Molecular cloning and deduced primary structures of acidic and basic phospholipases a2 from the venom of Deinagkistrodon acutus (original) (raw)
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Toxicon, 2005
Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A 2 (PLA 2 ) of basic nature (pI 8.5) was isolated from the venom of Amami-Oshima T. flavoviridis. Its amino acid sequence determined by the ordinary procedures was completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland, which was named PLA-B 0 . It consists of 122 amino acid residues and has aspartate at position 49. It induced edema in a mouse footpad assay and caused necrosis in mouse skeletal muscles. PLA-B 0 is similar in sequence to PLA-B (Tokunoshima) and PL-Y (Okinawa), both basic [Asp 49 ]PLA 2 s, with a few amino acid substitutions, indicating occurrence of interisland mutation. Although PLA 2 s of Crotalinae subfamily were phylogenetically classified into four types, PLA2 (acidic or neutral [Asp 49 ]PLA 2 ) type, basic [Asp 49 ]PLA 2 type, neurotoxic [Asp 49 ]PLA 2 type and [Lys 49 ]PLA 2 type, it was ascertained that PLA 2 s of PLA2 type and [Lys 49 ]PLA 2 type are most essential as toxic components for Crotalinae snake venoms and that basic [Asp 49 ]PLA 2 -type PLA 2 s are uniquely contained only in the venoms of T. flavoviridis species. Prediction of physiological activities of some PLA 2 s was made based on their location in the phylogenetic tree. Relationship of divergence of PLA 2 s via accelerated evolution followed by less rapid mutation and physiological activities was discussed. q
This study focuses on the structural and functional characterizations of novel venom phospholipases A 2 (PLA 2 s) from Trimeresurus gracilis, an endemic Taiwanese pitviper. The PLA 2 cDNAs were cloned from venom glands and sequenced. The majority of the clones encoded a Glu6-containing PLA 2 (designated as Tgc-E6) whose deduced amino acid sequence resembled those of other Crotalinae acidic PLA 2 s. Tgc-E6 was also purified and constituted about 6% (w/w) of the total venom proteins. For human platelet rich plasma, Tgc-E6 inhibited the ADP-and collagen-induced aggregation with an IC 50 of 272 nM and 518 nM, respectively. Like Ovophis okinavensis venom, T. gracilis venom did not contain any Lys49-PLA 2 s, although a cDNA encoding Lys49-PLA 2 has been cloned from each of the species. Their predicted protein sequences are 94% identical, and their pI values 8.3 are lower than those of other Lys49-PLA 2 s, mainly due to the acidic substitutions within positions 78-111, which are apparently more similar to those in Tgc-E6 than to those in other Lys49-PLA 2 s. This unique structural feature of the venom PLA 2 s thus render evidence for close phylogenetic relationship between both species. The structural variations in the venom acidic PLA 2 s of the two species possibly have resulted from adaptation to different prey ecology.
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.
Brazilian Journal of Medical and Biological Research, 2010
To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A 2 D49 (PLA 2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA 2). The cDNA encoding Cdc PLA 2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA 2 protein family. Cdc PLA 2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA 2 enzymes. Cdc PLA 2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA 2 showed the existence of two lines of N6-PLA 2 , denominated F24 and S24. Apparently, the sequences of the New World's N6-F24-PLA 2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA 2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2007
Venoms of Russell's vipers (genus Daboia) are known for their deadly coagulopathic and other effects. We herein studied various isoforms of venom phospholipases A 2 (PLAs) from two Daboia species at their geographic boundary. From Myanmar Daboia siamensis venom (designated as DsM), four PLAs (designated DsM-aI, aI', aII' and bI') were purified, and the cDNAs encoding two acidic (DsM-aI and aII) and two basic PLAs (DsM-bI and S1) were also cloned from its venom-glands. DsM-S1 is identical to the major venom PLA of southern India Daboia russelii, but the protein is absent from the venom. Additionally, four PLAs (designated DrK-aI, aII, bI and bII) were cloned from cDNA obtained from venom glands of a Kolkata D. russelii, and the PLAs were purified from the pooled venom (designated as DrK). The acidic DrK-aI is the most neurotoxic and lethal among these PLAs; DsM-aI which differs from DrK-aI by only the Phe2 substitution shows greatly reduced enzymatic activity and lethality. Both acidic PLAs do not form dimeric complex with basic PLAs in the same venoms. DsM-bI' is neurotoxic and lethal but its orthologous DrK-bI (97% identical to DsM-bI') is a much weaker toxin. Given the fact that most of the orthologous PLAs of DrK and DsM share 97-100% sequence identity, Daboia vipers of Myanmar and Kolkata must be closely related. Molecular phylogenetic analyses on 30 venom PLAs of Eurasian vipers' revealed co-evolution of five subtypes of venom PLAs in both Daboia and Vipera genera. Our results shed light on the intra-and inter-species variations and structure-function relationships of viperid venom PLAs.
Biomedical …, 2010
To examine the molecular diversity of the venom proteins of the Russell's viper (Daboia russellii russellii) and the Indian cobra (Naja naja) in Sri Lanka, we isolated 38 venom proteins through a combination of anion exchange chromatography followed by reversed-phase high performance liquid chromatography. From the venom of D. r. russellii we isolated 15 proteins: 5 isozymes of phospholipase A 2 (PLA 2), 4 serine proteases, 2 C-type lectin-like proteins, 2 l-amino acid oxidases, 1 cysteine-rich secretory protein (CRISP), and 1 metalloproteinase. From the venom of N. naja we isolated 23 proteins: 10 isoforms of cytotoxins (CTX), 7 PLA 2 isozymes, 2 muscarinic toxinlike proteins, 2 CRISPs, 1 nerve growth factor, and 1 new thrombin-like serine protease. Most of these proteins contained new amino acid sequences for each species, indicating molecular diversity in venom proteins. The entire amino acid sequences of PLA 2 3 from D. r. russellii and CTX7 from N. naja were determined. Additionally, the polymorphic amino acid residues of PLA 2 3 were preferentially localized on the potential antigenic sites. While 2 types of PLA 2 (N and S types) were found in D. r. russellii (India) and D. r. siamensis (Java), all the PLA 2 s from D. r. siamensis (Burma) were N type, and those from D. r. russellii (Sri Lanka) were primarily S type.
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.
Archives of Biochemistry and Biophysics, 2003
Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A 2 (PLA 2 s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA 2 s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA 2 s induced edema, but had no anti-platelet activity. There was also a common basic PLA 2 myotoxin in all the samples. We have cloned five acidic PLA 2 s and several hybrid-like nonexpressing PLA 2 s. Molecular masses and N-terminal sequences of the purified PLA 2 s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA 2 s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA 2 s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA 2 s in pit viper venom.