title: Utilization of a recombinant substrate rAgg1 to study the biochemical properties of aggrecanase in cell culture systems.
abstract: This paper describes the first report of the production and use of an artificial recombinant protein substrate to study "aggrecanase" activity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the "marker" sequences FLAGTM at the amino terminus and the human immunoglobulin G1 constant region at the carboxyl terminus. The expressed protein occurs as large multimolecular aggregates (>120 kDa) that, upon reduction, consist of a major isoform of 72 kDa (containing the IGD) and a minor 39-kDa species that through alternative splicing has had the IGD deleted. Using this recombinant substrate we developed a novel agarose cell culture system containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrecanase activities. These studies showed the following. (i) rAgg1 is a suitable substrate for aggrecanase proteolysis. (ii) Aggrecanase activity was specifically induced by exposing chondrocytes to retinoic acid. (iii) A considerable time period was required to synthesize and/or activate aggrecanase, with considerable differences in that found in rat chondrosarcoma versus bovine chondrocyte culture systems. (iv) Aggrecanase cleavage of the aggrecan IGD does not require the presence of the G1 or G2 globular domains or keratan sulfate post-translational modification in the IGD. (v) Aggrecanase is a diffusible activity that does not require association with the chondrocyte plasma membrane or immediate pericellular matrix for its action.
authors.affiliation: Connective Tissue Biology Laboratories, School of Molecular and Medical Biosciences, University of Wales, Cardiff CF1 3US, Wales, United Kingdom.
authors.forename: C E
authors.initials: CE
authors.lastname: Hughes
authors.forename: F H
authors.initials: FH
authors.lastname: Büttner
authors.forename: B
authors.initials: B
authors.lastname: Eidenmüller
authors.forename: B
authors.initials: B
authors.lastname: Caterson
authors.forename: E
authors.initials: E
authors.lastname: Bartnik
mesh_terms: D053674:Aggrecans; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D010450:Endopeptidases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D006801:Humans; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D012685:Sepharose; D013997:Time Factors
chemical_list: C508321:Acan protein, rat; D053674:Aggrecans; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D011994:Recombinant Proteins; D012685:Sepharose; D010450:Endopeptidases; C083641:aggrecanase