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.
Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan.
Yuta
Y
Sakaidani
Naoki
N
Ichiyanagi
Chika
C
Saito
Tomoko
T
Nomura
Makiko
M
Ito
Yosuke
Y
Nishio
Daita
D
Nadano
Tsukasa
T
Matsuda
Koichi
K
Furukawa
Tetsuya
T
Okajima
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology; D019143:Evolution, Molecular; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism*
D051880:Receptors, Notch; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine