Recombinant expression, and functional characterization of the non-catalytic domains of a P-III class metalloproteinase from Bothrops jararaca snake venom, using a cell-free expression system

Abstract

Proteases are major components of viperid snake venom proteomes. Among these, Snake Venom Metalloproteinases (SVMPs) are especially abundant components and are responsible for various pathological effects following envenomation. The SVMPs contain regulatory domains in additional to the catalytic domain which are involved in their mechanisms of interaction with the extracellular matrix proteins and integrins. Hemorrhagic factor 3 (HF3) is a highly hemorrhagic metalloproteinase isolated from the venom of Bothrops jararaca, and is comprised of a catalytic domain, a disintegrin-like domain (D), a cysteine-rich domain (C), and five putative N-glycosylation sites (three sites in the catalytic domain and two sites in the cysteine-rich domain). Previous studies on the role of these domains in the biological activity of HF3 revealed that recombinant proteins containing the cysteine-rich domain, obtained in E. coli in fusion with GST (Gluthationa S-transferase) (GST-DC- and GST-C), were able to inhibit collagen-induced platelet aggregation, and to induce pro-inflammatory response in mice. These activities were inhibited by integrin antibodies (anti-±M and anti-²2), suggesting a role for the C-domain in triggering the integrin-mediated pro-inflammatory effects of HF3. Analysis of the interaction of HF3 with extracellular matrix proteins and human plasma showed that HF3 interacts with collagen types I and VI, fibrinogen, fibronectin and laminin, which are important for maintaining the integrity of the capillaries and hemostasis. In this project our objective is obtain the recombinant protein DC/HF3 (wild-type) and containing site-directed mutations in the hyper-variable region (HVR) of the cysteine-rich domain (basic residues: K568A, K569A and K575A; acid residues: D573A, E579A, D580A, and D582A) using a cell-free synthesis system derived from E. coli to identify amino acid residues important for the interaction of HF3 with its substrates. The functional activity of the recombinant proteins will be evaluated by solid-phase binding assays, and by platelet-aggregation assays. The results of this study should advance knowledge on the interaction between metalloproteinases and their molecular targets. (AU)