Figure 1.

Detection of brevican isoforms and proteolytic degradation by endogenous proteases at specific cleavage sites. Brevican is secreted as a >145 kD protein bearing 1-3 CS chains (A). Brevican is also secreted as the holoprotein without CS chains at 145 kD (B). When probed on western blot with an N-terminal antibody (BD Biosciences, San Jose, CA) three immunoreactive bands appear;: a >145 smear (glycosylated brevican), the 145 kD core protein, and a ~55 kD proteolytic fragment (C, D, E and F). Arrows in (A) and (B) indicate proteolytic cleavage sites. Fragments of brevican are generated by endogenous proteases, the MMPs (D) and ADAMTSs (E). Each has a distinct, specific cleavage site sequence on the brevican protein. Shown here are the specific cleavage sequences for the MMPs and ADAMTSs in mouse, rat and human brevican (based on data from Nakamura et al. 2000). The MMP cleavage-site is 35 amino acid residues upstream from the ADAMTS-specific site (D and E). Distinct “neoepitope” antibodies recognize the MMP- and ADAMTS-derived cleavage fragments of brevican in mouse brain extracts subjected to Western blot on 4-20% gradient SDS-PAGE gels (F). Anti-SAHPSA recognizes the 53 kD, MMP-derived fragment of brevican (F; middle panel) whereas anti-EAMESE detects the 55 kD, ADAMTS-derived form (F; right panel). Mixing the two anti-bodies detects a “thicker” band in this region (F; left panel). “M” indicates molecular weight markers in (F). Antibodies recognize distinct products after proteolytic cleavage with hrADAMTS4 or hrMMP-2 (G). Proteoglycan purified from mouse brain was incubated with 50 nM hrADAMTS-4 (G, lane 1), 50 nM hrADAMTS4 + 5 mM EDTA (G, lane 2), 50 nM hrMMP-2 (G, lane 3) or 50 nM hrMMP-2 + 5 mM EDTA (G, lane 4) and immunoblotted for brevican. Note that the ADAMTS-derived brevican fragment was selectively recognized by anti-EAMESE and the brevican MMP product was recognized by anti-SAHPSA.