Trimeresurus Flavoviridis Research Articles

Response of the infrared receptors of a crotaline snake to ethanol

The pit organs of crotaline snakes can sense infrared (IR). The pit membrane has a finer, flatter, more convoluted vasculature than other sensory organs. Using extracellular recording from IR-sensitive trigeminal ganglion (TG) neurons (primary neurons) and tectal (OT) neurons of the crotaline snake Trimeresurus flavoviridis, we examined the IR response to ethanol (EtOH) in vivo. The response to EtOH was recorded in the TG and OT 20–80 s after 10% EtOH in Ringer's solution (100 μl/ 500 g body weight) was injected via the heart. The responses to EtOH and those to lower or higher temperature stimulation were additive. At a constant temperature (25 °C), EtOH significantly potentiated the IR-triggered discharges of IR-sensory pathways in this snake. These results suggest that the IR response to EtOH is due to either its vasodilatory effect on the abundant vasculature of the pit membrane or its chemical effect on temperature-sensitive receptors.

Degeneration of infrared receptor terminals of snakes caused by capsaicin

Capsaicin, the main pungent ingredient in hot peppers (genus Capsicum), caused degeneration of the infrared receptor terminals in infrared sensitive snakes, Trimeresurus flavoviridis, when it was applied perineurally to a branch of the trigeminal nerve. The degeneration of the terminals was found 6 h after the application. This finding suggests that capsaicin stimulates this infrared receptor terminal, a kind of warm receptor terminal.

Flavoxobin, a serine protease from Trimeresurus flavoviridis (habu snake) venom, independently cleaves Arg726-Ser727 of human C3 and acts as a novel, heterologous C3 convertase.

We have recently shown that crude Trimeresurus flavoviridis (habu snake) venom has a strong capability for activating the human alternative complement system. To identify the active component, the crude venom was fractionated and purified by serial chromatography using Sephadex G-100, CM-cellulose C-52, diethylaminoethyl-Toyopearl 650M, and Butyl-Toyopearl, and the active fractions were evaluated by the C3a-releasing and soluble membrane attack complex-forming activities. Two peak fractions with the highest activities were detected after gel filtration and ion exchange chromatography, and the first fraction was purified to homogeneity. The homogeneous protein was examined for its N-terminal amino acid sequence by Edman degradation. The determined sequence of 25 amino acids completely coincided with that of a previously reported serine protease with coagulant activity, flavoxobin, purified from the same snake venom. To elucidate the molecular mechanism of the complement activation, the reactive products of the mixture of the purified human C3 and flavoxobin were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The digesting pattern revealed that flavoxobin cleaves the alpha chain of the C3 molecule into two fragments. The N-terminal amino acid sequences for the remnant fragments of C3 disclosed that flavoxobin severs the human C3 at the Arg726-Ser727 site to form C3b and C3a the way C3bBb, the human alternative C3 convertase, does. In conclusion, flavoxobin acts as a novel, heterologous C3 convertase that independently cleaves human C3 and kick-starts the complement cascade.

Exotic snakes in São Paulo City, southeastern Brazil: why xenophobia?

Introduced exotic species cause environmental changes and threat public health in target sites. Illegal trade has enhanced this problem. To first report these risks in Brazil, exotic snakes found in Sao Paulo City (SPC) (23 ◦ 32 � S, 46 ◦ 38 � W), southeastern Brazil, and sent to Instituto Butantan between 1995 and 2000, were listed and characterized by their biological attributes. Seventy-six individuals of sixteen alien species were collected. Euriecians snakes, mainly booids, were predominant. Using multivariate tech- niques, their ecological niches were compared to those of 26 native species, as a way to point out the resource's availability. To evaluate the potential of successful implantation, two species absent in SPC and considered as problem snakes are included in these analyses: the brown treesnake Boiga irregularis and the habu Trimeresurus flavoviridis. There were niche similarities between these pest snakes, exotic booids and native viperids largely due to the similarities in the chosen prey (mammals), diel activity (nocturnal), color pattern (variegated) and body size (medium to large). To avoid predictable undesirable effects of implanted pest snakes, traffic control and punishment should be improved, as well as parallel environmental education programs.

Immunohistochemical analysis of neuronal nitric oxide synthase in the trigeminal ganglia of the crotaline snake Trimeresurus flavoviridis

The expression of neuronal nitric oxide synthase (nNOS) in the trigeminal ganglia (TG) of the infrared-sensitive crotaline snake Trimeresurus flavoviridis was studied immunohistochemically. The percentage of nNOS-positive (+) neurons in the TG was significantly higher (about 3.5-fold, P<0.001) in the mandibular division than in the infrared-sensory processing area (the maxillary division and ophthalmic ganglion). nNOS was found in varying sizes of TG neurons. However, nNOS (+) neurons were more abundant in small and large neurons than in medium-sized neurons, which include most of the infrared-sensitive neurons of the TG. These findings suggest that nNOS may be involved in normal physiological functions, such as the transmission of tactile, vibrotactile, and nociceptive sensations in the TG, rather than in infrared sensory processing in this species.

Trimeresurus flavoviridis (habu snake) venom induces human erythrocyte lysis through enzymatic lipolysis, complement activation and decreased membrane expression of CD55 and CD59.

Trimeresurus flavoviridis (habu snake) bites can be fatal to man because of its virulent venom, which is clinicopathologically classified as haemorrhagic, necrotic, and haemolytic toxins. Trimeresurus flavoviridis venom causes lysis of human erythrocytes in conditions where plasma is present as well as in plasma-free conditions in a dose-dependent manner. The haemolytic process requires Ca2+ and Mg2+ ions in the solution. Additionally, the venom initiates activation of the human complement cascade as evidenced by C3a and C5a releases, complement consumption indicated by CH50 and formation of soluble membrane attack complex. The insertion of membrane attack complex into the erythrocyte membranes is morphologically identified by electronmicroscopy. Immunofluorescence analysis reveals that incubation of erythrocytes with the venom decreased cell-surface expression of CD55 (decay accelerating factor) and CD59 (protectin), which renders erythrocyte more vulnerable to adherent C3 and C5 convertases and to polymerization of C9 into membranes, and may enhance autologous complement-mediated haemolysis triggered by the venom. Our data demonstrate that Trimeresurus flavoviridis venom induces haemolysis in the presence of plasma by three distinct mechanisms, direct lipolysis through PLA2 activity, activation of the human complement system, and cleavages of CD55 and CD59 from erythrocyte membranes.

Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human α2-macroglobulin and rabbit immunoglobulin

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human α2-macroglobulin (α2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human α2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by α2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with α2-M resulted in the formation of high molecular complex (∼180 000) and M r 90 000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human α2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action.

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A 2 from Bothrops asper snake venom

Myotoxic class II phospholipases A 2 from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115–129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1–15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A 2, showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins ( Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A 2, regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.

Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom

Two N-terminally truncated forms of the C-type natriuretic peptide (CNP) were isolated from the venom of habu snake, Trimeresurus flavoviridis, and their structures were determined by EMI-MS spectrometry and amino acid sequencing. Tf-CNP(6–22), the shorter peptide retaining the 17-membered ring structure formed by an intra-molecular disulfide bridge, has a vasorelaxant activity in rat aortic strips and a diuretic potency in anesthetized rats. Tf-CNP(3–22), the other 20 amino acid residues peptide, also comprised the 17- membered ring with a short N-terminal extention of 3 amino acid residues. Tf-CNP(6–22), the ring, is the shortest naturally occurring CNP peptide identified so far, and as potent as Tf-CNP(1–22), the supposedly intact CNP of 22 amino acid residues.

Bradykinin-potentiating peptides and C-type natriuretic peptides from snake venom

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)–C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP–CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP–CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (∼25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5–13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (∼100 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily.

Crystal structure of coagulation factor IX-binding protein from habu snake venom at 2.6 å: implication of central loop swapping based on deletion in the linker region

Coagulation factor IX-binding protein (IX-bp) isolated from the venom of the habu snake (Trimeresurus flavoviridis) is a disulfide-linked heterodimer consisting of homologous subunits A and B. The structure of IX-bp has been solved by X-ray crystallography at 2.6 Å resolution to a crystallographic R-value of 0.181. The main-chain fold of each subunit is homologous to the carbohydrate-recognition domain of C-type lectins (C-type CRDs) except for the extended central loop. The structure is almost identical with that of factors IX and X-binding protein (IX/X-bp) as expected from the high level of amino acid sequence homology. The functional difference in ligand recognition from IX/X-bp must reside in the amino acid differences. A continuity of different amino acid residues located from the C-terminal of the second α-helix to the following loop forms the local conformational difference in this region between the two proteins. This loop participates in the formation of the concave surface between the two subunits, the putative binding site for the Gla-domain (γ-carboxyglutamic acid-containing domain) of the coagulation factors. Another difference between the two proteins is in the relative disposition of subunits A and B. When the B subunits are superimposed, about a 6 ° rotation is required for the superposition of the A subunits.A calcium ion links the second α-helix region to the C-terminal tail in each subunit and helps to stabilize the structure for Gla-domain binding. The interface created by the central loop swapping in the dimer IX-bp is almost identical with that seen within the monomeric C-type CRDs. This dimer forms as the result of the amino acid deletion in the linker region of the central loop of the original C-type lectins. Such a dimerization disrupts the lectin active site and creates a Gla-domain binding site, imparting functional diversity.

Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein.

Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 +/- 0.7 (mean +/- SE, n = 6) and 103 +/- 10 microM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 amino acid residues and was 68% identical to the sequence of the A chain of IX/X-bp. The 15 kDa chain (B chain) consisted of 123 amino acid residues and was 87% identical to IX/X-bp. Three-dimensional model construction from the known fold of IX/X-bp showed that amino acid residues different from those of IX/X-bp are mostly on the molecular surface. Some of these are concentrated on a part of the concave surface which is considered to be the coagulation factor-binding site, presumably acting as a discriminator for ligand binding. These results indicated that X-bp isolated from D. acutus venom was a GD-binding protein, and the C-terminal region of GD peptide was critical for folding of the peptide.

The Bov-B LINEs found in Vipera ammodytes toxic PLA 2 genes are widespread in snake genomes

In the fourth intron of two toxic Vipera ammodytes PLA 2 genes a Ruminantia specific 5′-truncated Bov-B LINE element was identified. Southern blot analysis of Bov-B LINE distribution in vertebrates shows that, apart from the Ruminantia, it is limited to Viperidae snakes ( V. ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3′ end of Bov-B LINE in the V. ammodytes genome is between 62 000 and 75 000. At orthologous positions in other snake PLA 2 genes the Bov-B LINE element is absent, indicating that its retrotransposition in the V. ammodytes PLA 2 gene locus has occurred quite recently, about 5 Myr ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it should be a valuable phylogenetic marker. The neighbour-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments (1991–1997)

The properties of several factors — antihaemorrhagic, antineurotoxic, antimyotoxic — isolated from the blood serum or plasma of different animals are described with more emphasis placed on the structural differences and similarities among the factors of the snake ( Trimeresurus flavoviridis) and mammals ( Didelphis marsupialis and Herpestes edwardsii). Classification of antihaemorrhagic factors of snake and mammals according to structural homologies, and their effectiveness in neutralizing venom haemorrhagic activities in comparison with that of commercial antivenoms are also reviewed. The antineurotoxic factors isolated so far from the sera of viperid ( Vipera palestinae, Daboia r. siamensis), crotalid ( Crotalus d. terrificus, T. flavoviridis, Agkistrodon b. siniticus) and elapid ( Naja naja atra) snakes, as reviewed, are inhibitors of phospholipase A 2, and the amino acid sequences, particularly of those inhibitors from the sera of crotalid snakes, do not share significant sequence homology even within the same family Crotalidae. The amino acid sequences of antineurotoxic factors of the snake ( Crotalus d. terrificus) also are not homologous to those of the antihaemorrhagic factors from the blood of the snake ( T. flavoviridis) or mammals ( Didelphis virginiana, Herpestes edwardsii). The mechanism of action of antihaemorrhagic and antineurotoxic factors is briefly discussed as well as the possibility that crotalids and viperids might possess both of those endogenous neutralizing factors in their blood. Some recent findings on the antimyotoxic factors from the snake serum or plasma with inhibition properties against PLA 2 activity and myotoxicity of venoms or toxins are also shortly reviewed.

Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A 2 isozymes

Five inhibitors (PLI-I–V) against Trimeresurus flavoviridis ( Tf, habu snake, Crotalinae) venom phospholipase A 2 (PLA 2) isozymes have been isolated from its serum. PLI-I, which is composed of two repeated three-finger motifs, and PLI-IV and PLI-V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C-type lectins, were expressed in the forms fused with glutathione S-transferase (GST). The resulting GST-PLIs showed ability to bind to three Tf venom PLA 2 isozymes. The binding study with the truncated forms indicated that one of two three-finger motifs of PLI-I was able to bind to PLA 2 isozymes. The N-terminal 37-amino acid fragment and the CRD-like domain of PLI-IV and PLI-V were bound to PLA 2 isozymes. On the other hand, their C-terminal 12-amino acid segment also associated with PLA 2 isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C-terminal hydrophobic cores of PLI-IV and PLI-V were critically responsible for the binding to venom PLA 2 isozymes.

Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake's venom.

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

Evidence for functional involvement of asparagine 67 in substrate recognition by snake venom phospholipases A2.

Site-directed mutagenesis studies of recombinant Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) showed that the Asn residue at position 67 takes part in recognition of the substrate 2-arachidonoyl sn-glycero-3-phosphocholine in both monomeric and micellar states. The amount of arachidonate released from phosphatidylcholine mixed micelles was reduced to 30% for N67D and N67K mutants, and to 70% for N67G mutant, but remained unchanged for N67S mutant. In contrast, for monomeric substrate, the activity was decreased to 40% for N67D and N67G and to 60% for N67K but remained unchanged for N67S. These results suggest that the properties of the side chain of residue 67 exert a significant influence on recognition of 2-arachidonoyl sn-glycero-3-phosphocholine.

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

CDNA CLONING OF A HETEROGENEOUS TWO-CHAIN ANTICOAGULANT PROTEIN IX-BP

IX-bp and IX/X-bp are heterogeneous two-chain anticoagulant proteins isolated from the venom of habu snake, Trimeresurus flavoviridis. The amino acid sequence of one (B chain) of their two chains is identical. We recently reported the cloning of cDNA encoding the B chain and that of the A chain of IX/X-bp. Here, we report the isolation and characterization of cDNA clones encoding the A chain of IX-bp. The 697-bp sequence showed a putative ORF capable of encoding a 152-amino-acid protein containing a 23-amino-acid signal peptide. The overall amino acid sequence identity between the pre-A chain of IX-bp and that of IX/X-bp is 84%, whereas the cDNA sequence identity of the two ORFs is 91% indicating that habu snake acquired the variety of venom proteins by efficient mutations of antonymous sites. © 1997 Elsevier Science Ltd