Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms

Abstract

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

Anti-tumor effect investigation of obtustatin and crude Macrovipera lebetina obtusa venom in S-180 sarcoma bearing mice

“…The addition of MLO venom to single mixed-lipid giant unilamellar vesicles from brain lipids of rats shows noticeable changes, both distortions in the vesicle membrane and shrinking of the vesicle size. These effects are likely to disturb the local packing structure of the lipids and fluidity of the bilayer Ghazaryan et al, 2013Ghazaryan et al, , 2015. We decided to go further and develop these interesting results and to study how crude MLO venom and obtustatin influence was on the sarcoma model.…”

Extended snake venomics by top-down in-source decay: Investigating the newly discovered Anatolian Meadow viper subspecies,Vipera anatolica senliki

“…The comparison of native (13,639.9 Da) and reduced (13,654.0 Da) mass spectra showed a mass shift of Δ14.1 Da, that correlates to seven disulfide bridges and is a clear characteristic for snake venom PLA 2 ( Figure 2C ). 64, 65 We were able to further characterize the three dimeric DI proteoforms (13,982.8 Da, 13,999.8 Da and 14,001.8 Da) present in fractions 9 and 10 composed of four different subunits (6,979.0 Da, 6,998.0 Da, 7,022.0 Da and 7,023.9 Da) showing two inter-subunit (Δ2.01 Da) and twice four intra-subunit (Δ8.1 Da) disulfide bridges ( Figure 2D-I ). 66, 67 Moreover, we were able to identify small peptides, like the snake venom metalloproteinase inhibitor (svMP-i) pEKW by tandem MS ( SI- Figure 3 ).…”

Thermoprotective Properties of the Venom of the Transcaucasian Vipera (Macrovipera Lebetina Obtusa Dyigubsky, 1832)

“…By autors the isolated phospholipase A 2 were studied in more details and found to exert their toxins action both on lipid membranes [5] and on nicotinic acetylcholine receptor [6].…”