Regional differences in content of small basic peptide toxins in the venoms of Crotalus adamanteus and Crotalus horridus.

1. Reverse-phase HPLC and organic solvents were used to isolate small basic peptide (SBP) toxins from the venoms of Crotalus adamanteus, C. durissus terrificus, C. horridus, C. scutulatus scutulatus, C. viridis concolor, C. viridis helleri and C. viridis viridis. 2. Acid-DEP analyses indicated a high degree of toxin purity which was obtained with a single HPLC run. 3. The combined results of HPLC, immunodiffusion and electrophoresis analyses of venoms from different geographical regions indicate that the SBP toxin content in the venoms of Crotalus adamanteus, Crotalus horridus, Crotalus scutulatus and Crotalus viridis viridis may vary regionally.     

Snake venomics of Crotalus tigris: the minimalist toxin arsenal of the deadliest Neartic rattlesnake venom. Evolutionary Clues for generating a pan-specific antivenom against crotalid type II venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.     

Comparative enzymatic study of HPLC-fractionated Crotalus venoms

1. Ten venoms of the genus Crotalus (Crotalus adamanteus, Crotalus atrox, Crotalus durissus durissus, Crotalus horridus horridus, Crotalus lepidus, Crotalus polystictus, Crotalus molossus molossus, Crotalus pusillus, Crotalus scutulatus scutulatus, venom B, and Crotalus viridis lutosus) were fractionated using HPLC anion and cation exchange chromatography. 2. HPLC venom fractions were tested for hemorrhagic, hemolytic, and proteolytic activities. 3. Crude Virginia opossum (Didelphis virginiana) serum neutralized the hemorrhagic activity of HPLC fractions.     

Isolation of two phospholipases A2 from Mojave rattlesnake (Crotalus scutulatus scutulatus) venom and variation of immunologically related venom proteins in different populations

Two phospholipases A2 of mol. wt 14,500 (P1) and 14,400 (P2) and pI 9.2 and 7.4 respectively were isolated from Crotalus scutulatus scutulatus venom. The two isoenzymes cross-reacted immunologically with phospholipase A2 from C. adamanteus and C, atrox, but not with Mojave toxin, excluding them as the basic subunit of the Mojave toxin complex. C. s. scutulatus venoms from Arizona had two common bands recognized by anti-P2 which were absent in most C. s. scutulatus venoms from Texas, suggesting two genetically different populations east and west of the Continental Divide.     

Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus).

A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.     

Disintegrin, hemorrhagic, and proteolytic activities of Mohave rattlesnake, Crotalus scutulatus scutulatus venoms lacking Mojave toxin

Venom from the Mohave rattlesnake, Crotalus scutulatus scutulatus, has been reported to be either: (1) neurotoxic; (2) hemorrhagic, or both (3) neurotoxic and hemorrhagic. In this study, 14 Mohave rattlesnakes from Arizona and Texas (USA) were analyzed for the presence of disintegrins and Mojave toxin. All venom samples were analyzed for the presence of hemorrhagic, proteolytic and disintegrin activities. The venoms were each chromatographed by reverse phase and their fractions tested for disintegrin activity. All specimens containing Mojave toxin were the most toxic and lacked proteolytic, hemorrhagic and disintegrin activities. In contrast, the venoms containing these activities lacked Mojave toxin. Two disintegrin genes, scutustatin and mojavestatin, were identified by PCR of genomic sequences. Scutustatin is a highly conserved disintegrin, while mojavestatin shows low conservation to other known disintegrins. Venoms with the highest LD50 measurements lacked both disintegrin genes, while the specimens with intermediate and low LD50 contained both genes. The intermediate LD50 group contained Mojave toxin and both disintegrin genes, but lacked hemorrhagic and disintegrin activity. Our results raise the possibility that scutustatin and mojavestatin are not expressed in the intermediate LD50 group, or that they may not be the same disintegrins responsible for the disintegrin activity found in the venom. Therefore, it is possible that Mohave rattlesnakes may produce more than two disintegrins.     

Resurrexit, sicut dixit, alleluia. Snake venomics from a 26-year old polyacrylamide focusing gel

A 26-year-old dried polyacrylamide gel, cast in presence of an immobilized pH gradient and containing focused proteins from the venoms of a northern black-tailed rattlesnake (Crotalus molossus molossus), and of a western diamondback rattlesnake (Crotalus atrox) has been screened in order to see the feasibility of extracting the proteins, analyzing them by mass spectrometry (MS) and assessing their integrity. Nine gel bands were excised along the pH 3-10 gradient and the gel segments reswollen in warm acetonitrile. Upon digestion and MS analysis, all the bands could be identified and attributed to the respective venoms of the two rattlesnake species. Although a few peptides exhibited modified amino acids, the proteins were found to be well preserved even upon such a long storage at room temperature. The present data suggest the feasibility of identifying proteins from very old samples trapped in polyacrylamide gels, and analyzed in a pre-mass spectrometry era, thus of uncertain identity.     

Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona

Twenty-one Mojave rattlesnakes, Crotalus scutulatus scutulatus (C. s. scutulatus), were collected from Arizona and New Mexico U.S.A. Venom proteome of each specimen was analyzed using reverse-phase HPLC and SDS-PAGE. The toxicity of venoms was analyzed using lethal dose 50 (LD(50)). Health severity outcomes between two Arizona counties U.S.A., Pima and Cochise, were determined by retrospective chart review of the Arizona Poison and Drug Information Center (APDIC) database between the years of 2002 and 2009. Six phenotypes (A-F) were identified based on three venom protein families; Mojave toxin, snake venom metalloproteinases PI and PIII (SVMP), and myotoxin-A. Venom changed geographically from SVMP-rich to Mojave toxin-rich phenotypes as you move from south central to southeastern Arizona. Phenotypes containing myotoxin-A were only found in the transitional zone between the SVMP and Mojave toxin phenotypes. Venom samples containing the largest amounts of SVMP or Mojave toxin had the highest and lowest LD(50s), respectively. There was a significant difference when comparing the presence of neurotoxic effects between Pima and Cochise counties (p=0.001). No significant difference was found when comparing severity (p=0.32), number of antivenom vials administered (p=0.17), days spent in a health care facility (p=0.23) or envenomation per 100,000 population (p=0.06). Although not part of the original data to be collected, death and intubations, were also noted. There is a 10× increased risk of death and a 50× increased risk of intubations if envenomated in Cochise County.     

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus)

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.     

Molecular evolution of PIII-SVMP and RGD disintegrin genes from the genus Crotalus

Several types of disintegrins have been isolated from Crotalus spp rattlesnakes, including RGD disintegrins, and PIII-SVMPs. We isolated six cDNAs from snake venom glands using RT-PCR. Three RGD disintegrins (atroxatin, mojastin, and viridistatin) and three PIII-SVMPs (catroriarin, scutiarin, and viristiarin) cDNAs were isolated from the rattlesnakes Crotalus atrox, Crotalus scutulatus scutulatus, and Crotalus viridis viridis, respectively. Atroxatin and Viridistatin shared 90% amino acid identity to each other, and 87% identity to Mojastin. Scutiarin and Viristiarin were identical. All PIII-SVMPs isolated in this study shared the highest amino acid identity with Catrocollastatin. cDNA and protein sequences for RGD disintegrins, one MVD disintegrin, and PIII-SVMPs of the genus Crotalus (present in the NCBI database), were used in phylogenetic analysis. Neighbor-joining analysis of PIII-SVMP and RGD/MVD disintegrin-coding DNA sequences showed that these groups of genes separate into separate clades. A Phi(ST) pairwise comparison and Analysis of Molecular Variance (AMOVA) between PIII-SVMPs and RGD/MVD disintegrins showed significant genetic differences. Mutations observed in ten of the cDNAs analyzed did not affect Cys-coding sequences. Our K(A)/K(S) data suggest that rapid evolution occurred between the genes coding for PIII-SVMPs resulting, in the production of RGD disintegrin-coding genes. However, once these genes diverged, mutations in the PIII-SVMP-coding genes were accumulated less frequently.     

Soluble polymer-based isotopic labeling (SoPIL): a new strategy to discover protein biomarkers?

Much attention has been given to protein biomarker discovery in the field of proteomics in the past few years. Proteomic strategies for biomarker discovery normally include the identification of proteins that alter during the progression of a particular disease state in high throughput. To perform these studies requires the ability to measure changes of low-abundance proteins in highly complex mixtures from different biological states. Soluble polymer-based isotope labeling (SoPIL) is a new proteomics strategy that targets specific classes of proteins for isotopic labeling, efficient isolation and accurate quantitation by mass spectrometry. The method exploits the features of homogenous solution-phase reaction, simple solid-phase extraction and characteristic cell-permeable nanoparticles. Recent applications demonstrate that the SoPIL reagents are ideal for quantitative proteomics and phosphoproteomics, and could have the potential to discover disease markers in the most physiologically relevant settings.     

Soluble polymer-based isotopic labeling (SoPIL): a new strategy to discover protein biomarkers?

Much attention has been given to protein biomarker discovery in the field of proteomics in the past few years. Proteomic strategies for biomarker discovery normally include the identification of proteins that alter during the progression of a particular disease state in high throughput. To perform these studies requires the ability to measure changes of low-abundance proteins in highly complex mixtures from different biological states. Soluble polymer-based isotope labeling (SoPIL) is a new proteomics strategy that targets specific classes of proteins for isotopic labeling, efficient isolation and accurate quantitation by mass spectrometry. The method exploits the features of homogenous solution-phase reaction, simple solid-phase extraction and characteristic cell-permeable nanoparticles. Recent applications demonstrate that the SoPIL reagents are ideal for quantitative proteomics and phosphoproteomics, and could have the potential to discover disease markers in the most physiologically relevant settings.     

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.     

Snake venom dipeptidyl peptidase IV: taxonomic distribution and quantitative variation

The present study examined the taxonomic distribution of dipeptidyl peptidase IV (DPP IV) activity in venoms of 59 ophidian taxa, representing seven subfamilies of the Families Elapidae and Viperidae. DPP IV activity is extremely variable at all taxonomic levels. It ranged from essentially none in laticaudine, hydrophiine, and some bungarine and elapine venoms, to 10.72 μmol 4-methoxy-β-naphthylamine liberated per min per 200 μg venom, for Ophiophagus hannah. Intra- and interpopulational variation were examined among eight populations of prairie rattlesnakes (Crotalus viridis viridis), Great Basin rattlesnakes (Crotalus viridis lutosus) and southern Pacific rattlesnakes (Crotalus viridis helleri). Among these populations, the mean weighted range of variation was 4.9-fold, and even among litter mates of C. v. lutosus, DPP IV activity varied as much as 5.6-fold. The two most salient findings, the near ubiquity of DPP IV in snake venoms and its great quantitative variability, even among full siblings, are paradoxical. The widespread distribution of the enzyme suggests an important role in envenomation, while the variable activity levels suggest that DPP IV and by extension, other individual enzymatic constituents, may not be under much individual selective pressure.     

A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.     

The amino acid sequence of the acidic subunit B-chain of crotoxin

The B-chain of the acidic subunit of crotoxin proved refractory to Edman degradation. When subjected to sequence analysis using tandem mass spectrometry, pyroglutamate was found at the amino-terminal end, even though earlier attempts to de-block with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity with the corresponding segment from Mojave toxin, a homologous neurotoxin from Crotalus scutulatus scutulatus. The sequence of the last 24 residues of the B-chain is consistent with that previously published (Aird, S.D., Kaiser, I.I., Lewis, R.V. and Kruggel, W.G. (1985) Biochemistry 24, 7054-7058), except at position 20, where Edman degradation gave glycine and mass spectrometry gave glutamic acid.     

A comparative study of the biological activities of rattlesnake (genera Crotalus and Sistrurus) venoms.

1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and L-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.     

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.     

Wide distribution of cysteine-rich secretory proteins in snake venoms: isolation and cloning of novel snake venom cysteine-rich secretory proteins

Cysteine-rich secretory proteins (CRISPs) are found in epididymis and granules of mammals, and they are thought to function in sperm maturation and in the immune system. Recently, we isolated and obtained clones for novel snake venom proteins that are classified as CRISP family proteins. To elucidate the distribution of snake venom CRISP family proteins, we evaluated a wide range of venoms for immuno-cross-reactivity. Then we isolated, characterized, and cloned genes for three novel CRISP family proteins (piscivorin, ophanin, and catrin) from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus), king cobra (Ophiophagus hannah), and western diamondback rattlesnake (Crotalus atrox). Our results show the wide distribution of snake venom CRISP family proteins among Viperidae and Elapidae from different continents, indicating that CRISP family proteins compose a new group of snake venom proteins.     

The reproductive ecology of Mohave rattlesnakes

The reproductive ecology of Mohave rattlesnakes Crotalus scutulatus was investigated in the western Mohave Desert using radiotelemetry from August 2001 to November 2004. This paper documents reproductive behavior across successive seasons in the context of seasonal timing, mean daily movement, home range, body temperature and relationship with abiotic factors such as time of day, temperature, precipitation, photoperiod and microhabitat. This population of C. scutulatus used a bimodal mating system, with reproductive behavior occurring in late summer/fall (21 August to 7 October), interrupted by 4 months of cold weather, and concluding in the spring (16 March to 16 May). Drought apparently curtailed courtship and copulation during the 2002 activity season, but the pregnancy rates in 2002 and 2003 were not significantly affected. Communal denning was not detected and there was no indication of seasonal migration. Autumnal and vernal movements appeared to be driven by reproductive effort, predominantly males engaged in prolonged mate searching. Sexual maturity was achieved at 2.0 years/600 mm snout–vent length (SVL) for females, and 1.5 years/400 mm SVL for males.