The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis.
The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis.
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DOI：
10.1002/art.1780360906
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发表时间：
1993-09
影响因子：
--
通讯作者：
L. Lohmander;P. Neame;J. Sandy
中科院分区：
文献类型：
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作者：
L. Lohmander;P. Neame;J. Sandy
目的测定炎性关节炎、关节损伤和骨关节炎（OA）患者滑液中聚集蛋白聚糖（aggrecan）的蛋白裂解模式和N端序列。方法膝关节滑液取自关节损伤、OA、急性焦磷酸盐关节炎（假性痛风）、反应性关节炎、银屑病关节炎或幼年类风湿关节炎患者。通过氯化铯梯度离心纯化流体中存在的硫酸软骨素取代的聚集蛋白聚糖片段并酶促去糖基化。通过N-末端分析和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结合电印迹和单克隆抗体3B 3检测确定核心蛋白种类。结果：关节损伤、OA和炎性关节疾病患者的样本均显示出相似的3带模式，核心大小约为200 kd、170 kd和135 kd。在所有样本中，还观察到弥漫性免疫反应产物，表观大小> 250 kd。所有样品的核心制备物的N-末端分析显示一致的单一优势序列，从人聚集蛋白聚糖核心蛋白的丙氨酸374开始。结论：存在于各种炎性关节炎、关节损伤或OA患者关节液中的聚集蛋白聚糖片段是由于人聚集蛋白聚糖核心蛋白在G1和G2结构域之间的球间结构域内的谷氨酸373-丙氨酸374键处的主要裂解所致。在SDS-PAGE上看到的片段的一致模式和单一的主要N-末端序列表明在这些不同的关节条件下聚集蛋白聚糖的共同降解机制。然而，蛋白水解剂（聚集蛋白聚糖酶）的身份仍然未知。这些结果似乎具有重要的意义方面的治疗，以保护关节疾病患者的软骨退化的发展。
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 immunoreactive 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.