Calcium‐dependent binding of basement membrane protein BM‐40 (osteonectin, SPARC) to basement membrane collagen type IV

Abstract

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‐ionchelating or chaotropic agents, high salt and reduction of disulfide bonds in BM‐40. Fragment studies indicated that domains III (α‐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. Copyright © 1991, Wiley Blackwell. All rights reserved