Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A₂ class

Abstract

Phospholipases A₂ (PLA₂s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA₂s are well-characterized homologue PLA₂s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA₂s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA₂ (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA₂ conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA₂ enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA₂s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA₂s than to other Asp49-PLA₂s, thus allowing Crotalinae subfamily PLA₂s to be classified into two main branches: a catalytic and a myotoxic one.

Liposomes containing an ASP49-phospholipase A 2 from Bothrops jararacussu snake venom as experimental therapy against cutaneous leishmaniasis

“…In this study, the lipids phosphatidylcholine and phosphatidylserine, used to produce the liposomal structure showed a positive connection with the active site of the Asp49 complex. As affirmed by [25] the Asp49-PLA 2 is not able to make a connection with Ca + , indicating that it has low catalytic activity. Other authors demonstrated that it has no phospholipase activity as might be expected for any Asp49-PLA 2 s, as they retain all wastes considered important for catalytic and phospholipase activity.…”

“…These modifications prevent the binding of calcium ions and thus may be responsible for the low catalytic activity found in BthTX-II [24]. Still other literature [25] says that when experiments are performed in cell culture, BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA 2 variants.…”

Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

“…A recent phylogenetic study shows that snake venom PLA 2 s can be classified into two groups according to their evolutionary derivation: i) the calcium-dependent catalytically active enzymes, such as Asp49-, Asn49- and Gln49-PLA 2 s; and ii) the catalytically inactive PLA 2 s that exert their effects through a still unresolved calcium-independent mechanism (Lys49-, Arg49- and some Asp49-PLA 2 s) [27]. The former group usually includes acidic PLA 2 s that act as monomeric toxins whereas the latter includes basic PLA 2 s that adopt a homodimeric configuration [27].…”

Antitumoral Potential of Tunisian Snake Venoms Secreted Phospholipases A2

“…However, in some of the group IIA PLA 2 enzymes this aminoacid residue is replaced by lysine, serine, asparagine, or arginine and they are identified as K49 [10], S49 [11], N49 [12], or R49 [13] enzymes, respectively. Substitution of Asp in the forty-ninth position interrupts the binding of cofactor Ca 2+ to the Ca 2+ -binding loop, and hence “mutants” show low or no hydrolytic activity [10, 14, 15]. In addition, there are several substitutions in the Ca 2+ -binding loops of these mutant enzymes.…”