Jaovisidha Kanyakorn, Etim Ann, Yamakawa Koji, Masuda Ikuko, Gohr Claudia M, Wakim Bassam T, Boonapatcharoen Nimaradee, Ninomiya James, Rosenthal Ann K
Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee, 53295, USA.
J Rheumatol. 2006 Feb;33(2):318-25.
Trappins are small serine protease inhibitors bound to extracellular matrix (ECM) through the actions of transglutaminase (TGase) enzymes. Trappin-2 is present in many tissues and is upregulated at sites of injury. In osteoarthritis (OA), serine proteases contribute to articular cartilage destruction, and TGase activity is increased. Yet little is known about matrix-bound serine protease inhibitors or TGase substrates in articular cartilage. Our purpose was to determine if trappin-2 was present in OA cartilage and synovial fluid (SF).
OA knee articular cartilage and SF were assayed for trappin-2 protein by Western blotting, ELISA, and immunohistochemistry. Trappin-2 mRNA was detected with RT-PCR. The ECM components bound to trappin-2 were identified by 2-D gel electrophoresis and peptide fingerprinting.
Trappin-2 was detectable in OA articular cartilage extracts, cultured chondrocytes, conditioned media, and SF by Western blotting. OA cartilage protein extracts contained significantly higher quantities of trappin-2 than normal cartilage protein extracts (22.98 +/- 1.28 ng/mg wet weight vs 14.97 +/- 1.92 ng/mg wet weight; p < 0.01). RT-PCR confirmed the presence of trappin-2 mRNA in OA chondrocytes. Immunohistochemical studies of OA cartilage revealed trappin-2 protein in chondrocytes. Peptide mapping of trappin-2 binding partners showed that fibromodulin was bound to trappin-2 in cartilage.
We confirmed the presence of trappin-2 in OA cartilage and SF. Elevated levels of TGase activity in OA cartilage may increase levels of this serine protease inhibitor in response to injury.
捕集蛋白是一类通过转谷氨酰胺酶（TGase）的作用与细胞外基质（ECM）结合的小分子量丝氨酸蛋白酶抑制剂。捕集蛋白-2存在于多种组织中，且在损伤部位上调。在骨关节炎（OA）中，丝氨酸蛋白酶会导致关节软骨破坏，且TGase活性增加。然而，对于关节软骨中与基质结合的丝氨酸蛋白酶抑制剂或TGase底物知之甚少。我们的目的是确定捕集蛋白-2是否存在于OA软骨和滑液（SF）中。
通过蛋白质免疫印迹法、酶联免疫吸附测定法（ELISA）和免疫组织化学法检测OA膝关节软骨和SF中的捕集蛋白-2蛋白。用逆转录聚合酶链反应（RT-PCR）检测捕集蛋白-2 mRNA。通过二维凝胶电泳和肽指纹图谱鉴定与捕集蛋白-2结合的ECM成分。
通过蛋白质免疫印迹法可在OA关节软骨提取物、培养的软骨细胞、条件培养基和SF中检测到捕集蛋白-2。OA软骨蛋白提取物中捕集蛋白-2的含量明显高于正常软骨蛋白提取物（湿重22.98±1.28 ng/mg vs 14.97±1.92 ng/mg；p<0.01）。RT-PCR证实OA软骨细胞中存在捕集蛋白-2 mRNA。OA软骨的免疫组织化学研究显示软骨细胞中有捕集蛋白-2蛋白。捕集蛋白-与2结合伴侣的肽图谱分析表明，纤维调节素在软骨中与捕集蛋白-2结合。
我们证实了捕集蛋白-2存在于OA软骨和SF中。OA软骨中TGase活性的升高可能会在损伤时增加这种丝氨酸蛋白酶抑制剂的水平。
