A Recombinant Chimeric Epidermal Growth Factor-like Module with High Binding Affinity for Integrins

Abstract

Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing ␤ 1 and ␤ 3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with Targeting the appropriate cell type is a major challenge in cancer and cell therapy. Integrins, a family of cell receptors for plasma proteins, extracellular matrix proteins, and cell surface ligands (1-3), are potential targets for this purpose, because they are expressed by all cell types and are involved in many physiological functions including cell adhesion, proliferation, or differentiation, and pathological processes such as metastasis invasion, abnormal angiogenesis and vascularization, or thrombosis (4). In mammals, 18 ␣ and eight ␤ subunits assemble into 24 different non-covalent ␣␤ heterodimers that mediate bidirectional signals through the cell membrane. In response to cell activation, "inside-out" signals control the level of binding affinity for the ligand. In turn, ligand binding induces rearrangements in integrin structure that trigger "outside-in" signaling pathways (5). Extensive cross-talk also takes place between integrin and growth factor receptor signaling pathways (6, 7), influencing most integrin functions.The ␤ 1 integrin subset, comprising the major fibronectin receptor ␣ 5 ␤ 1 , is the most widely expressed. The ␤ 3 subset includes the fibrinogen receptor ␣ IIb ␤ 3 , present on platelets and megakaryocytes, and ␣ v ␤ 3 , originally described as the vitronectin receptor, which also binds a variety of matrix proteins including fibronectin and fibrinogen. Integrin ␣ v ␤ 3 is expressed on vascular cells during angiogenesis (8,9) and is also present in the adult on activated leukocytes, osteoclasts, and macrophages, where it participates in bone resorption and ingestion of apoptotic cells. The expression of various integrins has been shown to be deregulated in a number of cancer cells and invasive tumors and is thought to participate in the malignancy phenotypes (10 -14). Thus, a decreased expression of ␣ 5 ␤ 1 appears to increase the degree of tumorigenicity. In contrast, the level of ␣ v ␤ 3 is correlated with the survival and metastatic activity of tumor cells (10, 15) and has an important role in angiogenesis in tumors (11). Integrins, particularly ␣ v ␤ 3 , are therefore considered as appropriate targets for cancer therapy. Intrac...

Construction of epidermal growth factor fusion protein with cell adhesive activity

“…The improvement of the cell adhesion activities of the EGF module through the use of the RGD sequence was reported by Vella et al [36,41]. Reddy et al [3] reported the enhancement of the mitogenic potency of EGF through the use of specific mutations.…”

Integrin α5β1 Ligands: Biological Evaluation and Conformational Analysis

“…SPR has already been used for the investigation of different integrin–ligand interactions, like the binding between integrins α 1 β 1 and α 2 β 1 and collagen type I, or the interaction between a recombinant chimeric epidermal growth factor‐like module and integrins α 5 β 1 , α v β 3 and α IIb β 3 18. 19 Here we describe for the first time the application of SPR to the analysis of the interaction between fibronectin and integrin α 5 β 1 . SPR usually relies on the application of purified interaction partners.…”