title: 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.
abstract: OBJECTIVE
  To define the stromelysin cleavage site in the interglobular domain of rabbit aggrecan, and to determine whether the stromelysin-generated neoepitope can be used as a marker of matrix metalloproteinase (MMP) activity in vivo.
  
  
  METHODS
  The carboxy-terminus sequence of the stromelysin-generated hyaluronic acid-binding region (HABR) of rabbit aggrecan was determined by reverse transcription-polymerase chain reaction complementary DNA cloning and DNA sequence analysis, followed by purification and mass spectral protein sequence analysis of the HABR 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 immunofluorescence. Proteoglycan fragments in joint fluids were quantified by a dimethylmethylene blue dye-binding assay.
  
  
  RESULTS
  Stromelysin cleavage of rabbit aggrecan generated a 55-kd HABR fragment that terminated in the sequence FMDIPEN: An anti-FVDIPEN antibody recognized the FMDIPEN neoepitope in situ in cartilage from stromelysin-injected joints. The appearance of the FMDIPEN neoepitope corresponded to the release of cartilage proteoglycan fragments into the joint fluid, and could be inhibited by pretreatment of the rabbits with a synthetic stromelysin inhibitor.
  
  
  CONCLUSIONS
  These results indicate that the anti-FVDIPEN antibody can be used to assess the role of MMPs in cartilage degradation in vivo.
authors.affiliation: Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: E K
authors.initials: EK
authors.lastname: Bayne
authors.forename: K L
authors.initials: KL
authors.lastname: MacNaul
authors.forename: S A
authors.initials: SA
authors.lastname: Donatelli
authors.forename: A
authors.initials: A
authors.lastname: Christen
authors.forename: P R
authors.initials: PR
authors.lastname: Griffin
authors.forename: L A
authors.initials: LA
authors.lastname: Hoerrner
authors.forename: J R
authors.initials: JR
authors.lastname: Calaycay
authors.forename: J M
authors.initials: JM
authors.lastname: Ayala
authors.forename: K
authors.initials: K
authors.lastname: Chapman
authors.forename: W
authors.initials: W
authors.lastname: Hagmann
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002358:Cartilage, Articular / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D007270:Injections, Intra-Articular; D037181:Lectins, C-Type; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000008:administration & dosage / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000032:analysis / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D013582:Synovial Fluid / Q000737:chemistry / Q000187:drug effects
chemical_list: D053674:Aggrecans; D000906:Antibodies; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3