Glycoproteomics of NOTCH1 EGF repeat fragments overexpressed with different glycosyltransferases in HEK293T cells reveals insights into O-GlcNAcylation of NOTCH1
Glycoproteomics of NOTCH1 EGF repeat fragments overexpressed with different glycosyltransferases in HEK293T cells reveals insights into O-GlcNAcylation of NOTCH1
复制标题
HEK293T 细胞中用不同糖基转移酶过表达的 NOTCH1 EGF 重复片段的糖蛋白组学揭示了对 NOTCH1 O-GlcNAc 酰化的见解
DOI：
10.1093/glycob/cwac015
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发表时间：
2022
期刊：
影响因子：
4.3
通讯作者：
Okajima Tetsuya
中科院分区：
文献类型：
--
作者：
Tsukamoto Yohei;Ogawa Mitsutaka;Yogi Kentarou;Tashima Yuko;Takeuchi Hideyuki;Okajima Tetsuya
Notch受体的O-GlcNAc修饰以不同于Notch受体的表皮生长因子（EGF）样重复序列上的其他形式的O-聚糖的方式调节Notch配体相互作用。尽管除了Notch受体之外，许多蛋白质被预期通过EGF结构域特异性O-GlcNAc转移酶（EOGT）被O-GlcNAc酰化，但仅少数蛋白质已被报道在体内被修饰，并且延伸的O-GlcNAc聚糖尚未被广泛探索。为了扩展我们对聚糖修饰的特异性和多样性的看法，我们对哺乳动物中NOTCH 1上的O-GlcNAc聚糖进行了全面分析。对HEK 293 T细胞中表达的NOTCH 1片段的质谱分析显示，几个具有假定的O-GlcNAc化位点的EGF结构域几乎不被O-GlcNAc修饰。虽然修饰位点之前的氨基酸残基优先被芳香残基占据，但对于O-GlcNAc的表观修饰，Phe和Tyr优于Trp。此外，在EGF结构域的子集中检测到岩藻糖基化O-GlcNAc聚糖的次要形式。FUT 1、FUT 2或FUT 9表达可增强O-GlcNAc聚糖的岩藻糖基化。FUT 9依赖性刘易斯X表位通过使用抗刘易斯X抗体的免疫印迹来确认。正如O-Fuc和O-GlcNAc聚糖之间延伸结构的相似性所预期的，在与L-Fringe共表达的NOTCH 1片段上检测到Lexis X抗原，其介导O-Fuc聚糖的延伸。我们的研究结果完善了哺乳动物中EOGT依赖性O-GlcNAc修饰的推定共有序列，并揭示了功能性NotchO-聚糖的结构多样性。
O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms ofO-glycans on epidermal growth factor (EGF)-like repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specificO-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongatedO-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis ofO-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified withO-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification withO-GlcNAc. Furthermore, a minor form of fucosylatedO-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation ofO-GlcNAc glycans was enhanced byFUT1,FUT2, orFUT9expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the extended structures betweenO-Fuc andO-GlcNAc glycans, the Lexis X antigen was detected on NOTCH1 fragments co-expressed with L-Fringe, which mediates elongation ofO-Fuc glycans. Our results refined the putative consensus sequence for the EOGT-dependentO-GlcNAc modification in mammals and revealed the structural diversity of functional NotchO-glycans.