Isolation of a novel disintegrin containing Arg-Gly-Asp tripeptide sequence from Gloydius brevicaudus venom and investigation of its biological activities / 药学学报

ABSTRACT

Snake venom has special pharmacological activities and contains a array of small polypeptides that can antagonize integrins, therefore called disintegrins. Disintegrins can block integrin-dependent platelet aggregation, tumor growth, and tumor metastasis. A disintegrin fraction was isolated and purified from the venom of snake Gloydius brevicaudus (GBV). Its physical and chemical properties were characterized, and its biological activities were investigated. The crude venom of GBV were isolated by Superdex 75 gel filtration chromatography. The anti-platelet aggregation activity of the fractions was screened by the Born method. The fraction that shown anti-platelet activity was further purified with Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion exchange chromatography, and Lichrospher C18 reversed-phase chromatography respectively. The purity of the active component was analyzed with SDS-PAGE (Tris-Tricine system) and high-performance liquid-phase chromatography (HPLC), with protein concentration determined by the Bradford method. The molecular weight was evaluated by the gel imaging method and mass spectrometry, and the isoelectric point was measured by disc isoelectric focusing electrophoresis. The protease activity was measured with the Rick method. The phospholipase A activity was determined by the automatic potentiometric titration method. Amino acid sequencing results were subjected to homology comparison using the BLAST program. Seven fractions (Ⅰ-Ⅶ) were isolated from GBV by gel filtration chromatography on Superdex 75 column. The fraction Ⅳ inhibited the platelet aggregation induced by ADP with molecular weight lower than 10 000 Da, suggesting a disintegrin component. A disintegrin named GBV-Ⅳ4 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 shown as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) with molecular weight 8 746 Da as calculated by Image Master VDS system. The isoelectric point of GBV-Ⅳ4 was 6.3 by disc polyacrylamide gel electrophoresis. GBV-Ⅳ4 exhibited no detectable phospholipase A2 (PLA2) activity with the pH-stat technique or proteinase activity according to the method of Rick. GBV-Ⅳ4 is composed of 70 amino acids with RGD (Arg-Gly-Asp) active region and a molecular weight of 7 442 Dalton as assayed by Mass Spectrography. Characterization of GBV-Ⅳ4 is consistent with meta-chain disintegrin (70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank, GBV-Ⅳ4 has high homology with other disintegrins. We concluded that GBV-Ⅳ4 is a novel disintegrin contained RGD. GBV-Ⅳ4 showed dose-dependent inhibition of ADP- or thrombin-induced platelet aggregation with IC50 0.339 or 0.577 μg·mL-1 respectively. In conclusion, a new disintegrin derived from the GBV snake venom and named GBV-Ⅳ4 containing RGD tripeptide sequence could inhibit platelet aggregation.