UTILIZATION OF A RECOMBINANT SUBSTRATE RAGG1 TO STUDY THE BIOCHEMICAL-PROPERTIES OF AGGRECANASE IN CELL-CULTURE SYSTEMS

This paper describes the first report of the production and use of an artificial recombinant protein substrate to study ''aggrecanase'' acti vity. The substrate (rAgg1) is composed of the complete interglobular domain (IGD) of human aggrecan flanked by the ''marker'' sequences FLA G(TM) at the amino terminus and the human immunoglobulin GI constant r egion 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 spec ies that through alternative splicing has had the IGD deleted. Using t his recombinant substrate we developed a novel agarose cell culture sy stem containing either rat chondrosarcoma or bovine chondrocytes that could be used in studies of the biochemical characterization of aggrec anase activities. These studies showed the following. (i) rAgg1 is a s uitable substrate for aggrecanase proteolysis. (ii) Aggrecanase activi ty was specifically induced by exposing chondrocytes to retinoic acid. (iii) A considerable time period was required to synthesize and/or ac tivate aggrecanase, with considerable differences in that found in rat chondrosarcoma versus bovine chondrocyte culture systems. (iv) Aggrec anase cleavage of the aggrecan IGD does not require the presence of th e G1 or G2 globular domains or keratan sulfate post-translational modi fication in the IGD. (v) Aggrecanase is a diffusible activity that doe s not require association with the chondrocyte plasma membrane or imme diate pericellular matrix for its action.