Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

Abstract

Rumi O-glucosylates the EGF repeats of a growing list of proteins essential in metazoan development including Notch. Rumi is essential for Notch signaling, and Rumi dysregulation is linked to several human diseases. Despite Rumi’s critical roles, it is unknown how Rumi glucosylates a serine of many but not all EGF repeats. Here we report crystal structures of Drosophila Rumi as binary or ternary complexes with a folded EGF repeat and/or donor substrates. These structures provide insights into the catalytic mechanism, and show that Rumi recognizes structural signatures of the EGF motif, the U-shaped consensus sequence, C-X-S-X-(P/A)-C and a conserved hydrophobic region. We found that five Rumi mutations identified in cancers and Dowling-Degos disease are clustered around the enzyme active site and adversely affect its activity. Our study suggests that loss of Rumi activity may underlie these diseases, and the mechanistic insights may facilitate the development of modulators of Notch signaling.

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

“…Yun et al solved X-ray structures of the endoplasmic reticulum enzyme Xyloside α-1,3-xylosyltransferase (XXYLT1), a well conserved type II membrane protein that can extend O-glucose modifications within Notch EGF repeats. In addition they solved the structure of Rumi alone and in complex with an EGF repeat derived from the coagulation factor IX containing a Xyl-Glc disaccharide modification (Yu et al, 2016; Yu et al, 2015). These structures provided mechanistic insights into how these enzymes bind to and modify Notch.…”

“…Rumi/POGLUT1 is the glycosyltransferase that adds O-glucose moieties to serine residues within EGF repeats containing the consensus sequence C-X- S -X-(P/A)-C. The structure shows that Rumi is composed of two domains with βαβ folds (Rossmann fold), in which the EGF repeat binds within a cleft between the two domains (Yu et al, 2016) (Figure 1). The O-glucosylation consensus sequence C-X- S -X-(P/A)-C forms a U-shaped structural motif.…”

Integration of Drosophila and Human Genetics to Understand Notch Signaling Related Diseases

“…Considering that Notch regulates multiple aspects of melanocyte development[364], it is likely that defects in Notch signaling contribute to the pathogenesis of these diseases[365,366]. However, direct experimental evidence is necessary to test this hypothesis.…”

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.…”

“…, Ikenaka et al reported O-glucose glycans attached to the EGF repeats of bovine blood coagulation factors VII and IX(Hase et al, 1988). The O-glucose glycans are added to the serine residue between the first and second cysteine residue of the EGF domain (the consensus sequence C 1 -X-S-X-(P/A)-C 2 , where X is any amino acid) by POGLUT1 (rumi in Drosophila) that strictly recognizes the amino acid sequences and the folding status of EGF repeats(Figure 2b;Acar et al, 2008;Takeuchi et al, 2012;Yu et al, 2016). As for the biosynthetic elongation of O-glucose glycans, the first xylose is added to the O-glucose monosaccharide by GXYLT1 and GXYLT2 (Shams in Drosophila), and the second xylose is added by XXYLT1 (CG11388 in Drosophila;Sethi et al, 2012Sethi et al, , 2010 Figure 2b).…”