Immunochemical and tissue analysis of protease generated neoepitopes of BM-40 (osteonectin, SPARC) which are correlated to a higher affinity binding to collagens.
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.
Sasaki
T
T
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Miosge
N
N
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D003094:Collagen / Q000378:metabolism*; D003429:Cross Reactions; D010450:Endopeptidases; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D008297:Male; D051379:Mice; D009374:Neoplasms, Experimental / Q000276:immunology / Q000378:metabolism; D015676:Osteonectin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011863:Radioimmunoassay
D000939:Epitopes; D015676:Osteonectin; D003094:Collagen; D010450:Endopeptidases