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O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing, leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognitive function via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction. These findings suggest that blocking O-GlcNAc hydrolysis is a potential treatment strategy for OGT-CDG.Author summaryAttachment of single N-acetylglucosamine (GlcNAc) sugars to intracellular proteins has recently been linked to neurodevelopment and cognition. This link has been strengthened by discovery of O-GlcNAc transferase (OGT) missense mutations in intellectual disability. Most of these mutations lie outside the catalytic O-GlcNAc transferase domain and it is unclear how they affect cognitive function. Using the fruit fly Drosophila melanogaster as a model organism, we found that a balance in O-GlcNAc cycling is required for learning and neuronal development. Habituation, a fundamental form of learning, is affected in flies that carry patient-specific OGT mutations, and increasing O-GlcNAcylation genetically corrects the habituation deficit. Our work establishes a critical role for O-GlcNAc cycling in a cognition-relevant process, identifies defective O-GlcNAc transferase activity as a cause of intellectual disability, and proposes underlying mechanisms that can be further explored as treatment targets.

Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. The Drosophila melanogaster gene supersex combs (sxc), which encodes OGT, is a polycomb gene, whose null mutants display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved and the underlying mechanisms linking these phenotypes to embryonic development are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and by limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, among others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development.

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc (O-GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila, null mutants of the Polycomb gene O-GlcNAc transferase (OGT; also known as super sex combs (sxc)) display homeotic phenotypes. To dissect the requirement for O-GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxcK872M, was recessive lethal, whereas a second mutant, the hypomorphic sxcH537A, was homozygous viable. We observed that reduced total protein O-GlcNAcylation in the sxcH537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxcH537A and a null allele of Drosophila host cell factor (dHcf), encoding an extensively O-GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxcH537A flies lacking a copy of skuld (skd), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxcH537A enabled us to identify pleiotropic effects of globally reduced protein O-GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development.

O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O- N -acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development. * ACN, : acetonitrile; ConA, : concanavalin A; Cp OGA, : Clostridium perfringens OGA orthologue; dHCF, : Drosophila host cell factor; Dm OGT, : Drosophila OGT; ETD, : electron transfer dissociation; GalNAz, : N-azidoacetylgalactosamine; GlcNAc, : N -acetylglucosamine; Fmoc, : 9-fluorenylmethyloxycarbonyl; HA, : haemagglutinin; HCF, : host cell factor; HEK, : human embryonic kidney; hOGA, : human OGA; LB-Amp, : LB medium containing 50 μg/ml ampicillin; OGA, : O-GlcNAcase; OGT, : O-GlcNAc transferase; Ph, : polyhomoeotic; PNGase F, : peptide: N-glycosylase F; PTM, : post-translational modification; SPR, : surface plasmon resonance; sxc , : super sex combs ; TAB1, : transforming growth factor-beta-activated protein kinase 1- binding protein 1; TFA, : trifluoroacetic acid

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.

5‐hydroxy methyl cytosine (5hmC) is a modification identified in vertebrates several decades ago. More recently, a possible role of 5hmC as an epigenetic modifier and/or transcriptional regulator has started to emerge, with altered levels in early embryonic development, embryonic stem (ES) cell differentiation and tumours (Tahiliani et al , 2009; Yang et al , 2012). The balance between 5hmC and 5‐methyl cytosine (5mC) at gene promoters and CpG islands in the genome appears to be linked to pluripotency and lineage commitment of a cell (Ito et al , 2010). However, proteins with 5hmC binding capability have not yet been identified, and it has been proposed that 5hmC may only be a reaction intermediate in the process of demethylation (He et al , 2011; Ito et al , 2011). Over the last few years, ten‐eleven translocation (Tet) family proteins have been shown to be responsible for the conversion of 5mC to 5hmC (Iyer et al , 2009; Loenarz and Schofield, 2009; Tahiliani et al , 2009). However, how Tet family proteins and 5hmC are linked to transcriptional regulation is currently not clear. In the quest for finding the functional partners of Tet …

Glycosylation is essential for growth factor signaling through N-glycosylation of ligands and receptors and the biosynthesis of proteoglycans as co-receptors. Here, we show that protein O-GlcNAcylation is crucial for fibroblast growth factor (FGF) signaling in Drosophila . We found that nesthocker ( nst ) encodes a phosphoacetylglucosamine mutase and that nst mutant embryos exhibited low amounts of intracellular uridine 5′-diphosphate– N -acetylglucosamine (UDP-GlcNAc), which disrupted protein O-GlcNAcylation. Nst was required for mitogen-activated protein kinase (MAPK) signaling downstream of FGF but not MAPK signaling activated by epidermal growth factor. nst was dispensable for the function of the FGF ligands and the FGF receptor’s extracellular domain but was essential in the signal-receiving cells downstream of the FGF receptor. We identified the adaptor protein Downstream of FGF receptor (Dof), which interacts with the FGF receptor, as the relevant target for O-GlcNAcylation in the FGF pathway, suggesting that protein O-GlcNAcylation of the activated receptor complex is essential for FGF signal transduction.

In mammals, acquisition of fertilization competence of spermatozoa is dependent on the phenomenon of sperm capacitation. One of the critical molecular events of sperm capacitation is protein tyrosine phosphorylation. In a previous study, we demonstrated that a specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, tyrphostin-A47, inhibited hamster sperm capacitation, accompanied by a reduced sperm protein tyrosine phosphorylation. Interestingly, a high percentage of tyrphostin-A47-treated spermatozoa exhibited circular motility, which was associated with a distinct hypo-tyrosine phosphorylation of flagellar proteins, predominantly of Mr 45,000-60,000. In this study, we provide evidence on the localization of capacitation-associated tyrosine-phosphorylated proteins to the nonmembranous, structural components of the sperm flagellum. Consistent with this, we show their ultrastructural localization in the outer dense fiber, axoneme, and fibrous sheath of spermatozoa. Among hypo-tyrosine phosphorylated major proteins of tyrphostin-A47-treated spermatozoa, we identified the 45 kDa protein as outer dense fiber protein-2 and the 51 kDa protein as tektin-2, components of the sperm outer dense fiber and axoneme, respectively. This study shows functional association of hypo-tyrosine-phosphorylation status of outer dense fiber protein-2 and tektin-2 with impaired flagellar bending of spermatozoa, following inhibition of EGFR-tyrosine kinase, thereby showing the critical importance of flagellar protein tyrosine phosphorylation during capacitation and hyperactivation of hamster spermatozoa.

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.