Hideyuki Takeuchi

Rumi Is a CAP10 Domain Glycosyltransferase that Modifies Notch and Is Required for Notch Signaling

Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28 degrees C-30 degrees C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18 degrees C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.

Four-jointed Is a Golgi Kinase That Phosphorylates a Subset of Cadherin Domains

The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. We show that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed was essential for its kinase activity in vitro and for its biological activity in vivo. Our results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi.

Regulation of mammalian Notch signaling and embryonic development by the protein O -glucosyltransferase Rumi

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C1-X-S-X-P-C2 consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi−/− mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1+/− (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.

Significance of glycosylation in Notch signaling

Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.

Role of glycosylation of Notch in development

The Notch pathway is one of the major signaling pathways required for proper development in metazoans. Notch activity is regulated at numerous levels, and increasing evidence reveals the importance of protein glycosylation (modification of Notch receptors with sugars) for its regulation. In this review we summarize the significance of the Notch pathway in development and the players responsible for its glycosylation, and then discuss the molecular mechanisms by which protein glycosylation may regulate Notch function.

Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase.

Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila. Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O-glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O-glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O-xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O-glucose consensus sequence () of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence (), is also modified with either O-Xyl or O-glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O-Xyl but not O-glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used.

Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Significance The Notch pathway is a crucial metazoan cell–cell signaling pathway. The Notch receptor is regulated by O-glycosylation, a sugar modification that involves a series of enzyme-catalyzed additions to residues within EGF domains. Here, we demonstrate that the Fringe enzyme modification enhances the affinity of a receptor fragment for its ligand. X-ray crystallographic analysis demonstrates that the backbone structure does not change as a consequence of the modification, suggesting that the Fringe addition directly enhances ligand binding, although indirect effects cannot be ruled out. These data help to explain the Notch–Delta signaling increase seen in the presence of Fringe, but suggest that the inhibitory effects observed with the Jagged/Serrate ligand class are mediated by other regions of modified Notch. The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11–13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11–13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc–fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch–Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.

O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation

Thrombospondin type 1 repeat (TSR) superfamily members regulate diverse biological activities ranging from cell motility to inhibition of angiogenesis. In this study, we verified that mouse protein O-fucosyltransferase-2 (POFUT2) specifically adds O-fucose to TSRs. Using two Pofut2 gene-trap lines, we demonstrated that O-fucosylation of TSRs was essential for restricting epithelial to mesenchymal transition in the primitive streak, correct patterning of mesoderm, and localization of the definitive endoderm. Although Pofut2 mutant embryos established anterior/posterior polarity, they underwent extensive mesoderm differentiation at the expense of maintaining epiblast pluripotency. Moreover, mesoderm differentiation was biased towards the vascular endothelial cell lineage. Localization of Foxa2 and Cer1 expressing cells within the interior of Pofut2 mutant embryos suggested that POFUT2 activity was also required for the displacement of the primitive endoderm by definitive endoderm. Notably, Nodal, BMP4, Fgf8, and Wnt3 expression were markedly elevated and expanded in Pofut2 mutants, providing evidence that O-fucose modification of TSRs was essential for modulation of growth factor signaling during gastrulation. The ability of Pofut2 mutant embryos to form teratomas comprised of tissues from all three germ layer origins suggested that defects in Pofut2 mutant embryos resulted from abnormalities in the extracellular environment. This prediction is consistent with the observation that POFUT2 targets are constitutive components of the extracellular matrix (ECM) or associate with the ECM. For this reason, the Pofut2 mutants represent a valuable tool for studying the role of O-fucosylation in ECM synthesis and remodeling, and will be a valuable model to study how post-translational modification of ECM components regulates the formation of tissue boundaries, cell movements, and signaling.

Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of Notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats

Background: O-Glucosylation of EGF repeats occurs at high but variable stoichiometries on Notch. Results: In vitro assays revealed that the variability in glycosylation depends on the amino acid sequence and three-dimensional structure of individual EGF repeats. Conclusion: Proper folding and amino acid sequence of individual EGF repeats determine O-glucosylation efficiency. Significance: This work provides a regulatory mechanism for site-specific O-glucosylation on individual EGF repeats. O-Glucosylation of epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch is essential for Notch function. O-Glucose can be elongated by xylose to the trisaccharide, Xylα1–3Xylα1–3Glcβ1-O-Ser, whose synthesis is catalyzed by the consecutive action of three glycosyltransferases. A UDP-glucose:protein O-glucosyltransferase (Poglut/Rumi) transfers O-glucose to serine within the O-glucose consensus. Subsequently, either of two UDP-xylose:glucoside xylosyltransferases (Gxylt1 or Gxylt2) transfers xylose to O-glucose. Finally, a UDP-xylose:xyloside xylosyltransferase (Xxylt1) transfers xylose to Xylα1–3Glcβ1-O-EGF. Our prior site-mapping studies demonstrated that O-glucose consensus sites are modified at high but variable stoichiometries in mouse Notch1 and identified a novel glycosylation site with alanine in place of proline, suggesting a revised, broader consensus sequence (CXSX(P/A)C). Here we examined the molecular basis for this site specificity. A panel of EGF repeats from human coagulation factor 9 (FA9), mouse Notch1, and Notch2 were bacterially expressed and purified by reverse phase HPLC for use in in vitro enzyme assays. We demonstrate that proper folding of EGF repeats is essential for glycosylation by Poglut/Rumi, that alanine can substitute for proline in the context of coagulation factor 9 EGF repeat for O-glucose transfer, confirming the new consensus sequence, and that positively charged residues within the O-glucose consensus sequence reduce efficiency of glycosylation by Poglut/Rumi. Moreover, proper folding of EGF repeats is also important for the activities of Gxylt1, Gxylt2, and Xxylt1. These results indicate that protein folding and amino acid sequences of individual EGF repeats fundamentally affect both attachment and elongation of O-glucose glycans.

Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

A major remaining question in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xylosideα1–3 Xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly the Epidermal Growth Factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations where dysregulation of Notch is known to cause cancer or developmental disorders.