Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability.

The addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.

Impacts of ß-1, 3-N-acetylglucosaminyltransferases (B3GNTs) in human diseases.

Glycosylation modification of proteins is a common post-translational modification that exists in various organisms and has rich biological functions. It is usually catalyzed by multiple glycosyltransferases located in the Golgi apparatus. ß-1,3-N-acetylglucosaminyltransferases (B3GNTs) are members of the glycosyltransferases and have been found to be involved in the occurrence and development of a variety of diseases including autoimmunity diseases, cancers, neurodevelopment, musculoskeletal system, and metabolic diseases. The functions of B3GNTs represent the glycosylation of proteins is a crucial and frequently life-threatening step in progression of most diseases. In this review, we give an overview about the roles of B3GNTs in tumor, nervous system, musculoskeletal and metabolic diseases, describing the recent results about B3GNTs, in order to provide a research direction and exploration value for the prevention, diagnosis and treatment of these diseases.

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-ß-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

O-GlcNAc signaling increases neuron regeneration through one-carbon metabolism in Caenorhabditis elegans.

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

O-GlcNAcylation mediates H2O2-induced apoptosis through regulation of STAT3 and FOXO1.

The O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is a critical post-translational modification that couples the external stimuli to intracellular signal transduction networks. However, the critical protein targets of O-GlcNAcylation in oxidative stress-induced apoptosis remain to be elucidated. Here, we show that treatment with H2O2 inhibited O-GlcNAcylation, impaired cell viability, increased the cleaved caspase 3 and accelerated apoptosis of neuroblastoma N2a cells. The O-GlcNAc transferase (OGT) inhibitor OSMI-1 or the O-GlcNAcase (OGA) inhibitor Thiamet-G enhanced or inhibited H2O2-induced apoptosis, respectively. The total and phosphorylated protein levels, as well as the promoter activities of signal transducer and activator of transcription factor 3 (STAT3) and Forkhead box protein O 1 (FOXO1) were suppressed by OSMI-1. In contrast, overexpressing OGT or treating with Thiamet-G increased the total protein levels of STAT3 and FOXO1. Overexpression of STAT3 or FOXO1 abolished OSMI-1-induced apoptosis. Whereas the anti-apoptotic effect of OGT and Thiamet-G in H2O2-treated cells was abolished by either downregulating the expression or activity of endogenous STAT3 or FOXO1. These results suggest that STAT3 or FOXO1 are the potential targets of O-GlcNAcylation involved in the H2O2-induced apoptosis of N2a cells.

Immune skeletal dysplasia with neurodevelopmental abnormalities caused by a novel variant of EXTL3 gene in a Chinese family.

BACKGROUND: Immune skeletal dysplasia with neurodevelopmental abnormalities (ISDNA) is an extremely rare, autosomal recessive genetic disorder characterized by various skeletal abnormalities, neurodevelopmental deficits, and abnormal immune system function. ISDNA is caused by variation in the exostosin-like 3 (EXTL3) gene, located on chromosome 8p21.2, whose primary function is the biosynthesis of heparan sulfate (HS) skeleton structure. Only a few variations in the EXTL3 gene have been discovered so far. In these years of development, many pathogenic variants in genetic diseases with genetic and phenotypic heterogeneity have been investigated using whole-exome sequencing (WES) technology. METHODS: In this research, a novel EXTL3 variant was first detected in a patient using WES, which was validated from Sanger sequencing in this family. Family history and clinical information were then collected through comprehensive medical examinations and genetic counseling. In silico prediction was then utilized to confirm the pathogenicity of the variant. RESULTS: A novel homozygous variant, NM_001440: c.2015G>A (p.Arg672Gln) in the EXTL3 gene, was identified using WES, which has never been reported before. Sanger sequencing was performed to confirm that the variant segregated with the disease within the family. CONCLUSION: This research identified a novel pathogenic variant in the EXTL3 gene responsible for ISDNA in a Chinese family. It showed the potential diagnostic role of WES in ISDNA, expanded the EXTL3 gene variation spectrum, and demonstrated that the diagnosis of ISDNA using WES is feasible and effective. More comprehensive genetic counseling and precise prenatal diagnosis for the next pregnancy can also be provided to families with genetic disorders.

O-Linked N-Acetylglucosamine Transferase Ensures Survival of Mouse Fetal Liver Hematopoietic Progenitors Partly by Regulating Bcl-xL and Oxidative Phosphorylation.

O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.

Direct stimulation of de novo nucleotide synthesis by O-GlcNAcylation.

O-linked ß-N-acetyl glucosamine (O-GlcNAc) is at the crossroads of cellular metabolism, including glucose and glutamine; its dysregulation leads to molecular and pathological alterations that cause diseases. Here we report that O-GlcNAc directly regulates de novo nucleotide synthesis and nicotinamide adenine dinucleotide (NAD) production upon abnormal metabolic states. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1), the key enzyme of the de novo nucleotide synthesis pathway, is O-GlcNAcylated by O-GlcNAc transferase (OGT), which triggers PRPS1 hexamer formation and relieves nucleotide product-mediated feedback inhibition, thereby boosting PRPS1 activity. PRPS1 O-GlcNAcylation blocked AMPK binding and inhibited AMPK-mediated PRPS1 phosphorylation. OGT still regulates PRPS1 activity in AMPK-deficient cells. Elevated PRPS1 O-GlcNAcylation promotes tumorigenesis and confers resistance to chemoradiotherapy in lung cancer. Furthermore, Arts-syndrome-associated PRPS1 R196W mutant exhibits decreased PRPS1 O-GlcNAcylation and activity. Together, our findings establish a direct connection among O-GlcNAc signals, de novo nucleotide synthesis and human diseases, including cancer and Arts syndrome.

The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation.

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells in vitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.

EXTL3-Associated Immunoskeletal Dysplasia with Neurodevelopmental Abnormalities: A Lethal Phenotype.

Background: Immunoskeletal dysplasia with neurodevelopmental abnormalities (ISDNA) caused by Exostosin-Like Glycosyltransferase 3 (EXTL3) biallelic mutations is a very rare syndrome with only 16 cases reported in the literature. Skeletal dysplasia, neurodevelopmental delay, immunodeficiency, liver, and kidney cysts are the most common findings of this syndrome. Case Presentation: Here, we report on a patient who exhibited a lethal phenotype with clinical characteristics of this syndrome and had a homozygous pathogenic mutation in EXTL3 gene. Conclusions: ISDNA should be kept in mind in the differential diagnosis of patients presenting with neuro-immuno-skeletal dysplasia phenotype.

OGT-1 regulates synaptic assembly through the insulin signaling pathway.

The formation and maintenance of synapses are precisely regulated, and the misregulation often leads to neurodevelopmental or neurodegenerative disorders. Besides intrinsic genetically encoded signaling pathways, synaptic structure and function are also regulated by extrinsic factors, such as nutrients. O-GlcNAc transferase (OGT), a nutrient sensor, is abundant in the nervous system and required for synaptic plasticity, learning, and memory. However, whether OGT is involved in synaptic development and the mechanism underlying the process are largely unknown. In this study, we found that OGT-1, the OGT homolog in C. elegans, regulates the presynaptic assembly in AIY interneurons. The insulin receptor DAF-2 acts upstream of OGT-1 to promote the presynaptic assembly by positively regulating the expression of ogt-1. This insulin-OGT-1 axis functions most likely by regulating neuronal activity. In this study, we elucidated a novel mechanism for synaptic development, and provided a potential link between synaptic development and insulin-related neurological disorders.

Growing and dividing: how O-GlcNAcylation leads the way.

Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing posttranslational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase, O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor-suppressor p53 at Gap Phase 2 (G2). Finally, in mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1, O-GlcNAc control of DNA replication and repair, and finally, O-GlcNAc organization of mitotic progression and spindle dynamics.

Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation.

Protein O-GlcNAcylation is an evolutionary conserved post-translational modification catalysed by the nucleocytoplasmic O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). How site-specific O-GlcNAcylation modulates a diverse range of cellular processes is largely unknown. A limiting factor in studying this is the lack of accessible techniques capable of producing homogeneously O-GlcNAcylated proteins, in high yield, for in vitro studies. Here, we exploit the tolerance of OGT for cysteine instead of serine, combined with a co-expressed OGA to achieve site-specific, highly homogeneous mono-glycosylation. Applying this to DDX3X, TAB1, and CK2α, we demonstrate that near-homogeneous mono-S-GlcNAcylation of these proteins promotes DDX3X and CK2α solubility and enables production of mono-S-GlcNAcylated TAB1 crystals, albeit with limited diffraction. Taken together, this work provides a new approach for functional dissection of protein O-GlcNAcylation.

Sustained Increases in Cardiomyocyte Protein O-Linked ß-N-Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment.

Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O-linked ß-N acetylglucosamine (O-GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O-GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant-negative O-GlcNAcase (dnOGA) inducible cardiomyocyte-specific overexpression transgenic mouse model, we induced dnOGA in 8- to 10-week-old mouse hearts. We examined the effects of 2-week and 24-week dnOGA overexpression, which progressed to a 1.8-fold increase in protein O-GlcNAcylation. Two-week increases in protein O-GlcNAc levels did not alter heart weight or function; however, 24-week increases in protein O-GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24-week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA-sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24-week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O-GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2-week), which leads to cardiac remodeling (24-week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O-GlcNAcylation in heart.

Identification of glycogene-based prognostic signature and validation of B3GNT7 as a potential biomarker and therapeutic target in breast cancer.

BACKGROUND: Breast cancer is the most common cancer worldwide, with the fifth highest mortality rate among all cancers and high risk of metastasis. However, potential biomarkers and molecular mechanisms underlying the stratification of breast cancer in terms of clinical outcomes remain to be investigated. Therefore, we aimed to find a novel prognostic biomarker and therapeutic target for breast cancer patients. METHODS: Unsupervised hierarchical clustering was used to perform comprehensive transcriptomic study of total 185 glycogenes in public datasets of breast cancer with clinicopathological and survival information. A glycogene-based signature for subtype classification was discovered using Limma packages, and relevance to four known molecular features was identified by GSVA. Experimental verification was performed and biological functions of B3GNT7 were characterized by quantitative RT-PCR, western blot, transwell assays, and lectin immunofluorescence staining in breast cancer cells. RESULTS: A 23-glycogene signature was identified for the classification of breast cancer. Among the 23 glycogenes, B3GNTs showed significantly positive associations with ER-/Her2- subtype in breast cancer patients (n = 2655). Overexpressed B3GNT7 were correlated with poor prognosis in breast cancer patients based on public datasets. B3GNT7 depletion inhibited cell proliferation, migration, and invasion, and decreased global fucosylation in MDA-MB-231 and HCC1937 breast cancer cells. CONCLUSIONS: Herein, we discovered a unique 23-gene signature for breast cancer patient glycogene-type classification. Among these genes, B3GNT7 was shown to be a potential biomarker for unfavorable outcomes and therapeutic target of breast cancer.

Hexosamine biosynthetic pathway and O-GlcNAc cycling of glucose metabolism in brain function and disease.

Impaired brain glucose metabolism is considered a hallmark of brain dysfunction and neurodegeneration. Disruption of the hexosamine biosynthetic pathway (HBP) and subsequent O-linked N-acetylglucosamine (O-GlcNAc) cycling has been identified as an emerging link between altered glucose metabolism and defects in the brain. Myriads of cytosolic and nuclear proteins in the nervous system are modified at serine or threonine residues with a single N-acetylglucosamine (O-GlcNAc) molecule by O-GlcNAc transferase (OGT), which can be removed by ß-N-acetylglucosaminidase (O-GlcNAcase, OGA). Homeostatic regulation of O-GlcNAc cycling is important for the maintenance of normal brain activity. Although significant evidence linking dysregulated HBP metabolism and aberrant O-GlcNAc cycling to induction or progression of neuronal diseases has been obtained, the issue of whether altered O-GlcNAcylation is causal in brain pathogenesis remains uncertain. Elucidation of the specific functions and regulatory mechanisms of individual O-GlcNAcylated neuronal proteins in both normal and diseased states may facilitate the identification of novel therapeutic targets for various neuronal disorders. The information presented in this review highlights the importance of HBP/O-GlcNAcylation in the neuronal system and summarizes the roles and potential mechanisms of O-GlcNAcylated neuronal proteins in maintaining normal brain function and initiation and progression of neurological diseases.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth.

Gliomas are the most prevalent primary tumor of the central nervous system. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor type Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 capped O-mannosyl core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology, we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of human natural killer-1-capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Novel ß1,4 N-acetylglucosaminyltransferase in de novo enzymatic synthesis of hyaluronic acid oligosaccharides.

The efficiency of de novo synthesis of hyaluronic acid (HA) using Pasteurella multocida hyaluronate synthase (PmHAS) is limited by its low catalytic activity during the initial reaction steps when monosaccharides are the acceptor substrates. In this study, we identified and characterized a ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) derived from the O-antigen gene synthesis cluster of Escherichia coli O8:K48:H9. Recombinant ß1,4 EcGnT effectively catalyzed the production of HA disaccharides when the glucuronic acid monosaccharide derivative 4-nitrophenyl-ß-D-glucuronide (GlcA-pNP) was used as the acceptor. Compared with PmHAS, ß1,4 EcGnT exhibited superior N-acetylglucosamine transfer activity (~ 12-fold) with GlcA-pNP as the acceptor, making it a better option for the initial step of de novo HA oligosaccharide synthesis. We then developed a biocatalytic approach for size-controlled HA oligosaccharide synthesis using the disaccharide produced by ß1,4 EcGnT as a starting material, followed by stepwise PmHAS-catalyzed synthesis of longer oligosaccharides. Using this approach, we produced a series of HA chains of up to 10 sugar monomers. Overall, our study identifies a novel bacterial ß1,4 N-acetylglucosaminyltransferase and establishes a more efficient process for HA oligosaccharide synthesis that enables size-controlled production of HA oligosaccharides. KEY POINTS: • A novel ß-1,4-N-acetylglucosaminyl-transferase (EcGnT) from E. coli O8:K48:H9. • EcGnT is superior to PmHAS for enabling de novo HA oligosaccharide synthesis. • Size-controlled HA oligosaccharide synthesis relay using EcGnT and PmHAS.