Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin.
Domain IV of mouse perlecan, which consists of 14 immunoglobulin superfamily (IG) modules, was prepared from recombinant human cell culture medium in the form of two fragments, IV-1 (IG2-9, 100 kDa) and IV-2 (IG10-15, 66 kDa). Both fragments bound to a heparin column, being eluted at ionic strengths either below (IV-2) or above (IV-1) physiological level, and could thus be readily purified. Electron microscopy demonstrated an elongated shape (20-25 nm), and folding into a native structure was indicated by immunological assay and CD spectroscopy. Solid-phase and surface plasmon resonance assays demonstrated strong binding of fragment IV-1 to fibronectin, nidogen-1, nidogen-2 and the laminin-1-nidogen-1 complex, with Kd values in the range 4-17 nM. The latter binding apparently occurs through nidogen-1, as shown by the formation of ternary complexes. Only moderate binding was observed for fibulin-2 and collagen IV and none for fibulin-1 and BM-40. Fragment IV-2 showed a more restricted pattern of binding, with only weaker binding to fibronectin and fibulin-2. None of these activities could be demonstrated for recombinant fragments corresponding to the N-terminal perlecan domains I to III. This indicates a special role for domain IV in the integration of perlecan into basement membranes and other extracellular structures via protein-protein interactions.
Hopf
M
M
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Göhring
W
W
Kohfeldt
E
E
Yamada
Y
Y
Timpl
R
R
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D015374:Biosensing Techniques; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000235:genetics*; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007797:Laminin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; C084612:fibulin 2; C029967:nidogen; C071927:perlecan