FIGURE 7.
GDF-8 pd binds specifically to the GAG chains of domain V of perlecan. A, schematic diagram of the perlecan recombinant polypeptides used in this study. B, upper left graph, ELISA interaction assay with full-length perlecan (purified from mouse Engelbreth-Holm-Swarm sarcoma) coated on the wells showed specific interaction only with the GDF-8 pd. Pds of TGF-β superfamily members were titrated at different concentrations (0.5–0 μm) in solution, and anti-His6 antibody was used for detection of bound pds. Upper right graph, ELISA interaction study with coated recombinant polypeptides spanning full-length perlecan revealed that GDF-8 pd binds exclusively to domain V of perlecan (open triangles). Lower left graph, ELISA binding assay with coated domain V and nonmodified domain V (without GAG chains) confirmed that GDF-8 pd binds specifically to the GAG side chain and not to the protein core of domain V. Lower right sensorgrams, SPR with immobilized perlecan domain V and GDF-8 pd flowing in solution at different concentrations allowed the determination of a KD of 11 nm.