Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.
Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.
Fox
J W
JW
Max-Planck-Institut für Biochemie, Martinsried, FRG.
Mayer
U
U
Nischt
R
R
Aumailley
M
M
Reinhardt
D
D
Wiedemann
H
H
Mann
K
K
Timpl
R
R
Krieg
T
T
Engel
J
J
D000818:Animals; D015152:Blotting, Northern; D002850:Chromatography, Gel; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D007797:Laminin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D011487:Protein Conformation; D012313:RNA / Q000032:analysis; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection
D007797:Laminin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C029967:nidogen; D012313:RNA; D003094:Collagen; D004247:DNA