A Novel Phospholipase A2 (D49) from the Venom of the Crotalus oreganus abyssus (North American Grand Canyon Rattlesnake)

Currently, Crotalus viridis was divided into two species: Crotalus viridis and Crotalus oreganus. The current classification divides “the old” Crotalus viridis into two new and independent species: Crotalus viridis (subspecies: viridis and nuntius) and Crotalus oreganus (subspecies: abyssus, lutosus, concolor, oreganus, helleri, cerberus, and caliginis). The analysis of a product from cDNA (E6d), derived from the gland of a specie Crotalus viridis viridis, was found to produce an acid phospholipase A2. In this study we isolated and characterized a PLA2 (D49) from Crotalus oreganus abyssus venom. Our studies show that the PLA2 produced from the cDNA of Crotalus viridis viridis (named E6d) is exactly the same PLA2 primary sequence of amino acids isolated from the venom of Crotalus oreganus abyssus. Thus, the PLA2 from E6d cDNA is actually the same PLA2 presented in the venom of Crotalus oreganus abyssus and does not correspond to the venom from Crotalus viridis viridis. These facts highlight the importance of performing more studies on subspecies of Crotalus oreganus and Crotalus viridis, since the old classification may have led to mixed results or mistaken data.


Introduction
Crotalus viridis defines a large group of snakes, also named as Western Rattlesnakes, which inhabit the eastern region of the Rocky Mountains of the United States that stretch from southern Canada to northern Mexico ( Figure 1) [1,2]. Phylogenetic analyses on mitochondrial DNA sequences of snakes classified as Crotalus viridis show significant taxonomic variations between individuals from different areas of USA and indicate that this species has several subspecies [3]. To understand the variations in these subspecies, morphological analyses were carried out based on distance analysis of whole venom profiles and based on maximum parsimony (MP) analysis of cyt b and ND4 [1,4,5]. effects produced by PLA 2 , several studies have researched or developed natural or synthetic compounds to aid in the treatment of the snake bites to inhibit the toxic effects of PLA 2 [26][27][28][29][30][31][32][33]. In addition, the amino acid sequences of hundreds of PLA 2 s from snake venom have been determined [34][35][36].
Tsai et al. studied PLA 2 from glands obtained from different samples of C. viridis viridis, arising from several regions of the United States (Figure 1(a)) [37]. They purified and sequenced five acidic PLA 2 s sharing 78% or greater sequence identity. Interestingly, Tsai et al. observed that the product of the cDNA sequence named cvvE6d modified a PLA 2 with a molecular mass of 13782 ± 1 Da. This specific molecule of PLA 2 was found only in a unique snake from Southeastern Arizona. The authors correctly inferred and suggested that these individuals from Southeastern Arizona could actually represent a distinct population of Crotalus viridis viridis [37].
Recently, while studying the differences in total venoms from C. viridis and C. oreganus subspecies, Mackessy verified that all venoms display great variation, both in protein composition as well as in the activities of several enzymes, including the PLA 2 enzyme family [2]. The venom used by Mackssey was obtained from C. viridis and C. oreganus subspecies from the locations shown in Figure 1(b) [2].
According to Mackssey, as the Western Rattlesnake occurs across a broad geographical area, it represents an ideal species group to investigate variations in venom composition, and to understand how these differences evolve and how composition affects the biological role(s) of venom [2]. In this study, to further the understanding of the biological diversity of the subspecies of C. oreganus, we biochemically isolated and characterized a PLA 2 (D49) from C. oreganus abyssus venom. Moreover, we sequenced the primary structure of PLA 2 , performed pharmacological and biochemical characterization assays, and used molecular modeling to analyze the structure obtained.
To help remove any other impurities that might be present, the fraction with PLA 2 activity was again subjected to ultrafiltration using the MidJet apparatus (Ge Healthcare, USA), equipped with the UFP-10-C-MM01A cartridge (superficial area of 26 cm 2 , cut off: 10,000 Da-Ge Healthcare, USA). A PLA 2 named CoaPLA 2 was isolated, and the filtrate was lyophilized and rechromatographed to evaluate its purity, under the same conditions as described above (Figure 2). The fractions were monitored by spectrophotometry at 280 nm. The purity level of the CoaPLA 2 was also evaluated using native polyacrylamide gel (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [ 2.3.2. Phospholipase A 2 Activity. Enzymatic activity was measured by two methods using two different substrates; a nonmicellar (4-nitro-3-octanoyloxy benzoic acid-NOBA) and a micellar substrate (1-hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphoglycerol-HPGP).
(1) Phospholipase 2 Activity Measured Using a Nonmicellar Substrate (4-nitro-3-octanoyloxy benzoic acid-NOBA). The phospholipase A 2 activity of CoaPLA 2 (both in the isolated protein and in total venoms) was measured using the assay described by Holzer and Mackessy [43], but modified for 96well plates [17,[31][32][33][34]. The standard assay mixture contained 200 L of buffer (10 mM Tris-HCl, 10 mM CaCl 2 and 100 mM NaCl, pH 8.0), 20 L of substrate (3 mM 4-nitro-3-octanoyloxy benzoic acid), 20 L of water, and 20 L of PLA 2 (10 mg/mL) in a final volume of 260 L. After adding PLA 2 (or total venom) (20 g), the mixture was incubated for up to 40 min at 37 ∘ C, with the reading absorbance at intervals of 10 min until 60 min. Enzyme activity, expressed as the initial velocity of the reaction ( 0 ), was calculated based on the of absorbance at 20 min. After this time, the velocity did not change (maximum velocity was achieved). Enzyme activity was expressed as mean ± SD of three independent experiments and each experiment was carried out in triplicate.
(2) Phospholipase 2 Activity Measured Using a Micellar Substrate (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3phosphoglycerol-HPGP). The measurements of enzymatic activity using the substrate 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol (HPGP) were carried out using a microtiter plate assay [10,20,44]. One hundred L of solution A in assay buffer (27 M bovine serum albumin, 50 mM KCl, 1 mM CaCl 2 , 50 mM Tris-HCl pH 8.0) were added to a 96-well microtiter plate. Solution B presented the same composition as Solution A but with PLA 2 (0.5 g/mL) or total venom (1.0 g/mL) and was delivered in 100 L portions to four wells, except for the first one. As a control, instead of Solution B, an additional 100 L of Solution A was added to the first of the four wells in the assay. Solution B was prepared immediately prior to each set of assays to avoid loss of enzymatic activity. After the addition of Solution B, the assay was rapidly initiated by the addition of 100 L of Solution C (420 mM 1-hexadecanoyl-2-(1pyrenedecanoyl)-sn-glycero-3-phosphoglycerol vesicles in assay buffer) with a repeating pipette to all four wells. The fluorescence (excitation = 342 nm, emission = 395 nm) was read with a microtiter plate spectrophotometer (Fluorocount, Packard Instruments). Enzyme activity, expressed as the initial velocity of the reaction ( 0 ) was calculated based on the absorbance at 20 min. After this time, the velocity did not change (the maximum velocity was achieved). Enzyme activity was expressed as mean ± SD of three independent experiments and each experiment was carried out in triplicate.

Optimal pH and Temperature
Determination of the Enzymatic Activity. Optimal pH and optimal temperature of the PLA 2 activity (using methodology described in Section 2.3.2(1)) of the CoaPLA 2 were determined by incubating the enzyme in four buffers of different pH values (4)(5)(6)(7)(8)(9)(10) and at different temperatures (25,30,35,40, and 45 ∘ C), respectively, as described above (Section 2.3.2). The effect of substrate concentration (10, 5, 2.5, 1.25, 0.625, and 0.312 M) on enzyme activity was determined by measuring the increase of absorbance after 20 min and absorbance values at 425 nm were measured with a VersaMax 190 multiwell plate reader (Molecular Devices, S., CA). Enzyme activity was expressed as mean ± SD of three independent experiments and each experiment was carried out in triplicate.

Determination of Influence of Ca 2+ (and Other Ions)
on PLA 2 Activity. Three experiments were carried out to determine the influence of calcium ions on CoaPLA 2 activity (using methodology described in Section 2.3.2(1)). The activity was described above (Section 2.3.2). Initially, Ca 2+ concentrations of 0, 1, and 10 mM were used. After this procedure, the other three experiments were carried out: (1) without Ca 2+ , but in the presence of 10 mM of Mg 2+ , Cd 2+ , and Mn 2+ ;  BioMed Research International mean ± SD of three independent experiments and each experiment was carried out in triplicate.

Biological Activity
2.4.1. Animals. Groups of 6 Swiss male mice (6-8 weeks old) were matched for body weight (18-22 g). The animals were housed for at least one week before the experiment in laminar-flow cages maintained at a temperature of 22 ± 2 ∘ C and a relative humidity of 50-60%, under a 12 : 12 h lightdark cycle. The animal experiments were carried out with the approval of the Institutional Ethics Committee, in accordance with protocols following the recommendations of the Canadian Council on Animal Care. The mice used in this study were kept under specific pathogen-free conditions.

50% Lethal Dose.
To evaluate the 50% lethal dose (dose that causes death in 50% of animals) of CoaPLA 2 and the total venoms from C. o. abyssus, C. v. viridis, and C. v. nuntius, groups of six Swiss male mice (18−22 g) received an intravenous injection of 100 g of enzyme or total venom, dissolved in 100 L of PBS. As a control, six mice were similarly injected with 100 L of PBS alone. Animals were observed for up to 24 h after injection to record deaths. Lethal dose (LD) was expressed as mean ± SD of three independent experiments, performed in triplicate ( = 6) ( Figure 3) [17,18,28,29,31,33,34].

Edema-Inducing Activity.
Groups of six Swiss male mice (18-22 g) were injected in the subplantar region with various amounts of total venom or CoaPLA 2 (in a volume of 50 L) prepared in PBS, pH 7.2. Subsequently, paw volume was measured at different time intervals (30, 60, 120, and 180 min), subtracting the initial paw volume (time 0 h). Paw edema was measured with a low-pressure pachymeter (Mitutoyo, Japan). Edema-inducing activity was expressed as mean ± SD of three independent experiments and each experiment was carried out in triplicate ( = 6) ( Figure 4) [17,18,28,29,31,33,34].

Myotoxic Activity.
Groups of six Swiss male mice (18-22 g) were injected in the right gastrocnemius muscle with total venom or PLA 2 (50 mg/50 mL of PBS) or PBS alone (50 mL). After 3 h, blood was collected from the tail in heparinized capillary tubes and centrifuged for plasma separation. Activity of creatine kinase (CK) was then determined using 4 mL of plasma, which was incubated for 3 min at 37 ∘ C with 1.0 mL of the reagent according to the kinetic CK-UV protocol from Bioclin, Brazil. The activity was expressed in U/L, where one unit corresponds to the production of 1 mmol of NADH per minute ( Figure 6). Myotoxic activity was expressed as mean ± SD of three independent experiments and each experiment was carried out in triplicate ( = 6) ( Figure 5)    of 10 mg/mL sinapinic acid in 50% acetonitrile/miliQ water (v/v) and 0.1% trifluoroacetic acid (TFA). To calibrate the apparatus, a BSA standard solution was prepared following the same procedure, and 4 pmols were analyzed under the same conditions.  [50]. Protonation states of charged groups were set according to pH 7.0 and counter ions were added to neutralize the system. Gromos force field [51] was chosen to perform the MD simulation. The MD simulation was performed at constant temperature and pressure in a periodic truncated cubic box with a volume that was equal to 259.14 nm 3 and at a minimum distance of 5Å between any atom of the protein and the box wall. Sodium ions were added as counter ions to neutralize the system. Initially, an energy minimization using the steepest descent algorithm was performed. After, 20 ps of MD simulation with position restraints applied to the protein were performed at 300 K to relax the system. Finally, an unrestrained MD simulation was performed at 300 K during 10 ns to assess the stability of the structures. During the simulation, temperature and pressure (1.0 bar) were maintained by coupling to an external heat and an isotropic pressure bath.

Structural Analysis and Validation.
After the MD simulation, several tools of structural analysis contained in the GROMACS package were employed to evaluate the final 3D model. All figures were generated employing PyMOL 0.99c software [52]. Other validation methods were also used, such as a pseudoenergy profile, which was analyzed with Verify 3D [53,54] and ProSA-web [55,56], as well as the Ramachandran plot [57], ERRAT program [39], and ANOLEA web server [40].

Statistical Analysis.
Results are presented as mean ± SD obtained with the indicated number of animal samples or in vitro assays. The statistical significance of differences between groups was evaluated using the Student's unpaired -test and ANOVA analysis of variance. Significance levels were considered at a confidence interval of 0.1 > > 0.05.

Isolation and Purification of the Phospholipase A 2 from
Crotalus oreganus abyssus (CoaPLA 2 ). The process used to obtain the pure protein (CoaPLA 2 ) is shown in Figure 2. Gel filtration (Figure 2(a)) demonstrated the presence of fraction II containing PLA 2 activity, which was further purified. Figure 2(b) shows the HPLC profile obtained using a reverse phase C18 column and the detachment of the peak containing CoaPLA 2 . This peak was also further purified by rechromatography and subjected to electrophoresis (SDS-PAGE and PAGE). As shown in Figure 2(c), the purification process was efficiently purified. Nondenaturation electrophoresis showed that CoaPLA 2 was a dimeric protein with a molecular mass of approximately 28 kDa (lines 3 and 6), but under denaturing conditions, it was a monomer with a molecular mass of approximately 14 kDa (lines 2 and 5). This information was subsequently confirmed by MALDI-TOF mass spectrometry.

Biochemical Characterization of CoaPLA 2 .
We biochemically analyzed and characterized CoaPLA 2 . Figure 3 shows phospholipase A 2 activity under several conditions. We measured the phospholipase A 2 activity of the isolated enzyme and total venom (using two different methods related to substrate type), as well as the optimal temperature and pH, and the influence of ions on the activity of the enzyme. Interestingly, the enzymatic activity, obtained by Tsai et al., 2003, when cloning E6d was around 680 mol/min/mg and when using the micellar substrate L-dipalmitoyl-glycerophosphatidyl-choline, being relatively close to the value found in the present study. We used a different, but similar, micellar substrate (1-hexadecanoyl-2-(1-pyrenedecanoyl)-snglycero-3-phosphoglycerol) and obtained a value of enzymatic activity of approximately 590 mol/min/mg.
The optimal temperature of CoaPLA 2 was determined to be 37.3 ∘ C (Figure 3(d)) and optimal pH was 7.9 (Figure 3(c)). These values are in accordance with other PLA 2 measurements described in the literature . The influence ions on the enzyme activity was determined in the presence and absence of Ca 2+ and other divalent cations (also in the presence and absence of Ca 2+ ). Figure 3(e) shows that the PLA 2 activity of CoaPLA 2 is calcium-dependent. In the presence of 10 mM calcium, the PLA 2 activity was 45.8  nmols/min/mg. When the calcium concentration was 1 mM calcium, the phospholipase A 2 activity was slightly reduced to 38.1 nmols/min/mg. A complete absence of calcium ions drastically reduced the enzyme activity to values of approximately 3 nmols/min/mg. When 10 mM of other divalent cations (Mg +2 , Cd +2 and Mn +2 ) was employed, the activity of the PLA 2 was completely suppressed. However, phospholipase A 2 activity was recovered when Ca +2 was mixed with these divalent cations (Mg +2 , Cd +2 and Mn +2 ), both at concentrations of 1 mM and 10 mM (Figure 3(e)).

Biological
Characterization of CoaPLA 2 . The biological characterization of CoaPLA 2 , isolated from Crotalus oreganus abyssus, was carried out using measurements of lethal activity (LA 50% -dose that causes death in 50% of animal subjects), edema-inducing and myotoxic activities. We tested the lethal activity (LA 50% ) of CoaPLA 2 and of the total venom of Crotalus oreganus abyssus, Crotalus viridis viridis, and Crotalus viridis nuntius. Figure 4 shows that LA 50% of the venom from Crotalus oreganus abyssus is approximately 2.2 ± 0.4 g of venom/g of mouse and this value is bigger in relation to C. v. viridis and C. v. nuntius. CoaPLA 2 has a LA 50% at a dose of about 1.8 g ± 0.2 of venom/g mouse weight and was higher than that of the total venom of C. v. viridis and C. v. nuntius. The total C. o. abyssus venom is more lethal than CoaPLA 2 alone, as venom contains other enzymes that also exhibit lethality, such as serine proteases and metalloproteases .
The edema-inducing activity of CoaPLA 2 was measured using different dosages of the enzyme (25, 50, and 100 g). From Figure 5, we can see that the edema-inducing activity of CoaPLA 2 is dose-dependent. The increase in the amount of enzyme increases the percentage of edema formed, principally in the first 24 h. After this time, this edema-inducing activity is significantly reduced and the edema is suppressed.
Similarly to the edema-inducing activity results obtained, the myotoxic activity induced by CoaPLA 2 was also dosedependent. When we increased the quantity of CoaPLA 2 (25, 50 and 100 g) its myotoxic effects were augmented.
The phospholipases A 2 are a group of enzymes present in most venoms or oral secretions of snakes. In addition to the digestive function of the prey, these enzymes interfere with the physiological processes and cause many pharmacological and pathophysiological effects, such as neurotoxic, cardiotoxic, anticoagulant, antiplatelet, hemolytic, hemorrhagic, and inflammatory activities [59][60][61].
Both the crude venom of Crotalus oreganus abyssus and the isolated CoaPLA 2 , were able to induce experimental toxicity, such as myonecrosis, edema, and mortality. Due to the neurotoxic potential of this kind of snake, it was observed that the LD 50% of the crude venom of C. oreganus abyssus and its CoaPLA 2 showed low values of lethal doses, when compared to Bothrops genera venoms and its isolated PLA 2 s [36].
The CoaPLA 2 also induced myotoxic activity, similarly to other PLA 2 s isolated from snake venoms. The myotoxicity was evaluated by the activity levels of creatine kinase (CK) in the plasma of animals. Creatine kinase is an enzyme used in muscular energy metabolism and, in cases of cell damage, is released and can be detected in plasma as a marker [36,60]. The catalytic activity of the PLA 2 on the membrane suggests an important role these enzymes in the toxicity of snake venoms (svPLA 2 s). The breakdown of phospholipids causes severe changes in the structural and functional integrity of the plasmatic membrane with a consequent influx of calcium ions [62], release of calcium-dependent proteases [63], activation of endogenous PLA 2 s [64], and mitochondrial collapse [65]. The sum of all these molecular changes could lead to cell death.
The CoaPLA 2 was able to induce edema in mice paws. Local inflammation is a feature of poisoning by snakes of the subfamilies of Viperidae and Crotalidae [60,61]. The catalytically active mechanism by which PLA 2 induces edema is probably due to the release of precursors of eicosanoids due to the hydrolysis of phospholipids. Release of biogenic amines from mast cells is also proposed as a possible mechanism of induction of edema by PLA 2 [66,67].

Structural
Characterization of CoaPLA 2 . The molecular mass of CoaPLA 2 was analyzed by MALDI-TOF mass spectrometry ( Figure 7) and the mass determined was 13.793.8 Da. However, interestingly, Tsai et al. [37] found the cDNA E6d product to present a molecular mass of 13.782 Da. In addition, Tsai et al. [37], based on a phylogenetic analysis, also found that all cDNA products from all specimens studied showed biological relationships among themselves, with the exception of the cDNA E6d product. Authors inferred and suggested that the specimen, initially considered as Crotalus viridis viridis, which produced the E6d cDNA, in reality belonged to a distinct population present in Southwestern Arizona.
To extend our structural study of CoaPLA 2 , we determined its primary structure using LC/MS-MS, after insolution digestion (tryptic digestion). The fragments obtained were analyzed using the Proteome Discoverer  Table 1). The analysis using the ClustalW multiple sequence alignment showed that fragments recovered from the PLA 2 sequence produced by the cDNA E6d and described by Tsai et al. [37] displayed 94% sequential homology (Table 2), except for a unique fragment that was not found (VTDCNPK). From Table 2 we can see that this fragment is extremely conserved in all sequences analyzed and in the model proposed by us ( Figure 10). We have inserted the sequence VTDCNPK in the gap of the fragment as not found, as shown in the E6D sequence. The comparison between the E6D sequence and the CoaPLA 2 sequence obtained is interesting because both are exactly equal, except for the VTDCNPK fragment (not found in this study). Tsai et al., [37] suggested that the specimen, initially considered as Crotalus viridis viridis, may be a distinct population present in Southwestern Arizona, considered the natural habitat of Crotalus oreganus abyssus (Figure 1(b)). Thus, we infer that, probably, the specimen used by Tsai et al., [37] that produced the E6D cDNA was actually a Crotalus oreganus abyssus snake, and not a Crotalus viridis viridis. In addition to this information, and to enforce our conclusion, it should be remembered that the value of the enzymatic activity of CoaPLA 2 found in this work is very near to the value found by Tsai et al., 2003 (around 680 mol/min/mg for the E6d clone and approximately 590 mol/min/mg for CoaPLA 2 ).  This fact supports the need for more studies on Crotalus oreganus (all subspecies) because for many years all subspecies of Crotulus viridis and Crotalus oreganus were treated as a single serpent specimen. However, as subsequent studies have shown, the old classification was incorrect and the "old" Crotalus viridis can in fact be divided into two subspecies of Crotalus viridis (viridis and nuntius) and seven subspecies of Crotalus oreganus (abyssus, lutosus, concolor, oreganus, helleri, Cerberus, and caliginis).

Molecular Modeling.
To increase the understanding of the CoaPLA 2 structure, we conducted molecular modeling studies using Molecular Dynamics (MD) simulation. We calculated the values of root mean squared distance (RMSD) considering the protein backbone atoms, which are displayed in Figure 8. When analyzing these results, we noted that the PLA 2 model was stabilized after approximately 1300 ps of simulation.
From the root mean squared fluctuation (RMSF) values of the alpha carbons per residue, we can see that the fluctuation of PLA 2 residues from 1300 ps to 5000 ps is very low (except for the residues in the terminal loop, 119-133), indicating that there are no significant changes in the conformation of the residues (Figure 9).
Comparing the initial 3D model and the final model obtained after the MD simulation, it can be easily seen that the MD simulation is fundamental to refine the PLA 2 model. The ProSA energy profile indicates that both initial and final models have energy values per residue of lower than 0, indicating a good pseudoenergy profile. Table 3 displays the results obtained from ANOLEA, ERRAT, Verify 3D, ProSA, and Ramachandran analyses for the initial and final models.
The ANOLEA results indicate that the MD simulation decreased the number of high energy residues by 8%. With all high energy residues of the final model being located in the loop region (Figure 9(c)). The ERRAT results indicate that the MD simulation improved the quality of the structural model from 64% to 99%. From the Verify 3D results, the initial model had 17 residues with poor structural quality (score lower than 0) and all residues of the final model had score values of higher than 0. Finally, the Ramachandran plot analysis indicates that both initial and final models had 3 and 2 glycine residues located at an outlier region, respectively. Figure 9(a) shows the Stereoview of the final model. Figure 9(a) shows that our model displays the typical phospholipase conformation, containing three parallel -helixes and a -wing (one double-stranded antiparallel -sheet) [20].
Data reinforce the necessity of rearranging and clarifying all information available regarding the two subspecies of