Probing the role of substrate conformation in phospholipase A2 action on aggregated phospholipids using constrained phosphatidylcholine analogues

Phospholipase A2s hydrolyze aggregated phospholipid substrates much more rapidly than dispersed monomeric ones. Whether this is a consequence of interface-associated conformational changes of the enzyme or of the substrate, or of both, remains a key question in lipid enzymology. This problem is addressed herein using a rationally designed probe of substrate conformation. (1,3/2)-1-O-(phosphorylcholine)-2,3-O-dihexanoylcyclopentane-1,2,3 -triol is a novel short chain phosphatidylcholine analogue in which the glycerol-like backbone is part of a five-membered ring and therefore covalently constrained within a small defined range of conformations. To the extent that the constrained analogue resists aggregation-associated conformational changes, it provides a means for assessing the contribution of such changes to phospholipase A2 action on aggregated phospholipids. The monomeric (-)-cyclopentanoid analogue is a substrate for phospholipase A2s from Naja naja naja venom. However, when this constrained phospholipid is aggregated, its hydrolysis rate is not enhanced, in contrast to its unconstrained counterpart, 1,2-dihexanoyl-sn-glycero-3- phosphorylcholine. This lack of activation was not caused by a failure of the enzyme to bind the micellar, constrained analogue. While the constrained analogue does not show interfacial activation, it does show the activation of phosphatidylethanolamine hydrolysis typical of phosphorylcholine-containing lipids. Hence, these results strongly support the contention that specific packing-induced conformations of aggregated substrate play a substantial role in the large interfacial activations observed with phospholipase A2.     

Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.     

Tryptophan residue essential for activity of Naja naja atra phospholipase A2

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.     

Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2

Acidic phospholipase A2 (PLA2) from the venom of Chinese cobra (Naja naja atra) has high activity on zwitterionic membranes and contains six aromatic residues, including Tyr-3, Trp-18, Trp-19, Trp-61, Phe-64, and Tyr-110, on its putative interfacial binding surface. To assess the roles of these aromatic residues in the interfacial catalysis of N. n. atra PLA2, we mutated them to Ala and measured the effects on its interfacial catalysis. Enzymatic activities of the mutants toward various vesicle substrates and human neutrophils indicate that all but Trp-18 make significant contributions to interfacial catalysis. Among these aromatic residues, Trp-19, Trp-61, and Phe-64 play the most important roles. Binding affinities of the mutants for phospholipid-coated beads and their monolayer penetration indicate that Trp-19, Trp-61, and Phe-64 are critically involved in interfacial binding of N. n. atra PLA2 and penetrate into the membrane during the interfacial catalysis of N. n. atra PLA2. Further thermodynamic analysis suggests that the side chain of Phe-64 is fully inserted into the hydrophobic core of membrane whereas those of Trp-19 and Trp-61 are located in the membrane-water interface. Together, these results show that all three types of aromatic residues can play important roles in interfacial binding of PLA2 depending on their location and side-chain orientation. They also indicate that these aromatic side chains interact with membranes in distinct modes because of their different intrinsic preference for different parts of membranes.     

Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2

The full amino acid sequence of the acidic phospholipase A2 from Indian cobra (Naja naja naja) venom was determined and its tertiary structure examined by circular dichroism (CD). The sequence was aligned with other sequences of secreted phospholipase A2 from snakes of the genus Naja, using the progressive alignment method of Feng and Doolittle (J. Mol. Evol. (1987) 25, 351-360). The primary sequence of Naja naja naja phospholipases A2 shows up to 85% identity with the other acidic Naja phospholipase A2. CD studies indicate a 40-50% alpha-helical content in a tertiary structure which resists denaturation at high temperature, with or without chaotropic salts.     

Transesterification of phosphatidylcholine with eicosapentaenoic acid ethyl ester using phospholipase A2 in organic solvent

Phospholipase A₂-catalysed transesterification of phosphatidylcholine (PC, 99%) with eicosapentaenoic acid ethyl ester (EPAEE, 95%) was carried out in organic solvent. The maximum yield was 14.3% (w/w). The optimum reaction condition was 50°C, 48 h, initial water activity 0.25 and molar ratio of PC to EPAEE 1:10 in 5 ml toluene.     

Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Characterization and physical properties of the major form of phospholipase A2 from cobra venom (Naja naja naja) that has a molecular weight of 11,000.

The major form of phospholipase A2 from cobra venom (Naja naja naja) was prepared in 30% yield and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and on Sephadex G-100 chromatography. The monomer molecular weight is about 11,000 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ultracentrifugation and molecular sieve techniques were employed to confirm the molecular weight and to demonstrate a concentration-dependent aggregation of the enzyme. It was found that at concentrations below about 0.05 mg ml(-1), the enzyme exists predominantly in the monomeric form; kinetic studies are usually conducted in much more dilute solutions (0.2 mug ml(-1)). The amino acid composition of the enzyme is reported. Of special interest is the presence of five to six disulfide bonds, 1 tryptophan residue, and 1 histidine residue. It is stable at high temperatures and is unusually resistant to denaturing agents. The isoelectric point was found to be 4.95. The findings that the protein is unusually resistant to denaturing agents and that it undergoes a concentration-dependent aggregation help to explain some of the previous reports in the literature on the apparent multiple forms of the cobra enzyme and their separation.     

Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta 918, 175-188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2 activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group alpha to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom alpha to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25 degrees C ionic strength 0.1 in the presence of 3-10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of modification of tyrosine residues in cytotoxin-1 from Indian cobra venom. (Naja naja naja)

The role of tyrosine residues in the biological activity of cytotoxin-1 was evaluated using N-bromo succinimide. N-bromo succinimide effected the oxidation of tyrosine residues in cytotoxin-1 with an increase in absorption at 260 nm. N-chloro succinimide was ineffective in the oxidation of tyrosine residues in the toxin. Oxidation of a single tyrosine residue (at 3.50 equivalents of N-bromo succinimide/mole of the toxin) resulted in complete loss of lethal activity of the toxin. The lytic activity of the toxin (lysis of erythrocytes) remained uneffected even after three of the four tyrosine residues in the toxin were oxidised.     

Inhibition of Naja naja venom hyaluronidase: role in the management of poisonous bite

Hyaluronidase is present virtually in all snake venoms and has been known as a "spreading factor." The enzyme damages the extracellular matrix at the site of the bite, leading to severe morbidity. In this study, the benefits of inhibiting the hyaluronidase activity of Indian cobra (Naja naja) venom have been investigated. Anti-NNH1 and aristolochic acid both inhibited the in vitro activity of the purified hyaluronidase, (NNH1) and the hyaluronidase activity of whole venom in a dose-dependent manner. Both anti-NNH1 and aristolochic acid abolished the degradation of hyaluronan in human skin tissue sections by NNH1 and by whole venom. Aristolochic acid quenched the fluorescent emission of NNH1. A non-competitive mechanism of NNH1 inhibition was observed with aristolochic acid. NNH1 potentiates the toxicity of Daboia russellii VRV-PL-VIII myotoxin and hemorrhagic complex-I. However, the potentiation of toxicity was inhibited dose-dependently by anti-NNH1 and aristolochic acid. Further, mice injected with whole venom which had been preincubated with anti-NNH1/aristolochic acid, showed more than a two-fold increase in survival time, compared to mice injected with venom alone. A more moderate increase in survival time was observed when mice were injected with anti-NNH1/aristolochic acid 10 min after whole venom injection. This study illustrates the significance of venom hyaluronidase in the pathophysiology of snake venom poisoning and the therapeutic value of its inhibition.     

Expression of group IA phospholipase A2 in Pichia pastoris: identification of a phosphatidylcholine activator site using site-directed mutagenesis

Site-directed mutants of the group IA phospholipase A(2) from cobra venom were constructed and expressed in the methylotrophic yeast Pichia pastoris to probe for the proposed phosphatidylcholine (PC) activator site. Previous crystallographic and molecular modeling studies have identified two regions of the enzyme as likely candidates for this site. Residues Glu-55, Trp-61, Tyr-63, Phe-64, and Lys-65 were mutated to test the site advanced by Ortiz et al. [(1992) Biochemistry 31, 2887-2896] while Asp-23 and Arg-30 were mutated to assess the site proposed by Segelke et al. [(1998) J. Mol. Biol. 279, 223-232]. Expressed enzymes were purified by affinity chromatography and analyzed by SDS-PAGE, Western blotting, electrospray ionization mass spectroscopy, and circular dichroism. Both phospholipid headgroup specificity and rates of hydrolysis on monomeric PC substrates were determined and found to be similar for native, wild-type, and all of the mutant enzymes. These results suggest that all of the expressed enzymes were properly folded and contained functional catalytic sites. Mutations of the aromatic residues in the Ortiz site generally had little effect on PC activation, arguing against the importance of this region of the enzyme for PC activation; however, these aromatic amino acids appeared to be important for interfacial activation. In contrast, the D23N mutant in the Segelke site reduced PC activation by 10-fold without affecting activity toward micellar phosphatidylethanolamine substrates. Similar results were found with the D23N/R30M double mutant, suggesting that this region is critical for PC activation. These results provide evidence for the Segelke site as a PC activator site that is distinct from the catalytic site.     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

Design of specific peptide inhibitors for group I phospholipase A2: structure of a complex formed between phospholipase A2 from Naja naja sagittifera (group I) and a designed peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) at 1.9 A resolution reveals unique features

Phospholipase A(2) (PLA(2)) (E. C. 3.1.1.4) is a common enzyme in the two-way cascade mechanism leading to the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to the membrane surface and hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before its cleavage. To regulate the production of proinflammatory compounds, a specific peptide inhibitor Val-Ala-Phe-Arg-Ser (VAFRS) for the group I PLA(2) enzymes has been designed and synthesized. PLA(2) was isolated from Indian cobra (Naja naja sagittifera) venom and purified to homogeneity. The binding studies indicated the K(i) value of 1.02 +/- 0.10 x 10(-8) M. The purified PLA(2) samples and the designed inhibitor VAFRS were cocrystallized. The crystal structure of the complex was determined and refined to 1.9 A resolution. The peptide binds to PLA(2) at the active site and fills the hydrophobic channel completely. However, its placement with respect to the channel is in the opposite direction as compared to those observed in group II PLA(2)'s. Furthermore, the predominant intermolecular interactions involve strong electrostatic interactions between the side chains of peptide Arg and Asp 49 of PLA(2) together with a number of van der Waals interactions with other residues. A good number of observed interactions between the peptide and the protein indicate the significance of a structure-based drug design approach. The novel factor in the present sequence of the peptide is related to the introduction of a positively charged residue at the C-terminal part of the peptide.