the nociceptive toxin, (MitTx) α-subunit, but all six species had homologs to the β-subunit. The first non-neurotoxic, non-catalytic elapid phospholipase A₂s are reported. All are probably myonecrotic. Phylogenetic analysis indicates that the six taxa diverged 15-35 million years ago and that they split from their last common ancestor with Old World elapines nearly 55 million years ago. Given their early diversification, many cryptic micrurine taxa are anticipated.

Dataset Information

Effects of Mlx-8, a phospholipase A₂ from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

Background

Methods

Results

Conclusion

Publications

Effects of Mlx-8, a phospholipase A<sub>2</sub> from Brazilian coralsnake <i>Micrurus lemniscatus</i> venom, on muscarinic acetylcholine receptors in rat hippocampus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

Background

Methods

Results

Conclusion

Publications

Effects of Mlx-8, a phospholipase A<sub>2</sub> from Brazilian coralsnake <i>Micrurus lemniscatus</i> venom, on muscarinic acetylcholine receptors in rat hippocampus.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Effects of Mlx-8, a phospholipase A₂ from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.