USE OF AN ANTIBODY AGAINST THE MATRIX METALLOPROTEINASE-GENERATED AGGRECAN NEOEPITOPE FVDIPEN-COOH TO ASSESS THE EFFECTS OF STROMELYSIN IN A RABBIT MODEL OF CARTILAGE DEGRADATION

Objective, To define the stromelysin cleavage site in the interglobula r domain of rabbit aggrecan, and to determine whether the stromelysin- generated neoepitope can be used as a marker of matrix metalloproteina se (MMP) activity in vivo, Methods, The carboxy-terminus sequence of t he stromelysin-generated hyaluronic acid-binding region (HABR) of rabb it aggrecan was determined by reverse transcription-polymerase chain r eaction complementary DNA cloning and DNA sequence analysis; followed by purification and mass spectral protein sequence analysis of the HAB R fragment. Active stromelysin was injected into the stifle joints of rabbits, and a stromelysin-generated aggrecan neoepitope was analyzed by Western blotting and localized in situ by indirect immunofluorescen ce. Proteoglycan fragments in joint fluids were quantified by a dimeth ylmethylene blue dye-binding assay, Results, Stromelysin cleavage of r abbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN. An anti-FVDIPEN antibody recognized the FMDIPEN neoe pitope in situ in cartilage from stromelysin-injected joints. The appe arance of the FMDIPEN neoepitope corresponded to the release of cartil age proteoglycan fragments into the joint fluid, and could be inhibite d by pretreatment of the rabbits with a synthetic stromelysin inhibito r, Conclusion, These results indicate that the anti-FVDIPEN antibody c an be used to assess the role of MMPs in cartilage degradation in vivo .