Identification of aggrecanase activity in medium of cartilage culture.
Erosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10,000-fold. The enzyme cleaves at the aggrecanase site (Glu(373)-Ala(374)) but not at the MMP site (Asn(341)-Phe(342)) in the interglobular domain of the aggrecan. It also cleaves at Glu(1971)-Leu(1972), which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca(2+)-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase.
Sugimoto
K
K
Biological Research Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo, 140-8710, Japan.
Takahashi
M
M
Yamamoto
Y
Y
Shimada
K
K
Tanzawa
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002356:Cartilage / Q000187:drug effects / Q000201:enzymology*; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008954:Models, Biological; D009300:Nasal Septum; D009924:Organ Culture Techniques; D012965:Sodium Chloride / Q000494:pharmacology
D006025:Glycosaminoglycans; D061965:Matrix Metalloproteinase Inhibitors; D012965:Sodium Chloride; D010450:Endopeptidases; D020782:Matrix Metalloproteinases; C083641:aggrecanase; D002118:Calcium