FIG. 3.

Identification of the elastin-binding region of fibulin-5. A. Schematic representation of fibulin-5 mutants. C-terminal elastin-binding region (shaded yellow) is located within the fibulin module. The purple square indicates the signal peptides. The blue box indicates the first CB-EGF domain, separated from the remaining CB-EGF domain (aqua boxes) by a hinge region (H). An RGD motif is located in the first CB-EGF domain, and small red squares indicate glycosylation sites. B. Verification of various fibulin-5 mutant proteins and recombinant tropoelastin. Left panel: Western blot analysis of conditioned media (upper blot) or cell lysates (lower panel) containing recombinant fibulin-5 proteins detected by anti-V5 antibody. Right panel: bacterium-derived tropoelastin (lane 1) was purified through a nickel affinity column (lane 2) and stained with Coomassie blue. Western blots of these samples using antielastin antibody (BA4) is shown below. C, D. Solid-phase binding assays using tropoelastin (1 μg/well) as the solid phase and a serial dilution of conditioned medium (C) or cell lysates (D) as the soluble ligand. Note that wild-type fibulin-5 exhibits strong binding to tropoelastin in a dose-dependent manner. E, F. Binding of fibulin-5 mutants to tropoelastin expressed as a percentage of wild-type fibulin-5 binding. Data were analyzed within a linear range at a 1:4 dilution using conditioned media (E) or cell lysates (F). Note that point mutations of the integrin binding motif (G55S and D56E) did not affect tropoelastin binding; however, the binding was significantly reduced when the N-terminal CB-EGF motifs were sequentially deleted (E). Deletion of the C-terminal EB region (ΔEB) abolished tropoelastin binding. Data from five independent experiments using conditioned media and three independent experiments using cell lysates, performed in triplicate, are shown. G. Solid-phase binding assays using tropoelastin (TropoE) (1 μg/well) or alpha-elastin (αE) (1, 10, or 100 μg/well) as the solid phase and a serial dilution of conditioned medium containing wild-type fibulin-5 as the soluble ligand. Data from two independent experiments performed in triplicate are shown. H. Solid-phase binding assays using tropoelastin (1 μg/well) or alpha-elastin (100 μg/well) as the solid phase and wild-type fibulin-5 or deletion mutants as soluble ligands. Data from two independent experiments performed in triplicate are shown. Data are expressed as means ± standard errors.