Julio G. Soto

Proteolytic, hemorrhagic and hemolytic activities of snake venoms

Proteolytic, hemorrhagic and hemolytic activities were tested on 47 different venoms from the Crotalidae, Viperidae, Elapidae, and Hydrophiidae families. Antihemorrhagic activity of crude opossum (Didelphis virginiana) and woodrat (Neotoma micropus) serum was tested against the venoms that presented hemorrhagic activity. All venoms showed proteolytic activity when non-specific substrates such as hide powder and collagen were used. Members of the Crotalidae family had the highest hide powder, chymotrypsin-like and hemorrhagic activity. However, members of the Elapidae family had the highest collagen activity. Hemolytic activity was present in 85% of the snake venoms tested. The crude opossum and woodrat serum neutralized the hemorrhagic activity of all the hemorrhagic venoms. Of particular interest is the poor correlation between the venom activities measured here and the phylogenetic position of the snake that possess them. This is particularly true at the genus and species level. Differences in activities were found among individuals of the same genus. The significance of these differences among venoms of closely related snakes is unknown. They do not seem to be adaptive, however little is known of the physiology and habits of most venomous snakes.

Cloning, expression, and hemostatic activities of a disintegrin, r-mojastin 1, from the mohave rattlesnake (Crotalus scutulatus scutulatus).

Interactions with exposed subendothelial extracellular proteins and cellular integrins (endothelial cells, platelets and lymphocytes) can cause alterations in the hemostatic system associated with atherothrombotic processes. Many molecules found in snake venoms induce pathophysiological changes in humans, cause edema, hemorrhage, and necrosis. Disintegrins are low molecular weight, non-enzymatic proteins found in snake venom that mediate changes by binding to integrins of platelets or other cells and prevent binding of the natural ligands such as fibrinogen, fibronectin or vitronectin. Disintegrins are of great biomedical importance due to their binding affinities resulting in the inhibition of platelet aggregation, adhesion of cancer cells, and induction of signal transduction pathways. RT-PCR was used to obtain a 216 bp disintegrin cDNA from a C. s. scutulatus snake venom gland. The cloned recombinant disintegrin called r-mojastin 1 codes for 71 amino acids, including 12 cysteines, and an RGD binding motif. r-Mojastin 1 inhibited platelet adhesion to fibronectin with an IC50 of 58.3 nM and ADP-induced platelet aggregation in whole blood with an IC50 of 46 nM. r-Mojastin 1 was also tested for its ability to inhibit platelet ATP release using PRP resulting with an IC50 of 95.6 nM. MALDI-TOF mass spectrum analysis showed that r-mojastin has a mass of 7.95676 kDa.

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.

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.

Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells

Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220 μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68%+/- 3.09 of treated HUVEC, and 35.86% +/- 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

Regional variation of biochemical characteristics and antigeneity in great basin rattlesnake (Crotalus viridis lutosus) venom

1. Three pooled and 20 individual venom samples of Crotalus viridis lutosus from different localities in Utah and Arizona were screened and fractionated with HPLC-anion exchange. 2. Pooled venom samples and fractions were tested for hemorrhagic, collagenase, and phospholipase activities, and reactivity to a monoclonal antibody against a hemorrhagin from C. atrox venom (CA-P-8) using ELISA. 3. The 20 individual samples were organized into four groups based on their HPLC profiles. 4. ELISA results and specific hemorrhagic activity of the venom samples displayed a variation in latitidinal distribution although from the same species.

Identification of 3'UTR sequence elements and a teloplasm localization motif sufficient for the localization of Hro-twist mRNA to the zygotic animal and vegetal poles.

The early localization of mRNA transcripts is critical in sorting cell fate determinants in the developing embryo. In the glossiphoniid leech, Helobdella robusta, maternal mRNAs, such as Hro‐twist, localize to the zygotic teloplasm. Ten seven nucleotide repeat elements (AAUAAUA) called ARE2 and a predicted secondary structural motif, called teloplasm localization motif (TLM), are present in the 3′UTR of Hro‐twist mRNA. We used site‐directed mutagenesis, deletions, and microinjection of labeled, exogenous transcripts to determine if ARE2 elements, and the TLM, play a role in Hro‐twist mRNA localization. Deleting the poly‐A tail and the cytoplasmic polyadenylation element (CPE) had no effect on Hro‐twist mRNA localization. Site‐directed mutagenesis of nucleotides that altered ARE2 element sequences or the TLM suggest that the ARE2 elements and the TLM are important for Hro‐twist mRNA localization to the teloplasm of pre‐cleavage zygotes. Hro‐Twist protein expression data suggest that the localization of Hro‐twist transcripts in zygotes and stage two embryos is not involved in ensuring mesoderm specification, as Hro‐Twist protein is expressed uniformly in most cells before gastrulation. Our data may support a shared molecular mechanism for leech transcripts that localize to the teloplasm.