N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.

Biochemical Characteristics of Emicizumab Activity with Factors IXa, X, and VIIa

Introduction. Emicizumab (Hemlibra) is a bivalent antibody that binds to factor IXa and factor X; this binding can promote factor IXa activation of factor X. In patients on prophylaxis with emicizumab, breakthrough bleeding has been treated successfully with factor VIIa (eptacog alfa (activated) (NovoSeven)). Objective. Our goal was to study the biochemistry of the interaction of emicizumab with factor IXa and factor X. We further wanted to study how binding of emicizumab to factor X would impact factor X activation by factor VIIa. Background. Data from surface plasmon resonance binding studies has shown that solution phase interactions between emicizumab and factors IX, IXa, X, and Xa are in the micromolar range (supplement to Kitazawa et al Nature Med 2012; 18: 1570-1574). That same publication showed that a lipid surface is required for activity. Other studies have shown that emicizumab binds to EGF1 of factor IX and EGF2 of factor X (Kitazawa et al Thromb Haemost 2017; 117: 1348-1357). Methods. Lipids were large unilamellar vesicles with 14% phosphatidylserine. Factors IX and X were purified from plasma. The basic design of an assay is to vary one element while holding other elements constant. To determine the apparent Km for factor X, factor X was varied with constant factor IXa (0.1 nM), lipid (100 µM), and emicizumab (400 nM). To determine the apparent Kd for factor IXa, factor IXa was varied with constant factor X (135 nM), lipid (100 µM), and emicizumab (3, 10, or 30 nM). The binding of factor IXa was determined in the presence and the absence of plasma concentration (80 nM) factor IX. Factor VIIa/tissue factor activation of factor X was measured with 0.25 nM VIIa/TF complex with factor X (135 nM) and either no emicizumab, 400 nM, or 50 µM. Factor VIIa activation of factor X in the absence of tissue factor was measured with varied factor VIIa (25-100 nM), fixed factor X (135 nM) and lipid (100 µM) and either no emicizumab, 400 nM, or 50 µM. Results. In the presence of lipid, the apparent binding constants for formation of the IXa-emicizumab-X complex were tighter than the solution phase reactions. Under the conditions studied, the Km,app for factor X was about 25 nM. The Kd,app for factor IXa was about 5 nM. Surprisingly, when lipid and factor X were present, factor IX did not compete with factor IXa for activation of factor X. Factor VIIa/tissue factor activation of factor X was slowed considerably by emicizumab. By contrast, factor VIIa activation of factor X in the absence of tissue factor was not slowed. Conclusions. The binding of factor X to the factor VIIa/tissue factor complex involves multiple domains in factor X. Since emicizumab reduced factor X activation by the factor VIIa/tissue factor complex, it appears that binding of emicizumab to the second EGF domain of factor X interfered with formation of the activating complex. By contrast, activation of factor X by factor VIIa alone was not reduced by emicizumab suggesting that interactions between factor VIIa and the second EGF domain of factor X are not essential for formation of that lipid bound complex. We would predict from the solution phase binding constants that high concentrations of emicizumab would be required to form the complex with factors IXa and X. This is in contrast to the observation that relatively low concentrations of emicizumab give significant shortening of an aPTT assay. The tight binding constants in the presence of lipid may explain the results seen in clotting assays. Further, this significant effect of emicizumab in clotting assays is consistent with the surprising observation that factor IX does not compete with factor IXa in formation of the lipid bound complex of IXa-emicizumab-X. So even a small amount of factor IXa can form functional complexes that activate factor X. Disclosures Monroe: Novo Nordisk A/S: Honoraria, Research Funding. Hoffman:Novo Nordisk A/S: Consultancy, Honoraria, Research Funding.

EOGT and O-GlcNAc on secreted and membrane proteins

Here, we describe a recently discovered O-GlcNAc transferase termed EOGT for EGF domain-specific O-GlcNAc transferase. EOGT transfers GlcNAc (N-acetylglucosamine) to Ser or Thr in secreted and membrane proteins that contain one or more epidermal growth factor-like repeats with a specific consensus sequence. Thus, EOGT is distinct from OGT, the O-GlcNAc transferase, that transfers GlcNAc to Ser/Thr in proteins of the cytoplasm or nucleus. EOGT and OGT are in separate cellular compartments and have mostly distinct substrates, although both can act on cytoplasmic (OGT) and lumenal (EOGT) domains of transmembrane proteins. The present review will describe known substrates of EOGT and biological roles for EOGT in Drosophila and humans. Mutations in EOGT that give rise to Adams–Oliver Syndrome in humans will also be discussed.