Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation

Enhanced structural insights into the folding energy landscape of the N-terminal dimerization domain of Escherichia coli tryptophan repressor, [2-66]2 TR, were obtained from a combined experimental and theoretical analysis of its equilibrium folding reaction. Previous studies have shown that the three intertwined helices in [2-66]2 TR are sufficient to drive the formation of a stable dimer for the full-length protein, [2-107]2 TR. The monomeric and dimeric folding intermediates that appear during the folding reactions of [2-66]2 TR have counterparts in the folding mechanism of the full-length protein. The equilibrium unfolding energy surface on which the folding and dimerization reactions occur for [2-66]2 TR was examined with a combination of native-state hydrogen exchange analysis, pepsin digestion and matrix-assisted laser/desorption mass spectrometry performed at several concentrations of protein and denaturant. Peptides corresponding to all three helices in [2-66]2 TR show multi-layered protection patterns consistent with the relative stabilities of the dimeric and monomeric folding intermediates. The observation of protection exceeding that offered by the dimeric intermediate in segments from all three helices implies that a segment-swapping mechanism may be operative in the monomeric intermediate. Protection greater than that expected from the global stability for a single amide hydrogen in a peptide from the C-helix possibly and another from the A-helix may reflect non-random structure, possibly a precursor for segment swapping, in the urea-denatured state. Native topology-based model simulations that correspond to a funnel energy landscape capture both the monomeric and dimeric intermediates suggested by the HX MS data and provide a rationale for the progressive acquisition of secondary structure in their conformational ensembles.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

The crystal structure of Trypanosoma cruzi glucokinase reveals features determining oligomerization and anomer specificity of hexose-phosphorylating enzymes

Glucose is an essential substrate for Trypanosoma cruzi, the protozoan organism responsible for Chagas' disease. The glucose is intracellularly phosphorylated to glucose 6-phosphate. Previously, a hexokinase responsible for this phosphorylation has been characterized. Recently, we identified an ATP-dependent glucokinase in T. cruzi exhibiting a tenfold lower substrate affinity compared to the hexokinase. Both enzymes, which belong to very different groups of the same family, are located inside glycosomes, the peroxisome-like organelles of Kinetoplastida that are known to contain the first seven glycolytic steps as well as enzymes of the oxidative branch of the pentose phosphate pathway. Here, we present the crystallographic structure of T. cruzi glucokinase, in complex with glucose and ADP. The structure suggests a loose tetrameric assembly formed by the association of two tight dimers. TcGlcK was previously reported to exist in a concentration-dependent equilibrium of monomeric and dimeric states. Here, we used mass spectrometry analysis to confirm the existence of TcGlcK monomeric and dimeric states. The analysis of subunit interactions and comparison with the bacterial glucokinases give insights into the forces promoting the stability of the different oligomeric states. Each T. cruzi glucokinase monomer contains one glucose and one ADP molecule. In contrast to hexokinases, which show a moderate preference for the alpha anomer of glucose, the electron density clearly shows the d-glucose bound in the beta configuration in the T.cruzi glucokinase. Kinetic assays with alpha and beta-d-glucose further confirm a moderate preference of the T. cruzi glucokinase for the beta anomer. Structural comparison of the glucokinase and hexokinases permits the identification of a possible mechanism for anomer selectivity in these hexose-phosphorylating enzymes. The preference for distinct anomers suggests that in T. cruzi hexokinase and glucokinase are not directly competing for the same substrate and are probably both present because they exert distinct physiological functions.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

Summary The effect of phospholipase A₂ and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A₂ (PLA₂) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (P_i+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA₂, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and P_i+Mg media. Other detergents were ineffective.The ability of PLA₂ to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the P_i+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as P_i-oriented ouabain sites are inhibited by PLA₂.     

Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

Isolation and preliminary enzymatic characterization of a novel PLA2 from Crotalus durissus collilineatus venom

A crotoxin homolog was purified from the Crotalus durissus collilineatus venom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA₂ (Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificus venom had three PLA₂ isoforms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatus PLA₂ and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA₂ (Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nerve-diaphragm preparations when tested alone. However, when PLA₂ and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA₂ activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36°C. Full PLA₂ activity required the presence of a low Ca²⁺ concentration and was inhibited by Cu²⁺ and Zn²⁺ and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. These results indicate that crotoxin from C. d. collineatus venom is very similar enzymatically to crotoxin from C. d. terrificus.     

Assignment of the Disulfide Bridges in Bothropstoxin-I, a Myonecrotic Lys49 PLA2 Homolog from Bothrops jararacussu Snake Venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A₂ homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV₈) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV₈ digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA₂s.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

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.     

A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable

The anthranilate phosphoribosyltransferase from Sulfolobus solfataricus (ssAnPRT) forms a homodimer with a hydrophobic subunit interface. To elucidate the role of oligomerisation for catalytic activity and thermal stability of the enzyme, we loosened the dimer by replacing two apolar interface residues with negatively charged residues (mutations I36E and M47D). The purified double mutant I36E+M47D formed a monomer with wild-type catalytic activity but reduced thermal stability. The single mutants I36E and M47D were present in a monomer-dimer equilibrium with dissociation constants of about 1 μM and 20 μM, respectively, which were calculated from the concentration-dependence of their heat inactivation kinetics. The monomeric form of M47D, which is populated at low subunit concentrations, was as thermolabile as monomeric I36E+M47D. Likewise, the dimeric form of I36E, which was populated at high subunit concentrations, was as thermostable as dimeric wild-type ssAnPRT. These findings show that the increased stability of wild-type ssAnPRT compared to the I36E+M47D double mutant is not caused by the amino acid exchanges per se but by the higher intrinsic stability of the dimer compared to the monomer. In accordance with the negligible effect of the mutations on catalytic activity and stability, the X-ray structure of M47D contains only minor local perturbations at the dimer interface. We conclude that the monomeric double mutant resembles the individual wild-type subunits, and that ssAnPRT is a dimer for stability but not for activity reasons.