Immunochemical and Tissue Analysis of Protease Generated Neoepitopes of BM-40 (Osteonectin, SPARC) Which Are Correlated to a Higher Affinity Binding to Collagens

Matrix Biol. 1999 Oct;18(5):499-508. doi: 10.1016/s0945-053x(99)00041-4.

Abstract

Proteolytic cleavage at single sites in the extracellular calcium-binding module of BM-40/SPARC/osteonectin either by an unknown endogenous protease (L197-L198) or several matrix metalloproteinases (E196-L197) was previously shown to enhance collagen binding activity 10-fold. Polyclonal rabbit antibodies were now obtained against synthetic peptide antigens containing either an N-terminal L197 or L198 and characterized by radioimmunoassay, ELISA, immunoblots and immunohistology. These neoepitope-specific antibodies reacted with proteolytically processed but not with uncleaved mouse and human BM-40. The cross-reaction between the two different neoepitopes was < 1%, indicating the immunodominant role of the N-terminal residues. Analysis of a basement membrane producing mouse tumor demonstrated extensive cleavage at the L198 site, which correlated with a calcium-dependent binding to the matrix. A variable degree of this cleavage was also detected in BM-40 obtained from adult mouse bone and several other tissues. Negligible or much lower levels of conversion were detected at the MMP-specific L197 site, however. Immunogold staining of mouse heart and a basement membrane-producing mouse tumor showed a distinct extracellular labeling for BM-40 and the L198 neoepitope but only a very weak reaction for the L197 neoepitope. This strongly indicates that these neoepitopes are generated in vivo and emphasizes a specific biological role for the proteolytic activation of BM-40.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Binding Sites
  • Collagen / metabolism*
  • Cross Reactions
  • Endopeptidases
  • Epitopes / chemistry
  • Epitopes / genetics
  • Epitopes / metabolism
  • Extracellular Matrix / metabolism
  • Humans
  • Immunochemistry
  • Male
  • Mice
  • Neoplasms, Experimental / immunology
  • Neoplasms, Experimental / metabolism
  • Osteonectin / chemistry
  • Osteonectin / immunology*
  • Osteonectin / metabolism*
  • Protein Binding
  • Rabbits
  • Radioimmunoassay

Substances

  • Epitopes
  • Osteonectin
  • Collagen
  • Endopeptidases