November 28
PROGRAM08

Glycoscience : diversity and integration

O-GlcNAc modification ofEGF-like domains bya novel O-GlcNAc transferase, EGOT
Tetsuya Okajima, M.D., Ph.D.
Associate ProfessorNagoya University Graduate School of Medicine, JAPANTetsuya Okajima
ProfileDr. Okajima received both his M.D. andPh.D. degrees in 1996 and 2000 from Nagoya University School of Medicine. He
did his doctoral work in the laboratory ofDr. Koichi Furukawa on the isolation andcharacterization of mammalian glycosyltransferase genes, specifically those involved in glycolipid biosynthesis. He wenton to do post-doctoral work with Dr. KenIrvine at Rutgers University. In Dr. Irvine’s
laboratory he worked on the O-linked fucose (O-fucose) modification. His major
contribution was biological characterization of the enzyme responsible for additionof O-fucose to proteins, OFUT1. In 2005
he began his independent career as an assistant professor in the Department of Applied Molecular Biosciences, Nagoya University Graduate School of BioagriculturalSciences. There he has continued to workin unusual forms of protein O-glycosylation, namely the O-GlcNAc modificationsof extracellular protein domains. He waspromoted to lecturer in 2008 and then toassociate professor in 2009 in Biochemistry II, Nagoya University Graduate Schoolof Medicine.

O-Linked-N-acetylglucosamine(O-GlcNAc)
modification is a unique post-translational
modification that cooperates with phosphorylation to regulate a variety of cellular
processes, including intracellular signaling, cytokinesis, transcription, and protein
stability, and serves to modulate nutrient
sensing. O-GlcNAcylation is also involvedin epigenetic control of cell differentiationand is implicated in the etiology of several
human diseases, including type 2 diabetesand neurodegeneration. Although many
reports have described biological roles ofO-GlcNAcylation, they were limited to theintracellular protein functions, since themodification had been believed to be restricted to the cytoplasm and nucleus.
Our recent attempt to confirm the proposed structure for O-glycans on Notch
epidermal growth factor (EGF) repeats led
to the unexpected discovery of an novel
extracellular O-GlcNAc modification in
Drosophila S2 cells. Back in 1980’s, it wasreported that a small percentage of OGlcNAc containing proteins was detectedon the surface of lymphocytes. However,
the reexamination using improved methods could not detect O-GlcNAc bearing
protein on the cell surface of lymphocytes.
A few reports have identified O-GlcNAc in
the extracellular environment. Nevertheless, this could be attributed to a noncanonical secretion pathway for proteins thatbypass the endoplasmic reticulum (ER)and Golgi compartments. Moreover, extracellularly O-GlcNAcylated proteins had
not been molecularly identified, and allreported O-GlcNAcylated proteins wererestricted to the cytoplasmic and nuclearcompartment. Thus, the identification ofO-GlcNAc on Notch receptors provided
the first description of O-GlcNAcylated
proteins in the animal extracellular environment.
Intracellular O-GlcNAc modification is catalyzed by the cytoplasmic enzyme O-GlcNAc
transferase (OGT). This OGT cannot be
responsible for Notch O-GlcNAcylation
since the O-GlcNAc modifications on EGF
domains should occur during secretion by
the action of glycosyltransferases that are
typically localized in the ER or Golgi apparatus. Indeed, we could detect O-GlcNAc
transferase activity in the membrane fraction proteins prepared from S2 cells. To
isolate enzymes responsible for Notch OGlcNAcylation, we performed an RNAibased search for genes that specificallyaffect O-GlcNAcylation. We identified
one candidate gene, EOGT (EGF-domain
O-GlcNAc transferase), whose downregulation decreased the signals corresponding to O-GlcNAcylated EGF20 peptides.
EOGT is conserved from Drosophila tomammals, but shows no apparent homology to previously isolated GlcNActransferases, such as OGT or GlcNAcT
enzymes, but rather is phylogeneticallyrelated to the plant xylosyltransferase.
Although both OGT and EOGT are regulated by hexosamine flux, EOGT localizes
to the lumen of the endoplasmic reticulum
and transfers GlcNAc to EGF domains inan OGT-independent manner.

Figure 1. O-GlcNAcylation of EGF domains by EOGT.
A Amino acid sequence of EGF domains of human urinary plasminogen activator (uPA), tissue PA (tPA), blood clotting factors (VII, IX, and XII),
Protein Z, Cripto, EGF domain 12 from Drosophila and human Notch1, and Drosophila Notch EGF20, are aligned. The sites for O-fucosylation
or O-glucosylation are indicated by arrow. The site for O-GlcNAcylation found on EGF20 is indicated by arrowhead. Note that not all sites areexperimentally confirmed to be modified. The conserved cysteine residues are numbered.
B The proposed structure for O-GlcNAc glycans in mammals.

Genetic analysis in Drosophila revealed
that loss of Eogt gives phenotypes similar
to those caused by defects in the apical extracellular matrix. Dumpy, a membraneanchored extracellular protein, is OGlcNAcylated, and EOGT is required forDumpy-dependent epithelial cell-matrix
interactions. These results indicated thatO-GlcNAcylation of secreted and membrane glycoproteins is a novel mediator ofcell-cell or cell-matrix interactions at thecell surface.
To explore whether O-GlcNAcylationof EGF repeats is present in higher vertebrates, we have investigated Eogt1, amouse gene homologue of Drosophila
Eogt. We showed that as with Drosophila

EOGT, Eogt1 selectively utilizes UDPGlcNAc as a donor substrate, specificallymodifies the EGF domains, and the enzyme activity is enhanced in the presenceof manganese. Importantly, except for theslight difference in temperature dependence, most of the enzymatic activity ofEogt1 is indistinguishable from that of theDrosophila enzyme. This observation issupported by the fact that the expressionof Eogt1 in Drosophila rescued the celladhesion 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 togenerate a novel O-linked-N-acetyllactosamine structure.

Although Dumpy is shown to be a functionally important substrate for Drosophila Eogt, the mammalian genome does not
encode apparent Dumpy homologues or
structurally related proteins, and thus the
biological function of Eogt1 in mice, if any,
would rely on other EGF domain-containing substrates. Future studies, including
phenotypic analyses of Eogt1-deficient
mice, will be necessary to address the rolesof atypical O-GlcNAcylation in mammals.
Nonetheless, these considerations raise theintriguing possibility that the O-GlcNAcylation reaction in the secretory pathway
is evolutionarily conserved, whereas thesignificance of O-GlcNAcylated proteinsin the biological processes had changed
during the evolution.

References1) 	Matsuura A, Ito M, Sakaidani Y, Kondo T, Murakami K, Furukawa K, Nadano D, Matsuda T, Okajima T. O-linked GlcNAc is present on the extracellulardomain of Notch receptors. J. Biol. Chem. 283(51):35486-35495 (2008).
2) 	Sakaidani Y, Furukawa K, Okajima T. O-GlcNAc modification on EGF domains of Notch receptors. Methods Enzymol. 480:355-373 (2010).
3) 	Stanley, P and Okajima, T. Roles of glycosylation in Notch signaling. Curr.
top. dev. biol. 92:131-164 (2010).
4) 	Sakaidani Y, Nomura T, Matsuura A, Ito M, Suzuki E, Murakami K, Nadano34

the 20th Anniversary of the Mizutani Foundation for Glycoscience

D, Matsuda T, Furukawa K, Okajima T. O-linked-N-acetylglucosamine on
extracellular protein domains mediates epithelial cell-matrix interactions.
Nature Commun. 2:583 (2011).
5) 	Sakaidani Y, Ichiyanagi N, Saito C, Nomura T, Ito M, Nishio Y, Nadano D,Matsuda T, Furukawa K, Okajima T. O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase
Eogt1. Biochem. Biophys. Res. Commun. 419:14-19 (2012).

Commemorative Symposium

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