Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets
Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets
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DOI：
10.1073/pnas.1200425109
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发表时间：
2012-05-08
影响因子：
11.1
通讯作者：
Smith, Richard D.
中科院分区：
文献类型：
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作者：
Alfaro, Joshua F.;Gong, Cheng-Xin;Smith, Richard D.
O-连接的N-乙酰氨基葡萄糖（O-GlcNAc）是高等真核生物胞浆和核蛋白上丝氨酸和苏氨酸残基在O-GlcNAc转移酶（OGT）催化下的可逆翻译后修饰。O-GlcNAc最近被发现在Notch 1细胞外结构域上由EGF结构域特异性OGT催化。脑蛋白的异常O-GlcNAc修饰与阿尔茨海默病（AD）有关。然而，理解AD中O-GlcNAc化的特定功能受到蛋白质上O-GlcNAc位点表征困难的阻碍。在这项研究中，我们修改了化学/酶的光化学裂解方法富集O-GlcNAcylated肽的样品含有类似于100 μ g的胰蛋白酶肽从小鼠大脑皮层组织。共鉴定出274个O-GlcNAc酰化蛋白。其中，168个先前不知道被O-GlcNAc修饰。总共在195个蛋白质中鉴定了458个O-GlcNAc位点。许多修饰的残基是已知的磷酸化位点或位于已知磷酸化位点附近。这些发现支持了O-GlcNAc酰化和磷酸化之间的调节串扰。这项研究产生了最全面的O-GlcNAc蛋白质组的哺乳动物脑组织的蛋白质鉴定和O-GlcNAc位点分配。有趣的是，我们在五种膜蛋白的胞外结构域中观察到EGF样重复序列上的O-beta-GlcNAc，从而扩大了EGF结构域特异性OGT对胞外O-GlcNAc化的证据。我们还报告了多功能蛋白聚糖核心蛋白的EGF样重复序列上的GlcNAc-β-1，3-Fuc-alpha-1-O-Thr修饰，该核心蛋白是Fringe β-1，3-N-乙酰葡糖胺转移酶的建议底物。
O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing similar to 100 mu g of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-beta-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-beta-1,3-Fuc-alpha-1-O-Thrmodification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe beta-1,3-N-acetylglucosaminyltransferases.