Analysis of the cDNA for phospholipase A2 from honeybee venom glands. The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes (original) (raw)
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Toxicon, 1992
Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom. Toxicon 30, 1331Toxicon 30, -1341Toxicon 30, , 1992.-In addition to phospholipase A2-I (PLA 2-,) reported previously (ODA et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA,-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-,, PLA2II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLAA and PLA2-II for Ca 2+ . Secondary structure compositions of PLA2I, were similar to those of PLAA and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-,, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLAA and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLAA and Asn for PLA2-II).
Journal of Protein Chemistry
A novel basic phospholipase A 2 (PLA 2) 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 (DLWSWGQMIQETGLLP-SYTTY. . .) showed a high degree of homology with basic D49 PLA 2 myotoxins from other Bothrops venoms. Bj IV had high PLA 2 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 PLA 2 activity required Ca 2ϩ but was inhibited by Cu 2ϩ and Zn 2ϩ , and by Cu 2ϩ and Mg 2ϩ in the presence and absence of Ca 2ϩ , 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 PLA 2 was similar to that in the basic PLA 2 of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA 2 could partly explain the low PLA 2 activity of Bothrops venoms.
Enzymatic labelling of snake venom phospholipase A 2 toxins
Toxicon, 2019
Almost all animal venoms contain secretory phospholipases A 2 (PLA 2 s), 14 kDa disulfide-rich enzymes that hydrolyze membrane phospholipids at the sn-2 position, releasing lysophospholipids and fatty acids. These proteins, depending on their sequence, show a wide variety of biochemical, toxic and pharmacological effects and deserve to be studied for their numerous possible applications, and to improve antivenom drugs. The cellular localization and activity of a protein can be studied by conjugating it with a tag. In this work, we applied an enzymatic labelling method, using Streptomyces mobaraense transglutaminase, on three snake venom PLA 2 s: a recombinant neuro-and myotoxic group I PLA 2 from Notechis scutatus scutatus, and two myotoxic group II PLA 2 s from Bothrops asper-one of them a natural catalytically inactive variant. We demonstrate that TGase can be used to produce active mono-or bi-derivatives of these three PLA 2 s modified at specific Lys residues, and that all three of these proteins, conjugated with fluorescent peptides, are internalized in primary myotubes.
Novel Human Secreted Phospholipase A2 with Homology to the Group III Bee Venom Enzyme
Journal of Biological Chemistry, 2000
Venom and mammalian secreted phospholipases A 2 (sPLA 2 s) have been associated with numerous physiological, pathological, and toxic processes. So far, structurally related group I and II sPLA 2 s have been found in vertebrates such as mammals and snakes, whereas group III sPLA 2 s have mainly been found in venom from invertebrates such as bees and scorpions. Here we report the cloning and expression of a cDNA coding for a human group III (hGIII) sPLA 2. The full-length cDNA codes for a signal peptide of 19 residues followed by a protein of 490 amino acids made up of a central sPLA 2 domain (141 residues) flanked by large N-and C-terminal regions (130 and 219 residues, respectively). The sPLA 2 domain is 31% identical to bee venom sPLA 2 and displays all of the features of group III sPLA 2 s including 10 cysteines. The hGIII sPLA 2 gene consists of at least 7 exons and maps to chromosome 22q. By Northern blot analysis, a 4.4-kilobase hGIII transcript was found in kidney, heart, liver, and skeletal muscle. Transfection of hGIII sPLA 2 cDNA in COS cells led to accumulation of sPLA 2 activity in the culture medium, indicating that the cDNA codes for a secreted enzyme. Using small unilamellar vesicles as substrate, hGIII sPLA 2 was found to be a Ca 2؉-dependent enzyme showing an 11fold preference for phosphatidylglycerol over phosphatidylcholine and optimal activity at pH 8.
Characterization of the isoforms of phospholipase A2 from honeybee venom
Insect Biochemistry, 1991
These variants, termed PLA-16, PLA-18, and PLA-20, were isolated by lectin affinity chromatography and preparative polyacrylamide gel electrophoresis. The amino acid sequences of the N-terminal peptide portions of all three isoforms, as assessed by automated Edman degradation, were identical with that expected for honeybee phospholipase A2. Sequencing data suggest that, while PLA-18 and PLA-20 carry oligosaccharide residues at asparagine-13, PLA-16 has escaped glycosylation during biosynthesis. Release of the carbohydrate from PLA-18 and PLA-20 with peptide:N-glycosidase F abolished the molecular mass differences between the three isoforms of phospholipase. Differences in sensitivity to ~t-mannosidase and monosaccharide composition of PLA-18 and PLA-20 further indicate that their electrophoretic separation is based on structural features of the N-glycosidically linked oligosaccharide. Noticeably, PLA-20 contains N-acetylgalactosamine, a sugar not having yet been described as a constituent of insect glycoproteins.
Toxicon, 1999
Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60–84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60–65°C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.
Toxicon, 1991
acid sequence of a phospholipase AZ from the venom of Trimeresurus gramineus (green habu snake) . Toxicon 29, 157-166, 1991 .-Two phospholipases AZ, named phospholipases AZ-I and AZ-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake) . The complete amino acid sequence of phospholipase AZ-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases AZ from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases AZ and is assumed to be a part of the CaZ+-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases AZ. Secondary structure compositions of phospholipase AZI are similar to those of Crotalus atrox phospholipase AZ. No appreciable Cat+-induced difference spectrtun was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Cat+ binding site although CaZ+ is bound with affinity similar to that for T.~lavoviridis phospholipase AZ. 157