Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.

中文翻译：

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基因重新编码以剖析位点特异性蛋白 O-GlcNAcylation 的作用。

在 O-GlcNAc 转移酶 (OGT) 的催化下，用 O-GlcNAc 修饰核质蛋白的特定 Ser 和 Thr 残基是动物正常发育所必需的丰富的翻译后事件，并且在各种疾病中失调。由于单个 O-GlcNA 酶 (OGA) 的快速同时去除，在不影响整个 O-GlcNAc 蛋白质组的情况下，目前不可能在体内进行位点特异性 O-GlcNAc 修饰的精确功能解剖。利用 OGT 的偶然性，我们证明 S-GlcNAc 是 O-GlcNAc 的水解稳定且准确的结构模拟物，可以在哺乳动物系统中与 CRISPR-Cas9 在其他不受干扰的 O-GlcNAcome 中编码。使用这种方法，我们针对 OGA 上难以捉摸的 Ser 405 O-GlcNAc 位点，表明这种特定于位点的修改会影响 OGA 稳定性。