The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome.

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer's disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

Role of nutrient-driven O-GlcNAc-post-translational modification in pancreatic exocrine and endocrine islet development.

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKOPanc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.

UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification). To study the enzymology of OGT, independent of potential regulatory proteins within cytosol, we also developed and characterized an alternate OGT assay that uses antibody-purified OGT as the enzyme source. The major advantage of this assay lies in the ability to measure OGT in the absence of other cytosolic proteins.

Mucin biosynthesis: epidermal growth factor downregulates core 2 enzymes in a human airway adenocarcinoma cell line.

Enzymes which exhibit core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 beta1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I beta1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor alpha mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras-mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras-mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.

Modulation of the basal activity of phosphatidylinositol-3-kinase/protein kinase B signaling pathway in human hepatocarcinoma cells.

The modulation of GnT-V activity by signaling molecules in PI-3-K/PKB pathway in human hepatocarcinoma cell line 7721 was studied. GnT-V activity was determined after the transfection of sense or antisense cDNA of PKB into the cells, as well as the addition of activators, specific inhibitors, and the antibodies to the enzyme assay system or culture medium. It was found that the basal activity of GnT-V was up regulated by the sense and down regulated by the antisense cDNA of PKB transfected into 7721 cells. GnT-V was activated by PIP2, PIP3 or GTPgamma[S] added to the assay system, and the activation of PIP2 or GTPgamma[S] was abolished by LY2940002, a specific inhibitor of PI-3-K, but the activation of PIP3 was not attenuated by LY2940002. In addition, GnT-V activity in cultured parental or H-ras transfected cells was inhibited by the antibody against PKB or PI-3-K. These findings demonstrated the involvement of PI-3-K/PKB signaling pathway in the regulation of GnT-V. Moreover, ET18-OCH3, an inhibitor of Raf translocation and PI-PLC enzyme, which produces the activator of PKC, as well as the antibodies against Raf-1 or MEK also inhibited GnT-V activity in the parental and H-ras transfected cells. The inhibitory rates, however, were less in the transfected cells than those in the parental cells. These results reveal that in parental and H-ras transfected 7721 cells, the basal activity of GnT-V is also regulated by the Ras/Raf-1/MEK/MAPK cascade in addition to PI-3-K/PKB signaling pathway. The significance of these two pathways in the regulation of GnT-V and their relations to the activation of PKC previously reported by our laboratory (Ju TZ et al., 1995 Glyconjugate J 12, 767-772) was discussed.

Alterations in glycosyltransferases during myeloid and monocytoid differentiation of HL-60 cells.

HL-60, a human promyelocytic leukemia cell line, can be differentiated to myeloid lineage by all- trans retinoic acid (ATRA), dimethylsulfoxide (DMSO) and n -butyric acid (n -BA), or to monocytoid(monocytic/macrophagic) lineage by phorbol-12-myristate-13-acetate (PMA) and ganglioside GM(3). The activity alterations of N -acetylglucosaminyltransferase III and V (GnT-III, GnT-V) as well as alpha-1,6-fucosyl-tranferase (alpha1,6 Fuc T) were studied during the differentiation of HL-60 cells by the above-mentioned five inducers using the fluorescence (PA)-labeled glycan-HPLC method for GnT assays and biotin-labeled glycan-LCA affinity chromatography combined with the HRP-avidin colorimetric method for alpha1,6 Fuc T assay. It was observed that after 3 days, all three enzymes decreased in HL-60 cells induced by 1 micromol/l ATRA and 0.6 mmol/l n-BA, while GnT-III and alpha1,6 Fuc T increased, but GnT-V still decreased after induction by 1% DMSO. GnT-V and alpha1,6 Fuc T declined, while GnT-III was elevated after induction by 0.1 micromol/l PMA for 3 days. In contrast, GnT-III increased after the treatment with 50 micromol/l GM(3)for 3 or 6 days, but GnT-V was not appreciably changed and alpha1,6 FucT was elevated after 6 days of GM(3)treatment. It may be concluded that the decrease of GnT-V is the common change in myeloid differentiation and the increase of GnT-III is the general alteration in monocytoid differentiation. The changes in the activities of glycosyltransferases were consistent with the structural changes in surface N -glycans previously found in our laboratory, i.e. that the antennary number of N -glycans decreased during myeloid differentiation by ATRA, and the amount of bisecting GlcNAc in N -glycans increased during monocytoid differentiation by PMA.

Activity of UDP-GlcNAc:GlcNAc beta 1-->6(GlcNAc beta 1-->2) Man alpha 1-->R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) from the ovaries of Oryzias latipes (Medaka fish).

UDP-GlcNAc:GlcNAc beta 1-->(GlcNAc beta 1-->2)Man alpha 1-R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) activity was shown to be present in crude homogenates of Medaka fish (Oryzias latipes) ovaries using UDP-[14C]GlcNAc and synthetic GlcNAc beta 1-->6 (GlcNAc beta 1-->2)Man alpha 1-->6Glc beta 1-->octyl as substrates. Characterization of this activity showed a pH optimum at about pH 7.0 and an absolute requirement for divalent cations. The optimum concentration of Mn2+ was at about 25 mM. This finding is the first report on GnT VI activity in fish; the enzyme has previously been described only in avian tissues.

Regulation of N-acetylglucosaminyltransferase V by protein kinases.

When 7721 human hepatocarcinoma cells were treated with 100 nM phorbol-12-myristate-13-acetate (PMA), the activity of N-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, and D-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnTV were proportional to the concentrations of the two inhibitors. The activities of GnTV and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnTV activities were decreased. These results suggest that GnTV may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.