A sleep-inducing peptide from the venom of the Indian cone snail Conus araneosus

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins. (C) 2015 Elsevier Ltd. All rights reserved.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

AN ACTIVATOR OF BLOOD-COAGULATION FACTOR-X FROM THE VENOM OF BUNGARUS-FASCIATUS

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mel. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

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

Rong Gao, Yun Zhang, Qing-Xiong Meng, Wen-Hui Lee, Dong-Sheng Li, Yu-liang Xiong and Wan-Yu Wang. Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejneger ) venom. Toxicon 36, 457-467, 1998.-From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-l, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatograghy (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mel. wt of -50 000, 31 000 and 32 000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnfibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA. and stejnobin, stejnefibrase-l, -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 BE-chain while stejnefibrase-l and -3 cleaved concomitantly Ax and B beta-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-l. 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. (C) 1998 Elsevier Science Ltd. All rights reserved.

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

Two families of antimicrobial peptides from wasp (Vespa magnifica) venom

The hornet possesses highly toxic venom, which is rich in toxin, enzymes and biologically active peptides. Many bioactive substances were identified from wasp venom. Two families of antimicrobial peptides were purified and characterized from the venom of

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVl

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta(1)) and 22 kDa (beta(2)), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta(1) and beta(2) subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between 1 and 2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the U. and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against UNA inhibited the aggregation response to stejnulxin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha(2)beta(1) as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity

A novel L-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. Fifty percentage cytotoxic concentrations (CC50) of TSV-LAO on C8166 cells were 24 and 390 nM in the absence or presence of catalase (400nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs. (C) 2003 Elsevier Inc. All rights reserved.

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 precusors 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 nonenzymatic 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. (C) 2003 Elsevier Science Ltd. All rights reserved.

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 a 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 mug/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 mug/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. (C) 2003 Elsevier Science Ltd. All rights reserved.

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antib