Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV.
Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.
Mayer
U
U
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Aumailley
M
M
Mann
K
K
Timpl
R
R
Engel
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007124:Immunoenzyme Techniques; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000378:metabolism* / Q000648:ultrastructure; D011489:Protein Denaturation; D012513:Sarcoma, Experimental / Q000737:chemistry; D014357:Trypsin
D015676:Osteonectin; D003094:Collagen; D014357:Trypsin; D002118:Calcium