Kapphahn Rebecca J, Ethen Cheryl M, Peters Elizabeth A, Higgins LeeAnn, Ferrington Deborah A
Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Biochemistry. 2003 Dec 30;42(51):15310-25. doi: 10.1021/bi034774e.
Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.
晶状体蛋白是具有伴侣功能的小热休克蛋白，可防止热应激和氧化应激诱导的蛋白质聚集。这是首篇描述感觉视网膜中αA晶状体蛋白修饰的报告，包括其含量随衰老而改变以及截短情况。对成年、中年和老年费希尔344 布朗挪威大鼠的蛋白质进行了比较。采用蛋白质免疫印迹法评估αA晶状体蛋白含量，并在含有αA晶状体蛋白的二维凝胶上鉴定蛋白质斑点。通过质谱法鉴定多种翻译后修饰的类型和位点。我们发现，最老的大鼠中αA晶状体蛋白的含量显著降低。在二维凝胶上，视网膜晶状体蛋白分离为多个迁移改变的斑点，表明其内在电荷和/或截短情况发生了变化。鉴定出的翻译后修饰包括氧化、磷酸化、脱酰胺、乙酰化和截短。在所有年龄段大鼠的样本中，均发现了一个含有这些修饰的高度修饰的N端。我们还观察到N端和C端截短程度存在年龄依赖性差异。这些结果表明，老年视网膜中抵御应激诱导的蛋白质聚集的能力受损。
