Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies

The basement membrane protein, nidogen-1, was previously shown to consist o f three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, co llagen IV, perlecan and fibulins. In the present study, we have produced re combinant proteins of these predicted domains in mammalian cells and used t hese proteins for crystallographic and binding epitope analyses. These frag ments included G1, G2, the rod domain and a slightly larger G3 structure; a ll were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high aff inity binding (K-d = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate K-d for fibulin-1C. Domain G3 conta ined high affinity binding sites for the laminin gamma1 chain and collagen IV (K-d = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A mo derate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, t hese data indicate the potential of nidogen-1 for multiple interactions wit hin basement membranes. A similar binding repertoire was also identified fo r seven rat monoclonal antibodies that bound with K-d = 2-30 nM to either G 1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perleca n domain or laminin-1.