Shinako Kakuda

A Mutation in EGF Repeat-8 of Notch Discriminates Between Serrate/Jagged and Delta Family Ligands

Discerning a Difference Neighboring cells communicate via the Notch signaling pathway to make numerous decisions. Notch receptors are known to distinguish between two distinct ligand families, Delta and Serrate/Jagged, in different contexts. Posttranslational sugar modifications have been shown to play a role in this process, but it is not clear if other features of Notch are involved. Using a forward genetic approach in fruit flies, Yamamoto et al. (p. 1229) identified an evolutionarily conserved amino acid in the extracellular domain of Notch necessary for Serrate/Jagged signaling but dispensable for Delta signaling. A genetic screen identifies an extracellular motif in a conserved signaling receptor that confers ligand specificity. Notch signaling affects many developmental and cellular processes and has been implicated in congenital disorders, stroke, and numerous cancers. The Notch receptor binds its ligands Delta and Serrate and is able to discriminate between them in different contexts. However, the specific domains in Notch responsible for this selectivity are poorly defined. Through genetic screens in Drosophila, we isolated a mutation, Notchjigsaw, that affects Serrate- but not Delta-dependent signaling. Notchjigsaw carries a missense mutation in epidermal growth factor repeat-8 (EGFr-8) and is defective in Serrate binding. A homologous point mutation in mammalian Notch2 also exhibits defects in signaling of a mammalian Serrate homolog, Jagged1. Hence, an evolutionarily conserved valine in EGFr-8 is essential for ligand selectivity and provides a molecular handle to study numerous Notch-dependent signaling events.

Structural Basis for Acceptor Substrate Recognition of a Human Glucuronyltransferase, GlcAT-P, an Enzyme Critical in the Biosynthesis of the Carbohydrate Epitope HNK-1

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galβ1-4GlcNAc) but not lacto-N-biose (Galβ1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn2+, and an acceptor substrate analogue N-acetyllactosamine (Galβ1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an α/β protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp195-Asp196-Asp197). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val320 and Asn321, which are located on the C-terminal long loop from a neighboring molecule, and Phe245 contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C1XSXPC2. We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C1XSX(A/P)C2. We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity

Forces on Notch receptors can modulate receptor signaling. Tugging on Notch receptor tunes signaling Notch proteins are transmembrane receptors that are important in the regulation of cell fate. They are unusual in that their ligands are membrane-bound on adjacent cells. Luca et al. provide insights into the signaling interactions between Notch and its various ligands. The crystal structure of Notch complexed with a variant of its ligand Jagged1 revealed the interaction of Notch domains containing O-linked glycans with particular domains in Jagged1. Measurements of the effects of various forces on the interaction showed that application of force increases the lifetime of bonds between receptor and ligand. The studies help explain how low-affinity interactions of Notch with its ligands can lead to signaling and indicate that forces may differentially affect Notch-ligand interactions. Science, this issue p. 1320 Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.

HNK-1 Glyco-epitope Regulates the Stability of the Glutamate Receptor Subunit GluR2 on the Neuronal Cell Surface

HNK-1 (human natural killer-1) glyco-epitope, a sulfated glucuronic acid attached to N-acetyllactosamine on the nonreducing termini of glycans, is highly expressed in the nervous system. Our previous report showed that mice lacking a glucuronyltransferase (GlcAT-P), a key enzyme for biosynthesis of the HNK-1 epitope, showed reduced long term potentiation at hippocampal CA1 synapses. In this study, we identified an α-amino-3-hydroxy-5-methylisoxazole propionate (AMPA)-type glutamate receptor subunit, GluR2, which directly contributes to excitatory synaptic transmission and synaptic plasticity, as a novel HNK-1 carrier molecule. We demonstrated that the HNK-1 epitope is specifically expressed on the N-linked glycan(s) on GluR2 among the glutamate receptors tested, and the glycan structure, including HNK-1 on GluR2, was determined using liquid chromatography-tandem mass spectrometry. As for the function of HNK-1 on GluR2, we found that the GluR2 not carrying HNK-1 was dramatically endocytosed and expressed less on the cell surface compared with GluR2 carrying HNK-1 in both cultured hippocampal neurons and heterologous cells. These results suggest that HNK-1 stabilizes GluR2 on neuronal surface membranes and regulates the number of surface AMPA receptors. Moreover, we showed that the expression of the HNK-1 epitope enhanced the interaction between GluR2 and N-cadherin, which has important roles in AMPA receptor trafficking. Our findings suggest that the HNK-1 epitope on GluR2 regulates cell surface stability of GluR2 by modulating the interaction with N-cadherin.

Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi)

Haploinsufficiency for the Notch ligand JAG1 in humans results in an autosomal‐dominant, multisystem disorder known as Alagille syndrome, which is characterized by a congenital cholangiopathy of variable severity. Here, we show that on a C57BL/6 background, jagged1 heterozygous mice (Jag1+/−) exhibit impaired intrahepatic bile duct (IHBD) development, decreased SOX9 expression, and thinning of the periportal vascular smooth muscle cell (VSMC) layer, which are apparent at embryonic day 18 and the first postnatal week. In contrast, mice double heterozygous for Jag1 and the glycosyltransferase, Poglut1 (Rumi), start showing a significant improvement in IHBD development and VSMC differentiation during the first week. At P30, Jag1+/− mice show widespread ductular reactions and ductopenia in liver and a mild, but statistically, significant bilirubinemia. In contrast, P30 Jag1/Rumi double‐heterozygous mice show well‐developed portal triads around most portal veins, with no elevation of serum bilirubin. Conditional deletion of Rumi in VSMCs results in progressive arborization of the IHBD tree, whereas deletion of Rumi in hepatoblasts frequently results in an increase in the number of hepatic arteries without affecting bile duct formation. Nevertheless, removing one copy of Rumi from either VSMCs or hepatoblasts is sufficient to partially suppress the Jag1+/− bile duct defects. Finally, all Rumi target sites of the human JAG1 are efficiently glucosylated, and loss of Rumi in VSMCs results in increased levels of full‐length JAG1 and a shorter fragment of JAG1 without affecting Jag1 messenger RNA levels. Conclusions: On a C57BL/6 background, Jag1 haploinsufficiency results in bile duct paucity in mice. Removing one copy of Rumi suppresses the Jag1+/− bile duct phenotype, indicating that Rumi opposes JAG1 function in the liver. (Hepatology 2016;63:550–565)

HNK-1 (human natural killer-1) glyco-epitope is essential for normal spine morphogenesis in developing hippocampal neurons

The human natural killer-1 (HNK-1) glyco-epitope possesses a unique structural feature, a sulfated glucuronic acid attached to lactosamine on the non-reducing termini of glycans. The expression of HNK-1 is temporally and spatially regulated by glucuronyltransferase (GlcAT-P) in the brain. Our previous report showed that mice lacking GlcAT-P almost completely lost HNK-1 expression in the brain and exhibited reduced long-term potentiation (LTP) at hippocampal CA1 synapses. GlcAT-P-deficient mice also showed impaired hippocampus-dependent spatial learning. Although HNK-1 plays an essential role in synaptic plasticity and memory formation, it remains unclear how HNK-1 regulates these functions. In this study, we showed that loss of the HNK-1 epitope resulted in an increase of filopodium-like immature spines and a decrease of mushroom-like mature spines in both the early postnatal mouse hippocampus and cultured hippocampal neurons. However, HNK-1 had no influence on spine density or filopodium formation. Immunofluorescence staining revealed that loss of HNK-1 altered the distribution of postsynaptic proteins such as alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-type glutamate receptor subunit GluR2 and PSD-95 from spine heads onto dendritic shafts without affecting synapse formation, resulting in an increase of shaft synapses in cultured GlcAT-P-deficient neurons. GluR2, a major HNK-1 carrier glycoprotein in postsynaptic density, has the ability to promote spine morphogenesis. Overexpression of GluR2 promoted spine growth in both wild-type and GlcAT-P-deficient neurons, but the increase in GlcAT-P-deficient neurons was lower than that in wild-type neurons. This is the first evidence that HNK-1 is a key factor for normal dendritic spine maturation and is involved in the distribution of postsynaptic proteins.

O-fucosylation of the notch ligand mDLL1 by POFUT1 is dispensable for ligand function.

Fucosylation of Epidermal Growth Factor-like (EGF) repeats by protein O-fucosyltransferase 1 (POFUT1 in vertebrates, OFUT1 in Drosophila) is pivotal for NOTCH function. In Drosophila OFUT1 also acts as chaperone for Notch independent from its enzymatic activity. NOTCH ligands are also substrates for POFUT1, but in Drosophila OFUT1 is not essential for ligand function. In vertebrates the significance of POFUT1 for ligand function and subcellular localization is unclear. Here, we analyze the importance of O-fucosylation and POFUT1 for the mouse NOTCH ligand Delta-like 1 (DLL1). We show by mass spectral glycoproteomic analyses that DLL1 is O-fucosylated at the consensus motif C2XXXX(S/T)C3 (where C2 and C3 are the second and third conserved cysteines within the EGF repeats) found in EGF repeats 3, 4, 7 and 8. A putative site with only three amino acids between the second cysteine and the hydroxy amino acid within EGF repeat 2 is not modified. DLL1 proteins with mutated O-fucosylation sites reach the cell surface and accumulate intracellularly. Likewise, in presomitic mesoderm cells of POFUT1 deficient embryos DLL1 is present on the cell surface, and in mouse embryonic fibroblasts lacking POFUT1 the same relative amount of overexpressed wild type DLL1 reaches the cell surface as in wild type embryonic fibroblasts. DLL1 expressed in POFUT1 mutant cells can activate NOTCH, indicating that POFUT1 is not required for DLL1 function as a Notch ligand.

Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope

The HNK‐1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT‐P and GlcAT‐S) are involved in the biosynthesis of HNK‐1 carbohydrate. Our previous study on the crystal structure of GlcAT‐P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT‐S and GlcAT‐P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT‐S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT‐P and GlcAT‐S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT‐P residues for the recognition of acceptors, is a tryptophan in GlcAT‐S. In addition, Val320, which is located on the C‐terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT‐P dimer, is an alanine in GlcAT‐S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT‐S, which is further supported by site‐directed mutagenesis of GlcAT‐S and a computer‐aided model building of GlcAT‐S/substrate complexes. Proteins 2006.

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney.

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.