Abstract
Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.
Highlights
Glycosylation in the endoplasmic reticulum (ER)4 has long been associated with protein folding [1, 2]
To examine the effects of loss of O-fucose and O-glucose glycans on Notch1 functions, we genetically deleted POFUT1 or protein O-glucosyltransferase 1 (POGLUT1), which are solely responsible for the initial attachment of O-fucose or O-glucose to Epidermal growth factor-like (EGF) repeats, respectively, in
Flow cytometric analysis showed that the levels of endogenous Notch1 on the cell surface were reduced by a factor of 2 in either POFUT1 or POGLUT1
Summary
To examine the effects of loss of O-fucose and O-glucose glycans on Notch functions, we genetically deleted POFUT1 or POGLUT1, which are solely responsible for the initial attachment of O-fucose or O-glucose to EGF repeats, respectively, in HEK293T cells. Western blot analysis showed increased fulllength Notch proteins in the double KO cell lysates compared with the single KO cells (Fig. 1C) These results strongly suggested that both O-fucose and O-glucose glycans are required for proper Notch trafficking in HEK293T cells and that they function in a non-redundant, additive manner. In POGLUT1 KO cells, cotransfection of POGLUT1 rescued the secretion defect, whereas co-transfection of POFUT1 did not These results support the above notion that addition of O-fucose by POFUT1 and O-glucose by POGLUT1 to the EGF repeats in the Notch extracellular domain are both required for efficient protein folding and secretion in HEK293T cells. The O-glycan modifications showed similar effects on unfolding of hFA9 EGF repeat in the absence and presence of calcium ions (Fig. 2B compared with 2A). EGF28 of mouse Notch is a poorer substrate for POGLUT1, suggesting the efficiency of modification on EGF28 could vary depending on the levels of POGLUT1 in a particular cell [33]
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