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

Abstract

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 7.9509 kDa.

Anti-angiogenic activities of two recombinant disintegrins derived from the Mohave and Prairie rattlesnakes

“…r-Viridistatin 2 in our study is a monomeric, medium size disintegrin with 73 amino acids, and an RGDNP-motif region derived from the Prairie rattlesnake (Lucena et al, 2012). r-Mojastin 1 is a monomeric, medium size disintegrin with 71 amino acids, twelve cysteines and an RGDW-motif region derived from the Mohave rattlesnake (Sánchez et al, 2010) (Fig. 7).…”

“…Recombinant mojastin 1 and recombinant viridistatin 2 were expressed in E. coli and further purified by two-step chromatography using the method of Sánchez et al (2010)and Lucena et al (2012), respectively.…”

“…Recombinant disintegrin mojastin 1 (r-mojastin 1) derived from Crotalus scutulatus scutulatus , expressed using a prokaryotic host expression system in Escherichia coli BL21 cells, was shown to be highly active in inhibiting ADP-induced platelet aggregation using platelet-rich plasma and whole blood, platelet ATP release, and platelet adhesion to fibronectin (Sánchez et al, 2010). r-Mojastin 1 was also tested for its ability to inhibit some cellular function such as adhesion to extracellular matrices, migration and invasion in vitro and in vivo , using human urinary bladder carcinoma (T24), skin melanoma (SK-MEL-28), and murine melanoma (B16F10) cells (Lucena et al, 2011).…”

Functional characterizations of venom phenotypes in the eastern diamondback rattlesnake (Crotalus adamanteus) and evidence for expression-driven divergence in toxic activities among populations

“…We followed the approach of Suntravat et al (2010) and Sánchez et al (2010) to test the effects of each pooled venom on the coagulation cascade. Pooled normal citrated plasma was prepared by drawing a blood sample from M.J.M; 40 mL of blood was collected by gravity into a 50 mL plastic test tube containing 4 mL of 3.8% sodium citrate to prevent coagulation.…”

Inhibition of pancreatic tumoral cells by snake venom disintegrins

“…r-Mojastin 1 and r-viridistatin 2 were expressed in E.coli and further purified by two-step chromatography, using the method of Sánchez et al (2010) and Lucena et al (2012), respectively. Briefly, E. coli BL21 cells were grown, induced by 0.5 mM of isopropyl β-D thiogalactoside (IPTG) and centrifuged.…”