Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

doi:10.1016/j.bbrc.2006.02.071

Biochemical and Biophysical Research Communications

Volume 342, Issue 4, 21 April 2006, Pages 1027-1033

https://doi.org/10.1016/j.bbrc.2006.02.071 Get rights and content

Abstract

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A₂ (PLA₂) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A₂ activity of crude extract was inhibited by both a PLA₂-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA₂ was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15 kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA₂ obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A₂. This enzyme was stable at 4 °C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA₂ induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA₂ purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.

Section snippets

Materials and methods

Crude extract. Lonomia obliqua caterpillars were anesthetized with CO₂ and bristles were removed and maintained on ice. Phosphate-buffered saline (PBS), pH 7.4, at 4 °C was added to achieve a 10% final extract solution. Bristles were homogenized by shaking and then centrifuged to obtain a suspension [21] which was stored at −70 °C. Protein concentration of bristle extracts was determined colorimetrically [22], using bovine serum albumin as a standard.

PLA₂activity. PLA₂ activity was determined

Results

A dose–response curve was observed for PLA₂ activity in crude L. obliqua bristle extract, using soybean lecithin as substrate (Fig. 1A). This activity was augmented prominently by increasing CaCl₂ concentration in medium (Fig. 1B). PLA₂ activity in crude L. obliqua caterpillar bristle extract was inhibited 72% by 0.1 mM pBpb, 40% by 0.2 mM Na₂–EDTA, and 67% by 2.0 mM Na₂–EDTA (Fig. 2).

PLA₂ was purified by three chromatographic steps (Fig. 3). PLA₂ activity was detected in the first peak obtained

Discussion

Secreted PLA₂s are important for digestion and immobilization of prey, and these enzymes are responsible for some of the physiological disturbances observed in humans following bee, wasp, spider, and snake envenomations [14], [16], [30], [31], [32]. PLA₂ activity has been reported in the crude Euproctis caterpillar bristle extract [33] and, more recently, in the crude caterpillar bristle extract of L. obliqua[12]. In the present study, a secreted PLA₂ was purified to homogeneity from the crude

Acknowledgments

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP, Brazil (Proc. 00/11432-9 and 01/07643-7).

References (44)

A.C. Duarte et al.
Intracerebral haemorrhage after contact with Lonomia caterpillars
Lancet
(1996)
C.S. Seibert et al.
Intravascular hemolysis induced by Lonomia obliqua caterpillar bristle extract: an experimental model of envenomation in rats
Toxicon
(2004)
C.V. Reis et al.
A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (LOPAP) biochemical characterization
Tromb. Res.
(2001)
C.S. Seibert et al.
In vitro hemolytic activity of Lonomia obliqua caterpillar bristle extract on human and Wistar rat erythrocytes
Toxicon
(2003)
S. Chakraborti
Phospholipase A₂ isoforms: a perspective
Cell. Signal.
(2003)
E.A. Dennis
Diversity of group types, regulation, function of phospholipase A₂
J. Biol. Chem.
(1994)
E.A. Dennis
The growing phospholipase A₂ superfamily of signal transduction enzymes
TIBS
(1997)
S.M. Serrano et al.
A novel phospholipase A₂, BJ-PLA₂, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor
Arch. Biochem. Biophys.
(1999)
A. Fuly et al.
Purification and characterization of a phospholipase A₂ isoenzyme isolated from Lachesis muta snake venom
J. Biochem. Pharm.
(2002)
R. Doley et al.
Purification and characterization of an anticoagulant phospholipase A₂ from Indian monocled cobra (Naja kaouthia) venom
Toxicon
(2003)

M.H. Toyama et al.

Structural, enzymatic and biological properties of new PLA₂ isoform from Crotalus durissus terrificus venom

Toxicon

(2003)

M.L. Santoro et al.

Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake (Crotalus durissus terrificus, C. durissus cascavella and C. durissus collilineatus)

Comp. Biochem. Physiol. C

(1999)

O.H. Lowry et al.

Protein measurement with the folin phenol reagent

J. Biol. Chem.

(1951)

H. Schägger et al.

Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

Anal. Biochem.

(1987)

A.B. Veiga et al.

A catalog for the transcripts from the venomous structures of the caterpillar Lonomia obliqua: identification of the proteins potentially involved in the coagulation disorder and hemorrhagic syndrome

Gene

(2005)

J.A. Tischfield

A reassessment of the low molecular weight phospholipase A₂ gene family in mammals

J. Biol. Chem.

(1997)

A.M. Soares et al.

Chemical modifications of phospholipases A₂ from snake venoms: effects on catalytic and pharmacological properties

Toxicon

(2003)

H. Costa et al.

Agelotoxin: a phospholipase A₂ from the venom of neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae)

Toxicon

(2000)

L.C. Bastos et al.

Nociceptive and edematogenic responses elicited by crude bristle extract of Lonomia obliqua caterpillars

Toxicon

(2004)

M. Zannin et al.

Blood coagulation and fibrinolytic factors in 105 patients with hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillar in Santa Catarina, southern Brazil

Thromb. Haemost.

(2003)

E.A. Burdmann et al.

Severe acute renal failure induced by the venom of Lonomia caterpillars

Clin. Nephrol.

(1996)

A.C. Duarte et al.

Insuficiência renal aguda por acidentes com lagartas

J. Bras. Nefrol.

(1990)

Cited by (25)

Urine proteomic analysis reveals alterations in heme/hemoglobin and aminopeptidase metabolism during Lonomia obliqua venom-induced acute kidney injury
2021, Toxicology Letters
Citation Excerpt :
As mentioned above, an important mechanism that may underlie venom-induced kidney effects is hemoglobin and heme release. Hematuria is a common clinical feature observed in envenomed victims (Malaque et al., 2006), and L. obliqua venom has a phospholipase A2 toxin that is able to specifically target erythrocyte membrane-releasing free hemoglobin (Seibert et al., 2006). In experimental models, there has been a drop in red blood cell counts between 2 h and 6 h after venom injection, a corresponding increase in plasma hemoglobin levels at the same time, and an increase in plasma haptoglobin and reticulocyte numbers between 24 h and 48 h after envenomation (Seibert et al., 2004; Berger et al., 2013, 2019).
Accidental contact with the Lonomia obliqua caterpillar is a common event in southern Brazil. Envenomed victims present consumption coagulopathy, which can evolve to acute kidney injury (AKI). In the present study, we searched for AKI biomarkers and changes in molecular pathway signatures through urine proteomic analysis.
Male Wistar rats were injected with L. obliqua venom (1.5 mg/kg, via s.c.) or 0.9 % NaCl and distributed into metabolic cages. After 24 h, urine was obtained, and the set of differentially regulated proteins was analyzed by MudPIT technology in an OrbiTRAP mass spectrometer.
L. obliqua venom leads to an increase in urine output and water and electrolyte excretion and to an increase in the albumin to creatine ratio in urine. The proteomic analysis revealed an up-regulation of tubular injury biomarkers, such as neutrophil-gelatinase associated lipocalin (NGAL) and cystatin C, in urine from envenomed rats. Several components related to the heme scavenging system were up-regulated or exclusively identified in urine from envenomed animals. There was an increase in urinary heme levels and hemoglobin subunits, hemopexin, haptoglobin, and biliverdin reductase. Similarly, kinin- and angiotensin-generating/degrading peptidases, such as kallikreins, neprilysin, plasmin, dipeptidyl peptidase IV, cathepsin D, kininogen, and neutral, basic, glutamyl, and acidic aminopeptidases, were also up-regulated in urine.
L. obliqua envenomation induced tubular and glomerular injury, probably involving heme/hemoglobin toxicity and an imbalance in the kinin/angiotensin generating/degrading system.
Arthropod venoms: Biochemistry, ecology and evolution
2019, Toxicon
Comprising of over a million described species of highly diverse invertebrates, Arthropoda is amongst the most successful animal lineages to have colonized aerial, terrestrial, and aquatic domains. Venom, one of the many fascinating traits to have evolved in various members of this phylum, has underpinned their adaptation to diverse habitats. Over millions of years of evolution, arthropods have evolved ingenious ways of delivering venom in their targets for self-defence and predation. The morphological diversity of venom delivery apparatus in arthropods is astounding, and includes extensively modified pedipalps, tail (telson), mouth parts (hypostome), fangs, appendages (maxillulae), proboscis, ovipositor (stinger), and hair (urticating bristles). Recent investigations have also unravelled an astonishing venom biocomplexity with molecular scaffolds being recruited from a multitude of protein families. Venoms are a remarkable bioresource for discovering lead compounds in targeted therapeutics. Several components with prospective applications in the development of advanced lifesaving drugs and environment friendly bio-insecticides have been discovered from arthropod venoms. Despite these fascinating features, the composition, bioactivity, and molecular evolution of venom in several arthropod lineages remains largely understudied. This review highlights the prevalence of venom, its mode of toxic action, and the evolutionary dynamics of venom in Arthropoda, the most speciose phylum in the animal kingdom.
Cloning, purification and characterization of nigrelysin, a novel actinoporin from the sea anemone Anthopleura nigrescens
2019, Biochimie
Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family.
Improved purification and enzymatic properties of a mixture of Sticholysin i and II: Isotoxins with hemolytic and phospholipase A<inf>2</inf> activities from the sea anemone Stichodactyla helianthus
2014, Protein Expression and Purification
Citation Excerpt :
The hydrolysis of phospholipids takes place in the monolayer, but without a suitable method to remove free fatty acids from the monolayer to the subphase (e.g. by means of soluble ciclodextrins), the characteristic decrease in the surface pressure indicating phospholipid hydrolysis, cannot be registered [84]. The optimum pH = 8.0 for phospholipase activity of the StI–StII mixture (Fig. 8) is in agreement with the values reported for other secreted phospholipases, generally located in the 7–9 range [15,19,21,22,25,44,71,85]. Interestingly, the permeabilizing ability of StI in lipid vesicles shows an optimum pH in the range 8–9 and a marked decrease at pH 10 and 11, probably due to the effect of pH on protein structure [29].
Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10⁻³ U/mg of protein). In this work, a mixture StI–StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A₂ (PLA₂) activity of StI–StII was relatively high (1.85 U/mg) and dependent of Ca²⁺. The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40 mM Ca²⁺ at pH 8.0. This Ca²⁺ concentration did not exert any effect on binding of StI–StII with soybean PC monolayers. Then, PLA₂ activity seems not be required to binding to membranes.
Acute Lonomia obliqua caterpillar envenomation-induced physiopathological alterations in rats: Evidence of new toxic venom activities and the efficacy of serum therapy to counteract systemic tissue damage
2013, Toxicon
Citation Excerpt :
The case report of hemolysis-related AKI also indicated that treatment with ALS did not reduce hemoglobin levels to normal values and the patient did not completely recover renal function until 1 month after the accident, despite improvements in coagulation tests (Malaque et al., 2006). Until now, the main component with high in vitro hemolytic activity isolated from this venom was the phospholipase A2 enzyme, although the presence of proteolytic enzymes that act specifically on the membrane glycoproteins of erythrocytes cannot be ruled out (Seibert et al., 2006, 2010). Since myotoxins are commonly described in several snake, spider and bee venoms, the presence of myotoxic activity in L. obliqua was investigated using specific biochemical markers, in vitro experiments and histological analyses.
The clinical manifestations of Lonomia obliqua caterpillar envenomation are systemic hemorrhage and acute kidney injury. In an effort to better understand the physiopathological mechanisms of envenomation, a rat model was established to study systemic tissue damage during L. obliqua envenomation. An array of acute venom effects was characterized, including biochemical, hematological, histopathological, myotoxic and genotoxic alterations. Rapid increases in serum alanine and aspartate transaminases, γ-glutamyl transferase, lactate dehydrogenase, hemoglobin, bilirubin, creatinine, urea and uric acid were observed, indicating that intravascular hemolysis and liver and kidney damage had occurred. Treatment with a specific antivenom (antilonomic serum) for up to 2 h post-venom injection neutralized the biochemical alterations. However, treatment after 6 h post-venom injection failed to normalize all biochemical parameters, despite its efficacy in reversing coagulation dysfunction. The hematological findings were consistent with hemolytic anemia and neutrophilic leukocytosis. The histopathological alterations were mainly related to hemorrhage and inflammation in the subcutaneous tissue, lung, heart and kidneys. Signs of congestion and hemosiderosis were evident in the spleen, and hemoglobin and/or myoglobin casts were also detected in the renal tubules. Increased levels of creatine kinase and creatine kinase-MB were correlated with the myocardial necrosis observed in vivo and confirmed the myotoxicity detected in vitro in isolated extensor digitorum longus muscles. Significant DNA damage was observed in the kidneys, heart, lung, liver and lymphocytes. The majority of the DNA lesions in the kidney were due to oxidative damage. The results presented here will aid in understanding the pathology underlying Lonomia's envenomation.
Comparison of venom composition and biological activities of the subspecies Crotalus lepidus lepidus, Crotalus lepidus klauberi and Crotalus lepidus morulus from Mexico
2013, Toxicon
The rock rattlesnakes Crotalus lepidus comprise a group (lepidus, klauberi, morulus and maculosus) of poorly known mountain cold-tolerant snakes in Mexico. In particular, Crotalus lepidus morulus is a snake endemic of the northeast of Mexico, whereas Crotalus lepidus klauberi and C. l. lepidus are distributed in some regions of the north and central Mexico and southern U. S. Until now very little data are available from C. lepidus subspecies from Mexico, as the terrain inhabited by these snakes is generally steep and rugged. In this work, we have determined some biochemical and biological properties of C. l. morulus, C. l. klauberi and C. l. lepidus crude venoms. Some minor differences in venoms were noted in SDS-PAGE, HPLC profile and MALDI-TOF mass spectrometry analysis. Partial sequences of metalloproteinases, phospholipases A₂ (PLA₂) and galactose-specific lectins were identified in the venoms. Venoms of C. l. klauberi and C. l. lepidus had significantly higher hemorrhagic and lethal activities than C. l. morulus venom. Proteolytic activity in azocasein was higher in C. l. morulus venom, whereas gelatin hydrolysis was higher in C. l. klauberi. Fibrinogenolytic and PLA₂ activities were very similar in all venoms tested. The histological observations in the gastrocnemius muscle damaged by venoms from all the subspecies confirmed myonecrotic and hemorrhagic activities (at 3 and 24 h), which resulted in a poor regenerative response after 14 days. However, C. l. lepidus and C. l. klauberi venom induced a higher increase in the plasma activity of creatine kinase (CK), evidencing higher myotoxicity, whereas paw edema-inducing activity was higher in C. l. lepidus venom. The results indicate that the venoms from the three subspecies have similar protein profiles in electrophoresis, HPLC and molecular weight determinations. However, differences were found in the biological activities in mice. Notably, the venoms of C. l. lepidus and C. l. klauberi present higher toxicity (lower LD₅₀) and hemorrhagic activity than C. l. morulus venom.

View all citing articles on Scopus

View full text

Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Lancet

Toxicon

Tromb. Res.

Toxicon

Cell. Signal.

J. Biol. Chem.

TIBS

Arch. Biochem. Biophys.

J. Biochem. Pharm.

Toxicon

Toxicon

Comp. Biochem. Physiol. C

J. Biol. Chem.

Anal. Biochem.

Gene

J. Biol. Chem.

Toxicon

Toxicon

Toxicon

Blood coagulation and fibrinolytic factors in 105 patients with hemorrhagic syndrome caused by accidental contact with Lonomia obliqua caterpillar in Santa Catarina, southern Brazil

Thromb. Haemost.

Severe acute renal failure induced by the venom of Lonomia caterpillars

Clin. Nephrol.

Insuficiência renal aguda por acidentes com lagartas

J. Bras. Nefrol.

Purification of a phospholipase A₂ from Lonomia obliqua caterpillar bristle extract