Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. Methods: We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-ß) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. Results: We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-ß1 induced collagen expression/deposition. Conclusion: OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression.

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

N-glycosylation by N-acetylglucosaminyltransferase IVa enhances the interaction of integrin ß1 with vimentin and promotes hepatocellular carcinoma cell motility.

N-glycosylation has been revealed to be tightly associated with cancer metastasis. As a key transferase that catalyzes the formation of ß1,4 N-acetylglucosamine (ß1,4GlcNAc) branches on the mannose core of N-glycans, N-acetylglucosaminyltransferase IVa (GnT-IVa) has been reported to be involved in hepatocellular carcinoma (HCC) metastasis by forming N-glycans; however, the underlying mechanisms are largely unknown. In the current study, we found that GnT-IVa was upregulated in HCC tissues and positively correlated with worse outcomes in HCC patients. We found that GnT-IVa could promote tumor growth in mice; notably, this effect was attenuated after mutating the enzymatic site (D445A) of GnT-IVa, suggesting that GnT-IVa regulated HCC progression by forming ß1,4GlcNAc branches. To mechanistically investigate the role of GnT-IVa in HCC, we conducted GSEA and GO functional analysis as well as in vitro experiments. The results showed that GnT-IVa could enhance HCC cell migration, invasion and adhesion ability and increase ß1,4GlcNAc branch glycans on integrin ß1 (ITGB1), a tumor-associated glycoprotein that is closely involved in cell motility by interacting with vimentin. Interruption of ß1,4GlcNAc branch glycan modification on ITGB1 could suppress the interaction of ITGB1 with vimentin and inhibit cell motility. These results revealed that GnT-IVa could promote HCC cell motility by affecting the biological functions of ITGB1 through N-glycosylation. In summary, our results revealed that GnT-IVa is highly expressed in HCC and can form ß1,4GlcNAc branches on ITGB1, which are essential for interactions with vimentin to promote HCC cell motility. These findings not only proposed a novel mechanism for GnT-IVa in HCC progression but also revealed the significance of N-glycosylation on ITGB1 during the process, which may provide a novel target for future HCC therapy.

O-GlcNAcylation in cancer development and immunotherapy.

O-linked ß-D-N-acetylglucosamine (O-GlcNAc), as a posttranslational modification (PTM), is a reversible reaction that attaches ß-N-GlcNAc to Ser/Thr residues on specific proteins by O-GlcNAc transferase (OGT). O-GlcNAcase (OGA) removes the O-GlcNAc from O-GlcNAcylated proteins. O-GlcNAcylation regulates numerous cellular processes, including signal transduction, the cell cycle, metabolism, and energy homeostasis. Dysregulation of O-GlcNAcylation contributes to the development of various diseases, including cancers. Accumulating evidence has revealed that higher expression levels of OGT and hyper-O-GlcNAcylation are detected in many cancer types and governs glucose metabolism, proliferation, metastasis, invasion, angiogenesis, migration and drug resistance. In this review, we describe the biological functions and molecular mechanisms of OGT- or O-GlcNAcylation-mediated tumorigenesis. Moreover, we discuss the potential role of O-GlcNAcylation in tumor immunotherapy. Furthermore, we highlight that compounds can target O-GlcNAcylation by regulating OGT to suppress oncogenesis. Taken together, targeting protein O-GlcNAcylation might be a promising strategy for the treatment of human malignancies.

B4GALT1 promotes immune escape by regulating the expression of PD-L1 at multiple levels in lung adenocarcinoma.

BACKGROUND: Invasive adenocarcinoma (IAC), which is typically preceded by minimally invasive adenocarcinoma (MIA), is the dominant pathological subtype of early-stage lung adenocarcinoma (LUAD). Identifying the molecular events underlying the progression from MIA to IAC may provide a crucial perspective and boost the exploration of novel strategies for early-stage LUAD diagnosis and treatment. METHODS: Transcriptome sequencing of four pairs of MIA and IAC tumours obtained from four multiple primary lung cancer patients was performed to screen out beta-1,4-galactosyltransferase1 (B4GALT1). Function and mechanism experiments in vitro and in vivo were performed to explore the regulatory mechanism of B4GALT1-mediated immune evasion by regulating programmed cell death ligand 1 (PD-L1). RESULTS: B4GALT1, a key gene involved in N-glycan biosynthesis, was highly expressed in IAC samples. Further experiments revealed that B4GALT1 regulated LUAD cell proliferation and invasion both in vitro and in vivo and was related to the impaired antitumour capacity of CD8 + T cells. Mechanistically, B4GALT1 directly mediates the N-linked glycosylation of PD-L1 protein, thus preventing PD-L1 degradation at the posttranscriptional level. In addition, B4GALT1 stabilized the TAZ protein via glycosylation, which activated CD274 at the transcriptional level. These factors lead to lung cancer immune escape. Importantly, inhibition of B4GALT1 increased CD8 + T-cell abundance and activity and enhanced the antitumour immunity of anti-PD-1 therapy in vivo. CONCLUSION: B4GALT1 is a critical molecule in the development of early-stage LUAD and may be a novel target for LUAD intervention and immunotherapy.

High accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I gene suppression and mannosidase I gene overexpression in Nicotiana tabacum SR1.

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man5GlcNAc2 structure by combining N-acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N-glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man6-9GlcNAc2 structure (2.8%, 7.1%) and high abundance of the Man5GlcNAc2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man5GlcNAc2 structure, and Man I overexpression enhanced the conversion of Man6-9GlcNAc2 structures to the Man5GlcNAc2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins.

Chromatin-associated OGT promotes the malignant progression of hepatocellular carcinoma by activating ZNF263.

Reversible and dynamic O-GlcNAcylation regulates vast networks of highly coordinated cellular and nuclear processes. Although dysregulation of the sole enzyme O-GlcNAc transferase (OGT) was shown to be associated with the progression of hepatocellular carcinoma (HCC), the mechanisms by which OGT controls the cis-regulatory elements in the genome and performs transcriptional functions remain unclear. Here, we demonstrate that elevated OGT levels enhance HCC proliferation and metastasis, in vitro and in vivo, by orchestrating the transcription of numerous regulators of malignancy. Diverse transcriptional regulators are recruited by OGT in HCC cells undergoing malignant progression, which shapes genome-wide OGT chromatin cis-element occupation. Furthermore, an unrecognized cooperation between ZNF263 and OGT is crucial for activating downstream transcription in HCC cells. We reveal that O-GlcNAcylation of Ser662 is responsible for the chromatin association of ZNF263 at candidate gene promoters and the OGT-facilitated HCC malignant phenotypes. Our data establish the importance of aberrant OGT activity and ZNF263 O-GlcNAcylation in the malignant progression of HCC and support the investigation of OGT as a therapeutic target for HCC.