title: Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum.
abstract: Several members of the matrix metalloproteinase family have been reported to cleave aggrecan in the interglobular domain between Asn-341 and Phe-342. An antiserum was prepared against a peptide conjugate corresponding to the C-terminal sequence of the matrix metalloproteinase-generated aggrecan G1 fragment (Phe335-Val-Asp-Ile-Pro-Glu-Asn341). A quantitative radioimmunoassay, with a limit of detection of about 80 pM, was developed using this antiserum. This antiserum requires the free carboxyl group of the C-terminal asparagine for optimal recognition. If the C-terminal asparagine is excised from the sequence, replaced with closely related amino acids, or extended across the matrix metalloproteinase cleavage site, there is a 40-10,000-fold loss in detection. Using peptides cleaved from the N-terminus, it was determined that the antiserum requires the entire Phe-Val-Asp-Ile-Pro-Glu-Asn sequence for optimal recognition. The radioimmunoassay detects matrix metalloproteinase-generated G1 fragments with similar sensitivity to the Phe-Val-Asp-Ile-Pro-Glu-Asn peptide, but it does not recognize intact aggrecan. Immunoreactive aggrecan G1 fragments of molecular mass 50 kDa are generated by the matrix metalloproteinases stromelysin and gelatinase A. In contrast, under identical conditions, the closely related metalloproteinases, gelatinase B and collagenase, as well as cathepsin G, cathepsin B and human leucocyte elastase, did not generate a G1 fragment recognized by the antiserum. The anti-Phe-Val-Asp-Ile-Pro-Glu-Asn serum detects stromelysin-generated aggrecan G1 fragments from mouse, guinea pig, rabbit and human, indicating that the detection is not species-specific. This antiserum and radio-immunoassay should be useful for quantifying and characterizing matrix metalloproteinase-generated aggrecan G1 fragments in articular cartilage and synovial fluids from humans and various animal models of articular-cartilage destruction.
authors.affiliation: Department of Biochemical Pathology, Merck Research Laboratories, Rahway, NJ 07065, USA.
authors.forename: M W
authors.initials: MW
authors.lastname: Lark
authors.forename: H
authors.initials: H
authors.lastname: Williams
authors.forename: L A
authors.initials: LA
authors.lastname: Hoernner
authors.forename: J
authors.initials: J
authors.lastname: Weidner
authors.forename: J M
authors.initials: JM
authors.lastname: Ayala
authors.forename: C F
authors.initials: CF
authors.lastname: Harper
authors.forename: A
authors.initials: A
authors.lastname: Christen
authors.forename: J
authors.initials: J
authors.lastname: Olszewski
authors.forename: Z
authors.initials: Z
authors.lastname: Konteatis
authors.forename: R
authors.initials: R
authors.lastname: Webber
mesh_terms: D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002356:Cartilage / Q000737:chemistry; D002401:Cathepsin B / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D018093:Gelatinases / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007106:Immune Sera; D037181:Lectins, C-Type; D019272:Leukocyte Elastase; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011509:Proteoglycans / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D013045:Species Specificity
chemical_list: C508320:Acan protein, mouse; D053674:Aggrecans; D004792:Enzyme Precursors; D016326:Extracellular Matrix Proteins; D007106:Immune Sera; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D002401:Cathepsin B; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2