Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin

The myotoxic mechanism for PLA₂-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA₂-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA₂-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA₂-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA₂-like toxins in general.     

The synthetic varespladib molecule is a multi-functional inhibitor for PLA2 and PLA2-like ophidic toxins

BackgroundThe treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A₂ (PLA₂) and PLA₂-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA₂s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA₂ and PLA₂-like toxins.MethodsIn vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence.ResultsVarespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA₂-like myotoxin, presenting an oligomeric-independent function.ConclusionResults suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA₂ and PLA₂-like toxins.General significanceVarespladib appears to be a promissory molecule in the treatment of local effects led by PLA₂ and PLA₂-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.     

Synergism between Basic Asp49 and Lys49 Phospholipase A2 Myotoxins of Viperid Snake Venom In Vitro and In Vivo

Two subtypes of phospholipases A₂ (PLA₂s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca²⁺ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA₂, an acidic non-toxic PLA₂ from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA₂ enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA₂ activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA₂ myotoxins acting synergistically in viperid venoms.     

Conservation of group XII phospholipase A2 from bacteria to human

Vertebrate group XII phospholipases A₂ (GXII PLA₂, conserved domain pfam06951) are proteins with unique structural and functional features within the secreted PLA₂ family. In humans, two genes (GXIIA PLA₂ and GXIIB PLA₂) have been characterised. GXIIA PLA₂ is enzymatically active whereas GXIIB PLA₂ is devoid of catalytic activity. Recently, putative homologues of the vertebrate GXII PLA₂s were described in non-vertebrates. In the current study a total of 170 GXII PLA₂ sequences were identified in vertebrates, invertebrates, non-metazoan eukaryotes, fungi and bacteria. GXIIB PLA₂ was found only in vertebrates and the searches failed to identify putative GXII PLA₂ homologues in Archaea. Comparisons of the predicted functional domains of GXII PLA₂s revealed considerable structural identity within the Ca^2 +-binding and the catalytic sites among the various organisms suggesting that functional conservation may have been retained across evolution. The preservation of GXII PLA₂ family members from bacteria to human indicates that they have emerged early in evolution and evolved via gene/genome duplication events prior to Eubacteria. Gene duplicates were identified in some invertebrate taxa suggesting that species-specific duplications occurred. The analysis of the GXII PLA₂ homologue genome environment revealed that gene synteny and gene order are preserved in vertebrates. Conservation of GXII PLA₂s indicates that important functional roles involved in species survival and were maintained across evolution and may be dependent on or independent of the enzyme's phospholipolytic activity.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A2-like from Bothrops brazili venom

Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A₂-like (Lys49-PLA₂s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA₂s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA₂s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA₂s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration.     

Modification of Lys-6 and Lys-65 Affects the Structural Stability of Taiwan Cobra Phospholipase A2

To assess whether chemical modification of phospholipase A₂ (PLA₂) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA₂ were selectively modified with trinitrobenzene sulfonate and pyridoxal-5′-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA₂ activity, but the Ca²⁺-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA₂ when thermal unfolding was conducted in the presence of 20 mM Ca²⁺. Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca²⁺. Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca²⁺ pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA₂, our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA₂ activity.     

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A₂ (PLA₂). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA₂ toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA₂. Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA₂. Moreover, it was found that PLA₂ and N-terminally mutated PLA₂ adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA₂ and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA₂, our data indicate that sphingomyelin modulates the mode of membrane binding of PLA₂ at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA₂.     

Neurotoxic,Myotoxic and Cytolytic Activities of the New Basic PLA<Subscript>2</Subscript> Isoforms BmjeTX-I and BmjeTX-II Isolated from the <Emphasis Type="Italic">Bothrops marajoensis</Emphasis> (Marajó Lancehead) Snake Venom

The BmjeTX-I and BmjeTX-II isoforms of PLA₂ were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS–PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA₂ show high degree of homology with basic PLA₂ myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 μM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA₂. These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA₂ also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA₂ exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes.     

Structural basis of phospholipase A2-like myotoxin inhibition by chicoric acid,a novel potent inhibitor of ophidian toxins

Background

Specific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A₂-like (PLA₂-like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA₂-like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently.

Phospholipases A2 isolated from snake venoms block acetylcholine-elicited currents in identified Lymnaea stagnalis neurons

Phospholipases A₂ (PLA₂s) are the most abundant family of snake venom proteins and play a significant role in prey envenomation. Their content in venoms is rather high. PLA₂s not only have enzyme activity but exhibit other types of biological activities including neurotoxicity. We have earlier shown that a protein bitanarin from the venom of the puff adder Bitis arietans is capable to block the responses of Lymnaea stagnalis neurons to acetylcholine and represents an active PLA₂ at the same time. Further investigation of PLA₂s isolated from the venoms of snakes of two families revealed their capability to interact with nicotinic acetylcholine receptors (nAChRs): PLA₂ from Vipera ursinii (Viperidae family), Naja kaouthia, and Bungarus fasciatus (Elapidae family) suppressed acetylcholine-induced current in identified neurons of L. staganlis. The effect was evident at PLA₂ concentration in the range of tens micromoles. The data obtained suggest the presence in a PLA₂ molecule of a site interacting with nAChR and a possible involvement of nAChR block in toxic action of PLA₂s.     

Amyloid-type fiber formation in control of enzyme action: interfacial activation of phospholipase A2

The lag-burst behavior in the action of phospholipase A₂ (PLA₂) on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was investigated at temperatures slightly offset from the main phase transition temperature T_m of this lipid, thus slowing down the kinetics of the activation process. Distinct stages leading to maximal activity were resolved using a combination of fluorescence parameters, including Förster resonance energy transfer between donor- and acceptor-labeled enzyme, fluorescence anisotropy, and lifetime, as well as thioflavin T fluorescence enhancement. We showed that the interfacial activation of PLA₂, evident after the preceding lag phase, coincides with the formation of oligomers staining with thioflavin T and subsequently with Congo red. Based on previous studies and our findings here, we propose a novel mechanism for the control of PLA₂, involving amyloid protofibrils with highly augmented enzymatic activity. Subsequently, these protofibrils form “mature” fibrils, devoid of activity. Accordingly, the process of amyloid formation is used as an on-off switch to obtain a transient burst in enzymatic catalysis.     

Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms

Summary The amino acid sequences of 40 secreted phospholipase A₂'s (PLA₂) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect venom types of PLA₂. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, repectively, indicating that at least two PLA₂ genes existed in the vertebrate line before the divergence of reptiles and mammals about 200–300 million years ago. This allows resolution for the first time of major genetic events in the evolution of current PLA₂'s and the relationship of human PLA₂'s to those of snake venom, many of which are potent toxins. Implications for possible mechanisms of regulation of mammalian intra- and extracellular PLA₂'s are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.     

Release of GPI-Anchored Zn2+-Glycerophosphocholine Cholinephosphodiesterase as an Amphiphilic Form from Bovine Brain Membranes by Bee Venom Phospholipase A2

Enzymatic release of Zn²⁺-glycerophosphocholine (GPC)cholinephosphodiesterase, as an amphiphilic form, from bovine brain membranes was examined. Of various membrane hydrolases, bee PLA₂ was the most effective in the release of the GPC cholinephosphodiesterase (amphiphilic form, 63–70%) from membrane. Compared to pancreatic PLA₂, bee PLA₂ was more efficient in the release of GPC cholinephosphodiesterase. In pH-dependent release of GPl-anchored phosphodiesterase, there was a similar pH-release profile between PLA₂-mediated release and spontaneous one, implying the involvement of membrane disruption in the PLA₂ action. The PLA₂-mediated release showed a limited time-dependence (until 45 min) and a limited dose dependence (up to 3 units / ml), characteristic of a receptor-type binding. An ionic binding of PLA₂ to membrane may be alluded from the interfering effect of anionic phospholipids on the PLA₂ action. In support of an interaction between PLA₂ and membrane glycoproteins, the PLA₂ action was found to be blocked by lectins, wheat germ agglutinin or concanavalin A. In combination with detergent, the PLA₂-mediated release was found to be enhanced synergistically by saponin, a cholesterol-complexing agent. Meanwhile, an additive interaction between PLA₂ and lysolecithin suggests that PLA₂ action is independent of lysolecithin. It is suggested that the binding of PLA₂ to specific sites of membranes, probably rich in GPI-anchored glycoproteins, may be related to the facilitated release of GPI-anchored proteins as amphiphilic form.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Biochemical,Pharmacological and Structural Characterization of Two PLA<Subscript>2</Subscript> Isoforms Cdr-12 and Cdr-13 from <Emphasis Type="Italic">Crotalus durissus ruruima</Emphasis> Snake Venom

Cdr-12 and Cdr-13 isoforms of PLA₂, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on μ-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA₂ was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA₂. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA₂s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA₂ isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA₂, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA₂ family, which presents the typical bioactivities described for such proteins.     

Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance

sPLA₂ is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA₂. Affinity of hesperidin towards PLA₂ isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA₂ with IC₅₀ of 5.1?µM. Molecular modeling studies revealed the association of hesperidin with the docking scores ?6.90, ?9.53, ?5.63 and ?8.29?kcal for isozymes Group IB, IIA, V and X of PLA₂ respectively. Their binding energy values were calculated as ?20.25, ?21.63, ?21.66 and ?33.43?kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA₂ and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA₂ isozyme specific to various diseases, with IC₅₀ value of therapeutic significance.     

Rapid Evolution by Positive Selection and Gene Gain and Loss: PLA<Subscript>2</Subscript> Venom Genes in Closely Related <Emphasis Type="Italic">Sistrurus</Emphasis> Rattlesnakes with Divergent Diets

Rapid evolution of snake venom genes by positive selection has been reported previously but key features of this process such as the targets of selection, rates of gene turnover, and functional diversity of toxins generated remain unclear. This is especially true for closely related species with divergent diets. We describe the evolution of PLA₂ gene sequences isolated from genomic DNA from four taxa of Sistrurus rattlesnakes which feed on different prey. We identified four to seven distinct PLA₂ sequences in each taxon and phylogenetic analyses suggest that these sequences represent a rapidly evolving gene family consisting of both paralogous and homologous loci with high rates of gene gain and loss. Strong positive selection was implicated as a driving force in the evolution of these protein coding sequences. Exons coding for amino acids that make up mature proteins have levels of variation two to three times greater than those of the surrounding noncoding intronic sequences. Maximum likelihood models of coding sequence evolution reveal that a high proportion (∼30%) of all codons in the mature protein fall into a class of codons with an estimated d _N /d _S (ω) ratio of at least 2.8. An analysis of selection on individual codons identified nine residues as being under strong (p < 0.01) positive selection, with a disproportionately high proportion of these residues found in two functional regions of the PLA₂ protein (surface residues and putative anticoagulant region). This is direct evidence that diversifying selection has led to high levels of functional diversity due to structural differences in proteins among these snakes. Overall, our results demonstrate that both gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.