Tetsuya Okajima

Regulation of Notch Signaling by O-Linked Fucose

Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. By using RNAi to decrease Ofut1 expression in Drosophila, we demonstrate that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for Ofut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. The transcription of Ofut1 is developmentally regulated, and surprisingly, overexpression of Ofut1 inhibits Notch signaling. Together, these results indicate that OFUT1 is a core component of the Notch pathway, which is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development.

Molecular Cloning of a Novel α2,3-Sialyltransferase (ST3Gal VI) That Sialylates Type II Lactosamine Structures on Glycoproteins and Glycolipids

A novel member of the human CMP-NeuAc:β-galactoside α2,3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of α2,3-sialyltransferase toward Galβ1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity,i.e. it utilized Galβ1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.

Molecular Basis for the Progeroid Variant of Ehlers-Danlos Syndrome IDENTIFICATION AND CHARACTERIZATION OF TWO MUTATIONS IN GALACTOSYLTRANSFERASE I GENE

Progeroid type Ehlers-Danlos (E-D) syndrome was reported to be caused by defects in galactosyltransferase I (EC2.4.1.133), which is involved in the synthesis of common linkage regions of proteoglycans. Recently, we isolated cDNA of the galactosyltransferase I (XGalT-1) (Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) J. Biol. Chem.274, 22915–22918). Therefore, we analyzed mutations in this gene of a patient with progeroid type E-D syndrome by reverse transcription polymerase chain reaction and direct sequencing. Two changes of G and T to A and C at 186 and 206, respectively, were detected. Then, we determined the genomic DNA sequences encompassing the A186D and L206P mutations, revealing that the unaffected parents and two siblings were heterozygous for either one of the two different mutations and normal, while the patient had both of two different mutant genes. Enzymatic functions of cDNA clones of XGalT-1 containing the individual mutations were examined, elucidating that L206P clone completely lost the activity, while A186D retained ∼50% or 10% of the activity when analyzed with extracts from cDNA transfectant cells or recombinant soluble enzymes, respectively. Moreover, L206P enzyme showed diffuse staining in the cytoplasm of transfectant cells, while the wild type or A186D clones showed Golgi pattern. These results indicated that the mutations in XGalT-1 were at least one of main molecular basis for progeroid type E-D syndrome.

Human Homolog of Caenorhabditis elegans sqv-3 Gene Is Galactosyltransferase I Involved in the Biosynthesis of the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

A cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated from a human melanoma line library. The new cDNA sequence encoded a type II membrane protein with 327 amino acid sequence and showed 38% homology to theCaenorhabditis elegans sqv-3 gene involved in the vulval invagination and oocyte development. Extracts from L cells transfected with the galactosyltransferase cDNA in an expression vector and a fusion protein with protein A exhibited marked galactosyltransferase activity specific forp-nitrophenyl-β-d-xylopyranoside. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis of galactosyltransferase I-deficient Chinese hamster ovary mutant pgsB-761 cells. Analysis of the enzyme product by β-galactosidase digestion, mass spectroscopy, and NMR spectroscopy revealed that the reaction product was formed via β-1,4 linkage, indicating that the enzyme is galactosyltransferase I (UDP-galactose:O-β-d-xylosylprotein 4-β-d-galactosyltransferase, EC 2.4.1.133) involved in the synthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

O-Linked N-Acetylglucosamine Is Present on the Extracellular Domain of Notch Receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-β-GlcNAc by galactosyltransferase labeling and β-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.

O-Linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions

The O-linked-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the aetiology of diabetes and neurodegeneration. This intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, OGT. Here we report a novel OGT, EOGT, responsible for extracellular O-GlcNAcylation. Although both OGT and EOGT are regulated by hexosamine flux, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. Loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy (Dp), a membrane-anchored extracellular protein, is O-GlcNAcylated, and EOGT is required for Dp-dependent epithelial cell-matrix interactions. Thus, O-GlcNAcylation of secreted and membrane glycoproteins is a novel mediator of cell-cell or cell-matrix interactions at the cell surface.

Roles of glycosylation in notch signaling

Notch and the DSL Notch ligands Delta and Serrate/Jagged are glycoproteins with a single transmembrane domain. The extracellular domain (ECD) of both Notch receptors and Notch ligands contains numerous epidermal growth factor (EGF)-like repeats which are post-translationally modified by a variety of glycans. Inactivation of a subset of genes that encode glycosyltransferases which initiate and elongate these glycans inhibits Notch signaling. In the formation of developmental boundaries in Drosophila and mammals, in mouse T-cell and marginal zone B-cell development, and in co-culture Notch signaling assays, the regulation of Notch signaling by glycans is to date a cell-autonomous effect of the Notch-expressing cell. The regulation of Notch signaling by glycans represents a new paradigm of signal transduction. O-fucose glycans modulate the strength of Notch binding to DSL Notch ligands, while O-glucose glycans facilitate juxta-membrane cleavage of Notch, generating the substrate for intramembrane cleavage and Notch activation. Identifying precisely how the addition of particular sugars at specific locations on Notch modifies Notch signaling is a challenge for the future.

MOLECULAR CLONING OF BRAIN-SPECIFIC GD1ALPHA SYNTHASE (ST6GALNAC V) CONTAINING CAG/GLUTAMINE REPEATS

A novel member of the mouse CMP-NeuAc: β-N-acetylgalactosaminide α2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc V, was identified by BLAST analysis of expressed sequence tags. The sequence of the longest cDNA clone of ST6GalNAc V encoded a type II membrane protein with 8 amino acids comprising the cytoplasmic domain, 21 amino acids comprising the transmembrane region, and 306 amino acids comprising the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III and IV, with common amino acid sequences in sialyl motifs L and S among these three enzymes. Eleven CAG repeats were found in the stem region. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc V in a expression vector showed enzyme activity of α2,6-sialyltransferase almost exclusively for GM1b, but not toward glycoproteins. Sialidase treatment and thin layer chromatography immunostaining revealed that the product was GD1α. Northern blotting revealed that three transcripts of the gene were expressed specifically in brain tissues. It is concluded that this enzyme is involved in the synthesis of GD1α in the nervous tissues, and the CAG repeats may have implications in neurodegenerative diseases.

O-linked- N -acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.

Molecular cloning and expression of mouse GD1α/GT1aα/GQ1bα synthase (ST6GalNAc VI) gene

A novel member of the mouse CMP-NeuAc:β-N-acetylgalactosaminide α2,6-sialyltransferase (ST6GalNAc) subfamily, designated ST6GalNAc VI, was identified by BLAST analysis of expressed sequence tags. The sequence of the cDNA clone of ST6GalNAc VI encoded a type II membrane protein with 43 amino acids composing the cytoplasmic domain, 21 amino acids composing the transmembrane region, and 269 amino acids composing the catalytic domain. The predicted amino acid sequence showed homology to the previously cloned ST6GalNAc III, IV, and V, with common amino acid sequences in sialyl motif L and S among these four enzymes. A fusion protein with protein A and extracts from L cells transfected with ST6GalNAc VI in an expression vector showed enzyme activity of α2,6-sialyltransferase for GM1b, GT1b, and GD1a but not toward glycoproteins. Thin layer chromatography-immunostaining revealed that the products were GD1α, GQ1bα, and GT1aα. Northern blotting revealed that this gene was expressed in a wide range of mouse tissues such as colon, liver, heart, spleen, and brain. It is concluded that this enzyme is a novel sialyltransferase involved in the synthesis of α-series gangliosides in the nervous tissues and many other tissues.