Anti-metastatic activity of the recombinant disintegrin, r-mojastin 1, from the Mohave rattlesnake

Cancer is the uncontrollable growth of cell, which may spread to other parts of the body. The interaction of cancer cells with extracellular matrix (ECM) is essential for metastasis, which is the principal cause of death in cancer patients. Disintegrins are naturally occurring low molecular weight peptides found in the venoms of many snakes. Disintegrins were first used to inhibit platelet aggregation, but more recently have been used to inhibit cancer cell growth, adhesion, migration, invasion and/or angiogenesis. The purpose of this study was to determine the anti-tumor properties of recombinant mojastin 1 (r-mojastin 1) and r-mojastin-GST, cloned from the venom glands of the Mohave rattlesnake (Crotalus scutulatus scutulatus). Human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-ML-28) and murine skin melanoma (B16F10) cell lines were used. r-Mojastin 1 inhibited SK-MEL-28 cell adhesion to fibronectin, but was not able to inhibit T24 cell adhesion to fibronectin. However, r-Mojastin-GST inhibited SK-MEL-28 and T24 cells adhesion to fibronectin. r-Mojastin-GST and r-mojastin 1 decreased the ability of SK-MEL-28 cells to migrate after 24 h of incubation but were not able to inhibit T24 cell migration. r-Mojastin 1 and r-mojastin-GST inhibited invasion of T24 and SK-MEL-28 cancer cells in vitro, and r-Mojastin 1 inhibited lung tumor colonization of B16F10 cells in mice in vivo. In conclusion, our studies suggest that r-mojastin could be a useful tool to develop novel anti-tumor agents by virtue of its ability to inhibit tumor cell adhesion, migration and invasion.     

The distribution among ophidian venoms of a toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus)

A toxin analogous to Mojave toxin or protein K' was isolated from venom of the Mojave rattlesnake (Crotalus s. scutulatus) by anion exchange and gel permeation chromatography. This toxin has an apparent native molecular weight of 20,000-22,000, a subunit molecular weight of 14,000 and a pI of 4.9-5.0. The i.p. LD50 is 0.094 mg/kg for mice. A wide variety of ophidian venoms (crotaline, viperine, elapid, hydrophid and colubrid) were examined for the presence of this toxin using Ouchterlony, immunoelectrophoresis, ELISA and Western transfer. High concentrations were found in 4 of 6 C. scutulatus venom samples, 2 of 3 C. durissus samples and samples from C. viridis concolor and C. tigris. A moderate concentration was found in 1 of 3 C. durissus samples and low to trace concentrations in 1 C. durissus sample, 1 C. scutulatus sample, 2 of 12 C. atrox samples and a Trimeresurus flavoviridis sample, the latter being the only instance of detection of the toxin in a snake other than a rattlesnake. The toxin appears in at least two phylogenetic lines of rattlesnakes, and its geographic distribution in North American rattlesnake species resembles a mosaic.     

Pharmacological aspects of Vipera xantina palestinae venom

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation.     

Mojave rattlesnake (Crotalus scutulatus scutulatus) venom: in vitro effect on platelets, fibrinolysis, and fibrinogen clotting

Rattlesnake envenomation commonly produce defects in the hemostatic mechanism. However, Mojave rattlesnake (Crotalus scutulatus scutulatus) envenomation has been reported not to cause a systemic bleeding diathesis. In this study, whole venom from the Mojave rattlesnake was tested in vitro for fibrinogen clotting activity, ability to induce platelet aggregation, and for fibrinolytic activity. The Mojave venom caused no fibrinogen clotting and it displayed very weak ability to cause platelet aggregation and fibrinolytic activity. These in vitro studies support the clinical observation that Mojave envenomation does not cause a coagulopathy.     

Variation in the antigenic characteristics of venom from the Mojave rattlesnake (Crotalus scutulatus scutulatus).

Venoms from 31 specimens of the Mojave rattlesnake (Crotalus scutulatus scutulatus) were examined to further characterize reported differences among venoms of this species. Twenty-two venoms were recognized by a monoclonal antibody to Mojave toxin, CSS12. Nine venoms were recognized by CA-P-8, a monoclonal antibody produced against the hemorrhagic venom of C. atrox. Seven of these produced strong hemorrhage in mice and were also recognized by polyclonal antibodies (anti-F5) produced against a fraction of Mojave rattlesnake venom that inactivates serum complement. Fractionated venom revealed that CA-P-8 and anti-F5 recognized different proteins. Two of the venoms recognized by CA-P-8 were not recognized by anti-F5 and produced minimal hemorrhage in mice. This suggests that more than one factor may be necessary to induce strong hemorrhage.     

Anti-invasive and anti-adhesive activities of a recombinant disintegrin, r-viridistatin 2, derived from the Prairie rattlesnake (Crotalus viridis viridis)

Snake venom disintegrins inhibit platelet aggregation and have anti-cancer activities. In this study, we report the cloning, expression, and functional activities of a recombinant disintegrin, r-viridistatin 2 (GenBank ID: JQ071899), from the Prairie rattlesnake. r-Viridistatin 2 was tested for anti-invasive and anti-adhesive activities against six different cancer cell lines (human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorectal adenocarcinoma (CaCo-2), human breast adenocarcinoma (MDA-MB-231) and murine skin melanoma (B16F10)). r-Viridistatin 2 shares 96% and 64% amino acid identity with two other Prairie rattlesnake medium-sized disintegrins, viridin and viridistatin, respectively. r-Viridistatin 2 was able to inhibit adhesion of T24, SK-MEL-28, HT-1080, CaCo-2 and MDA-MB-231 to various extracellular matrix proteins with different affinities. r-Viridistatin 2 decreased the ability of T24 and SK-MEL-28 cells to migrate by 62 and 96% respectively, after 24 h of incubation and the invasion of T24, SK-MEL-28, HT-1080 and MDA-MB-231 cells were inhibited by 80, 85, 65 and 64% respectively, through a reconstituted basement membrane using a modified Boyden chamber. Finally, r-viridistatin 2 effectively inhibited lung colonization of murine melanoma cells in BALB/c mice by 71%, suggesting that r-viridistatin 2 could be a potent anti-cancer agent in vivo.     

Mojave toxin in venom of Crotalus helleri (Southern Pacific Rattlesnake): molecular and geographic characterization.

Mojave toxin (MT) was detected in five of 25 Crotalus helleri (Southern Pacific rattlesnake) sampled using anti-MT antibodies and nucleotide sequence analysis. All of the venoms that were positive for MT were collected from Mt San Jacinto in Riverside Co., California. Since this population is geographically isolated from C. scutulatus scutulatus (Mojave rattlesnake), it is unlikely that this finding is due to recent hybridization. MT concentration differences between C. helleri and C. s. scutulatus reflected the presence of 'isoforms' of the toxin in the venom. Whereas C. s. scutulatus generally has several isoforms of the toxin (detected by Western blotting), only one 'isoform' that focused at pI 5.1 was detected in C. helleri. Both acidic and basic subunits of MT sequences were obtained from C. helleri DNA with primers specific for MT, but only from snakes that had MT in their venom. The sequence identity of the C. helleri acidic subunit to the C. s. scutulatus subunit was 84.9%, whereas the sequence identity of the C. helleri basic subunit was 97% to the C. s. scutulatus basic subunit. Using casein, fibrin, and hide powder azure as substrates, assays for proteolytic activity suggested that C. helleri possesses several different types of metalloproteinases in their venom. However, proteolytic activity was not detected, or present in reduced amounts, in specimens having MT. Clinical neurotoxicity following envenomation by certain populations of C. helleri may be due to MT.     

Factors in snake venoms that increase capillary permeability

Capillary permeability increasing (CPI) activity is a phenomenon of the microvasculature caused by many agents such as snake venoms, histamine, 5-hydroxytryptamine (5-HT), prostaglandins and leukotrienes. Since no systematic study has been done to determine what components of snake venom cause CPI activity, a CPI factor from Naja naja atra (Taiwan cobra) venom was isolated using intravenous injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja naja kaouthia (Thailand cobra) and Mojave toxin from Crotalus scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity. Postsynaptic neurotoxins from an elapid and a hydrophid and myotoxin a from Crotalus viridis viridis (prairie rattlesnake) showed no CPI activity at the dose studied. The purified CPI active component from Naja naja atra venom was found to have cardiotoxic activity. Therefore, Elapidae cardiotoxins are CPI active factors. However, CPI activity is not due to cardiotoxins alone as the presynaptic neurotoxin, Mojave toxin, also showed CPI activity. Selective inhibitors were used to indicate possible mechanisms of action on the capillaries by Naja naja atra venom and Crotalus scutulatus scutulatus venom. The histamine H1-receptor blockers diphenhydramine, promethazine, and cyproheptadine were effective against both venoms in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms.     

Intergradation of two different venom populations of the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona

Two distinct venom populations of Crotalus scutulatus scutulatus exist in Arizona. The venom of one population (venom A) contains the toxin 'Mojave toxin' and is lacking in hemorrhagic and specific proteolytic activities. The other population (venom B) does not contain Mojave toxin but does produce hemorrhagic and proteolytic activities. The venoms of 15 Crotalus scutulatus scutulatus from regions between the venom A and venom B populations in Arizona were examined for the presence of Mojave toxin by immunochemical assay, lethality by mouse i.p. LD50, proteolytic activity and hemorrhagic activity in mice. Venom protein constituents were analyzed using reverse-phase HPLC. Seven venoms contained both the Mojave toxin of venom A and the proteolytic and hemorrhagic activities of venom B. The i.p. LD50 values of the A + B venoms were 0.4-2.6 mg/kg, compared to 0.2-0.5 mg/kg for venom A individuals and 2.1-5.3 mg/kg for the venom B individuals. HPLC illustrated that the A + B venoms exhibited a combined protein profile of venom A and venom B. These data indicate that an intergrade zone exists between the two venom types which arcs around the western and southern regions of the venom B population. Within these regions, three major venom types can occur in Crotalus s. scutulatus.     

Amplification of the classical complement pathway by the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus)

The lytic attack of human red blood cells by complement was enhanced by the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus). Lysis, however, of sheep red blood cells by complement was not enhanced by this venom. Western diamondback rattlesnake (Crotalus atrox) venom was anticomplementary. Enhanced lysis of human red blood cells by Mojave venom required that the red blood cells be sensitized with antibodies and that the complement system be intact. Heat inactivation of complement (56°C, 30 min) prevented lysis of red blood cells. Moreover, heating of the Mojave venom (66°C, 60 min) prevented enhanced lysis. Lytic enhancement was time, dose and temperature dependent. It is postulated that membrane active venom components enhance the efficiency of the complete complement cascade for red blood cell lysis to occur, because venom increased the lytic reaction rate.     

Characterization of two arginine ester hydrolases from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom

Two arginine ester hydrolases, designated AAEI and AAEII, from the venom of Crotalus scutulatus scutulatus have been investigated. The amino acid content of both enzymes were very similar and both esterases contained carbohydrate. Following treatment of AAEI and AAEII with neuraminidase, both enzymes migrated identically in two electrophoresis systems and one electrofocusing system. The esterase activities of both enzymes were optimally active in the range pH 8.0-8.5. Neither esterase hydrolyzed casein, hemoglobin (Hb) or alpha-N-benzoyl-DL-arginine-p-nitroaniline (BAPNA), yet both AAEI and AAEII hydrolyzed alpha-N-benzoyl-L-arginine ethyl ester (BAEE), alpha-N-benzoyl-L-arginine methyl ester (BAME), p-tosyl-L-arginine methyl ester (TAME) and acetylphenylalanylarginine methyl ester (Ac-Phe-Arg-OMe). The esterase activities of the two enzymes were inhibited by serine specific reagents and benzamide, but not by EDTA or soybean trypsin inhibitor. The Km values for each enzyme with alpha-N-benzoyl-L-arginine ethyl ester and acetylphenylalanylarginine methyl ester were determined. Neither esterase displayed thrombin-like or fibrinolytic activities. Both AAEI and AEII possessed kinin releasing activity as shown by the twitch response of an isolated rat uterus. The N-terminal sequences of AAEI and AAEII were identical and both enzymes sequences were similar to other arginine esterases from crotalid venoms. The properties of AAEI and AAEII are compared to several other arginine esterases possessing kallikrein-like activities which have been isolated from snake venoms.     

Primary structure of brevilysin L4, an enzymatically active fragment of a disintegrin precursor from Gloydius halys brevicaudus venom.

Brevilysin L4 (L4) is a non-hemorrhagic P-I class metalloprotease (MP) isolated from Gloydius halys brevicaudus venom. Its complete amino acid sequence has been determined. L4 is a single-chain polypeptide and highly homologous to those of other snake venom MPs. A zinc-binding motif, HExxHxxGxxH, is located at residues 142-152. A characteristic feature of L4 is the presence of a spacer sequence (LRTDTVS) at the C-terminal that links metalloprotease and disintegrin domains and is usually removed by post-translational proteolysis, suggesting that L4 is expressed together with a spacer region and a disintegrin domain at the C-terminal. The nucleotide sequence of a cDNA clone encoding L4 has revealed that L4 is a disintegrin precursor and produced as a P-II class MP. The disintegrin coded after L4 sequence was brevicaudin 1, a disintegrin previously isolated from the same venom. P-II class MPs have been suspected to undergo autoproteolysis to release disintegrins. Although being P-I class MP, L4 itself autocatalytically degrades with a half-life of 30min at pH 8.5 and 37 degrees C in the absence of Ca(2+). Sequence analysis of several fragment peptides produced during the autolysis of L4 indicated that more than 40 peptide bonds were split, and the cleavages of Ser(60)-Asn(61), Thr(99)-Ala(100), and Phe(103)-Asp(104) bonds may trigger the autoproteolysis. Addition of Ca(2+) completely suppressed the cleavage of these particular bonds, resulting in a marked prevention of autoproteolysis. Thus, L4 provides a good model for the investigation of autolysis of some MPs.     

蛇毒解离素的结构特征及其与生物学活性的关系

目前已从蛇毒中分离出 80余种解离素多肽,根据其结构及来源可分为五大类,即短链、中链、长链解离素、金属蛋白酶P Ⅲ的类解离素区释放的解离素以及二聚体解离素。不同解离素的分子结构共同点包括RGD活性中心、半胱氨酸残基配对形成的二硫键、RGD三肽的C 端和N 端的氨基酸序列以及解离素多肽分子的C 末端。这些结构决定了解离素的抑制血小板聚集、抑制细胞粘附以及血管形成等生物学活性。     

cDNA cloning of a novel P-I lebetase isoform Le-4.

In order to better understand the function of fibrinolytic enzyme lebetase isoforms and the synthesis of disintegrins we have isolated a cDNA encoding the most basic isoform (Le-4) from the cDNA library prepared from the poly(A)(+) RNA of the venomous gland of an individual Vipera lebetina snake. The truncated 5'-sequence of 1112 basepairs encodes the mature protein with 203 amino acid residues with calculated isoelectric point and size of 5.6 and 22,930 Da, respectively. Multiple comparison of the deduced amino acid sequence of the metalloprotease part of Le-4 is related to short reprolysins, identities were within the range of 60--87%. The two lebetase isoforms are synthesized in different way: Le-4 is synthesized with metalloprotease domain only; Le-3 is synthesized with metalloprotease and disintegrin-like domain and processed posttranslationally. The sequence of the disintegrin-like part of Le-3 is identical to A-chain of the heterodimeric disintegrin VLO5 from Vipera lebetina obtusa venom (Calvete et al., 2003).     

Characterization and amino acid sequences of two lethal peptides isolated from venom of Wagler's pit viper, Trimeresurus wagleri

Two lethal toxins were isolated from Trimeresurus wagleri venom by fast protein liquid chromatography (molecular sieve) and high performance liquid chromatography (reverse phase). The toxins (termed peptide I and II) had mol. wt of 2504 and 2530, respectively, pIs of 9.6-9.9 and lacked phospholipase A, proteolytic, and hemolytic activity. Lethal peptide I had a murine i.p. LD50 of 0.369 mg/kg, while lethal II had a murine i.p. LD50 of 0.583 mg/kg. Peptide I retained full toxicity after autoclaving at 121 degrees C for 40 min. The lethal activity was found to represent less than 1% of the total venom protein, which was only 62-65% of crude venom. The amino acid sequence of peptide I revealed a proline-rich (over 30% of total sequence) sequence unique among snake venom toxins. Lethal peptide II showed the same sequence except for a second tyrosine in the position of histidine (residue No. 10) in peptide I. The toxin lacked antigenic identity with a number of representative neurotoxins and myotoxins. The crude venom shared at least one antigen with Crotalus scutulatus scutulatus venom. This antigen was not Mojave toxin. The toxin appears symptomatologically suggestive of a vasoactive peptide or neurotoxin.     

Isolation and partial characterization of a phospholipase A2 from the venom of Crotalus scutulatus salvini

A phospholipase A₂ (EC 3.1.1.4) has been isolated and purified from the venom of Crotalus scutulatus salvini in 33% yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was in the same range (30,000) whether determined by gel filtration or ultracentrifugation. On polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate and β-mercaptoethanol, the enzyme migrated into a single protein band with a mobility corresponding to about 14,000 molecular weight, indicating that the native enzyme was a dimer. Amino acid composition of the enzyme is reported. Two NH₂-amino acid terminals were found which indicates that the dissociable monomers of the native enzyme were non-identical. Antiserum against the purified enzyme completely inhibited the phospholipase A₂ activity and immunodiffusion of the antiserum against both purified phospholipase A₂ and crude venom of Crotalus scutulatus salvini gave single precipitin lines indicating a lack of contaminating antigens.     

Inhibition of lung tumor colonization and cell migration with the disintegrin crotatroxin 2 isolated from the venom of Crotalus atrox.

Disintegrins are low molecular weight proteins (4-15 kDa) with an RGD binding region at their binding loop. Disintegrin and disintegrin-like proteins are found in the venom of four families of snakes: Atractaspididae, Elapidae, Viperidae, and Colubridae. This report describes the biological activity of a disintegrin, crotatroxin 2, isolated by a three-step chromatography procedure from the venom of the Western diamondback rattlesnake (Crotalus atrox). The intact molecular mass for crotatroxin 2 was 7.384 kDa and 71 amino acids. Crotatroxin 2 inhibited human whole blood platelet aggregation with an IC(50) of 17.5 nM, inhibited cell (66.3p) migration by 63%, and inhibited experimental lung tumor colonization in BALB/c mice at 1000 microg/kg. Our data suggest that while crotatroxin 2 inhibits platelet aggregation, cancer cell migration, and lung tumor colonization, it is done via different integrins.     

Isolation of a hemorrhagic toxin from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom

A hemorrhagic toxin was isolated from Mojave rattlesnake venom. The isoelectric point of the toxin was 4.7 and its mol. wt was 27,000. Concentrations as low as 2 micrograms injected s.c. in mice caused hemorrhage greater than 5 mm in diameter. The toxin was fibrinogenolytic and hydrolyzed hide powder azure, casein and collagen. The toxin also partially inactivated complement. It had no activity against elastin, fibrin, and the chromogenic substrates S-2805, S-2302 and S-2238. Its esterolytic activity was 3% of the activity of the unfractionated venom. The enzymatic and hemorrhagic activities were inhibited by EDTA. The hemorrhagic toxin was absent or in low quantities in Mojave rattlesnake venoms containing Mojave toxin. Chromatography by HPLC easily distinguishes Mojave rattlesnake venoms into two types by the presence or absence of the hemorrhagic toxin.     

Envenomation by the Mojave rattlesnake (Crotalus scutulatus scutulatus) in southern Arizona,U.S.A.

Fifteen cases of envenomation by the Mojave rattlesnake (Crotalus scutulatus scutulatus) are reviewed. Systemic effects were observed in eight patients, consisting of early hypotension (3), decreased plasma fibrinogen (3) and platelets (2), elevated fibrinolytic split products (3) and eyelid ptosis (1). Local venom effects were most common and included swelling (15), ecchymosis (10), bleb formation (6) and necrosis (3). Effects upon neuromuscular transmission were neither common nor a clinical problem. Treatment consisted of i.v. crystalloid solution (15) and antivenin (12).     

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.