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.

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.

Gene expression profiling of the venom gland from the Venezuelan mapanare (Bothrops colombiensis) using expressed sequence tags (ESTs).

BACKGROUND: Bothrops colombiensis is a highly dangerous pit viper and responsible for over 70% of snakebites in Venezuela. Although the composition in B. colombiensis venom has been identified using a proteome analysis, the venom gland transcriptome is currently lacking. RESULTS: We constructed a cDNA library from the venom gland of B. colombiensis, and a set of 729 high quality expressed sequence tags (ESTs) was identified. A total number of 344 ESTs (47.2% of total ESTs) was related to toxins. The most abundant toxin transcripts were metalloproteinases (37.5%), phospholipases A2s (PLA2, 29.7%), and serine proteinases (11.9%). Minor toxin transcripts were linked to waprins (5.5%), C-type lectins (4.1%), ATPases (2.9%), cysteine-rich secretory proteins (CRISP, 2.3%), snake venom vascular endothelium growth factors (svVEGF, 2.3%), L-amino acid oxidases (2%), and other putative toxins (1.7%). While 160 ESTs (22% of total ESTs) coded for translation proteins, regulatory proteins, ribosomal proteins, elongation factors, release factors, metabolic proteins, and immune response proteins. Other proteins detected in the transcriptome (87 ESTs, 11.9% of total ESTs) were undescribed proteins with unknown functions. The remaining 138 (18.9%) cDNAs had no match with known GenBank accessions. CONCLUSION: This study represents the analysis of transcript expressions and provides a physical resource of unique genes for further study of gene function and the development of novel molecules for medical applications.