Drusen proteome analysis: An approach to the etiology of age-related macular degeneration
Drusen proteome analysis: An approach to the etiology of age-related macular degeneration
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DOI：
10.1073/pnas.222551899
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发表时间：
2002-11-12
影响因子：
11.1
通讯作者：
Hollyfield, JG
中科院分区：
文献类型：
--
作者：
Crabb, JW;Miyagi, M;Hollyfield, JG
drusen是细胞外沉积物，在Bruch膜上积聚在视网膜色素上皮下方，是发展与年龄相关的黄斑变性（AMD）的危险因素。如果可以调节DRUSEN的形成，则AMD的进展可能会减慢或停止。为了朝着对DRUSEN形成的分子理解，我们开发了一种分离微克量的Drusen和Bruch膜进行蛋白质组分析的方法。来自18个正常供体和5个AMD供体的DRUSEN制剂的液相色谱串联MS分析鉴定出129种蛋白质。到目前为止，免疫细胞化学研究已将这些蛋白的16％局部局部局部。组织金属蛋白酶抑制剂3，簇蛋白，玻璃纤维和血清白蛋白是正常供体drusen中最常见的蛋白质，而在AMD供体drusen中更频繁地检测到结晶蛋白。在AMD和正常供体的DRUSEN中发现了多达65％的蛋白质。然而，还观察到氧化蛋白修饰，包括组织金属蛋白酶抑制剂3和玻璃体蛋白的明显交联物种，以及羧乙基吡咯蛋白加合物。羧乙基金刚加合物是由含二十六烯酸酯的脂质的氧化产生的。通过西方分析，它们在AMD中比正常的Bruch膜更丰富，并被发现与Drusen蛋白有关。在drusen中还检测到羧甲基赖氨酸是另一种氧化性修饰。这些数据强烈支持氧化损伤有助于AMD发病机理的假设，并表明氧化蛋白修饰可能在DRUSEN形成中具有关键作用。
Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximate to16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.