ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan
ADAMTS4 (aggrecanase-1) interaction with the C-terminal domain of fibronectin inhibits proteolysis of aggrecan
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DOI：
10.1074/jbc.m314216200
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发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
Okada, Y
中科院分区：
文献类型：
--
作者：
Hashimoto, G;Shimoda, M;Okada, Y
ADAMTS 4（聚集蛋白聚糖酶-1）是ADAMTS（一种具有血小板反应蛋白基序的去整合素和金属蛋白酶）基因家族的一种分泌酶，被认为在骨关节炎和类风湿性关节炎中软骨蛋白聚糖（聚集蛋白聚糖）的降解中起关键作用。为了克隆与ADAMTS 4结合的分子，我们使用ADAMTS 4间隔区作为诱饵，通过酵母双杂交系统筛选人软骨细胞cDNA文库，并获得来自纤连蛋白的cDNA克隆。ADAMTS 4和纤连蛋白之间的相互作用通过化学交联来证明。使用野生型纤连蛋白和ADAMTS 4及其突变体的酵母双杂交测定和固相结合测定证明纤连蛋白的C-末端结构域能够结合ADAMTS 4的C-末端间隔结构域。野生型ADAMTS 4与纤连蛋白共定位，如通过共聚焦显微镜在表达ADAMTS 4的稳定的293 T转染子的细胞表面上所确定的，尽管ADAMTS 4缺失突变体，包括DeltaSp（DeltaArg（693）-Lys（837），缺乏间隔区结构域）显示可忽略的定位。野生型ADAMTS 4的聚集蛋白聚糖酶活性受到纤连蛋白的剂量依赖性抑制（IC 50 = 110 nM），而DeltaSp没有观察到抑制作用。C-末端40-kDa纤连蛋白片段也抑制野生型ADAMTS 4的活性（IC 50 = 170 nM）。这些数据首次证明ADAMTS 4的聚集蛋白聚糖酶活性通过与其C-末端结构域的相互作用被纤连蛋白抑制，并表明ADAMTS 4活性的这种细胞外调节机制可能对关节炎软骨中聚集蛋白聚糖的降解很重要。
ADAMTS4 (aggrecanase-1), a secreted enzyme belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, is considered to play a key role in the degradation of cartilage proteoglycan (aggrecan) in osteoarthritis and rheumatoid arthritis. To clone molecules that bind to ADAMTS4, we screened a human chondrocyte cDNA library by the yeast two-hybrid system using the ADAMTS4 spacer domain as bait and obtained cDNA clones derived from fibronectin. Interaction between ADAMTS4 and fibronectin was demonstrated by chemical cross-linking. A yeast two-hybrid assay and solid-phase binding assay using wild-type fibronectin and ADAMTS4 and their mutants demonstrated that the C-terminal domain of fibronectin is capable of binding to the C-terminal spacer domain of ADAMTS4. Wild-type ADAMTS4 was co-localized with fibronectin as determined by confocal microscopy on the cell surface of stable 293T transfectants expressing ADAMTS4, although ADAMTS4 deletion mutants, including DeltaSp (DeltaArg(693)-Lys(837), lacking the spacer domain), showed negligible localization. The aggrecanase activity of wild-type ADAMTS4 was dose-dependently inhibited by fibronectin (IC50 = 110 nM), whereas no inhibition was observed with DeltaSp. The C-terminal 40-kDa fibronectin fragment also inhibited the activity of wild-type ADAMTS4 (IC50 = 170 nM). These data demonstrate for the first time that the aggrecanase activity of ADAMTS4 is inhibited by fibronectin through interaction with their C-terminal domains and suggest that this extracellular regulation mechanism of ADAMTS4 activity may be important for the degradation of aggrecan in arthritic cartilage.