Tortorella M D, Malfait A M, Deccico C, Arner E
DuPont Pharmaceuticals Company, Wilmington, DE 19880-0400, USA.
Osteoarthritis Cartilage. 2001 Aug;9(6):539-52. doi: 10.1053/joca.2001.0427.
Cleavage of aggrecan between residues Glu(373)-Ala(374), which is believed to be a key event in aggrecan destruction in arthritic diseases, has been attributed to an enzymatic activity, aggrecanase. Two cartilage aggrecanases have been identified, aggrecanase-1 (ADAM-TS4) and aggrecanase-2 (ADAM-TS5) and both enzymes have been shown very efficiently to cleave soluble aggrecan at the Glu(373)-Ala(374) site.
To determine whether ADAM-TS4 and/or ADAM-TS5 are the aggrecanases responsible for aggrecan catabolism following interleukin-1 (IL-1) and tumor necrosis factor (TNF) treatment of bovine articular cartilage.
(1) IL-1- and TNF-stimulated release of aggrecan was associated with cleavage of aggrecan within the C-terminus at the ADAM-TS4 and ADAM-TS5-sensitive sites, Glu(1480)-Gly(1481), Glu(1667)-Gly(1668), and Glu(1871)-Leu(1872). (2) The order of cleavage following IL-1 stimulation of cartilage explants was the same as when soluble aggrecan is digested with recombinant human ADAM-TS4 and ADAM-TS5. (3) Both constitutive and stimulated cleavage of aggrecan at the ADAM-TS4 and ADAM-TS5-sensitive sites in cartilage was blocked by a general metalloproteinase inhibitor but not by a MMP-specific inhibitor, and this inhibition correlated with inhibition of aggrecan release from cartilage. (4) PCR and Western blot analysis indicated that both ADAM-TS proteases are expressed in cartilage explants; ADAM-TS5 is constitutively expressed whereas ADAM-TS4 is induced following IL-1 and TNF treatment. (5) Immunodepletion of both ADAM-TS4 and ADAM-TS5 from bovine articular cartilage cultures following IL-1 stimulation resulted in a 90% reduction of aggrecanase activity in the culture medium.
在关节炎疾病中，聚集蛋白聚糖在谷氨酸（373）-丙氨酸（374）残基之间的裂解被认为是聚集蛋白聚糖破坏的关键事件，这一过程归因于一种酶活性，即聚集蛋白聚糖酶。已鉴定出两种软骨聚集蛋白聚糖酶，聚集蛋白聚糖酶-1（ADAM-TS4）和聚集蛋白聚糖酶-2（ADAM-TS5），并且已证明这两种酶都能非常有效地在谷氨酸（373）-丙氨酸（374）位点裂解可溶性聚集蛋白聚糖。
确定ADAM-TS4和/或ADAM-TS5是否是白细胞介素-1（IL-1）和肿瘤坏死因子（TNF）处理牛关节软骨后负责聚集蛋白聚糖分解代谢的聚集蛋白聚糖酶。
（1）IL-1和TNF刺激引起的聚集蛋白聚糖释放与聚集蛋白聚糖在C末端ADAM-TS4和ADAM-TS5敏感位点，即谷氨酸（1480）-甘氨酸（1481）、谷氨酸（1667）-甘氨酸（1668）和谷氨酸（1871）-亮氨酸（1872）处的裂解有关。（2）IL-1刺激软骨外植体后的裂解顺序与用重组人ADAM-TS4和ADAM-TS5消化可溶性聚集蛋白聚糖时相同。（3）软骨中ADAM-TS4和ADAM-TS5敏感位点的聚集蛋白聚糖组成型和刺激型裂解均被一种通用金属蛋白酶抑制剂阻断，但未被MMP特异性抑制剂阻断，且这种抑制与软骨中聚集蛋白聚糖释放的抑制相关。（4）PCR和蛋白质印迹分析表明，两种ADAM-TS蛋白酶均在软骨外植体中表达；ADAM-TS5组成型表达，而ADAM-TS4在IL-1和TNF处理后被诱导表达。（5）IL-1刺激后从牛关节软骨培养物中免疫去除ADAM-TS4和ADAM-TS5导致培养基中聚集蛋白聚糖酶活性降低90%。
