Xi Jinghua, Farjo Rafal, Yoshida Shigeo, Kern Timothy S, Swaroop Anand, Andley Usha P
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Vis. 2003 Aug 28;9:410-9.
Crystallins are expressed at high levels in lens fiber cells. Recent studies have revealed that several members of the alpha, beta, and gamma-crystallin family are also distributed in many non-lens tissues, though at lower levels. We observed that the use of retinal RNA as target for both custom I-Gene microarrays and Affymetrix GeneChips revealed significant expression of many crystallin genes. This prompted us to undertake a comprehensive investigation to delineate the baseline expression of crystallin genes in the adult mouse retina.
Quantitative RT-PCR was carried out using gene specific primers (derived from the mouse genomic sequence) for each crystallin gene. Immunofluorescence studies using frozen sections of the mouse retinas were performed with crystallin-specific antibodies. Retinal lysates were analyzed by immunoblotting using antibodies specific to alphaA and alphaB crystallins and those produced against total beta-crystallin and gamma-crystallin fractions of bovine lenses.
Microarray analysis followed by quantitative RT-PCR revealed that mouse retinal cells express transcripts for 20 different members of the crystallin gene family; these are alphaA, alphaA-INS, alphaA-nov1, alphaB, betaA1, betaA2, betaA3, betaA4, betaB1, betaB2, betaB3, gammaA, gammaC, gammaD, gammaE, gammaF, gammaS, mu, zeta, and lambda-crystallin. The gene products of alphaA, alphaB, beta-, and gamma-crystallins are detected in the outer and inner nuclear layers of the retina by immunofluorescence analysis. In addition, alpha and beta-crystallins are detected in the photoreceptor inner segments. Retinal expression of these proteins was further confirmed by immunoblot analysis. Interestingly, our studies also showed a significant animal-to-animal variation in the expression level of some of the crystallins.
Our results establish the expression of many crystallins in the adult mouse retina. Detection of crystallins in the retinal nuclear layers, though surprising, is consistent with their proposed role in cell survival and genomic stability. We suggest that crystallins play vital functions in protecting retinal neurons from damage by environmental and/or metabolic stress.
晶状体蛋白在晶状体纤维细胞中高水平表达。最近的研究表明，α、β和γ晶状体蛋白家族的几个成员也分布于许多非晶状体组织中，不过表达水平较低。我们观察到，将视网膜RNA用作定制I-Gene微阵列和Affymetrix基因芯片的靶标时，发现许多晶状体蛋白基因有显著表达。这促使我们进行全面研究，以描绘成年小鼠视网膜中晶状体蛋白基因的基础表达情况。
使用针对每个晶状体蛋白基因的基因特异性引物（源自小鼠基因组序列）进行定量逆转录聚合酶链反应（RT-PCR）。用晶状体蛋白特异性抗体对小鼠视网膜冰冻切片进行免疫荧光研究。通过使用针对αA和αB晶状体蛋白的特异性抗体以及针对牛晶状体总β晶状体蛋白和γ晶状体蛋白组分产生的抗体进行免疫印迹分析视网膜裂解物。
微阵列分析随后进行定量RT-PCR显示，小鼠视网膜细胞表达晶状体蛋白基因家族20个不同成员的转录本；这些成员是αA、αA-INS、αA-nov1、αB、βA1、βA2、βA3、βA4、βB1、βB2、βB3、γA、γC、γD、γE、γF、γS、μ、ζ和λ晶状体蛋白。通过免疫荧光分析在视网膜的外核层和内核层中检测到αA、αB、β和γ晶状体蛋白的基因产物。此外，在光感受器内节中检测到α和β晶状体蛋白。这些蛋白质在视网膜中的表达通过免疫印迹分析得到进一步证实。有趣的是，我们的研究还显示，某些晶状体蛋白的表达水平在不同动物之间存在显著差异。
我们的结果证实了许多晶状体蛋白在成年小鼠视网膜中的表达。在视网膜核层中检测到晶状体蛋白，尽管令人惊讶，但与它们在细胞存活和基因组稳定性中所起的作用一致。我们认为，晶状体蛋白在保护视网膜神经元免受环境和/或代谢应激损伤方面发挥着至关重要的作用。
