期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2018
卷号：115
期号：36
页码：E8395-E8402
DOI：10.1073/pnas.1804005115
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The Notch-signaling pathway is normally activated by Notch–ligand interactions. A recent structural analysis suggested that a novel O -linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O -glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O -glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O -glucose ( O -Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2 , POGLUT3 , or both genes showed that either POGLUT2 or POGLUT3 can add this novel O -Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O -glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O -Glc modification site on EGF11 (serine 435) in combination with sensitizing O -fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.
关键词：glycosyltransferases ; Notch ; signal transduction ; development ; O -glucose
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