Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis
Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis
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DOI：
10.1016/j.ab.2018.02.028
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发表时间：
2018-05-01
影响因子：
2.9
通讯作者：
Tsuchiya, Mikako
中科院分区：
文献类型：
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作者：
Osago, Harumi;Kobayashi-Miura, Mikiko;Tsuchiya, Mikako
关节软骨包含透明基质中的胶原蛋白、蛋白聚糖和糖胺聚糖（GAG）以及水。关节软骨对蛋白水解溶解有抵抗力，部分原因是胶原纤维与热不稳定氢键的组装。在这项研究中，我们使用热稳定蛋白酶嗜热菌蛋白酶在70 ℃下消化固体关节软骨中的胶原蛋白，并将胶原蛋白消化和GAG提取的效率与50 ℃下胶原酶消化的效率进行比较。用嗜热菌蛋白酶过夜消化可完全溶解软骨，而蛋白水解活性高出10倍以上的胶原酶可消化< 20%的胶原蛋白。在嗜热菌蛋白酶处理后，几乎所有的GAG都从软骨中提取，而胶原酶消化后仅提取了56%的GAG。提取的GAG链的二糖分析显示，与使用胶原酶的< 60%提取效率相比，根据GAG类别，从嗜热菌蛋白酶处理的软骨中提取几种GAG类别的效率> 98%。这些结果表明，嗜热菌蛋白酶允许从固体关节软骨中完全提取GAG，并且需要完全溶解才能准确且可重复地分析软骨GAG。因此，嗜热菌蛋白酶提供了一种在全面GAGomic分析之前完全溶解软骨的工具，并且可能适用于其他富含胶原蛋白的组织，如韧带、皮肤和血管。
Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 degrees C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 degrees C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with > 10-times higher proteolytic activity digested < 20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed > 98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with < 60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.