Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Characterization of phospholipases A2 of Naja melanoleuca snake venom modified at the N-terminal region

In phospholipase A2 from Naja melanoleuca snake venom all four lysines were converted into the epsilon-amidinated derivatives without reaction of the alpha-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity. Starting from AMPA, chemical modification reactions were carried out at the alpha-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Asn1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine residue. Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1%), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.     

Kinetics of the hydrolysis of micellar substrates catalyzed by snake venom phospholipases A2.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by a cobra (Naja naja atra) (Group I) and a Habu (Trimeresurus flavoviridis) (Group II) PLA2s, were studied and compared with the results reported for other Group I and II enzymes. The substrate bindings to Group I enzymes were independent of the Ca2+ binding, whereas the substrate bindings to Group II enzymes were facilitated more than 10 times by the Ca2+ binding to the enzymes. The result for Group II enzymes, but not Group I enzymes, seemed compatible with the hypothesis for interpreting the catalytic mechanism that an intermediate complex should be stabilized by the coordination of the bound Ca2+ with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. The pH dependence of the kinetic parameters for the hydrolysis of the mixed micellar diC16PC, catalyzed by the cobra (N. naja atra) (Group I) and Habu (T. flavoviridis) (Group II) PLA2s, was also studied. The pK values of the catalytic group, His 48, and Tyr 52 for N. naja atra PLA2, shifted from 7.25 to 7.70 and from 10.30 to 10.85, respectively, and the corresponding values for T. flavoviridis PLA2 shifted from 5.80 to 6.95 and from 10.10 to 10.76, respectively, on binding of the micellar substrates to the enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A₂ (PLA₂), and BaltTX-II, an Asp49 catalytically active PLA₂ isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA₂ BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA₂, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA₂s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.     

The relationship between high-affinity noncatalytic binding of snake venom phospholipases A2 to brain synaptic plasma membranes and their central lethal potencies

The basic phospholipase A2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom, following injection into the right lateral ventricle of the brain of rats. When radiolabeled with 125I, these phospholipases A2 retained enzymatic activities and lethal potencies. Both enzymes bound with high affinity and specificity to brain synaptic plasma membrane preparations in vitro even in the absence of calcium, suggesting a non-catalytic binding. The acidic enzyme, in a calcium-free medium, had two binding components with Kd values of 1 X 10(-10) and 2.75 X 10(-8) M and Bmax values of 6 X 10(-13) and 3.4 X 10(-11) mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A2 in a calcium-free medium (Bmax = 3.4 X 10(-11) mol/mg). The levels of specific and total binding were 150 pmol/mg and 450 pmol/mg, respectively, for the comparatively toxic enzyme and 15 pmol/mg and 35 pmol/mg, respectively, for the comparatively nontoxic enzyme at a concentration of 2.5 X 10(-8) M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A2 (0.5 and 5.0 micrograms/rat for the N. nigricollis and N. naja atra phospholipases A2, respectively), suggesting a possible relationship between binding and lethal potency. Carbamylation of lysines reduced the levels of binding and the lethal potencies of both enzymes to a greater extent than their enzymatic activities. Pretreatment with high temperature, proteinases, phospholipases A2 or C suggested that radiolabeled phospholipase A2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A2 manifested a strict dependence on a divalent cation (Ca2+ or Sr2+) for most of its binding. The N. nigricollis enzyme demonstrated a much lower rate of dissociation from synaptic plasma membranes than did N. naja atra phospholipase A2, suggesting that hydrophobic interactions are more important in the binding of the more toxic enzyme as compared to the less toxic enzyme. It is proposed that differences in the extent of high-affinity noncatalytic binding to membrane phospholipids may be at least partly responsible for the marked difference in central toxicities of these two phospholipases A2.     

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.     

Leukocyte recruitment induced by snake venom metalloproteinases: Role of the catalytic domain

Snake venom metalloproteinases (SVMPs) are key toxins involved in local inflammatory reactions after snakebites. This study aimed to investigate the effect of SVMP domains on the alterations in leukocyte-endothelium interactions in the microcirculation of mouse cremaster muscle. We studied three toxins: BnP1, a PI-toxin isolated from Bothrops neuwiedi venom, which only bears a catalytic domain; Jararhagin (Jar), a PIII-toxin isolated from Bothrops jararaca venom with a catalytic domain, as well as ECD-disintegrin and cysteine-rich domains; and Jar-C, which is produced from the autolysis of Jar and devoid of a catalytic domain. All these toxins induced an increase in the adhesion and migration of leukocytes. By inhibiting the catalytic activity of Jar and BnP1 with 1.10-phenanthroline (oPhe), leukocytes were no longer recruited. Circular dichroism analysis showed structural changes in oPhe-treated Jar, but these changes were not enough to prevent the binding of Jar to collagen, which occurred through the ECD-disintegrin domain. The results showed that the catalytic domain of SVMPs is the principal domain responsible for the induction of leukocyte recruitment and suggest that the other domains could also present inflammatory potential only when devoid of the catalytic domain, as with Jar-C.     

Crystal structure of a CRISP family Ca2+ -channel blocker derived from snake venom

The cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals, reptiles, amphibians and secernenteas, and are involved in a variety of biological reactions. Here we report the crystal structure of triflin, a snake venom derived blocker of high K(+)-induced artery contraction, at 2.4A resolution. Triflin consists of two domains. The first 163 residues form a large globular body with an alpha-beta-alpha sandwich core, which resembles pathogenesis-related proteins of group-1 (PR-1). Two glutamic acid-associated histidine residues are located in an elongated cleft. A Cd(2+) resides in this binding site, and forms a five-coordination sphere. The subsequent cysteine-rich domain adopts a rod-like shape, which is stabilized by five disulfide bridges. Hydrophobic residues, which may obstruct the target ion-channel, are exposed to the solvent. A concave surface, which is surrounded by these two domains, is also expected to play a significant role in the binding to the target receptor, leading to ion channel blockage. The C-terminal cysteine-rich region has a similar tertiary structure to voltage-gated potassium channel blocker toxins, such as BgK and ShK. These findings will contribute toward understanding the functions of the widely distributed CRISP family proteins.     

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic $\text{Naja}$$\text{nigricollis}$ and the acidic relatively non-toxic $\text{Naja}$$\text{naja}$$\text{atra}$ phospholipases A₂. Following methylation a very low residual enzymatic activity (0.4 -- 1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzyme's ability to bind monomeric of micellar substrates or Ca²⁺, the results suggest that the pharmacologicallly active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Exploring the binding mechanism and kinetics of Piperine with snake venom secretory Phospholipase A2

Secreted venom Phospholipase A₂ is highly responsible for pharmacological effects like neurotoxicity, myotoxicity, hemolytic, anti-coagulation, and platelet aggregation. Neutralization of these pharmacological behaviors is one of the challenges existing for many decades and a potent drug compound for this is very much needed to control local effects of venom sPLA₂. In this study, we investigated binding mechanism and kinetics of inhibition of Piperine (major constitute of Piper nigrum) with sPLA₂ using DFT, MD simulation, MM-PBSA, and SPR method. Frontier MO properties were suggested that it procured better chemical reactivity and druglikeness and binding mode of Piperine with EcPLA₂ defined that it occupied well in N-terminal hydrophobic cleft. The persistence of Piperine interactions with and without calcium ion was analyzed and confirmed by MD simulation analysis. The dPCA-based FEL shows the nature of apo- and Piperine-bound conformational behavior of EcPLA₂ including intermediate forms. Further, binding energy of Piperine was calculated by high-throughput MM-PBSA which states that calcium ion presence enhances the Piperine binding by additional electrostatic interactions. Finally, kinetics of inhibition between Piperine and EcPLA₂ implied that it secured better binding affinity (K_D: as 1.708 pM) and the result gives clear evidence for the binding mechanism and binding energy calculated. In conclusion, Piperine was authenticated with better drug ability, entrenched binding interaction, and robust kinetics of inhibition with EcPLA₂ through which it can become an exceeding drug candidate for pharmacological as well as catalytic activity of sPLA₂.     

Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2

A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A2, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission at 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A2, the emission maximum shifted to about 470 nm. PPC showed a critical micellar concentration around 5 microM, with evidence of premicellar aggregation above 1 microM. Binding of PPC to Crotalus adamanteus phospholipase A2 was evidenced by an increase in emission at 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 microM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants for PPC complexes with phospholipases A2 from Naja naja naja and porcine pancreas and the prophospholipase A2 from porcine pancreas were 0.509, 0.107, and 0.114 microM, respectively. PPC was shown to inhibit the activity of pancreatic phospholipase A2 in thio-PC-sodium cholate mixed micelles. Inhibition studies were complicated because PPC can also serve as an activator of the snake venom enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of mechanisms of Ca2+-signalization. Role of Ca2+ in regulation of specialized cell functions

The review describes peculiarities of Ca²⁺-signalization in electro-excitable cells of higher eukaryotes. The light has been shed on problems of Ca²⁺-dependent mechanisms of regulation of muscle contractility and of neuronal synaptic plasticity in the higher vertebrate animals. A particular attention has been paid to analysis of contribution of such poorly studied components of Ca²⁺-signalization as non-selective TRPC-channels, Orai channels, sensory STIM1 proteins, Ca²⁺-controlled K⁺-channels of large and small conductance, and neuronal Ca²⁺-sensors (NCS).     

Evolution of mechanisms of Ca2+-signaling. Role of Ca2+ in regulation of fundamental cell functions

The review considers mechanisms of Ca2+-dependent regulation of cell growth, differentiation, and apoptosis in cells of the higher eukaryotes by modulation of the signal Ras-MAPK pathway.     

Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom.

Two basic myotoxic phospholipases A2 were purified to homogeneity from the venom of Bothrops godmani from Costa Rica by ion-exchange chromatography on CM-Sephadex. They have molecular weights of 14,300 (myotoxin I) and 13,400 (myotoxin II) and isoelectric points of 8.2 (myotoxin I) and 8.9 (myotoxin II). They behave as amphiphilic proteins in charge-shift electrophoresis and have similar amino acid compositions. Both toxins induce drastic myotoxic effects when injected in the gastrocnemius muscle of mice and induce release of peroxidase trapped in negatively charged liposomes. In addition, myotoxin I has high phospholipase A2 activity and is anticoagulant at doses higher than 0.3 microgram/ml, whereas myotoxin II has a very low phospholipase A2 activity and exerts anticoagulant effect only at concentrations higher than 50 micrograms/ml. Immunochemical data indicate that both toxins are immunologically related to Bothrops asper myotoxins, although B. godmani myotoxin II gives a stronger cross-reactivity when tested with antisera raised against B. asper myotoxins I and II.     

The role of intramembrane Ca2+ in the hydrolysis of the phospholipids of Escherichia coli by Ca2+-dependent phospholipases

Ca2+-dependent phospholipases A require Ca2+ concentrations in the millimolar range for optimal activity toward artificial substrates. Because Ca2+-dependent phospholipases A2 degrade the phospholipids of Escherichia coli, treated with the membrane-active antibiotic polymixin B equally well with and without added Ca2+ (Weiss, J., Beckerdite-Quagliata, S., and Elsbach, P. (1979) J. Biol. Chem. 254, 11010-11014), we have examined the possibility that intramembrane Ca2+ can provide the Ca2+ needed for phospholipase action. We studied the effect of Ca2+ depletion on the hydrolysis of the phospholipids of polymixin B-killed E. coli by 1) added pig pancreas phospholipase A2 in E. coli S17 (a phospholipase A-lacking mutant) and 2) endogenous Ca2+-dependent phospholipase A1 in the parent strain E. coli S15. Transfer of E. coli from nutrient broth (Ca2+ concentration approximately 3 X 10(-5) M) to Ca2+-depleted medium (Ca2+ concentration less than 10(-6)M) reduced polymixin B-induced hydrolysis by 50-75%, in parallel with a reduction of bacterial Ca2+ from 19.6 +/- 2.8 to 3.9 +/- 0.6 nmol (mean +/- standard error) per 3 X 10(10) bacteria. The bacterial Ca2+ content was repleted and the sensitivity of the bacterial phospholipids to hydrolysis by both exogenous phospholipase A2 (E. coli S17) and endogenous phospholipase A (E. coli S15) was restored by adding Ca2+ back to the suspensions. Complete restoration occurred at low Ca2+ levels in the reaction mixture (3 X 10(-5) - 10(-4) M) and required time, suggesting that hydrolysis was restored because bacterial Ca2+ stores were gradually replenished and not because extracellular Ca2+ concentrations were raised to levels that were still at least 10X lower than needed for optimal phospholipase A activity. This conclusion is supported by the finding that Ca2+ depletion or addition caused respectively decreased and increased release of lipopolysaccharides by EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), suggesting that the bacterial Ca2+ pool bound to lipopolysaccharides in the outer membrane shrinks or expands depending on extracellular Ca2+ levels. Thus, the cationic membrane-disruptive polymixin B, thought to compete with Mg2+ and Ca2+ for the same anionic sites on lipopolysaccharides, may liberate the Ca2+ near where the phospholipids are exposed to phospholipase.