Cloning And Characterization Of Adinbitor, A Novel Disintegrin From The Snake Venom Of Agkistrodon Halys Brevicaudus Stejneger

Disintegrins are a family of small proteins mainly derived from snake venoms. Most of the disintegrins contain RGD or KGD sequence which is the structural motif recognized by the platelet fibrinogen receptor αIIbβ3, and also act as potent antagonists of several integrins including αVβ3 and α5β1 which are expressed on vascular endothelial cells and some tumor cells. In addition to their potential antiplatelet activity, studies of disintegrins such as Rhodostomin[1], Accutin[2], Salmosin[3] [4], Saxatilin[5] and Contortrostatin[6] etc., have revealed a new application in inhibiting angiogenesis and tumor growth [1-9]. Because disintegrins can inhibit the binding of ECM to integrins according to the RGD or KGD sequence, their signaling pathways are close related with integrins' pathways.Integrins are a family of heterodimeric cell surface receptors. The known 18 α and 8 β subunits combine to form at least 24 αβ heterodimers[10- 12]. Most cells express more than one type of integrin heterodimer. Integrin expression profiles are unique for distinct cell types, and change with development stage and physiological conditions within a cell type. The common binding site of ligands is composed of the N-terminal of α and β subunits. Because most of cell integrins recognize RGD sequence and most of ECM molecules contain the sequence RGD, the RGD sequence has been already considered as the binding site of ECM molecules to integrins [21]. Although the intracellular part of integrins is much shorter, they can form links to the cytoskeleton through α-actinin, talin and vinculin.At sites of integrin activation and clustering, proteins aggregate termed focal complexes and focal adhesions assemble on the intracellular surface. These complexes are the sites where the cell can generate tension with respect to its surroundings, allowing the cell to alter its shape and carry out complex processes such as migration and cell division [10].The signaling proteins found at focal complexes and focal adhesions associate with integrin cytoplasmic domains to form a nexus for stimulating intracellular signaling cascades. At these sites, signaling from outside to inside the cell occurs, which contributes to the regulation of diverse cellular processes including entry into the cell cycle, apoptosis, gene transcription, regulation of intracellular pH, differentiation, and migration. Signaling through integrins from the inside to the outside of the cell regulates adhesive strength through affinity and avidity modulation, and helps in the remodeling of the ECM during tissue development and tissue invasion [13].The 125kD kinase termed focal adhesion kinase (FAK) is the best characterized of the integrin-associated signaling proteins. FAK binds to integrin receptors and plays a central role in assembling complexes of signaling proteins at the cell surface.FAK has five tyrosine phosphorylation sites (Tyr397,407, 576, 577, 681, and 925) [14]. Tyr397 is an autophosphorylation site that generates a high-affinity binding site for the SH2 domain of Src-family NRPTKs. The interaction of Src with the FAK autophosphorylation site then leads to phosphorylation of Tyr407, 576 and 577, which maximizes the kinase activity of FAK in vitro. Interaction with Src also results in the phosphorylation of Tyr925 on FAK, which creates a Grb2-binding site and therefore links FAK to the Ras/mitogen-activated protein kinase (MAPK) pathway [15]. Tyr681 is an autophosphorylation site of Src[16]. These data suggest that phosphorylated FAK can bind and integrate multiple signaling pathways in response to signals from the external environment. FAK is also implicated in controlling cell cycle progression and preventing apoptosis through a pathway involving protein kinase C, phospholipase A2, and p53 [12, 17].Referenced as the reports about the bioactivities and functional principles of disintegrins, we studied the disintegrin from Chinese snake (Agkistrodon halys brevicaudus stejneger). Adinbitor is cloned from Agkistrodon halys brevicaudus stejne...