Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin – a Novel Feature in Lys49-PLA2s Protein Class

Abstract

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys.

BmajPLA 2 -II, a basic Lys49-phospholipase A 2 homologue from Bothrops marajoensis snake venom with parasiticidal potential

“…However, BmajPLA 2 -II presents itself in dimeric and tetrameric forms, as seen by dynamic light scattering assays (Tables 1 and 2), which is an uncommon behavior for snake venom PLA 2 s (svPLA 2 s). Similar aggregates were observed for MjTX-I from B. moojeni, showing monomeric, dimeric, trimeric, tetrameric and pentameric forms in gel electrophoresis depending on the presence or absence of reducing agents [61,62]. Oligomerization can occur in high concentrations of the toxin, when the PLA 2 is interacting with a phospholipid substrate, achieving a more efficient enzymatic action, or in the presence of certain ions [63][64][65][66].…”

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like

“…Statistical comparison of the indirectly evoked contractions t 1/2 of the MjTX-II with those of other Lys49 PLA 2 s, such as PrTX-I from Bothrops pirajai (t 1/ 2 ¼ 49.0 AE 6.9 min, n ¼ 8) and BthTX-I from Bothrops jararacussu (t 1/2 ¼ 40.3 AE 3.5 min, n ¼ 6), obtained at same experimental conditions, indicates that these toxins have similar potency (Cavalcante et al, 2007). In contrast, MjTX-II presents a more potent neuromuscular blockade when compared to MjTX-I from B. moojeni, since at 1.0 mM this toxin depressed in only about 20% the twitches amplitude after 90 min of contact with the preparation (Salvador et al, 2013). Then, these results indicate MjTX-II has similar or superior neuromuscular blockade action compared to other Lys49-PLA 2 s.…”

“…moojeni that presents unusual oligomeric characteristics and displays lower myotoxic activity when compared to all other bothropic Lys49-PLA 2 s that have already been structurally and functionally characterized (Andriao-Escarso et al, 2000;Salvador et al, 2013).…”

Structural Basis for the Inhibition of a Phospholipase A2-Like Toxin by Caffeic and Aristolochic Acids

“…In addition,three PEG 4000 moleculeswere noted in the PrTX-I/CA structure,with two PEGs inside the hydrophobic channels from both monomersand the third PEG moleculeinteracting with Lys7 on monomer B. The PEG molecules found in these sites are found in other Lys49-PLA 2 s structures [ 12 , 18 , 46 ]. Finally, in the PrTX-I/CA structure, a sulfate molecule establishedhydrogen bonds with Arg33 in monomer A, similarly to sulfate ions found in other Lys49-PLA 2 s structures [ 18 , 35 ].…”