Evaluation of the biological activity of the disintegrin-like and cysteine-rich domains of Bothropasin, a metalloproteinase from Bothrops jararaca

Abstract

Bothropasin is a 48kDa hemorrhagic class III Snake Venom Metalloproteinase (SVMP). As a multi-domain protein, it contains catalytic metalloproteinase (M), disintegrin-like (D), and cysteine rich domains (C). Integrins belong to the superfamily of transmembrane cell adhesion receptor that interact and bind to soluble ligands, cell-surface ligands, and extracellular matrix ligands such as collagen. The integrin-binding motif of bothropasin unlike the P-II members (that contain RGD-motif) is an ECD tripeptide. It was previously reported that cysteine (C) and aspartate (D) of the ECD were responsible for disulfide formation and calcium interaction respectively, while the glutamate (E) was found exposed to solvent and free to make interaction. Thus, we propose that (E) may also be responsible for integrin binding. Furthermore, in the current project, we intend to further ascertain the role of glutamate in integrin binding via the ability of this protein to inhibit adhesion of HUVECs (cell lines that can also express α2β1 integrin) to collagen1-coated wells. Previously in our group two clones were built to express the bothropasin DC domains (BDC) containing ECD motif (BDC-ECD protein) and the mutant ACD (BDC-ACD protein). The clones were transformed in E. coli (BL21(DE3) and DH5α) and the expression was induced by IPTG. The soluble fractions of the proteins were purified with affinity chromatography, analyzed with 15 % SDS-PAGE and western blotting; and we investigated the effect of glutamate mutation on the ability of these proteins (in fusion with GST) to inhibit cell adhesion. The result showed that the proteins did not inhibit adhesion of HUVECs to collagen 1. Although the result indicate that pretreatment of HUVEC cells with GST GST only did not affect protein activities, there is a possibility that GST in fusion with this protein could interfere with protein native folding and culminate in protein inactivities. The current data thus suggest the use of proteins free from GST for further studies.