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PubMed2002

Molecular cloning of HR1a and HR1b, high molecular hemorrhagic factors, from Trimeresurus flavoviridis venom.

HR1a and HR1b are two high molecular weight (P-III class) hemorrhagic factors in the venom of Trimeresurus flavoviridis. In this study, we cloned cDNAs of the HR1a and HR1b precursors and analyzed their nucleotide sequences. The cDNA for HR1a was 2368 nucleotides in length and encoded an open reading frame (ORF) of 609 amino acids; that for HR1b was 2237 nucleotides and encoded an ORF of 614 amino acids. Both cDNAs belonged to the N-III class consisting of signal, pro, metalloproteinase, disintegrin-like and cysteine-rich regions, and shared strong amino acid sequence similarity (74.4%). The HR1b precursor was found to have an additional seven amino acid sequence at the carboxyl terminus compared with a mature form of HR1b.

Amino Acid Sequence↗
PubMed2003

Purification and characterization of a thrombin like enzyme, elegaxobin II, with lys-bradykinin releasing activity from the venom of Trimeresurus elegans (Sakishima-Habu).

A thrombin like enzyme, named elegaxobin II, with Lys-bradykinin releasing activity was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel-filtration on Sephadex G-100, and ion-exchange chromatography on the Q-Sepharose Fast Flow. By this procedure, about 9mg of purified enzyme was obtained from 1.1g of the venom. The purified enzyme showed a single protein band, the molecular weight of which was estimated to be about 35,000Da by sodium dodecyl sulfate-PAGE) under reducing condition, and this enzyme was found to contain a carbohydrate moiety. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 250 TAME units/mg of protein. This enzyme clotted only rabbit fibrinogen, whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin conversion, this enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas it did not release fibrinopeptide B. Furthermore, elegaxobin II released Lys-bradykinin when the enzyme was incubated with bovine plasma. The esterase activity was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), suggesting that this enzyme is a serine protease. The N-terminal sequence (Val-Ile-Gly-Gly) of this enzyme was identical to the typical sequence of serine proteinases.

Animals↗
PubMed2003

Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri.

A blood coagulation factor IX-binding protein (TSV-FIX-BP) was isolated from the snake venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-FIX-BP showed a single band with an apparent molecular weight of 23,000 under non-reducing conditions, and two distinct bands with apparent molecular weights of 14,800 and 14,000 under reducing conditions. cDNA clones containing the coding sequences of TSV-FIX-BP were isolated and sequenced to determine the structure of the precursors of TSV-FIX-BP subunits. The deduced amino acid sequences of two subunits of TSV-FIX-BP were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. TSV-FIX-BP was a non-enzymatic C-type lectin-like anti-coagulant. The anti-coagulant activity of TSV-FIX-BP was mainly caused by its dose dependent interaction with blood coagulation factor IX but not with blood coagulation factor X.

Amino Acid Sequence↗
PubMed2003

Molecular cloning and characterization of a platelet glycoprotein Ib-binding protein from the venom of Trimeresurus stejnegeri.

A platelet glycoprotein Ib-binding protein, termed TSV-GPIb-BP, was isolated from the venom of Trimeresurus stejnegeri. On SDS-polyacrylamide gel electrophoresis, TSV-GPIb-BP showed a single band with an apparent molecular weight of 28,000 and two distinct bands with apparent molecular weights of 16,000 and 15,000 under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for both TSV-GPIb-BP subunits were isolated and sequenced. The deduced amino acid sequences of TSV-GPIb-BP subunits were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting. Interestingly, the alpha subunit of TSV-GPIb-BP is identical to that of alboaggregin-B, and the sequence identity of their beta subunits is 94.3%. TSV-GPIb-BP inhibited ristocetin-induced human platelet agglutination in platelet-rich plasma under lower dosages (<5 microg/ml). On the other hand, it directly aggregated washed human platelets in the absence of additional Ca2+ or any other cofactors under higher dosages (>5 microg/ml). This platelet aggregation activity was dose-dependently inhibited by specific GPIbalpha antibodies, but not by those antibodies against platelet GPIa, GPIIa, GPIIb and GPIIIa.

Amino Acid Sequence↗
PubMed1998

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom.

From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-1, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatography (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mol. wt of 50000, 31000 and 32000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnefibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA and stejnobin, stejnefibrase-1, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially Bbeta-chain while stejnefibrase-1 and -3 cleaved concomitantly Aalpha and Bbeta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-1. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases.

Animals↗
PubMed1998

Effect of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom.

Trimucrotoxin, the basic phospholipase A2 from Trimeresurus mucrosquamatus venom, is neurotoxic and myotoxic, and structurally similar to crotoxin B subunit. To investigate the amino acid residues responsible for its neurotoxicity, we have mutated its interface-recognition residues including a conserved Asn6 in all the Crotalinae neurotoxic phospholipases. The wild-type and the mutants were expressed in E. coli as fusion-proteins and activated in vitro by factor Xa cleavage after folding. The completion of folding and activation were checked with electrospray ionization mass spectrometry and circular dichroism measurement. Enzymatic activities and neurotoxicities toward the chick tissue of four trimucrotoxin mutants (N6A, N6E, N6R and 6E7T8L) were compared with those of the wild type which was as active as that was isolated from the venom. Mutants N6A and N6E retained more than half of the original enzymatic activity but their neurotoxicities reduced to 33% and 10% that of the wild type, respectively. Mutants N6R and 6E7T8L retained 20-25% of the enzyme activity toward the anionic micellar substrate but were inactive toward the zwitterionic micellar substrate, and their neurotoxicities were less than 3% of that of the wild type. These results demonstrate the importance of residues 6-8 in trimucrotoxin for its neuronal specificity and the specificity toward potential substrates.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.

Amino Acids↗
PubMed2000

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii.

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin, was purified by DEAE A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH(2)-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg. The fibrinopeptides released, identified by HPLC, consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it, indicating it is venom serine protease.

Amino Acid Sequence↗
PubMed2002

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.

Animals↗
PubMed2003

Immunohistochemical localization of the delta subspecies of protein kinase C in the trigeminal sensory system of Trimeresurus flavoviridis, an infrared-sensitive snake.

We examined the expression of the protein kinase C (PKC) delta subspecies in the trigeminal sensory system of the infrared-sensitive snake Trimeresurus flavoviridis. In the trigeminal ganglion (TG), diffuse low-intensity PKC delta immunoreactivity was found in TG neurons and fibers, while intense reactions were observed mainly in medium-sized neurons, which include most of the infrared-sensitive neurons. In the brainstem, intense PKC delta immunoreactivity was present in the intermediate layer of the optic tectum of the midbrain and in the nucleus descendens lateralis n. trigemini of the medulla oblongata; these areas are related to the infrared sensory pathway. In the pit organ (the infrared receptor), PKC delta immunoreactivity was present in terminal nerve masses in the pit membrane. These findings suggest that the PKC delta subspecies is involved in the infrared sensory pathway in the trigeminal sensory system of the infrared-sensitive snake.

Afferent Pathways↗
PubMed1997

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake.

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

Amino Acid Sequence↗
PubMed2003

Apoptotic effect in the glioma cells induced by specific protein extracted from Okinawa Habu (Trimeresurus flavoviridis) venom in relation to oxidative stress.

Okinawa Habu (Trimeresurus flavoviridis) venom is well known for its toxic efficacy, from which one kind of specific protein, Okinawa Habu apoxin protein-1 (OHAP-1) has been extracted. The purpose of this study was to investigate whether OHAP-1 could induce apoptosis in some glioma cells, and if so, to elucidate the possible mechanism involved. Three malignant glioma cell lines were tested. The malignant glioma cell lines were rat C6 and human RBR 17T, U251. OHAP-1 inhibited growth of all cell lines. Whether or not the apoptosis had been induced was determined by using DNA gel electrophoresis, DNA flow cytometry and TUNEL assay. After OHAP-1 treatment, DNA fragmentation, an increase in the percentage of subdiploid DNA content, and TUNEL positive cells were found in the C6, RBR17T, and U251 cells. Furthermore, OHAP-1 showed L-amino acid oxidase (LAAO) activity. In order to study the mechanism of apoptosis induced by OHAP-1, the changes of intracellular reactive oxygen species (ROS) were measured using flow cytometry, and the expression of p53 protein was examined using immunohistochemistry. OHAP-1 was found to generate ROS and increase the expression of p53 protein in glioma cells. The inhibiting effect of OHAP-1 on three tested cells was reversed when an antioxidant of either catalase or reduced glutathione (GSH) was added; its apoptotic effect correspondingly became weaker. In this study, the apoptotic effect of OHAP-1 on some malignant glioma cells was confirmed, and it could be that this effect might be mediated through promoting the generation of intracellular ROS and p53 protein expression in glioma cells. It was suggested that OHAP-1 is promising as a potential candidate for clinical tumor therapy.

Amino Acid Oxidoreductases↗
PubMed2002

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen.

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K(m) and the catalytic rate constant K(cat) of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen.

Animals↗
PubMed2002

The stratagem utilized by the plasminogen activator from the snake Trimeresurus stejnegeri to escape serpins.

The plasminogen activator isolated from the venom of the snake Trimeresurus stejnegeri (TSV-PA) triggers plasmin production, along with tissue-type plasminogen activators (t-PA) and urokinase (u-PA). The half-life of TSV-PA in plasma is remarkable. We unveil in this paper two of the molecular mechanisms allowing TSV-PA to escape inhibition by plasma serpins. The first involves a phenylalanine at position 193 (chymotrypsinogen numbering system). Phe(193) distinguishes TSV-PA from nearly all trypsin-like proteinases, having glycine at this position. A mutant of TSV-PA (F193G), in which Phe(193) had been replaced by a glycine, was inactivated by plasminogen activator inhibitor 1 (PAI-1) and alpha(2)-antiplasmin 100-fold more rapidly than the wild-type enzyme. The second mechanism originates from the 37-loop of TSV-PA. Swapping the 37-loop of TSV-PA for either that of t-PA or that of u-PA also increased dramatically the rate of inactivation by PAI-1. Loop swapping and F193G mutations were additive, resulting in a rate of inactivation by PAI-1 that was 4 orders of magnitude higher than for the wild-type enzyme. The potential role of Phe(193) and of the 37-loop in the immunity of TSV-PA toward alpha(1)-antitrypsin and antithrombin is also discussed.

Amino Acid Sequence↗
PubMed2000

Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan.

Conventional chromatographic analysis showed that phospholipase A(2) (PLA(2)) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys(49)]PLA(2) isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA(2) isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavovirdis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA(2) isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.

Animals↗
PubMed2004

Crystal structure of a platelet-agglutinating factor isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus).

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbalpha and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Mucrocetin is a GPIbalpha agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbalpha antagonist) on GPIbalpha, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 A (1 A=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four alphabeta heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer 'head-to-tail' disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the alpha-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the beta-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb-IX-V complex.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus).

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.

Amino Acid Sequence↗