title: cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects.
abstract: A 5' truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5'-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, Crotalus atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC(50) of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC(50)s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders.
authors: National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX 78363, USA. montamas@hotmail.com
authors: Montamas
authors: M
authors: Suntravat
authors: Ying
authors: Y
authors: Jia
authors: Sara E
authors: SE
authors: Lucena
authors: Elda E
authors: EE
authors: Sánchez
authors: John C
authors: JC
authors: Pérez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000187:drug effects; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003435:Crotalid Venoms / Q000737:chemistry / Q000201:enzymology*; D017839:Crotalus / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D019483:Disintegrins / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology; D015723:Gene Library; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013045:Species Specificity
chemical_list: D003435:Crotalid Venoms; D018076:DNA, Complementary; D019483:Disintegrins; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D045726:Metalloproteases