Diversity of astacin-like metalloproteases identified by transcriptomic analysis in Peruvian Loxosceles laeta spider venom and in vitro activity characterization.

Loxosceles spiders are found in almost all countries of South America. In Peru, Loxosceles laeta species is the main responsible for the accidents caused by poisonous animals, being known as "killer spiders", due to the large number of fatal accidents observed. Astacin-like metalloproteases, named LALPs (Loxosceles astacin-like metalloproteases) are highly expressed in Loxosceles spiders venom gland. These proteases may be involved in hemorrhage and venom spreading, being relevant to the envenoming proccess. Thus, the aim of this work was to analyze Peruvian L. laeta venom gland transcripts using bioinformatics tools, focusing on LALPs. A cDNA library from Peruvian L. laeta venom glands was constructed and sequenced by MiSeq (Illumina) sequencer. After assembly, the resulting sequences were annotated, seeking out for similarity with previously described LALPs. Nine possible LALPs isoforms from Peruvian L. laeta venom were identified and the results were validated by in silico and in vitro experiments. This study contributes to a better understanding of the molecular diversity of Loxosceles venom and provide insights about the action of LALPs.

Ureases as multifunctional toxic proteins: A review.

Ureases are metalloenzymes that hydrolyze urea into ammonia and carbon dioxide. They were the first enzymes to be crystallized and, with them, the notion that enzymes are proteins became accepted. Novel toxic properties of ureases that are independent of their enzyme activity have been discovered in the last three decades. Since our first description of the neurotoxic properties of canatoxin, an isoform of the jack bean urease, which appeared in Toxicon in 1981, about one hundred articles have been published on "new" properties of plant and microbial ureases. Here we review the present knowledge on the non-enzymatic properties of ureases. Plant ureases and microbial ureases are fungitoxic to filamentous fungi and yeasts by a mechanism involving fungal membrane permeabilization. Plant and at least some bacterial ureases have potent insecticidal effects. This entomotoxicity relies partly on an internal peptide released upon proteolysis of ingested urease by insect digestive enzymes. The intact protein and its derived peptide(s) are neurotoxic to insects and affect a number of other physiological functions, such as diuresis, muscle contraction and immunity. In mammal models some ureases are acutely neurotoxic upon injection, at least partially by enzyme-independent effects. For a long time bacterial ureases have been recognized as important virulence factors of diseases by urease-producing microorganisms. Ureases activate exocytosis in different mammalian cells recruiting eicosanoids and Ca(2+)-dependent pathways, even when their ureolytic activity is blocked by an irreversible inhibitor. Ureases are chemotactic factors recognized by neutrophils (and some bacteria), activating them and also platelets into a pro-inflammatory "status". Secretion-induction by ureases may play a role in fungal and bacterial diseases in humans and other animals. The now recognized "moonlighting" properties of these proteins have renewed interest in ureases for their biotechnological potential to improve plant defense against pests and as potential targets to ameliorate diseases due to pathogenic urease-producing microorganisms.

Synergism between basic Asp49 and Lys49 phospholipase A2 myotoxins of viperid snake venom in vitro and in vivo.

Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, 'Asp49' and 'Lys49' myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp(92) and Asp(96) as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS.

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

Structural analysis of trimeric phospholipase A2 neurotoxin from the Australian taipan snake venom.

Snake pre-synaptic neurotoxins endowed with phospholipase A(2) activity are potent inducers of paralysis through the specific disruption of the neuromuscular junction pre-synaptic membrane and represent a valuable tool for investigating neuronal degeneration and recovery. They have different structural complexity and a wide range of lethal potency and enzymatic activity, although they share a similar mechanism of action. Although no correlation has been reported between neurotoxicity and enzymatic activity, toxicity increases with structural complexity and phospholipase A(2) oligomers show 10-fold lower LD(50) values compared to their monomeric counterparts. To date, no structural study has been performed on multimeric SPANs with the aim of shedding light on the correlation between structural complexity and neurotoxicity. In the present study, we investigated the structure of taipoxin, a trimeric phospholipase A(2) neurotoxin, as well as that of its subunits, by X-ray crystallography and small angle X-ray scattering analysis. We present the high-resolution structure of two isoforms of the taipoxin ß subunit, which show no neurotoxic activity but enhance the activity of the other subunits in the complex. One isoform shows no structural change that could justify the lack of activity. The other displays three point mutations in critical positions for the catalytic activity. Moreover, we designed a model for the quaternary structure of taipoxin under physiological conditions, in which the three subunits are organized into a flat holotoxin with the substrate binding sockets exposed on the same side of the complex, which suggests a role for this interface in the toxin-membrane interaction.

Chromatography, mass spectrometry, and molecular modeling studies on ammodytoxins.

The ammodytoxins (Atxs) are neurotoxic phospholipases which occur in Vipera ammodytes ammodytes (Vaa) snake venom. There are three Atx isoforms, A, B, and C, which differ in only five amino acid positions at the C-terminus but differ substantially in their toxicity. The objective of this study was to establish an analytical method for unambiguous identification of all three isoforms and to use the method to assess a procedure for purification of the most toxic phospholipase, AtxA, from the venom. Isolation procedure for AtxA consisted of isolation of Atx-cross-reactive material (proteins recognized by anti-Atx antibodies), by use of an affinity column, then cation exchange on CIM (Convective Interaction Media) disks. The purification procedure was monitored by means of reversed-phase chromatography (RPC) and mass spectrometry (MS). Although previous cation exchange of the pure isoforms enabled separate elution of AtxA from B and C, separation of AtxA from Atxs mixture was not accomplished. RPC was not able to separate the Atx isoforms, whereas an MS based approach proved to be more powerful. Peptides resulting from tryptic digestion of Atxs which enable differentiation between the three isoforms were successfully detected and their sequences were confirmed by post-source decay (PSD) fragmentation. Separation of Atx isoforms by ion-exchange chromatography is most presumably prevented by Atxs heterodimer formation. The tendency of Atxs to form homodimers and heterodimers of similar stability was confirmed by molecular modeling.

Hypersensitivity to mGluR5 and ERK1/2 leads to excessive protein synthesis in the hippocampus of a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is caused by loss of the FMR1 gene product FMRP (fragile X mental retardation protein), a repressor of mRNA translation. According to the metabotropic glutamate receptor (mGluR) theory of FXS, excessive protein synthesis downstream of mGluR5 activation causes the synaptic pathophysiology that underlies multiple aspects of FXS. Here, we use an in vitro assay of protein synthesis in the hippocampus of male Fmr1 knock-out (KO) mice to explore the molecular mechanisms involved in this core biochemical phenotype under conditions where aberrant synaptic physiology has been observed. We find that elevated basal protein synthesis in Fmr1 KO mice is selectively reduced to wild-type levels by acute inhibition of mGluR5 or ERK1/2, but not by inhibition of mTOR (mammalian target of rapamycin). The mGluR5-ERK1/2 pathway is not constitutively overactive in the Fmr1 KO, however, suggesting that mRNA translation is hypersensitive to basal ERK1/2 activation in the absence of FMRP. We find that hypersensitivity to ERK1/2 pathway activation also contributes to audiogenic seizure susceptibility in the Fmr1 KO. These results suggest that the ERK1/2 pathway, and other neurotransmitter systems that stimulate protein synthesis via ERK1/2, represent additional therapeutic targets for FXS.

Neurotoxic, myotoxic and cytolytic activities of the new basic PLA(2) isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (Marajó Lancehead) snake venom.

The BmjeTX-I and BmjeTX-II isoforms of PLA(2) were purified from Bothrops marajoensis venom by ion-exchange chromatography and reverse phase HPLC. Both isoforms showed a molecular mass of 13808.89 Da (BmjeTX-I) and 13863.97 Da (BmjeTX-II) determined by based on the determined primary structures and SDS-PAGE and confirmed experimentally by MALDI-TOF mass spectrometry. Multiple alignment of BmjeTX-I and BmjeTX-II isoforms of PLA(2) show high degree of homology with basic PLA(2) myotoxins from other Bothrops venoms. Ex vivo, both isoforms caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In chick preparations, contractures to exogenous acetylcholine (55 and 110 microM) or KCl (13.4 mM) were unaltered after complete blockade for the both isoforms BmjeTX-I and BmjeTX-II of PLA(2). These results, which strongly suggested a presynaptic mechanism of action for these toxins. In mice, both isoforms induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Both isoforms BmjeTX-I and BmjeTX-II of PLA(2) also induced moderate marked paw edema, evidencing the local increase in vascular permeability. Since both isoforms of PLA(2) exert a strong proinflammatory effect, the enzymatic hydrolysis of phospholipids might be relevant for this phenomenon and produced cytotoxicity in murine skeletal muscle C2C12 myoblasts and myotubes.

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: biochemical and toxicological characterization.

Phospholipases A(2) (PLA(2)) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA(2)s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA(2)-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA(2)-II is monomeric, with a mass of 14,212 +/- 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA(2)-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA(2)-II also differed from other acidic PLA(2)s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 microg (5.9 microg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA(2)-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA(2)-II revealed that acidic and basic PLA(2)s form two different antigenic groups in B. asper venom.

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes.

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.

Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa).

Bothrops snake venoms contain a variety of phospholipases (PLA(2)), some of which are myotoxic. In this work, we used reverse-phase HPLC and mass spectrometry to purify and sequence two PLA(2) from the venom of Bothrops insularis. The two enzymes, designated here as BinTX-I and BinTx-II, were acidic (pI 5.05 and 4.49) Asp49 PLA(2), with molecular masses of 13,975 and 13,788, respectively. The amino acid sequence and molecular mass of BinTX-I were identical to those of a PLA(2) previously isolated from this venom (PA2_BOTIN, SwissProt accession number ) while those of BinTX-II indicated that this was a new enzyme. Multiple sequence alignments with other Bothrops PLA(2) showed that the amino acids His48, Asp49, Tyr52 and Asp99, which are important for enzymatic activity, were fully conserved, as were the 14 cysteine residues involved in disulfide bond formation, in addition to various other residues. A phylogenetic analysis showed that BinTX-I and BinTX-II grouped with other acidic Asp49 PLA(2) from Bothrops venoms, and computer modeling indicated that these enzymes had the characteristic structure of bothropic PLA(2) that consisted of three alpha-helices, a beta-wing, a short helix and a calcium-binding loop. BinTX-I (30 microg/paw) produced mouse hind paw edema that was maximal after 1h compared to after 3h with venom (10 and 100 microg/paw); in both cases, the edema decreased after 6h. BinTX-1 and venom (40 microg/ml each) produced time-dependent neuromuscular blockade in chick biventer cervicis preparations that reached 40% and 95%, respectively, after 120 min. BinTX-I also produced muscle fiber damage and an elevation in CK, as also seen with venom. These results indicate that BinTX-I contributes to the neuromuscular activity and tissue damage caused by B. insularis venom in vitro and in vivo.

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.

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.

In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA(2) D49 purified from the Bothrops jararacussu venom. The isoforms 6-1 and 6-2 had a sequence of amino acids of 121 amino acid residues 6-1: DLFEWGQMIL KETGKNPFPY YGAYGCYCGW GGRGKPKDKD TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C and pI value 7.83 and 6-2: DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C with a pI value of 7.99. Skeletal muscle preparations from the young chicken have been used previously in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. Both isoforms have produced neuromuscular blockade in young chicken biventer cervicis nerve-muscle preparations in presence or absence of crotapotin crotalic (F3 and F4) indicating that catalytic activity was not essential for neuromuscular action in this preparation.

Prostanoids, not reactive oxygen species, mediate COX-2-dependent neurotoxicity.

The prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2) has emerged as a critical pathogenic factor in brain diseases associated with activation of N-methyl-D-aspartate (NMDA) receptors, including stroke and neurodegenerative diseases. However, the COX-2 reaction products responsible for these deleterious effects have not been identified. In particular, the relative contribution to the neurotoxicity of COX-2-derived prostanoids and reactive oxygen species has not been defined. We found that the brain damage produced by direct injection of NMDA into the somatosensory cortex is attenuated by the COX-2 inhibitor NS-398 or in COX-2-null mice, but that the associated production of free radicals is not. Furthermore, COX-2 inhibition reduces the lesions even if the deleterious effects of free radicals are eliminated by the scavenger superoxide dismutase. The protection exerted by NS-398 is counteracted by a stable analog of prostaglandin E2. The findings directly implicate COX-2-derived prostanoids, rather then radicals, in the COX-2-dependent component of the damage mediated by NMDA receptors and strengthen the rationale for using COX-2 inhibitors in the treatment of neurological diseases associated with glutamate neurotoxicity.

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.

Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic.

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v-ras-transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from -7 for RNase Sa to -1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are potential targets for ribonuclease-based drugs.

Phospholipases A2 from Callosellasma rhodostoma venom gland cloning and sequencing of 10 of the cDNAs, three-dimensional modelling and chemical modification of the major isozyme.

Callosellasma rhodostoma (Malayan pitviper) is a monotypic Asian pitviper of medical importance. Three acidic phospholipases A2 (PLA2s) and one basic PLA2-homolog were purified from its venom while 10 cDNAs encoding distinct PLA2s were cloned from venom glands of a Thailand specimen of this species. Complete amino-acid sequences of the purified PLA2s were successfully deduced from their cDNA sequences. Among the six un-translated PLA2 cDNAs, two apparently result from recombination of its Lys49-PLA2 gene with its Asp49-PLA2 genes. The acidic PLA2s inhibit platelet-aggregation, while the noncatalytic PLA2-homolog induces local edema. This basic PLA2-homolog contains both Asp49 and other, unusual substitutions unique for the venom Lys49-PLA2 subtype (e.g. Leu5, Trp6, Asn28 and Arg34). Three-dimensional modelling of the basic protein revealed a heparin-binding region, and an abnormal calcium-binding pocket, which may explain its low catalytic activity. Oxidation of up to six of its Met residues or coinjection with heparin reduced its edema-inducing activity but methylation of its active site His48 did not. The distinct Arg/Lys-rich and Met-rich region at positions 10-36 of the PLA2 homolog presumably are involved in its heparin-binding and the cell membrane-interference leading to edema and myotoxicity.

Purification and characterization of the venom phospholipases A2 from Asian monotypic crotalinae snakes.

Phospholipases A2 were purified from the venoms of Asian monotypic crotalinae snakes including Callosellasma, Hypnale, Deinagkistrodon, and Tropidolaemus by a combination of gel filtration and reversed-phase chromatographic methods. One to four isoforms of the enzyme were found in each of the venoms. The venom enzymes were subjected to N-terminal sequencing up to the 30th amino acids, and their molecular weights were analyzed by electrospray-ionization mass spectrometry. Homologous antiplatelet phospholipase with a conserved Glu 6 residue was found in each of the venoms. Basic phospholipases with Trp 6 (W6) but without detectable enzyme activities were also isolated from the venom of C. rhodostoma, H. hypnale, and T. wagleri. These W6 enzymes showed strong heparin-binding affinity and capable of inducing edema in rat paws. The fact that the venoms of Callosellasma and Hypnale contain similar types of phospholipases is in accord with recent reports that these two taxa formed a clade. Deinagkistrodon venom does not contain phospholipase variants other than the Glu-6 subtype as Trimeresurus, Agkistrodon, and Protobothrops venoms do. Interestingly, the Glu-6 enzyme from T. wagleri venom has a molecular weight of 15,319 Daltons, higher than those of most other venom phospholipases. Our results show that new types of the enzyme are more likely to be found in the venom of monotypic species; the amino acid sequence data or the subtypes of venom-phospholipases are potentially useful as markers or a character system for studying higher-order systematics of venomous snakes.

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A2.

Two phospholipases A2 (PLA2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA2s show close sequence homologies to those of PLA2s from other Elapid snakes (60-84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60-65 degrees C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA2 (H1) shows higher toxicity to albino rats (LD50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.