title: Monoclonal antibodies that specifically recognize neoepitope sequences generated by 'aggrecanase' and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro.
abstract: Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, 'aggrecanase', and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of 'aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that 'aggrecanase' activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than 'aggrecanase' are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited 'aggrecanase' activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.
authors.affiliation: Division of Orthopaedics, University of North Carolina at Chapel Hill.
authors.forename: C E
authors.initials: CE
authors.lastname: Hughes
authors.forename: B
authors.initials: B
authors.lastname: Caterson
authors.forename: A J
authors.initials: AJ
authors.lastname: Fosang
authors.forename: P J
authors.initials: PJ
authors.lastname: Roughley
authors.forename: J S
authors.initials: JS
authors.lastname: Mort
mesh_terms: D053674:Aggrecans; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D002813:Chondrosarcoma / Q000378:metabolism; D017077:Culture Media, Conditioned; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D016326:Extracellular Matrix Proteins; D006801:Humans; D037181:Lectins, C-Type; D008666:Metalloendopeptidases / Q000378:metabolism*; D011509:Proteoglycans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: C508321:Acan protein, rat; D053674:Aggrecans; D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D010450:Endopeptidases; D008666:Metalloendopeptidases; C083641:aggrecanase