cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

Braz J Med Biol Res, March 2010, Volume 43(3) 262-270

cDNA and deduced primary structure of basic phospholipase A₂ with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

F.H.R. Fagundes¹, M. Oliveira², S. Huancahuire-Vega¹, F.F. Romero-Vargas¹, L.A. Ponce-Soto¹ and S. Marangoni¹

¹Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil

²Feldmann Laboratories, São Paulo, SP, Brasil

^{Correspondence and Footnotes}

Abstract

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 A₂ D49 (PLA₂ D49), from the venom of Crotalus durissus collilineatus (Cdc PLA₂). The cDNA encoding Cdc PLA₂ from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA₂ protein family. Cdc PLA₂ has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA₂ enzymes. Cdc PLA₂ 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 PLA₂ showed the existence of two lines of N6-PLA₂, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA₂ are similar to those of the agkistrodotoxin from

the Asian genus Gloydius. The sequences of N6-S24-PLA₂ are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Key words: Cdc PLA₂; cDNA; Chick biventer cervicis; Crotalus durissus collilineatus; Mass spectrometry MALDI-TOF; Neurotoxic agents

Introduction

Phospholipase A₂ (PLA₂; EC 3.1.1.4) enzymes are small proteins widespread in nature and frequently the major toxic component of snake venoms (1). Despite their similar structures and enzymatic mechanisms for the catalysis of the hydrolysis of sn-2 bonds in phosphoglycerides in a Ca²⁺-dependent manner, they have evolved to acquire a wide range of activities, including neurotoxic, myotoxic, cardiotoxic, edema-forming, platelet-aggregating or -inhibiting, anticoagulant, convulsive, and hypotensive effects (2). These activities are attributed primarily to crotoxin and crotoxin B (PLA₂), the principal toxin of Crotalus durissus terrificus (South American rattlesnake) venom, which is also present in other subspecies such as C. durissus collilineatus, C. durissus ruruima, and C. durissus cumanensis (3-5).

The cDNAs and genes encoding PLA₂ have been cloned; however, comparison of their nucleotide sequences led to a novel discovery that Darwinian-type accelerated evolution has occurred and diverse physiological activities have been acquired (6-8). Therefore, the study of the neurotoxic protein PLA₂ D49, derived from the venom of a rattlesnake subspecies, C. durissus collilineatus (Cdc PLA₂), is of particular interest. When comparing the chromatography profiles of other crotalic venoms with the venom of C. durissus collilineatus, the absence of crotamine in C. durissus collilineatus venom is particularly interesting since this toxin has myotoxin and neurotoxin activities. These findings suggest that the crotoxin and PLA₂ F6 from C. durissus collilineatus probably play an important role in this venom’s action (3), suggesting a molecular microevolution. However, acquiring information on the molecular genetics of precursor structures derived from cDNAs involves sacrificing the specimens for subsequent dissection of the venom gland (9,10).

We describe here a simple and rapid technique for the synthesis of venom gland protein cDNAs from lyophilized venom samples that does not require the sacrifice and dissection of the specimen. The venom from Cdc was chosen because, although it is a clinically important species of rattlesnake found in central Brazil, few studies have been carried out on its venom and its main toxin, a crotoxin homolog (11).

Material and Methods

Venom, chemicals and reagents

Lyophilized Cdc venom was obtained from "Centro de Extração de Toxina Animal" (CETA) snakes farmed in Morungaba, SP, Brazil. Crotoxin and Cdc PLA₂ were purified from this venom as described by Ponce-Soto et al. (3). All chemicals and reagents used were of analytical, sequencing or molecular grade.

Animals

Male HY-LINE W36 chickens (Gallus gallus), 4-8 days old were supplied by Granja Ito S/A (Sumaré, SP, Brazil). Animals had free access to food and water.

mRNA isolation and cDNA construction

Lyophilized venoms (10 mg) were reconstituted with 0.25 mL Trizol LS (Gibco-BRL, USA), 50 µL chloroform (-20°C) was added and the preparation was incubated at 4°C for 5 min. Samples were centrifuged at 16,000 g for 5 min at 4°C. Following centrifugation, the upper aqueous phase, containing all RNAs, was transferred to a fresh tube. The RNA was precipitated with 300 µL cold isopropyl alcohol and collected by centrifugation at 16,000 g for 5 min at 4°C. The supernatant was removed and the RNA pellet was washed once with 400 µL 75% ethanol and collected by centrifugation at 10,000 g for 2 min at 4°C. The supernatant was discarded and the pellet was resuspended in 8 µL diethyl pyrocarbonate water for cDNA synthesis.

Reverse transcription polymerase chain reaction (RT-PCR) was conducted using RETROTOOLS cDNA/DNA polymerase (BIOTools, USA), according to manufacturer protocols. RT-PCR was carried out using oligo (dT) as primer. DNAs were amplified by PCR using total RT-PCR products as template and two oligonucleotide primers. The two primers were designed according to the reported nucleotide sequences of venom PLA₂ (12). The forward primer was designed around the start codon: primer 1 (5’ tct gga ttg agg agg ATG 3’); the reverse primer was designed around the stop codon: primer 2 (5’ cAT GCC TGC AGA GAC TTA 3’). Both primers corresponded to the conserved non-coding regions of venom PLA₂. PCR conditions were: 95°C/3 min (1 cycle), 94°C/30 s, 52°C/30 s, and 72°C/30 s (25 cycles), and 72°C/10 min (1 cycle). The amplified DNA fragment obtained was analyzed on 1.5% (w/v) agarose gel stained with ethidium bromide and visualized under UV light.

cDNA sequencing

PCR products were sequenced directly using a big dye terminator cycle sequencing kit (Applied Biosystems, USA) and an ABIPrism 377 Sequencer automated DNA sequencer (Perkin Elmer, USA). The structure of the product was compared to that of the GenBank nucleotide and protein data bases using the Blastn and Blastx programs available at the National Center for Biology Information (NCBI) for identification (www.ncbi.nlm.nih.gov).

Phylogenetic tree of the N6-PLA₂ and G6-PLA₂

The amino acid sequences of the PLA₂ Cdcoll cDNAs were aligned to PLA₂ isoform sequences from related viperid species: Crotoxin B1 and B2 (P07517, P24027) C. durissus terrificus, Sistruxin B (AAR14164) Sistrurus catenatus tergeminus, Agkistrotoxin (P14421) Gloydius halys, Smstoxin (AAR14160) S. miliarius streckeri, Cvvtoxin (AAQ13337) C. viridis viridis, Trimucrotoxin (Q90W39) Protobothrops mucrosquamatus, Cgtoxin (AAR14161) Cerrophidion godmani, Bstoxin (AAR14162) Bothriechis schlegelii, TfPLA-N (BAC56893) Trimeresurus flavoviridis, Batoxin (S09314) Bothrops asper, Bjtoxin (AAO27454) B. jararacussu, using the Lasergene software (DNASTAR, USA) and a phylogenetic tree constructed for the latter sequences using the CLUSTAL W program (13). The W6 PLA₂ D49 of Calloselasma rhodostoma venom (14) was used as out-group. Bootstrap values higher than 50 at each node support the robustness of the cladogram.

The predicted isoelectric point of PLA₂ examined here was determined using the Lasergene software (Protean, DNASTAR).

Measurement of PLA₂ activity

PLA₂ activity was measured using the assay described by Holzer and Mackessy (15) in the use of a chromogenic substratum, modified for 96-well plates (3). The standard assay mixture contained 200 µL buffer (10 mM Tris-HCl, 10 mM CaCl₂, 100 mM NaCl, pH 8.0), 20 µL chromogenic lipid substrate 4-nitro-3-octanoyloxy-benzoic acid, 20 µL water, and 20 µL PLA₂ in a final volume of 260 µL. After the addition of PLA₂ (20 µg), the mixture was incubated for up to 40 min at 37°C, and the chromophore, 4-nitro-3-hydroxy-benzoic acid, detected by absorbance measurements at 425 nm, at 10-min intervals. Enzyme activity, reported as the initial reaction velocity, was calculated on the basis of the increase of absorbance at 425 nm after 20 min (3).

MALDI-TOF mass spectrometric analysis

The molecular mass of Cdc PLA₂ was determined by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry using a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems). One microliter of sample (Cdc PLA₂) in 0.1% TFA was mixed with 2 µL of the matrix a-cyano-4-hydroxycinnamic acid, 50% acetonitrile, and 0.1% TFA (v/v). The matrix was prepared with 30% acetonitrile and 0.1% (v/v) TFA. Ion masses were determined under the following conditions: accelerate voltage 25 kV, the laser operated at 2890 µJ/com2, delay 300 ns and in the linear analysis mode (16).

N-terminal amino acid sequence

One milligram of purified Cdc PLA₂ was dissolved in 6 M guanidine hydrochloride containing 0.4 M Tris-HCl and 2 mM EDTA at a final pH of 8.15. After reduction with DTT the products were carboxymethylated with ¹⁴C-iodoacetic acid (16). The mixture was desalted on a Sephadex G25 column equilibrated with 1 M acetic acid at 25°C and the reduced and carboxymethylated proteins were lyophilized. The N-terminal amino acid sequence of Cdc PLA₂ protein was determined by automated Edman degradation in a model Procise automatic sequencer (Applied Biosystems). PTH amino acids were identified with a model 120A PTH amino acid analyzer (Applied Biosystems) based on their retention times. Cdc PLA₂ was sequenced to double check the N-terminal sequence of up to 42 residues in order to confirm and compare it with the PLA₂ sequence deduced from nucleotide sequencing.

Effect on the chick biventer cervicis preparation

Animals were anesthetized with halothane and sacrificed by exsanguination. The biventer cervicis muscles were removed and mounted under a tension of 0.5 g, in a 5-mL organ bath (Automatic organ multiple-bath LE01 Letica Scientific Instruments, Spain) at 37°C containing aerated (95% O₂-5% CO₂) Krebs solution, pH 7.5, of the following composition: 118.7 mM NaCl, 4.7 mM KCl, 1.88 mM CaCl₂, 1.17 mM KH₂PO₄, 1.17 mM MgSO₄, 25.0 mM NaHCO₃ and 11.65 mM glucose. Contraction to exogenously applied acetylcholine (ACh; 55 and 110 µM for 60 s) and KCl (20.1 mM for 130 s) was obtained in the absence of field stimulation, prior to the addition of a single dose of venom or crotoxin. A bipolar platinum ring electrode was placed around the tendon, which runs along the nerve trunk supplying the muscle. Indirect stimulation was performed with a MAIN BOX LE 12404 Panlab s.l. stimulator (Powerlab AD Instruments, Spain; 0.1 Hz, 0.2 ms, 3-4 V). Muscle contractions and contractures were isometrically recorded by force-displacement transducers (Model MLT0201 Force transducer 5 mg-25 g Panlab s.l. AD Instruments Pty Ltd.) connected to a PowerLab/4SP (OUAD Bridge AD Instruments, Spain).

Statistical analysis

Data are reported as the mean ± SEM of n experiments. The significance of differences between means was assessed by analysis of variance followed by the Dunnett test when several experimental groups were compared with the control group. The confidence limit for significance was 5%.

Results

mRNA isolation and cDNA construction

The single PCR product generated was within the expected size of approximately 450 bp (Figure 1) and nucleotide sequencing, as shown by trawling unedited data on the database, which unequivocally identified the product, demonstrating that the polyadenylated mRNAs constituted representative products of the venom secretion of a neurotoxic venomous snake.

cDNA sequencing

The cDNA of PLA₂ D49 from Cdc has 451 bp, which includes the start codon ATG and the termination codon TAA (Figure 2). By comparing this with other PLA₂ cDNAs, we deduced that this cDNA encodes a putative signal peptide of 16 amino acids and a mature protein of 122 residues. There are 14 cysteines in the mature peptide, its isoelectric point was estimated to be 8.5 and its molecular mass to be about 14 kDa.

Purification of PLA₂ and measurement of PLA₂ activity

Purification of PLA₂ from Cdc venom was performed according to the procedure described by Ponce-Soto et al. (3). Reverse-phase-HPLC of the crotoxin of the Cdc venom resulted in one PLA₂ peak (Cdc PLA₂) that eluted at 35 min, and any other isoforms of PLA₂ were absent. The specific activity of purified PLA₂ was 9.87 nmol·min^-1·mg^-1.

N-terminal amino acid sequence of the expressed protein

The complete amino acid sequence deduced was confirmed up to the 42nd residue by direct protein N-terminal sequencing (HLLQF NKMIK FETRR NAIPF YAFYG CYCGW GGRGR PKDAT DR). Amino acid analysis was deduced as the sequence of cDNA of the protein, using the Lasergene software (Protean, DNASTAR, USA), and revealed the following composition of Cdc PLA₂: A/6, C/14, D/6, E/5, F/6, G/11, H/2, I/5, K/7, L/6, M/2, N/3, P/4, Q/3, R/13, S/6, T/7, V/2, W/3, and Y/11.

MALDI-TOF mass spectrometric analysis

MALDI-TOF results showed that Cdc PLA₂ has a molecular mass of 14,276.90 Da (Figure 3), confirming the results obtained by analysis of the amino acids deduced from the cDNA (14,340.54 Da). The molecular mass of Cdc PLA₂ was confirmed by MALDI-TOF mass spectrometry, indicating the protonated protein species of homodimer with a molecular mass of 28,395.68 Da (2M+2H⁺), monomers of 14,185.48 Da (M+H⁺) and half of the mass of 7,105.18 Da (MH₂²⁺) showed in the mass spectrum (Figure 3).

Effects on the chick biventer cervicis preparation

The neuromuscular effect of Cdc PLA₂ was studied using chick biventer cervicis nerve-muscle preparations. Concentrations of 10 and 20 µg/mL Cdc PLA₂ affected neuromuscular transmission, and the times required to reach a 50% blockade were 42.61 min for 10 µg/mL and 36.14 min for 20 µg/mL (Figure 4). In both cases, the blockade was irreversible. PLA₂ did not block ACh and KCl-induced contractions significantly compared to control values. In control preparations, the contractions induced by ACh and KCl remained stable after 120 min of indirect stimulation compared to control values (data not shown).

Phylogenetic tree of the N6-PLA₂ and G6-PLA₂

We constructed the phylogenetic tree (Figure 5) based on the protein sequences of neurotoxic N6-PLA₂ and G6 or A6-myotoxic PLA₂ (17-20) from pit viper venom (Figure 5). Molecular phylogeny of basic crotalid PLA₂ D49 includes all sequences listed in Figure 6. The comparison of Cdc PLA₂ sequences revealed a high level of conservation within the PLA₂ protein family and showed that there are extremely conserved positions in the PLA₂ (Figure 6).

Figure 1.
Reverse transcription polymerase chain reaction amplification of mRNA encoding protein transcripts from lyophilized venom. Lane 1, RT-PCR products from cDNA phospholipase A₂ primers, around 450 bp. Lane M contains a standard DNA ladder, each band representing 100-bp increments.

Figure 2.
The cDNA sequence determined by sequencing and the deduced amino acid sequence of Cdc PLA₂. One-letter codes of amino acids are used and the numbering is shown below the sequences. PCR primers are in bold and the signal peptide is underlined. The sequence confirmed by direct n-terminal sequence is double underlined.

Figure 3.
Determination of the molecular mass of Cdc PLA₂ by MALDI-TOF mass spectrometry, using a Voyager DE PRO MALDI-TOF mass spectrometry (Applied Biosystems, USA). The ionic MH⁺ corresponds to the real mass of the Cdc PLA₂ and MH₂²⁺ and 2MH⁺ species are shown in the mass spectrum. The value of the real mass of Cdc PLA₂ corresponds to the average of several spectrum (±7.8 x 10^-5).

Figure 4.
Neurotoxic activity of Cdc PLA₂ on the chick biventer cervicis preparation. Control (circles) without PLA₂. Doses of 10 (squares) and 20 µg/mL (triangles) of Cdc PLA₂. Each point represents the average of five experiments ± SEM. P < 0.05 compared to control (Dunnett test).

Figure 5.
Molecular phylogeny of basic crotalid PLA₂ D49. The data used include all sequences listed in Figure 6. Clustal X (DNA Star program) was employed for the comparison of Cdc PLA₂ sequence with other Crotoxin B1 and B2 (P07517, P24027) Crotalus durissus terrificus, Sistruxin B (AAR14164) Sistrurus catenatus tergeminus, Agkistrotoxin (P14421) Gloydius halys, Smstoxin (AAR14160) Sistrurus miliarius streckeri, Cvvtoxin (AAQ13337) Crotalus viridis viridis, Trimucrotoxin (Q90W39) Protobothrops mucrosquamatus, Cgtoxin (AAR14161) Cerrophidion godmani, Bstoxin (AAR14162) Bothriechis schlegelii,
TfPLA-N (BAC56893) Trimeresurus flavoviridis, Batoxin (S09314) Bothrops asper, Bjtoxin (AAO27454) Bothrops jararacussu. The phylogeny relationship of Cdc PLA₂ to other PLA₂ isoforms was statistically evaluated by the Bootstrap method.

Figure 6.
Amino acid sequence and homology of Cdc PLA₂ with other basic PLA₂ D49. The PLA₂, with GenBank accession numbers and the venom species are: Crotoxin B1 and B2 (P07517, P24027) Crotalus durissus terrificus, Sistruxin B (AAR14164) Sistrurus catenatus tergeminus, Agkistrotoxin (P14421) Gloydius halys, Smstoxin (AAR14160) Sistrurus miliarius streckeri, Cvvtoxin (AAQ13337) Crotalus viridis viridis, Trimucrotoxin (Q90W39) Protobothrops mucrosquamatus, Cgtoxin (AAR14161) Cerrophidion godmani, Bstoxin (AAR14162) Bothriechis schlegelii, TfPLA-N (BAC56893) Trimeresurus flavoviridis, Batoxin (S09314) Bothrops asper, Bjtoxin (AAO27454) Bothrops jararacussu.

Discussion

Venoms from many invertebrates and vertebrates are rich in amphipathic/cationic peptides that have been shown to possess nucleic acid binding properties and indeed to afford protection against degradation (21,22). The presence of these peptides permits the retrieval of these RNAs in a RT-PCR-compatible form.

Chen et al. (23) reported that it was impossible to recover high quality mRNAs from reptile venom for RT-PCR using the precipitation method with organic solvents. We succeeded in recovering these mRNAs by conventional techniques of precipitation with organic solvents. For the construction of the PLA₂ cDNA encoder from Cdc, we then used samples of total RNA extracted from the venom. These findings show that it is unnecessary to separate the mRNA from other RNAs to construct the cDNA.

The single PCR product generated was within the expected size, approximately 450 bp (Figure 1), and nucleotide sequencing unequivocally identified the product, demonstrating that the polyadenylated mRNAs constituted representative products of the venom gland transcriptome of a neurotoxic venomous snake.

The cDNA of PLA₂ D49 from Cdc has 451 bp, include the start codon ATG and the termination codon TAA (Figure 2). By comparing this with other PLA₂ cDNAs, we deduced that this cDNA encodes a putative signal peptide of 16 amino acids and a mature protein of 122 residues. The deduced signal peptide agrees with the characteristics of a general signal peptide: i) the central core is rich in hydrophobic amino acid residues (Leu), which can form a transmembrane α-helix; ii) the signal peptide cleavage site is present after an apolar residue (Gly) at position 16 (Figure 2). There are 14 cysteines in the mature peptide, which can form 7 disulfide bridges, its isoelectric point is estimated to be 8.5 and molecular mass is about 14 kDa. Since position 49 in the mature peptide is Asp, we conclude that it is a form of PLA₂ D49.

A pattern of biochemical and genetic variation exists within any species with respect to the protein fractions expressed in their venom (24). Therefore, we opted to isolate the mRNA and PLA₂ from the same extracted venom sample to avoid differences among samples due to mixing the venom of more than one individual animal.

Purification of PLA₂ from Cdc venom was performed in accordance with the procedure described by Ponce-Soto et al. (3) for the comparison of its deduced molecular structure with its primary structure and with its neurotoxic activity.

In comparison with the chromatographic profiles of other crotalid venoms, the venom of Cdc lacks the fraction corresponding to crotamine. The absence of crotamine in Cdc venoms is interesting because this toxin is typically found in C. durissus terrificus venom. This toxin is responsible for myotoxic and neurotoxic activities and in this rattlesnake species (C. durissus terrificus) it accounts for 20% of the dry venom. These findings suggest that the crotoxin homolog in Cdc probably plays an important role in the neurotoxic action of this venom (3).

The effect of Cdc PLA₂ on neuromuscular preparations was studied using chick biventer cervicis nerve-muscle preparations. The blockade was irreversible (Figure 4). PLA₂ did not block Ach and KCl-induced contractions significantly compared to control values. In control preparations, contractions induced by ACh and KCl remained stable after 120 min of indirect stimulation compared to control values (data not shown). These results confirm that isolated Cdc PLA₂ may be considered a presynaptic neurotoxin that presents different times of blockade. Furthermore, these results suggest that its pharmacological activity is dose-dependent.

The side chain of F24 has proven to be important for the toxicity of ammodytoxins A and C (25), and modification of Y22 by tetranitromethane greatly reduces the specific binding of crotoxin to synaptosomal vesicles (26). In addition, residues 6-8 have been shown to be essential for the neurotoxicity of trimucrotoxin by site-directed mutagenesis (27). Therefore, the neurotoxic effects induced by Cdc PLA₂ may be related to the presence of residue F at position 24, making it a member of the group of F24-N6-PLA₂. Moreover, the importance of residues F24, Y22 and residues 6-8 is to preserve the neurotoxic activity of Cdc PLA₂ (Figure 5).

Recently, an R34Q mutation in basic PLA₂ from Chinese pit viper venom reduced its hemolytic activity in vitro (28), suggesting that R34 in the group II PLA₂ is favorable for membrane binding while Q34 may be more favorable for binding to specific neuronal proteins or receptors. In Figure 5, the presence of an arginine residue can be clearly seen at position 34. PLA₂ Cdc may exert some hemolytic activity in addition to its neurotoxic activity.

Rattlesnakes (genus Crotalus) and other pit vipers of the New World are probably the descendants of some species of Asian pit vipers (29). The group II PLA₂ in pit viper venom have been classified into different pharmacological subtypes that play different roles, e.g., as platelet aggregation inhibitors, anticoagulants, neurotoxins, or myotoxins (30,31). One of these subtypes is the myotoxic/neurotoxic PLA₂ D49 with an Asn 6 substitution (hereafter designated as N6-PLA₂). The known members of this venom PLA₂ subtype include the basic subunits of crotoxin and Mojave toxin, agkistrodotoxin from Gloydius halys brevicaudus (32), trimucrotoxin from Protobothrops mucrosquamatus (33), Tf-PLA-N from flavoviridis (34), and the myotoxin from C. viridis viridis (designated as Cvv-N6) (14,35).

The phylogenetic tree based on protein sequences of neurotoxic N6-PLA₂ and G6 or A6-miotoxic PLA₂ (18-20) from pit viper venom classifies the group of N6-PLA₂, separately from G6-PLA₂ D49 and the existence of two lineages of N6-PLA₂, denominated F24 and S24, according to the presence of residue at position 24 of PLA₂. In addition to the residue at position 24 that can be distinguished in the N-terminal, the amino acids N1, I11, K14, and T/S23, conserved in the S24-subtype, and the amino acids, H/S1, F11, R14, and A23, conserved in the F24-subtype, can be found (Figure 5).

Apparently, the sequences of the New World’s N6-F24-PLA₂ are similar to those of agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA₂, also from the New World, are similar to the sequence of trimucrotoxin from Protobothrops, found in the Old World (Figure 6). Therefore, ancient pit vipers found in the Old World and related to the present day Gloydius and Protobothrops, possibly migrated to the New World and evolved into various species of rattlesnakes.

While proteome analysis has shown the secretion to be non-lethal, transcriptome analysis has until now required sacrificing the specimens to dissect the venom gland for cDNA library construction. Here, we have shown that polyadenylated mRNAs encoding proteins from venom are present in the secreted venom. Therefore, secretory proteome and transcriptome analyses can be performed simultaneously without sacrificing the specimen.

The simple protocol described here should circumvent the need to sacrifice snakes hitherto killed for their venom glands in the pursuit of what are essentially molecular biological approaches to the structural characterization of toxic venom proteins. In addition, the way has been paved to facilitate accelerated acquisition of this information for modern biotechnological applications.

References

1. Kini RM. Phospholipase A₂: a complex multifuncional protein puzzle. In: Kini RM (Editor), Venom phospholipase A₂ enzymes: Structure, function and mechanism. Chichester: Wiley; 1997. p 1-28.

2. Kini RM. Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon 2003; 42: 827-840.

3. Ponce-Soto LA, Toyama MH, Hyslop S, Novello JC, Marangoni S. Isolation and preliminary enzymatic characterization of a novel PLA2 from Crotalus durissus collilineatus venom. J Protein Chem 2002; 21: 131-136.

4. Ponce-Soto LA, Baldasso PA, Romero-Vargas FF, Winck FV, Novello JC, Marangoni S. Biochemical, pharmacological and structural characterization of two PLA₂ isoforms Cdr-12 and Cdr-13 from Crotalus durissus ruruima snake venom. Protein J 2007; 26: 39-49.

5. Romero-Vargas FF, Ponce-Soto LA, Martins-de-Souza D, Marangoni S. Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom. Comp Biochem Physiol C Toxicol Pharmacol 2010; 151: 66-74.

6. Nakashima K, Ogawa T, Oda N, Hattori M, Sakaki Y, Kihara H, et al. Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes. Proc Natl Acad Sci U S A 1993; 90: 5964-5968.

7. Nakashima K, Nobuhisa I, Deshimaru M, Nakai M, Ogawa T, Shimohigashi Y, et al. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc Natl Acad Sci U S A 1995; 92: 5605-5609.

8. Ohno M, Menez R, Ogawa T, Danse JM, Shimohigashi Y, Fromen C, et al. Molecular evolution of snake toxins: Is the functional diversity of snake toxins associated with a mechanism of accelerated evolution? In: Moldave K (Editor), Progress in nucleic acid research and molecular biology. New York: Academic Press; 1998. p 307-364.

9. Silveira de Oliveira J, Rossan de Brandao Prieto da Silva A, Soares MB, Stephano MA, de Oliveira Dias W, Raw I, et al. Cloning and characterization of an alpha-neurotoxin-type protein specific for the coral snake Micrurus corallinus. Biochem Biophys Res Commun 2000; 267: 887-891.

10. Junqueira de Azevedo I, Farsky SH, Oliveira ML, Ho PL. Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins. J Biol Chem 2001; 276: 39836-39842.

11. Lennon BW, Kaiser II. Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus). Comp Biochem Physiol B 1990; 97: 695-699.

12. Bouchier C, Boulain JC, Bon C, Menez A. Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor. Biochim Biophys Acta 1991; 1088: 401-408.

13. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22: 4673-4680.

14. Tsai IH, Wang YM, Chen YH, Tu AT. Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis. Arch Biochem Biophys 2003; 411: 289-296.

15. Holzer M, Mackessy SP. An aqueous endpoint assay of snake venom phospholipase A2. Toxicon 1996; 34: 1149-1155.

16. Ponce-Soto LA, Lomonte B, Rodrigues-Simioni L, Novello JC, Marangoni S. Biological and structural characterization of crotoxin and new isoform of crotoxin B PLA(2) (F6a) from Crotalus durissus collilineatus snake venom. Protein J 2007; 26: 221-230.

17. Ponce-Soto LA, Bonfim VL, Rodrigues-Simioni L, Novello JC, Marangoni S. Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation. Protein J 2006; 25: 147-155.

18. Andriao-Escarso SH, Soares AM, Fontes MR, Fuly AL, Correa FM, Rosa JC, et al. Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom. Biochem Pharmacol 2002; 64: 723-732.

19. Bonfim VL, Toyama MH, Novello JC, Hyslop S, Oliveira CR, Rodrigues-Simioni L, et al. Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom. J Protein Chem 2001; 20: 239-245.

20. Soares AM, Andriao-Escarso SH, Bortoleto RK, Rodrigues-Simioni L, Arni RK, Ward RJ, et al. Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom. Arch Biochem Biophys 2001; 387: 188-196.

21. Wachinger M, Kleinschmidt A, Winder D, von Pechmann N, Ludvigsen A, Neumann M, et al. Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression. J Gen Virol 1998; 79 (Part 4): 731-740.

22. Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry 1997; 36: 3008-3017.

23. Chen T, Bjourson AJ, Orr DF, Kwok H, Rao P, Ivanyi C, et al. Unmasking venom gland transcriptomes in reptile venoms. Anal Biochem 2002; 311: 152-156.

24. Nkinin SW, Chippaux JP, Pietin D, Doljansky Y, Tremeau O, Menez A. [Genetic origin of venom variability: impact on the preparation of antivenin serums]. Bull Soc Pathol Exot 1997; 90: 277-281.

25. Petan T, Krizaj I, Gubensek F, Pungercar J. Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity. Biochem J 2002; 363: 353-358.

26. Tzeng MC, Yen CH, Hseu MJ, Tseng CC, Tsai MD, Dupureur CM. Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity. Toxicon 1995; 33: 451-457.

27. Tsai IH, Wang YM. Effect of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom. Toxicon 1998; 36: 1591-1597.

28. Jiao HM, Jin Q, Zhao JJ, Feng B, Wu XF, Zhou YC. [The hemolytic site of the basic phospholipase A(2) from Agkistrodon halys pallas]. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao 2002; 34: 383-387.

29. Parkinson CL. Molecular systematics and biogeographical history of pit vipers as determined by mitochondria ribosomal DNA sequences. Copeia 1999; 576-586.

30. Danse JM, Gasparini S, Menez A. Molecular biology of snake venom phospholipases A₂. In: Kini RM (Editor), Venom phospholipase A₂ enzyme: Structure, function and mechanism. Chichester: Wiley; 1997. p 29-71.

31. Tsai IH. Phospholipase A₂ of Asian snake venoms. J Toxicol Toxin Rev 1997; 16: 79-114.

32. Kondo K, Zhang J, Xu K, Kagamiyama H. Amino acid sequence of a presynaptic neurotoxin, agkistrodotoxin, from the venom of Agkistrodon halys Pallas. J Biochem 1989; 105: 196-203.

33. Tsai IH, Lu PJ, Wang YM, Ho CL, Liaw LL. Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus). Biochem J 1995; 311 (Part 3): 895-900.

34. Chijiwa T, Hamai S, Tsubouchi S, Ogawa T, Deshimaru M, Oda-Ueda N, et al. Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of Crotalinae group II phospholipases A2. J Mol Evol 2003; 57: 546-554.

35. Ownby CL, Colberg TR, White SP. Isolation, characterization and crystallization of a phospholipase A2 myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis). Toxicon 1997; 35: 111-124.

Acknowledgments

The authors thank Paulo A. Baldasso and Marino Forner for general technical help and Granja Ito S/A (Sumaré, SP, Brazil) for supplying male, 4-8-day-old HY-LINE W36 chickens (Gallus gallus). Research supported by FAPESP (#06/51566-0).

Address for correspondence: L.A. Ponce-Soto, Departamento de Bioquímica, Instituto de Biologia, UNICAMP, Caixa Postal 6109, 13083-970 Campinas, SP, Brasil. Fax: [phone]. E-mail: [e-mail]

Received October 1, 2009. Accepted February 3, 2010. Available online February 19, 2010. Published March 12, 2010.

The Brazilian Journal of Medical and Biological Research is partially financed by

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  09 Mar 2010
• Date of issue
  Mar 2010

History

• Accepted
  03 Feb 2010
• Received
  01 Oct 2009