Department of Protein Science, Helmholtz Zentrum Mùnchen - German Research Center for Environmental Health, Neuherberg, Germany.
Mol Cell Proteomics. 2010 Oct;9(10):2292-305. doi: 10.1074/mcp.M110.001073. Epub 2010 Jul 4.
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Müller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis.
自身免疫性葡萄膜炎是一种致盲性疾病，其特征是针对眼特异性蛋白的自身抗体以及浸润和攻击免疫特惠靶组织视网膜的自身攻击性 T 细胞。使 T 细胞能够浸润和攻击组织的分子事件仍然难以捉摸。病变和健康阶段之间膜蛋白表达模式的变化尤其有趣，因为疾病的起始事件很可能发生在膜上。由于疾病的进展伴随着血视网膜屏障的破坏，血清来源的蛋白质掩盖了潜在的靶组织相关变化。为了克服这一限制，我们使用了来自唯一可用的自发性动物模型马复发性葡萄膜炎的视网膜中提取的富含膜的级分，并通过无标记的 LC-MSMS 基于策略与健康对照样本进行了表达水平比较。我们可以轻松识别总共 893 种马蛋白，其中 57%归因于基因本体项目术语“膜”。其中，在马复发性葡萄膜炎组织中发现有 179 种蛋白表达差异。途径富集分析表明与抗原处理和呈递、TNF 受体信号转导、整合素细胞表面相互作用和黏附斑相关的蛋白增加。此外，还观察到反映视力下降的视网膜特异性蛋白的丢失，以及神经胶质细胞特异性蛋白的增加，表明神经胶质反应性。选择的蛋白候选物（小窝蛋白 1、整合素 alpha 1 和黏着斑激酶）通过免疫组织化学进行了验证，组织染色模式表明这些蛋白在外限膜（血视网膜外屏障的一部分）水平上的显著增加。综上所述，膜的富集与基于 LC-MSMS 的无标记定量相结合，极大地提高了比较组织分析的灵敏度，并检测到与马复发性葡萄膜炎相关的新分子途径。
