Differences in primary structure among five phospholipases A2 from Heloderma suspectum

Five increasingly anionic phospholipases A2 (Pa1-Pa5) exist in the venom of the lizard Heloderma suspectum. We recently elucidated the sequence of Pa5, the most abundant and most active variant, towards emulsified phosphatidylcholines. Here we present the primary structures of Pa2, Pa3 (subvariants a and b) and Pa4, based on Edman degradation of tryptic, endoproteinase Arg-C and chymotryptic fragments of the reduced and S-carboxymethylated proteins. Pa1-Pa5, considered collectively, belong to an original class of secretory phospholipases A2 with 141-143 residues, a short hydrophobic N-terminus, 10 half-cystine residues and an extended C-terminus. The only known phospholipase A2 with characteristics close enough to be a member of the same class is that present in the venom from the insect Apis mellifera. More specifically, the sequences of Pa3 and Pa5 are almost identical, and those of Pa2 and Pa4 are also quite similar. Both groups diverge enough to indicate the translation of two mRNA species in the venom gland. The primary structure of Pa3 reveals the existence of subvariants a and b, the sequence of which is identical to that previously defined for Pa5, except that the C-terminal tripeptide GEG in Pa5 is replaced by the dipeptide GE in Pa3a and the tetrapeptide GEGR in Pa3b, Pa4, when compared to Pa5, shows 21 substitutions with a cluster of five modified amino acids in positions 40-44, immediately after the catalytic segment amino acids 30-39, and added changes scattered before the C-terminus. Pa2 differs from Pa4 only by the absence of the Gly142 C-terminal residue. The 15% difference in primary structure observed between the Pa3-Pa5 and Pa2-Pa4 subgroups might be largely responsible for their distinct biological properties.     

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.     

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann)

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)     

日本蝮蛇蛇毒碱性磷脂酶A2同源物的分离及鉴定

We purified and characterizated a phospholipase A2 homologue from Agkistrodon blomhoffii ussurensis snake venom. We used Hitrap SP cation exchange and Superdex 75 columns chromatography to obtain a basic protein, used SDS-PAGE to analyse molecular mass, and IEF (Isoelectric focusing electrophoresis) IEF to identify isoelectric point. The molecular mass was 16 kDa, and the isoelectric point was 8.56. We detected its phospholipase A2 activity on egg yolk phospholipids, hemolytic activity on washed erythrocytes, and anticoagulant effect on pig platelet-rich plasma, as well as the N-terminal sequence with protein sequencer. The results showed that it had no phospholipase A2 activity and hemolytic activity, but had obvious anticoagulant effect on in witro. The N terminal sequence (21 amino acid residues) compared with other phospholipases A2 demonstrated that the protein was homogenous with BPLA2s from Agkistrodon halys Palls.     

Properties of phospholipase A2 from the venom of the large hornet Vespa orientalis

Some properties (catalytic and hemolytic activity, pH and temperature optima, stability, substrate specificity, effects of detergents and metal ions, N-terminal sequence, chemical modification of histidine in the enzyme active center, etc.) of phospholipase A2 from hornet (Vespa orientalis) venom were studied. It was shown that phospholipase A2 from hornet venom differs essentially from other enzymes of this species in terms of stability, catalytic properties and structural features. The active center of the enzyme contains an essential histidine residue, similar to other phospholipases A2 from various sources. Unlike other known forms of phospholipase A2, the enzyme under study exerts a pronounced hemolytic action. The hemolysis is inhibited by Ca2+ at concentrations capable of inducing the activation of the hydrolytic activity of the enzyme.     

Characterization of phospholipase A2 from the venom of Horned viper (Cerastes cerastes)

Phospholipase A2 has been purified from the venom of Horned viper (Cerastes cerastes) by gel permeation chromatography followed by reverse-phase HPLC. The primary structure was established by sequence analysis of the intact protein and its enzymic peptides. The structure has 120 residues, properties like other group IIB phospholipases, but only 45-55% identity with the enzyme from other viperid species, and large variations even within the species (26% residue differences at known positions in another form).     

Purification of a cellular (granulocyte) and an extracellular (serum) phospholipase A2 that participate in the destruction of Escherichia coli in a rabbit inflammatory exudate

A granule-associated phospholipase A2 from rabbit polymorphonuclear leukocytes and a closely similar phospholipase A2 from rabbit serum have been purified to near homogeneity by ion-exchange and reverse-phase chromatography. The cellular (polymorphonuclear leukocyte) phospholipase A2 has been purified greater than 100,000-fold and the extracellular (serum) phospholipase A2 approximately 60,000-fold. The NH2-terminal amino acid sequence of the ascitic fluid phospholipase A2 that we have recently purified from inflammatory exudates produced in rabbits is nearly identical (15 of 16 residues) to that of the polymorphonuclear leukocyte phospholipase A2 and completely identical (19 of 19 residues) to that of the purified serum phospholipase A2. The functional properties of these three phospholipases A2 are indistinguishable. Each enzyme is active against Escherichia coli killed by the bactericidal/permeability-increasing protein of polymorphonuclear leukocyte, a property shared only by a subset of phospholipases A2. The presence of structurally and functionally very closely similar phospholipases A2 in the cellular and extracellular compartments of an inflammatory exudate is consistent with the apparent role of these enzymes in the destruction of certain microbial invaders during the acute inflammatory response.     

Ostrich pancreatic phospholipase A(2): purification and biochemical characterization

Ostrich pancreatic phospholipase A(2) (OPLA(2)) was purified from delipidated pancreases. Pure protein was obtained after heat treatment (70 degrees C), precipitation by ammonium sulphate and ethanol, respectively followed by sequential column chromatography on MonoQ Sepharose and size exclusion HPLC column. Purified OPLA(2), which is not a glycosylated protein, was found to be monomeric protein with a molecular mass of 13773.93 Da. A specific activity of 840U/mg for purified OPLA(2) was measured at optimal conditions (pH 8.2 and 37 degrees C) in the presence of 4 mM NaTDC and 10 mM CaCl(2) using PC as substrate. This enzyme was also found to be able to hydrolyze, at low surface pressure, 1,2-dilauroyl-sn-glycero-3 phosphocholine (di C(12)-PC) monolayers. Maximal activity was measured at 5-8 mNm(-1). The sequence of the first 22 amino-acid residues at the N-terminal extremity of purified bird PLA(2) was determined by automatic Edman degradation and showed a high sequence homology with known mammal pancreatic secreted phospholipases A(2).     

Group IB phospholipase A2 from Pseudonaja textilis

Pseudonaja textilis, an Australian Elapid, is known to produce a highly toxic venom. Both protein profiling and N-terminal sequence analysis showed the presence of four new phospholipases A(2) in this venom. Besides being non-lethal, the phospholipase A(2) proteins were found to be moderately active enzymes and they showed procoagulant property. cDNA cloning and characterization indicated the presence of two isoforms of PLA(2) proteins in a single snake, each containing the "pancreatic loop," characteristic of group IB phospholipase A(2). The genomic cloning also confirmed the presence of two genes each containing four exons that are interrupted by three introns. Phylogenetic analysis showed that the venom group IB PLA(2) gene is primitive and could have evolved from the same ancestor as the mammalian and venom group IA PLA(2) genes. In the present study, we report that the Pt-PLA2 gene could be responsible for the production of PL1, 2, and 3 possibly via RNA editing process.     

A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2

Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.     

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.     

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.     

Purification and characterization of a phospholipase A2 from the venom of the coral snake, Micrurus fulvius microgalbineus (Brown and Smith).

A phospholipase A2 was purified from the Mexican coral snake Micrurus fulvius microgalbieus (Brown and Smith). Gel filtration of the soluble crude venom on Sephadex g-50 resolved five fractions, of which fraction II had 98% of the total phospholipase activity. This fraction was rechromatographed on a CM-cellulose column that resolved eight fractions, four of which had an important phospholipase activity. The first fraction (II-1) was homogeneous by polyacrylamide-gel electrophoresis and displayed a phospholipase specific activity of 920 units/mg of protein. The apparent molecular weight as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 14000. The amino acid analysis revealed the presence of 119 amino acid residues, with 12 half-cystines. the N-terminal sequence was shown to be Ser-Leu-Leu-Asx-Phe-Lys-Asx-Met-Ile-Glu-Ser-Thr..., which is homologous with that of phospholipases from other snake venoms.     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of phospholipase A2 of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

The amino-acid sequence of phospholipase A2 from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) is presented. The enzyme consists of 122 amino-acid residues including 7 disulfide bonds and thus belongs to phospholipases A2 group IIA. The sequence was determined by automatic Edman degradation of the intact chain and of the peptides obtained after tryptic hydrolysis of the oxidized chain. The short cleavage time of 30 min and another limited tryptic digestion of the oxidized and citraconylated chain provided overlapping peptides. Sequencing was done with liquid- and gas-phase sequenators. The complete alignment of all peptides was facilitated by the high degree of homology with known viperid venom phospholipases A2. In common with mammalian phospholipases, the tryptophan residue in position 30 (essential for enzymatic activity) as well as the histidine in position 47 in the active site are present. Vipoxin phospholipase A2 shows 53.3% homology with another phospholipase A2 from Vipera ammodytes ammodytes venom (Ammodytoxin B), whereas 62% homology was found between both subunits of vipoxin phospholipase A2 and its inhibitor. This high degree of identity can be accounted for in terms of a common origin by gene duplication.     

Isolation, cloning and characterization of Polistes dominulus venom phospholipase A1 and its isoforms

Polistes dominulus is known to be an allergenically important social wasp. Its venom has four major allergens (ziv. phospholipase A1, hyaluronidase, antigen 5 and a serine-protease). Amino acid sequences of its serine-protease and Antigen 5 have been published. In this paper, the partial amino acid sequence of its venom phospholipase native protein is reported. Also, we give an account to the complete nucleotide sequence of Polistes dominulus venom phospholipase A1 gene, its isoforms and their complete deduced amino acid sequences. Their similarity to the other phospholipases A1 of the family: Vespidae is discussed.     

Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.     

Purification from bee venom of melittin devoid of phospholipase A2 contamination

Conditions for the inactivation of phospholipase A₂ which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A₂ activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A₂ still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.     

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Inhibition of [3H]nitrendipine binding by phospholipase A2

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.