The structure of aggrecan fragments in human synovial fluid. evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis

Authors

  • L. Stefan Lohmander MD, PhD,

    Corresponding author
    1. Department of Orthopedics, University Hospital, Lund, Sweden; and Shriners Hospital for Crippled Children, Tampa Unit, and the Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa.
    • Department of Orthopedics, University Hospital, S-22185 Lund, Sweden
    Search for more papers by this author
  • Peter J. Neame PhD,

    1. Department of Orthopedics, University Hospital, Lund, Sweden; and Shriners Hospital for Crippled Children, Tampa Unit, and the Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa.
    Search for more papers by this author
  • John D. Sandy PhD

    1. Department of Orthopedics, University Hospital, Lund, Sweden; and Shriners Hospital for Crippled Children, Tampa Unit, and the Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa.
    Search for more papers by this author

Abstract

Objective. To determine the proteolytic fragmentation patterns and N-terminal sequence of aggrecan fragments in human synovial fluid from patients with inflammatory arthritides, joint injury, or osteoarthritis (OA).

Methods. Knee synovial fluid was obtained from patients with joint injury, OA, acute pyrophosphate arthritis (pseudogout), reactive arthritis, psoriatic arthritis, or juvenile rheumatoid arthritis. Chondroitin 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 analysis and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with electroblotting and detection with monoclonal antibody 3B3.

Results. Samples from patients with joint injury, OA, and inflammatory joint disease all showed a similar 3-band pattern, with core sizes of approximately 200 kd, 170 kd, and 135 kd. In all samples, diffuse immuno-reactive products were also seen, with an apparent size of >250 kd. N-terminal analysis of core preparations of all samples showed a consistent single predominant sequence, beginning at alanine 374 of the human aggrecan core protein.

Conclusion. The aggrecan fragments present in joint fluids from patients with various inflammatory arthritides, joint injury, or OA result from a predominant cleavage of the human aggrecan core protein at the glutamate 373–alanine 374 bond within the interglobular domain, between the G1 and G2 domains. The consistent 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 agent (aggrecanase), however, remains unknown. These results appear to have important implications with regard to the development of therapies to protect cartilage from degradation in patients with joint disease.

Ancillary