Abstract
Background:Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17.Methods:Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.Results:The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.
Highlights
Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones
The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by polyethylene glycol (PEG)-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation
We suggest that the cytolytic effect was due to a pore-forming protein in the venom after analyzing the osmotic protectant effect of polyethylene glycol (PEG) and mass spectrometry
Summary
Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. The phylum Cnidaria is an ancient group with four main classes including corals, jellyfish, hydrozoans, and sea anemone [2,3]. The principal characteristic of this group is the production of nematocysts that are distributed in different tissues [4]. The components of the venom have been determined by different strategies, e.g., electrical stimulation of the organism [8], isolation of nematocysts from the tentacles [9,10], and homogenization of the entire organism (extract) [11]. Several sea anemone toxins have been studied for the development of drugs to serve as therapeutic agents with antitumor activity, as well as vasodilators, and ion channel modulators [17,18,19,20,21,22]
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