Effects Of Adinbitor, A Novel Disintegrin From The Snake Venom Of Gloydius Blomhoffi Brevicaudus, On Integrin α_vβ₃-mediated Signaling Pathway

Snake venom is one of the most complicated mixture of functional proteins in nature. 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 receptorαIIbβ3, and also act as potent antagonists of several integrins includingαVβ3 andα5β1 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].Integrins are a family of heterodimeric receptors on cell surface. The known 18αand 8βsubunits combine to form at least 24αβheterodimers[11-12]. Most cells express more than one type of integrin heterodimer. Integrin expression profiles are unique for distinct cell types, and change with development stages and physiological conditions within a cell type. The common binding site of ligands is composed of the N-terminal ofαandβsubunits. 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 [13]. Although the intracellular part of integrins is much shorter, they can form links to the cytoskeleton throughα-actinin, talin and vinculin. Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling playessential roles in cell adhesion, migration, and morphogenesis during cancer metastasis[14]. Interestingly, IntegrinαVβ3 mediates signaling, which contributes to cancer development. Researchers suggested that integrinαVβ3 is an important target for the integrin-dependent regulation of cancer development. At sites of integrin activation and clustering, proteins aggregate into so called focal complexes and focal adhesions assemble on the intracellular surface. These complexes are the sites where the cell can generate tension in response to its surroundings, allowing the cell to alter its shape and carry out complex processes such as migration and cell division [10].Integrin signaling pathway is activated by tyrosine phosphorylation, which triggers a intracellular signaling cascades. Integrin ligand binding leads to the formation of focal adhesions where integrins link the ECM to intracellular cytoskeletal complexes and signaling proteins. These proteins such as FAK, Ras, MEK, Erk, JNK, p38MAPK and so on play important roles in stabilizing cell adhesion and regulating cell shape and motility. Focal adhesion kinase (FAK) is the best characterized integrin-associated signaling proteins. FAK binds to integrin receptors and plays a central role in assembling complexes of signaling proteins at the cell surface[15]. IntegrinαVβ3 also mediates cell cycle, apoptosis via activating integrin linked kinase (ILK)[16].FAK has six tyrosine phosphorylation sites (Tyr397, 407, 576, 577, 681 and 925), which play a critical role in signaling pathway[17]. Stimulated integrin receptor induces an autophosphorylation of FAK, which is then capable of stimulating signaling pathways through son of sevenless protein (SOS), Ras and Raf proteins, and subsequently mitogen-activated protein kinases (MAPK), extracellular-signal regulated kinase 1(ERK1), and kinase 2(ERK2)[18].Obviously,the inhibition of integrin signaling pathway may act on cell adhesion and signaling. Disintegrin from snake venom have been used as integrin antagonist in the fields of tumor biology and angiogenesis. Referenced as the reports about the bioactivities and functional principles of disintegrins, we studied the disintegrin from the snake (Gloydius blomhoffi brevicaudus) in Dalian.Adinbitor was cloned from Gloydius blomhoffi brevicaudus and characterized as a novel disintegrin. In this study, we designed two oligonucleotide primers. Total RNA was extracted from venom gland and cDNA with 219 bp long was produced by RT-PCR. The encoded protein composed of 73 amino acids including 12 cysteines and an RGD motif, the signature motif of disintegrin. Recombinant Adinbitor (rAdinbitor) was expressed in E.coli and purified with His-Bind affinity chromatography.According to the results above, in this study, we investigated the molecular mechanism of Adinbitor action. There were some studies we operated shown below.1. The expression of Adinbitor in E.coli was induced by IPTG, the target protein was purified by using the His-Bind affinity chromatography, and the 9KD soluble homogeneous protein was obtained.2. rAdinbitor could dose-dependently inhibited the basic fibroblast growth factor (bFGF) induced proliferation of C6 glioma cells, and the median inhibitory concentration (IC50) was 2.75μmol/L; Studies also revealed that rAdinbitor could act on integrinαVβ3 where the action of rAdinbitor was similar to anti-integrinαVβ3 antibody, and could significantly induce C6 glioma cells apoptosis through caspase-3 signaling pathway.3. Investigation of rAdinbitor on expression and phosphorylation of FAK, Ras, MEK, and ERK revealed that rAdinbitor could significantly inhibit the expression of FAK, MEK and ERK, but intend to promote the expression of Ras. These indicated that rAdinbitor could inhibit integrin's signal transduction. rAdinbitor could inhibit block sites of ECM to integrin and focal complexes formation, and induce a series of signaling reactions about apoptosis.In a word, we obtained purified rAdinbitor, and did researches on the molecular mechanism of rAdinbitor on Ras-MAPK signaling pathway. All the results indicated that rAdinbitor as a novel member of disintegrins would be valuable for developing clinical antithrombotic and anti-tumor agents.