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 of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, 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 perlecan domain or laminin-1.
Ries
A
A
Max-Planck-Institut für Biochemie, Am Klopferspitz 18 A, D-82152 Martinsried, Germany.
Göhring
W
W
Fox
J W
JW
Timpl
R
R
Sasaki
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001483:Base Sequence; D001485:Basement Membrane / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C029967:nidogen