Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.     

Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)₁₈-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.     

Amino acid sequence of a basic aspartate-49-phospholipase A2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A2.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A2 (PLA2) of basic nature (pI 8.5) was isolated from the venom of Amami-Oshima T. flavoviridis. Its amino acid sequence determined by the ordinary procedures was completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland, which was named PLA-B'. It consists of 122 amino acid residues and has aspartate at position 49. It induced edema in a mouse footpad assay and caused necrosis in mouse skeletal muscles. PLA-B' is similar in sequence to PLA-B (Tokunoshima) and PL-Y (Okinawa), both basic [Asp49]PLA2s, with a few amino acid substitutions, indicating occurrence of interisland mutation. Although PLA2s of Crotalinae subfamily were phylogenetically classified into four types, PLA2 (acidic or neutral [Asp49]PLA2) type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys49]PLA2 type, it was ascertained that PLA2s of PLA2 type and [Lys49]PLA2 type are most essential as toxic components for Crotalinae snake venoms and that basic [Asp49]PLA2-type PLA2s are uniquely contained only in the venoms of T. flavoviridis species. Prediction of physiological activities of some PLA2s was made based on their location in the phylogenetic tree. Relationship of divergence of PLA2s via accelerated evolution followed by less rapid mutation and physiological activities was discussed.     

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms.

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes.     

Immunological comparison of hemorrhagic principles present in venoms of the Crotalinae and Viperinae subfamilies

Antibodies were raised against hemorrhagic factors HF1, HF2 and HF3 isolated from the venom of Bothrops jararaca and NHFa,b from the venom of Bothrops neuwiedi. Crude venoms of different species of snakes were assayed with the rabbit antisera specific for the hemorrhagic factors. Results of immunodiffusion, neutralization of hemorrhagic activity and micro-complement fixation indicated that there is an immunological relationship between the venom hemorrhagic components of the Bothrops species and those of other species of the Crotalinae subfamily. The factors of Bothrops species seem to be structurally similar. The hemorrhagic proteins from the venoms of Lachesis, North American Crotalus, Asian Trimeresurus and Agkistrodon species show some resemblance to the Bothrops factors. The venom hemorrhagic principles from snakes of the Viperinae subfamily (Bitis and Vipera species) might have few epitopes similar to those of Bothrops species as the only relation shown was the partial neutralization by the immune sera.     

Molecular cloning and sequence analysis of cDNA encoding flavoridin, a disintegrin from the venom of Trimeresurus flavoviridis.

We isolated a cDNA of 2001bp encoding the full-length precursor of flavoridin, which is one of the four disintegrins in the venom of Trimeresurus flavoviridis, and analyzed the cDNA nucleotide sequence. The deduced amino acid sequence of the open reading frame consisted of a pro-domain (190 residues), a metalloproteinase domain (205 residues), a spacer domain (18 residues) and a disintegrin (flavoridin) domain (70 residues), thus indicating that the flavoridin precursor belongs to the P-II class of snake venom metalloproteinases. The unknown metalloproteinase domain shared strong sequence similarity with HR2a (71.2% identity) and H(2)-proteinase (74.1% identity), a low molecular mass hemorrhagic metalloproteinase and a non-hemorrhagic metalloproteinase in the same snake venom, respectively.     

Cysteine-rich venom proteins from the snakes of Viperinae subfamily - Molecular cloning and phylogenetic relationship

Cysteine-rich proteins found in animal venoms (CRISP-Vs) are members of a large family of cysteine-rich secretory proteins (CRISPs). CRISP-Vs acting on different ion channels were found in venoms or mRNA (cDNA) encoding CRISP-Vs were cloned from snakes of three main families (Elapidae, Colubridae and Viperidae). About thirty snake CRISP-Vs were sequenced so far, however no complete sequence for CRISP-V from Viperinae subfamily was reported. We have cloned and sequenced for the first time cDNAs encoding CRISP-Vs from Vipera nikolskii and Vipera berus vipers (Viperinae). The deduced mature CRISP-V amino acid sequences consist of 220 amino acid residues. Phylogenetic analysis showed that viper proteins are closely related to those of Crotalinae snakes. The presence of CRISP-V in the V. berus venom was revealed using a combination of gel-filtration chromatography, electrophoresis and MALDI mass spectrometry. The finding of the putative channel blocker in viper venom may indicate its action on prey nervous system.     

Properties and cDNA cloning of antihemorrhagic factors in sera of Chinese and Japanese mamushi (Gloydius blomhoffi).

An antihemorrhagic protein has been isolated from the serum of Chinese mamushi (Gloydius blomhoffi brevicaudus) by using a combination of ethanol precipitation and a reverse-phase high-performance liquid chromatography (HPLC) on a C8 column. This protein-designated Chinese mamushi serum factor (cMSF)-suppressed mamushi venom-induced hemorrhage in a dose-dependent manner. It had no effect on trypsin, chymotrypsin, thermolysin, and papain but inhibited the proteinase activities of several snake venom metalloproteinases (SVMPs) including hemorrhagic enzymes isolated from the venoms of mamushi and habu (Trimeresurus flavoviridis). A similar protein (Japanese MSF, jMSF) with antihemorrhagic activity has also been purified from the sera of Japanese mamushi (G. blomhoffi). The N-terminal 70 and 51 residues of the intact cMSF and jMSF were directly analyzed; a similarity between the sequences of two MSFs to that of antihemorrhagic protein (HSF) from habu serum was noticed. To obtain the complete amino acid sequences of MSFs, cDNAs encoding these proteins were cloned from the liver mRNA of Chinese and Japanese vipers based on their N-terminal amino acid sequences. The mature forms of both MSFs consisted of 305 amino acids with a 19-residue signal sequence, and a unique 17-residue deletion was detected in their His-rich domains.     

Characterization, amino acid sequence and evolution of edema-inducing, basic phospholipase A2 from Trimeresurus flavoviridis venom.

Two phospholipases A2 (PLA2s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp49]PLA2) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA2s in Fig. 8), thus belonging to an [Asp49]PLA2 subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the beta-sheet region. On the other hand, PLA-A had beta-Asp-Gly and alpha-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X', whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a beta-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA2 isozymes indicated that PLA-B and PL-X' diverged from PLA2 after branching of [Asp49]PLA2 forms and [Lys49]PLA2 forms.     

Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake)

Two phospholipases A2, named phospholipases A2-I and A2-II, were purified to homogeneity from the venom of Trimeresurus gramineus (green habu snake). The complete amino acid sequence of phospholipase A2-I was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I, clostripain, and chymotrypsin) and chemical (hydroxylamine) cleavages of the S-pyridylethylated derivative of the protein. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid snakes which belong to the category of Group II. A most striking feature of this protein is that tyrosine at the 28th position which is common in phospholipases A2 and is assumed to be a part of the Ca2(+)-binding loop is replaced by phenylalanine. Such replacement is the first finding in Group II phospholipases A2. Secondary structure compositions of phospholipase A2-I are similar to those of Crotalus atrox phospholipase A2. No appreciable Ca2(+)-induced difference spectrum was observed, due probably to the absence of the effective chromophoric groups in the neighborhood of the Ca2+ binding site although Ca2+ is bound with affinity similar to that for T. flavoviridis phospholipase A2.     

Comparison of amino terminal region of three isoenzymes of phospholipases A2 (TFV PL-Ia, TFV PL-Ib, TFV PL-X) from Trimeresurus flavoviridis (habu snake) venom and the complete amino acid sequence of the basic phospholipase, TFV PL-X

Three phospholipases (PLA2s) isolated from Japanese habu snake (Trimeresurus flavoviridis) venom differ from each other in the physiological symptoms induced in animals. The amino acid compositions and amino terminal sequences of these isoenzymes have been compared. TFV PL-Ia and TFV PL-Ib resemble each other very closely, but TFV PL-X has a distinctly different amino acid composition and amino terminal region. The complete amino acid sequence of the basic PLA2, TFV PL-X, was determined by sequencing the four peptides obtained by the cleavage of Asp-Pro bonds. The overlapping peptide was obtained by cleaving tryptophanyl bond and the carboxy terminal region was determined by sequencing the chymotryptic peptide. TFV PL-X consisted of a single chain of 122 amino acid residues, including 14 half-cystine residues. It is a group II A PLA2 and is homologous with the phospholipase super family. However, it has a distinctly different sequence compared with the other PLA2s from the venom of Trimeresurus species.     

Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.     

Purification and amino acid sequence of basic protein I, a lysine-49-phospholipase A2 with low activity, from the venom of Trimeresurus flavoviridis (Habu snake)

A basic protein (pI 10.2), named basic protein I, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides produced by chemical (cyanogen bromide and formic acid) and enzymatic (chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid and viperid snakes. A most striking feature of this protein is that aspartic acid at the 49th position common in phospholipases A2 is replaced by lysine. When the protein acted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, oleic acid was preferentially released, indicating that the protein has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was 1.5% that of T. flavoviridis phospholipase A2 isolated previously. The fact that both affinity to Ca2+ and reactivity to p-bromophenacyl bromide of basic protein I are approximately one order of magnitude lower than those of T. flavoviridis phospholipase A2 might explain the low activity of basic protein I.     

Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland.

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from this venom. In this work, we cloned two cDNAs of P-III metalloproteinase precursors, designated as stejnihagin-A and stejnihagin-B, respectively, from T. stejnegeri venom gland. Both cDNAs encode an opening reading frame of 600 amino acid residues, containing a signal sequence, a proprotein domain, a metalloproteinase domain, a disintegrin-like domain and a cystetine-rich domain. Sequence analysis suggested that these two sequences shared highest similarity to the hemorrhagic toxin HR1b from T. flavoviridis. Aligning the deduced mature protein sequences of stejnihagin-A and stejnihagin-B with other snake venom metalloproteinases (SVMPs), we observed that stejnihagin-A and stejnihagin-B, together with HR1b shared the common cysteinyl residue at the position 100 in the metalloproteinase domain. In combination with the phylogenetic analysis, we presumed that stejnihagin-A, stejnihagin-B and HR1b might constitute a novel subclass of P-III SVMPs, named P-IIIc.     

Molecular cloning of serine proteinases from Bothrops jararaca venom gland.

Snake venom is known to contain an abundance of enzyme isoforms, and various disorders associated with envenomation have been ascribed partially to their diversified functions. Crude venom of Bothrops jararaca was subjected to conventional two-dimensional SDS-PAGE, followed by immunoblot analysis using an antiserum raised against KN-BJ 2, a serine proteinase previously isolated from this venom. A number of immunoreactive proteins with comparable molecular masses and different pIs emerged, implying the venom contains yet-unknown serine proteinases. A B. jararaca venom gland cDNA library was subsequently screened with a labeled KN-BJ 2 cDNA as a probe. Among a number of positive cDNA clones, three--HS112, HS114, and HS120--were selected and sequenced. These clones each had an open reading frame of 759-774 bp, and their deduced amino acid sequences illustrated considerable similarities to that of KN-BJ 2 as well as to those of serine proteinases of different origins. However, no apparent match to any of the deposited sequences was found in the current GenBank/EMBL databases, indicating that each of these cDNA clones encodes a serine proteinase distinct from the known enzymes. Analyses of the nucleotide and amino acid sequences of these cDNA clones support the accelerated evolution hypothesis proposed for snake venom enzymes.     

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure-function relationship of BPPs

Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.     

Inhibition of hemorrhagic and edematogenic activities of snake venoms by a broad-spectrum protease inhibitor, murinoglobulin; the effect on venoms from five different genera in Viperidae family.

In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 degrees C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family.     

Snake venom glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency.     

Molecular cloning and characterization of ecto-5′-nucleotidase from the venoms of Gloydius blomhoffi

A Gloydius blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences from known ecto-5′-nucleotidases (ecto-5′-NTs). Molecular cloning of ecto-5′-NT from G. blomhoffi brevicaudus venom predicted that it was a glycosyl phosphatidylinositol-anchored membrane protein containing 588 amino acid residues with 7 potential N-linked glycosylation sites. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian ecto-5′-NT sequences. This is the first report of the primary structure of ecto-5′-NT from a reptile. Gel-filtration chromatography of fresh venom from Gloydius blomhoffi blomhoffi, a subspecies of G. blomhoffi, revealed that at least a part of ecto-5′-NT is bound to exosome-like vesicles.     

Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.