Riedle B, Kerjaschki D
Section of Ultrastructural Pathology, University of Vienna, Austria.
Am J Pathol. 1997 Jul;151(1):215-31.
Reactive oxygen species (ROS) are produced and released into the extracellular spaces in numerous diseases and contribute to development and progression, for example, of inflammatory diseases, proteinuria, and tumor invasion. However, little is known about ROS-induced chemical changes of interstitial matrix proteins and their consequences for the integrity of the matrix meshwork. As basement membranes and other matrices are highly cross-linked and complex, the relatively simple matrix produced by Engelbreth-Holm-Swarm (EHS) sarcoma, and proteins isolated therefrom, were incubated in vitro with defined concentrations of ROS that were generated by the Fenton or xanthine oxidase/xanthine reactions. This resulted in two counter-current effects. Although up to approximately 15% of the EHS matrix proteins were released into the supernatant in a ROS dose-response relationship, the residual insoluble matrix was partially cross-linked by ROS. Matrix proteins released into the supernatants were examined by rotary shadowing, quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, and fluorospectrometry for loss of tryptophans and formation of bityrosine residues. At relatively low ROS concentrations, selective liberation of morphologically intact laminin/entactin was found that, however, failed to reassociate and showed oxidative damage of its tryptophan residues. At higher ROS concentrations, laminin and entactin were progressively disintegrated, partially fragmented, and eventually completely degraded. At this point oligomers of type IV collagen predominated in the supernatant, and proteoglycans were not encountered at any concentration of ROS. Similar gradual molecular changes were also obtained when fractions of isolated soluble EHS matrix proteins were incubated with graded concentrations of ROS. In these experiments, the formation of covalently linked oligomers and aggregates paralleled the ROS-dependent formation of cross-linking bityrosine groups. ROS scavengers pinpointed to the hydroxyl radical as the most damaging radical species. Protease inhibitor experiments suggested that degradation of matrix proteins was caused primarily by the direct action of ROS and not by proteolysis by potentially contaminating proteases. Collectively, these results provide evidence that EHS matrix proteins show differential sensitivity to ROS-induced damage in a reproducible, sequential pattern, in the order entactin > laminin > type IV collagen, and that ROS cause partial dissociation and cross-linking of the EHS matrix.
活性氧（ROS）在多种疾病中产生并释放到细胞外空间，促进疾病的发展和进展，例如炎症性疾病、蛋白尿和肿瘤侵袭。然而，关于ROS诱导的间质基质蛋白化学变化及其对基质网络完整性的影响，人们知之甚少。由于基底膜和其他基质高度交联且复杂，因此将Engelbreth-Holm-Swarm（EHS）肉瘤产生的相对简单的基质及其分离出的蛋白质，与通过芬顿反应或黄嘌呤氧化酶/黄嘌呤反应产生的特定浓度的ROS在体外孵育。这产生了两种相反的效应。尽管高达约15%的EHS基质蛋白以ROS剂量反应关系释放到上清液中，但残留的不溶性基质被ROS部分交联。通过旋转阴影法、定量十二烷基硫酸钠聚丙烯酰胺凝胶电泳、免疫印迹和荧光光谱法检测释放到上清液中的基质蛋白，以检测色氨酸的损失和双酪氨酸残基的形成。在相对较低的ROS浓度下，发现形态完整的层粘连蛋白/巢蛋白有选择性释放，然而，它们未能重新结合，并显示出色氨酸残基的氧化损伤。在较高的ROS浓度下，层粘连蛋白和巢蛋白逐渐解体、部分片段化，最终完全降解。此时，上清液中IV型胶原寡聚体占主导地位，在任何ROS浓度下均未检测到蛋白聚糖。当将分离的可溶性EHS基质蛋白组分与分级浓度的ROS孵育时，也获得了类似的逐渐分子变化。在这些实验中，共价连接的寡聚体和聚集体的形成与ROS依赖性的交联双酪氨酸基团的形成平行。ROS清除剂指出羟基自由基是最具破坏性的自由基种类。蛋白酶抑制剂实验表明，基质蛋白的降解主要是由ROS的直接作用引起的，而不是由潜在污染的蛋白酶的蛋白水解作用引起的。总的来说，这些结果提供了证据，表明EHS基质蛋白对ROS诱导的损伤表现出可重复的、顺序性的差异敏感性，顺序为巢蛋白>层粘连蛋白>IV型胶原，并且ROS导致EHS基质的部分解离和交联。
