N-Glycans on EGF domain-specificO-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization
N-Glycans on EGF domain-specificO-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization
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DOI：
10.1074/jbc.ra119.012280
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发表时间：
2020-06-19
影响因子：
4.8
通讯作者：
Okajima, Tetsuya
中科院分区：
文献类型：
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作者：
Alam, Sayad Md. Didarul;Tsukamoto, Yohei;Okajima, Tetsuya
表皮生长因子（EGF）结构域特异性O-GlcNAc转移酶（EOGT）是内质网（ER）驻留蛋白，其修饰Notch受体的EGF重复序列，从而调节Delta样配体介导的Notch信号传导。在癌症数据库中记录了几个可能影响假定的N-糖基化共有位点的EOGT突变，但EOGT中N-聚糖的存在和功能尚未得到表征。在此，我们鉴定了小鼠EOGT中的N-糖基化位点，并阐明了它们的分子功能。三个预测的EOGT N-糖基化共有序列在哺乳动物中高度保守。在这些位点中，我们发现Asn-263和Asn-354，而不是Asn-493，被N-聚糖修饰。凝集素印迹，内切糖苷酶H消化，和MS分析表明，这两个残基的修饰与寡甘露糖N-聚糖。在HEK 293 T细胞中，EOGT上单个N-聚糖的丢失并不影响其内质网（ER）定位、酶活性和O-GlcN酰化Notch 1的能力。然而，同时取代两个N-糖基化位点影响EOGT的成熟和表达水平，而酶活性没有明显的变化，这表明在一个单一的网站N-糖基化是足够的EOGT的成熟和表达。因此，与WT EOGT相比，在与N263 Q/N354 Q变体共表达的Notch 1中观察到O-GlcNAc化学计量的减少。此外，N263 Q/N354 Q变体在HEK 293 T细胞中ER内的亚细胞分布发生改变，表明EOGT的N-糖基化是其ER定位于细胞外周所必需的。这些结果表明，N-聚糖在维持O-GlcNAc转移酶功能中的关键作用，既通过维持EOGT水平，又通过确保其在ER中的适当亚细胞定位。
Epidermal growth factor (EGF) domain-specificO-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. SeveralEOGTmutations that may affect putativeN-glycosylation consensus sites are recorded in the cancer database, but the presence and function ofN-glycans in EOGT have not yet been characterized. Here, we identifiedN-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predictedN-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified withN-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannoseN-glycans. Loss of an individualN-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability toO-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of bothN-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting thatN-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease inO-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating thatN-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles ofN-glycans in sustainingO-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.