Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)
Murine Retinal Citrullination Declines With Age and is Mainly Dependent on Peptidyl Arginine Deiminase 4 (PAD4)
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DOI：
10.1167/iovs.18-24118
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发表时间：
2018-08-01
影响因子：
4.4
通讯作者：
Iannaccone, Alessandro
中科院分区：
文献类型：
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作者：
Hollingsworth, T. J.;Radic, Marko Z.;Iannaccone, Alessandro
目的。瓜氨酸化是一种具有多种正常生理功能的翻译后修饰（PTM）。研究表明，这种ptm以及催化酶肽基精氨酸脱亚胺酶（pad）的表达在自身免疫和年龄相关疾病中增加。先前在大鼠和人的视网膜中有记录显示PAD2的表达。在此，我们报道了PAD2、PAD4和视网膜瓜氨酸化在小鼠视网膜中随年龄增长的表达水平和模式。野生型（WT）和Pad4(-/-) （PAD4KO）小鼠分别在0.5、0.75、1、3、6和9个月大时安乐死和眼球摘除。用western blot检测3月龄小鼠视网膜裂解液中PAD4的含量。固定全眼，冷冻切片，用抗pad2 /4抗体（Ab）、特异性抗pad4抗体和F95抗瓜氨酸肽Ab进行探针检测。用ImageJ.RESULTS定量荧光强度。WT视网膜在不同的视网膜层中显示出不同水平的PAD4表达，其中视网膜内层的标记最强烈，而PAD4KO小鼠缺乏视网膜PAD4。使用非特异性抗PAD2/4抗体，在PAD4KO小鼠中观察到的PAD反应性归因于PAD2。在WT中，PAD2和PAD4的表达水平随着年龄的增长而显著下降，而在PAD4KO小鼠中，PAD2的表达水平仍很低。WT视网膜中的瓜氨酸化水平与PAD4表达平行，PAD4KO小鼠的瓜氨酸化水平一直很低。在小鼠视网膜中，PAD2和PAD4的表达和瓜氨酸化随年龄的增长而降低。然而，在缺乏PAD4的情况下，视网膜瓜氨酸化几乎被废除，这表明在生理条件下，PAD4是视网膜瓜氨酸化的主要效应器。
PURPOSE. Citrullination is a post-translational modification (PTM) that serves many normal physiological functions. Studies have shown that this PTM-along with expression of the catalyzing enzymes, peptidyl arginine deiminases (PADs)-are increased in autoimmune and age-related pathologies. PAD2 retinal expression has been previously documented in rat and human. Herein, we report on the expression levels and patterns of PAD2, PAD4, and retinal citrullination in the murine retina with age.METHODS. Wild-type (WT) and Pad4(-/-) (PAD4KO) mice ages 0.5, 0.75, 1, 3, 6, and 9 months were investigated after euthanasia and eye enucleation. Retinal lysates from 3-month-old mice were probed for PAD4 by western blot. Whole eyes were fixed, cryosectioned, and probed using an anti-PAD2/4 antibody (Ab), a specific anti-PAD4 Ab, and F95 anti-citrullinated peptide Ab. Fluorescent intensities were quantified with ImageJ.RESULTS. WT retinas show different levels of PAD4 expression in distinct retinal layers, with the most intense labeling in inner retinal layers, while PAD4KO mice lacked retinal PAD4. Using a nonspecific anti-PAD2/4 Ab, PAD reactivity observed in PAD4KO mice was attributed to PAD2. In WT, both PAD2 and PAD4 expression levels decrease significantly with age while low-level residual PAD2 expression was seen in PAD4KO mice. Citrullination levels in WT retinas paralleled PAD4 expression, with PAD4KO mice exhibiting consistently minimal citrullination.CONCLUSIONS. Both PAD2 and PAD4 expression and citrullination decrease with age in the murine retina. However, in the absence of PAD4, retinal citrullination is nearly abolished, indicating that PAD4 is a main effector for retinal citrullination under physiological conditions.