title: Impaired O-linked N-acetylglucosaminylation in the endoplasmic reticulum by mutated epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine transferase found in Adams-Oliver syndrome.
abstract: Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT(R377Q) were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT(R377Q) was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.
authors.affiliation: From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, the Department of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829.
authors.forename: Mitsutaka
authors.initials: M
authors.lastname: Ogawa
authors.affiliation: From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065.
authors.forename: Shogo
authors.initials: S
authors.lastname: Sawaguchi
authors.affiliation: From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065.
authors.forename: Takami
authors.initials: T
authors.lastname: Kawai
authors.affiliation: the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601.
authors.forename: Daita
authors.initials: D
authors.lastname: Nadano
authors.affiliation: the Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, and.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yagi
authors.affiliation: the Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, and the Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.affiliation: From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065.
authors.forename: Koichi
authors.initials: K
authors.lastname: Furukawa
authors.affiliation: From the Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, tokajima@med.nagoya-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Okajima
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004476:Ectodermal Dysplasia / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics*; D014644:Genetic Variation; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D006595:Hexosamines / Q000378:metabolism; D006801:Humans; D017880:Limb Deformities, Congenital / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012536:Scalp Dermatoses / Q000151:congenital* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D006595:Hexosamines; D051880:Receptors, Notch; D011994:Recombinant Proteins; D025801:Ubiquitin; D004815:Epidermal Growth Factor; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D046988:Proteasome Endopeptidase Complex; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000117:Acetylglucosamine
keywords: Adams-Oliver Syndrome; EOGT; Endoplasmic Reticulum (ER); Glycoconjugate; Glycosyltransferase; Hexosamine Pathway; Notch Receptor; O-GlcNAcylation