Purification, Characterization And Biological Activities Of A Disintegrin From Gloydius Brevicaudus Venom

Disintegrin which originate of snake and leech venom ,contains the amino acid sequence Arg-Gly-Asp(RGD) or Lys-Gly-Asp(KGD). RGD is the common sequence which exists in extracellular matrix(ECM),as fibrinogen,fibronectin,thrombospondin,osteopontin and von willebrand factor(vWF) etc, has been implicated as a recognition site in interactions between integrin and its ligands.It exhibit a wide array of biological activities, such as inhibit platelet aggregation, angiogenesis and tumor migration.This study was designed to isolate and purify a disintegrin from Gloydius brevicaudus venom and analyse its characterization and biological activities.1.Purification and characterization of the disintegrin from Gloydius brevicaudus Venom(Gbv)Seven fractions(Ⅰ～Ⅶ) were isolated from Gbv by Gel filtration chromatography on Superdex 75 colum . The fractionⅣinhibited the platelet aggregations induced by ADP with molecular weight lower than 10000 dalton,so it suggested as a contained disintegrin component. A disintegrin named GbvⅣ4 was purified from the fraction by Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE,Tris-Tricine system) with molecular weight 8746 dalton as calculated by Image Master VDS system.The isoelectric point of GbvⅣ4 was pI 6.3 by disc polyacrylamide gel eletrophoresis. GbvⅣ4 exhibited no detectable phospholipase A2(PLA2)activity with the pH-stat technique and proteinase activity according to the method of Rick. GbvⅣ4 is composed of 70 amino acid sequence with RGD active region and molecular weight 7442 dalton as assayed by Mass Spectrography. Characteration of GbvⅣ4 is consistent with meta-chain disintegrin's(70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank , GbvⅣ4 has high homology with other disintegrins .Above all,we conclude that GbvⅣ4 is a new kind of disintegrn.2. The effects of GbvⅣ4 on the human platelet aggregation.According to the Born's method, the effects of GbvⅣ4 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that GbvⅣ4 could dose-dependently inhibite ADP- and thrombin-induced platelet aggregation with IC50 0.339 and 0.577μg/ml respectively.3.The effect of GbvⅣ4 on the proliferation of human umbilical vein ECV304 cellsThe inhibition of GbvⅣ4 on cultured ECV304 cells was observed and IC50 value was assayed by SRB method.The result showed that GbvⅣ4 inhibited the proliferation of ECV304 cells in a dose-dependent manner with IC50(24h) 25.42μg/ml. Furthermore,The ECV304 cells undergo remarkable morphological change by treatment with GbvⅣ4:the cells retracted ,turn to ground, formed gaps and multicelluar aggregation.4.The effect of GbvⅣ4 on angiogenesis of chick chorioallantoic membrane(CAM)The antiangiogenesis effect in vivo of GbvⅣ4 on CAM assay was observed and the the ratio of area of blood vessel and area of CAM in measure area (VA/CAM) was used to asses the inhibition.The result showed that GbvⅣ4(3.098,11.79μg)inhibited the angiogenesis in vivo: the morphology of blood vessels on CAM were basically normal,but the blood vessels distributed scarcely .The VA/CAM was less than control group significantly.5. The effects of GbvⅣ4 on B16 cells'adhesive capability to fibronection (Fn)Cell-matrix adhesion assay was used to study the adhesive ability of B16 cells.With phase microscope observation we found the B16 cells looked like epidermoid cells and were polygonal in shape with the typical cobblestone growing pattern, but the B16 cells treated with GbvⅣ4 undergo remarkable morphological change :the cells retracted ,turn to ground,formed gaps and multicelluar aggregation. According to MTT method, different concentration of GbvⅣ4 showed inhibitory effects, and the inhibited effects was dose dependent, the inhibition ratio was 59.34% at the concentration of 16μg/ml.6. The effects of GbvⅣ4 on B16 cells'migration on Fibronection.The migration of B16 cells was observed by erasion trace test.The results showed that GbvⅣ4 could inhibit B16 cells from moving on Fibronection effectively.7. The effects of GbvⅣ4 on B16 and B16-F10 cells'invasion on MatrigelTranswell chamber assay was applied to measure the effect of GbvⅣ4 on invasion of B16 and B16-F10 cells. After 48 h, the invasion of B16 and B16-F10 cells was less than those in control groups, and the number of invasive cells was dose dependent.8. Inhibitory effects of GbvIV on metastases of mouse B16 melanomaThe effects of GbvIV on metatasis were studied by using an experimental metastasis model of mouse BI6-F10 melanoma cell line In mice that received i.v.injection of mouse B 16-F10 melalloma cells,daily i.p.GbvIV remakably reduced lung weight, and number of lung metastases.Lung metastasis inhibition rates of GbvIV(50,100μg/ml) and cyclophosphamide(50 mg/kg) on B16 -F10 melanoma cells were 67.8% ,82.8% ,and 48.1% ,respectively.ConclusionsA disintegrin GbvⅣ4 was purified from Gloydius brevicaudus venom with molecular weight 7442 dalton and pI 6.3.It is composed of 70 amino acid sequence with RGD active region . It was a potent platelet aggregation inhibitor. GbvⅣ4 inhibited proliferation of human umbilical vein endothelial cells and antiangiogenesis of CAM. GbvⅣ4 has strong activity of anti-tumor cell adhesion,migration,invasion and metastases.