cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom (original) (raw)
Related papers
Journal of Molecular Evolution, 1995
The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5′ and 3′ untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.
Biochemical Journal, 1980
The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by ge...
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.
Molecular cloning of phospholipases A2 from venom glands of Echis carpet vipers
Toxicon, 2003
Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A 2 (PLA 2 ) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA 2 protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA 2 enzymes. Comparison of the PLA 2 sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA 2 -function distinctions between these Echis PLA 2 isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA 2 -neutralizing antibodies with polyspecific potential. q
Toxicon, 2003
During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, several putative toxins, including a phospholipase A 2 homologue cDNA (clone V2), were identified. The V2 cDNA clone codes for a potential coral snake toxin with a signal peptide of 27 amino acid residues plus a predicted mature protein with 119 amino acid residues. The deduced protein is highly similar to known phospholipases A 2 , with seven deduced S-S bridges at the same conserved positions. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies, which recognized the recombinant protein in Western blot. This antiserum was used to screen a large number of venoms, showing a ubiquitous distribution of immunorelated proteins in all elapidic venoms but not in the viperidic Bothrops jararaca venom. This is the first description of a complete primary structure of a phospholipase A 2 homologue deduced by cDNA cloning from a coral snake. q
The phospholipase A 2 superfamily encompasses 15 groups that are classified into: secreted PLA 2 (sPLA 2 ); cytosolic PLA 2 (cPLA 2 ); Ca 2+ -independent intracellular PLA 2 (iPLA 2 ); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA 2 . Currently, approximately 700 PLA 2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA 2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA 2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA 2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA 2 reveals that Cdc PLA 2 clustered with other acidic type IIA PLA 2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA 2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA 2 purification and analysis.
Protein Journal, 2004
In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III – AY145836) is related to an acidic PLA2 sharing similarity, within the range 55–81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.
Toxicon, 1995
Localization and expression of phospholipases A2 in Trimeresurusflavoviridis (habu snake) venom gland. Toxicon 33, 1645-1652, 1995.--The localization and expression profiles of phospholipases A2 (PLA2 s) in Trimeresurus flavoviridis venom gland were studied by means of in situ hybridization and immunohistochemical techniques. Venom gland cells are tightly arrayed in a single layer along the inlet-like lumens in which venom proteins are stored, mRNAs for PLA2s were detected at the high level in cytoplasm. Using the immunohistochemical technique with polyclonal anti-Asp-49-PLA2 antibody, Asp-49-PLA2 and, possibly, its isozymes were detected in intracellular granules and in venom lumens. The intracellular granules containing PLA2 proteins appear to be transferred from the nucleus towards the outer membrane facing the lumen, and then to be secreted.