title: Multiple low-affinity interactions support binding of human osteopontin to integrin αXβ2.
abstract: Integrin α(X)β(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)β(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)β(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)β(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)β(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)β(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)β(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)β(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)β(2), which seem to differ in principles considerably from other OPN receptors.
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark; Dept. of Biomedicine, Denmark.
authors.forename: Eva
authors.initials: E
authors.lastname: Kläning
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark.
authors.forename: Goran
authors.initials: G
authors.lastname: Bajic
authors.affiliation: Institute for Storage Ring Facilities Aarhus (ISA), Dept. of Physics and Astronomy & Center for Storage Ring Facilities Aarhus, Denmark.
authors.forename: Søren V
authors.initials: SV
authors.lastname: Hoffmann
authors.affiliation: Institute for Storage Ring Facilities Aarhus (ISA), Dept. of Physics and Astronomy & Center for Storage Ring Facilities Aarhus, Denmark.
authors.forename: Nykola C
authors.initials: NC
authors.lastname: Jones
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark.
authors.forename: Morten M
authors.initials: MM
authors.lastname: Callesen
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark.
authors.forename: Gregers R
authors.initials: GR
authors.lastname: Andersen
authors.affiliation: Dept. of Molecular Biology and Genetics Aarhus University, Aarhus, Denmark; Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
authors.affiliation: Dept. of Biomedicine, Denmark; Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus Denmark; MEMBRANES Research Center, Aarhus University, Aarhus, Denmark. Electronic address: vorup-jensen@biomed.au.dk.
authors.forename: Thomas
authors.initials: T
authors.lastname: Vorup-Jensen
mesh_terms: D020816:Amino Acid Motifs; D002448:Cell Adhesion; D006801:Humans; D016167:Integrin alphaXbeta2 / Q000737:chemistry* / Q000378:metabolism; D007962:Leukocytes / Q000737:chemistry / Q000378:metabolism; D053495:Osteopontin / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D016167:Integrin alphaXbeta2; D053495:Osteopontin
keywords: Cell adhesion; Integrin; Integrin I domain; Osteopontin; Phosphorylation; Surface plasmon resonance