title: Expression, purification, and analysis of three recombinant ECD disintegrins (r-colombistatins) from P-III class snake venom metalloproteinases affecting platelet aggregation and SK-MEL-28 cell adhesion.
abstract: Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases.
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.
authors.forename: Montamas
authors.initials: M
authors.lastname: Suntravat
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Helmke
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.
authors.forename: Chairat
authors.initials: C
authors.lastname: Atphaisit
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.
authors.forename: Esteban
authors.initials: E
authors.lastname: Cuevas
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Lucena
authors.affiliation: Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas 1041, Venezuela.
authors.forename: Nestor L
authors.initials: NL
authors.lastname: Uzcátegui
authors.affiliation: National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA; Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA. Electronic address: elda.sanchez@tamuk.edu.
authors.forename: Elda E
authors.initials: EE
authors.lastname: Sánchez
authors.affiliation: Laboratorio de Inmunoquímica y Ultraestructura, Instituto Anatómico de la Universidad Central de Venezuela, Caracas 1041, Venezuela.
authors.forename: Alexis
authors.initials: A
authors.lastname: Rodriguez-Acosta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D003435:Crotalid Venoms / Q000201:enzymology*; D019483:Disintegrins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D045726:Metalloproteases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
chemical_list: D003435:Crotalid Venoms; D019483:Disintegrins; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D045726:Metalloproteases
keywords: Bothrops colombiensis; Colombistatins; ECD disintegrin-like domain; SK-Mel-28; Tumor