THE STRUCTURE OF AGGRECAN FRAGMENTS IN HUMAN SYNOVIAL-FLUID - EVIDENCE THAT AGGRECANASE MEDIATES CARTILAGE DEGRADATION IN INFLAMMATORY JOINT DISEASE, JOINT INJURY, AND OSTEOARTHRITIS

Objective. To determine the proteolytic fragmentation patterns and N-t erminal sequence of aggrecan fragments in human synovial fluid from pa tients with inflammatory arthritides, joint injury, or osteoarthritis (OA). Methods. Knee synovial fluid was obtained from patients with joi nt injury, OA, acute pyrophosphate arthritis (pseudogout), reactive ar thritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondr oitin sulfate-substituted aggrecan fragments present in the fluid were purified by cesium chloride gradient centrifugation and enzymatically deglycosylated. Core protein species were determined by N-terminal an alysis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresi s (SDS-PAGE) with electroblotting and detection with monoclonal antibo dy 3B3. Results. Samples from patients with joint injury, OA, and infl ammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, dif fuse immunoreactive products were also seen, with an apparent size of >250 kd. N-terminal analysis of core preparations of all samples showe d a consistent single predominant sequence, beginning at alanine 374 o f the human aggrecan core protein. Conclusion. The aggrecan fragments present in joint fluids from patients with various inflammatory arthri tides, joint injury, or OA result from a predominant cleavage of the h uman aggrecan core protein at the glutamate 373-alanine 374 bond withi n the interglobular domain, between the G1 and G2 domains. The consist ent pattern of fragments seen on SDS-PAGE and the single predominant N -terminal sequence suggest a common degradative mechanism of aggrecan in these different joint conditions. The identity of the proteolytic a gent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapie s to protect cartilage from degradation in patients with joint disease .