Structural basis of Notch O-glucosylation and O–xylosylation by mammalian protein–O-glucosyltransferase 1 (POGLUT1)

Abstract

Protein O-glucosyltransferase 1/Rumi-mediated glucosylation of Notch epidermal growth factor-like (EGF-like) domains plays an important role in Notch signaling. Protein O-glucosyltransferase 1 shows specificity for folded EGF-like domains, it can only glycosylate serine residues in the C1XSXPC2 motif, and it possesses an uncommon dual donor substrate specificity. Using several EGF-like domains and donor substrate analogs, we have determined the structures of human Protein O-glucosyltransferase 1 substrate/product complexes that provide mechanistic insight into the basis for these properties. Notably, we show that Protein O-glucosyltransferase 1’s requirement for folded EGF-like domains also leads to its serine specificity and that two distinct local conformational states are likely responsible for its ability to transfer both glucose and xylose. We also show that Protein O-glucosyltransferase 1 possesses the potential to xylosylate a much broader range of EGF-like domain substrates than was previously thought. Finally, we show that Protein O-glucosyltransferase 1 has co-evolved with EGF-like domains of the type found in Notch.

Two novel protein O -glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling

“…Neither enzyme required divalent cations for activity (SI Appendix, Fig. S7A), similar to POGLUT1 (24,30). Both enzymes showed enhanced activity below pH 6 (SI Appendix, Fig.…”

“…1A). Recent cocrystal structures of two of these enzymes [POGLUT1 (24,30) and POFUT1 (32)], each in complex with an EGF repeat, highlight the reason why the EGF repeats need to be folded and have an appropriate consensus sequence to be modified. The combination of the consensus sequence and the 3D fold of the EGF repeat, bound in a particular orientation within a large pocket on the surface of these enzymes, places the hydroxyl group to be modified precisely in the proper place to serve as a nucleophile in the transfer reaction.…”

Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1

“…The presence of a Xylo_C domain is a hallmark of XTs involved in GAG biosynthesis; the domain is not found in other XTs ( Li et al., 2017 , Yu et al., 2015 , Yu et al., 2016 ). The role of the Xylo_C domain remains unclear.…”

Effects of Notch glycosylation on health and diseases

“…The original discovery of the Notch gene in Drosophila identified 36 epidermal growth factor-like (EGF) repeats consecutively present in the Notch receptor extracellular domain (NECD; Figure 1a; Wharton, Johansen, Xu, & Artavanis-Tsakonas, 1985). Recent visualization of the structures of Notch-decorating glycosyltransferases and the frosted Notch receptors in complex with Notch ligands has advanced our understanding of the significance of glycosylation in Notch regulation (Kovall, Gebelein, Sprinzak, & Kopan, 2017;Li et al, 2017;Luca et al, 2015Luca et al, , 2017Taylor et al, 2014;Yu et al, 2016Yu et al, , 2015. Recent visualization of the structures of Notch-decorating glycosyltransferases and the frosted Notch receptors in complex with Notch ligands has advanced our understanding of the significance of glycosylation in Notch regulation (Kovall, Gebelein, Sprinzak, & Kopan, 2017;Li et al, 2017;Luca et al, 2015Luca et al, , 2017Taylor et al, 2014;Yu et al, 2016Yu et al, , 2015.…”