Covalent immobilization of recombinant human alphavbeta3 integrin on a solid support with retention of functionality

Alphavbeta3 is the major receptor mediating the attachment of osteoclasts to bone surface and plays a critical role in bone resorption and remodeling. Interfering with alphavbeta3 binding inhibits osteoclast-mediated bone resorption, and thus demonstrates the potential utility of alphavbeta3 antagonists for therapy of osteoporosis. This report describes the generation of an alphavbeta3 affinity column which was created to enable screening of collections of large numbers of ligands, e.g., combinatorial libraries (previously prepared by us), to sort and identify ligands with the highest affinity for alphavbeta3. We demonstrate that covalent immobilization of the heterodimeric alphavbeta3 receptor can be achieved with retention of characteristic ligand binding properties. Human alphavbeta3 was isolated from human embryonic kidney cells (HEK 293) that stably express high levels of the recombinant receptor and then affinity purified to homogeneity. Purified alphavbeta3 receptor was linked covalently to activated CH-Sepharose 4B beads. Quantification of immobilized functional receptor was determined by Scatchard analysis. The immobilized functional receptor maintains binding properties similar to the membrane-embedded and soluble receptor. The immobilized receptor also can be used to select the highest affinity ligand from among a mixture of peptides which differ in their binding affinity, structure, and hydrophobicity, both when the peptides are loaded in equimolar concentrations in a mixture and when the concentration of the highest affinity ligand is reduced 10-fold. Liquid chromatography-mass spectrometry was utilized to confirm selective ligand binding and to demonstrate that preferential binding was not due to nonspecific hydrophobic interactions with immobilized alphavbeta3 receptor or the affinity column. This approach may be of general use for affinity-based screening of ligands for other integrins and should enable practical screening of combinatorial libraries containing large numbers of potential ligands for the human alphavbeta3 integrin receptor, including linear peptides, cyclic peptides, and peptidomimetics.