IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation.

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1+ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1+ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1+ progenitor cells.

High core 1ß1,3-galactosyltransferase 1 expression is associated with poor prognosis and promotes cellular radioresistance in lung adenocarcinoma.

PURPOSE: Core 1ß1,3-galactosyltransferase 1 (C1GALT1) exhibits elevated expression in multiple cancers. The present study aimed to elucidate the clinical significance of C1GALT1 aberrant expression and its impact on radiosensitivity in lung adenocarcinoma (LUAD). METHODS: The C1GALT1 expression and its clinical relevance were investigated through public databases and LUAD tissue microarray analyses. A549 and H1299 cells with either C1GALT1 knockdown or overexpression were further assessed through colony formation, gamma-H2A histone family member X immunofluorescence, 5-ethynyl-2'-deoxyuridine incorporation, and flow cytometry assays. Bioinformatics analysis was used to explore single cell sequencing data, revealing the influence of C1GALT1 on cancer-associated cellular states. Vimentin, N-cadherin, and E-cadherin protein levels were measured through western blotting. RESULTS: The expression of C1GALT1 was significantly higher in LUAD tissues than in adjacent non-tumor tissues both at mRNA and protein level. High expression of C1GALT1 was correlated with lymph node metastasis, advanced T stage, and poor survival, and was an independent risk factor for overall survival. Radiation notably upregulated C1GALT1 expression in A549 and H1299 cells, while radiosensitivity was increased following C1GALT1 knockdown and decreased following overexpression. Experiment results showed that overexpression of C1GALT1 conferred radioresistance, promoting DNA repair, cell proliferation, and G2/M phase arrest, while inhibiting apoptosis and decreasing E-cadherin expression, alongside upregulating vimentin and N-cadherin in A549 and H1299 cells. Conversely, C1GALT1 knockdown had opposing effects. CONCLUSION: Elevated C1GALT1 expression in LUAD is associated with an unfavorable prognosis and contributes to increased radioresistance potentially by affecting DNA repair, cell proliferation, cell cycle regulation, and epithelial-mesenchymal transition (EMT).

Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.

Characterization of galactosyltransferase and sialyltransferase genes mediating the elongation of the extracellular O-GlcNAc glycans.

O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.

Molecular cloning, characterisation and molecular modelling of two novel T-synthases from mollusc origin.

The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.

Downregulation of B4GALT5 attenuates cardiac fibrosis through Lumican and Akt/GSK-3ß/ß-catenin pathway.

Virtually all forms of cardiac disease exhibit cardiac fibrosis as a common trait, which ultimately leads to adverse ventricular remodeling and heart failure. To improve the prognosis of heart disease, it is crucial to halt the progression of cardiac fibrosis. Protein function is intricately linked with protein glycosylation, a vital post-translational modification. As a fundamental member of the ß1,4-galactosyltransferase gene family (B4GALT), ß1,4-galactosyltransferase V (B4GALT5) is associated with various disorders. In this study, significant levels of B4GALT5 expression were observed in cardiac fibrosis induced by transverse aortic constriction (TAC) or TGFß1 and the activation of cardiac fibroblasts (CFs). Subsequently, by administering AAV9-shB4GALT5 injections to TAC animals, we were able to demonstrate that in vivo B4GALT5 knockdown decreased the transformation of CFs into myofibroblasts (myoFBs) and reduced the deposition of cardiac collagen fibers. In vitro tests revealed the same results. Conversely, both in vivo and in vitro experiments indicated that overexpression of B4GALT5 stimulates CFs activation and exacerbates cardiac fibrosis. Initially, we elucidated the primary mechanism by which B4GALT5 regulates the Akt/GSK-3ß/ß-catenin pathway and directly interacts with laminin, thereby affecting cardiac fibrosis. Our findings demonstrate that B4GALT5 promotes cardiac fibrosis through the Akt/GSK-3ß/ß-catenin pathway and reveal laminin as the target protein of B4GALT5.

B3GALT6 promotes dormant breast cancer cell survival and recurrence by enabling heparan sulfate-mediated FGF signaling.

Breast cancer mortality results from incurable recurrences thought to be seeded by dormant, therapy-refractory residual tumor cells (RTCs). Understanding the mechanisms enabling RTC survival is therefore essential for improving patient outcomes. Here, we derive a dormancy-associated RTC signature that mirrors the transcriptional response to neoadjuvant therapy in patients and is enriched for extracellular matrix-related pathways. In vivo CRISPR-Cas9 screening of dormancy-associated candidate genes identifies the galactosyltransferase B3GALT6 as a functional regulator of RTC fitness. B3GALT6 is required for glycosaminoglycan (GAG) linkage to proteins to generate proteoglycans, and its germline loss of function in patients causes skeletal dysplasias. We find that B3GALT6-mediated biosynthesis of heparan sulfate GAGs predicts poor patient outcomes and promotes tumor recurrence by enhancing dormant RTC survival in multiple contexts, and does so via a B3GALT6-heparan sulfate/HS6ST1-heparan 6-O-sulfation/FGF1-FGFR2 signaling axis. These findings implicate B3GALT6 in cancer and nominate FGFR2 inhibition as a promising approach to eradicate dormant RTCs and prevent recurrence.

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase.

Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.

Transcriptional Mechanism of the Mouse ß4-Galactosyltransferase 6 Gene in Mouse Neuroblastoma Cell Line Neuro-2a.

Lactosylceramide (Lac-Cer) constitutes the backbone structure of various gangliosides whose abnormal expression is associated with malignancy of neuroblastoma. The understanding of the regulatory mechanism of Lac-Cer contributes to the development of neuroblastoma therapy. In this study, the transcriptional mechanism of mouse ß4-galactosyltransferase (ß4GalT) 6, which is one of Lac-Cer synthase, was analyzed using mouse neuroblastoma cell line Neuro-2a. The -226 to -13 region relative to the most downstream transcriptional start site was determined to be the promoter region by luciferase assay using the 5'-deletion constructs. The mutation into the activating protein (AP) 4-binding site -110/-101 drastically decreased the promoter activity, indicating that this site is mainly implicated in the transcription. Furthermore, the mutation into the GATA-binding site -210/-201 or another AP4-binding site -202/-193 partially decreased the promoter activity. The study suggests that the mouse ß4GalT6 gene is transcriptionally regulated by AP4 in cooperation with GATA family transcription factor in neuroblastoma.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression.

BACKGROUND: Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression. METHODS: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins. RESULTS: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC. CONCLUSION: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

Up-regulation of Core 1 Beta 1, 3-Galactosyltransferase Suppresses Osteosarcoma Growth with Induction of IFN-γ Secretion and Proliferation of CD8+ T Cells.

AIM: Abnormal glycosylation often occurs in tumor cells. T-synthase (core 1 beta 1,3- galactosyltransferase, C1GALT1, or T-synthase) is a key enzyme involved in O-glycosylation. Although T-synthase is known to be important in human tumors, the effects of T-synthase and T-antigen on human tumor responses remain poorly defined. METHODS: In this study, a T-synthase-specific short hairpin RNA (shRNA) or T-synthase-specific eukaryotic expression vector(pcDNA3.1(+)) was transfected into murine Osteosarcoma LM8 cells to assess the effects of T-synthase on T cells and cytokines. RESULTS: The up-regulation of T-synthase promoted the proliferation of osteosarcoma cells in vitro, but it promoted the proliferation of tumor initially up to 2-3 weeks but showed significant growth inhibitory effect after 3 weeks post-implantation in vivo. Osteosarcoma cells with high T-synthase expression in vitro promoted the proliferation and inhibited the apoptosis of CD8+ T cells. Further, T-synthase upregulation promoted CD8+ T-cell proliferation and the increased production of CD4+ T cell-derived IFN-γ cytokines to induce the increased tumor lethality of CTLs. CONCLUSION: Our data suggest that high T-synthase expression inhibits tumor growth by improving the body's anti-tumor immunity. Therefore, using this characteristic to prepare tumor cell vaccines with high immunogenicity provides a new idea for clinical immunotherapy of osteosarcoma.

B3galt5 deficiency attenuates hepatocellular carcinoma by suppressing mTOR/p70s6k-mediated glycolysis.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with high morbidity and mortality. Beta-1,3-galactosyltransferase 5 (b3galt5) plays crucial roles in protein glycosylation, but its function in HCC remains unclear. Here, we investigated the role and underlying mechanism of b3galt5 in HCC. We found that b3galt5 is highly expressed and associated with a poor prognosis in HCC patients. In vitro studies showed that b3galt5 promoted the proliferation and survival of HCC cells. We also demonstrated that b3galt5 deficiency suppressed hepatocarcinogenesis in DEN/TCPOBOP-induced HCC. Further investigation confirmed that b3galt5 promoted aerobic glycolysis in HCC. Mechanistically, b3galt5 promoted glycolysis by activating the mTOR/p70s6k pathway through O-linked glycosylation modification on mTOR. Moreover, p70s6k inhibition reduced the expression of key glycolytic enzymes and the glycolysis rate in b3galt5-overexpressing cells. Our study uncovers a novel mechanism by which b3galt5 mediates glycolysis in HCC and highlights the b3galt5-mTOR/p70s6k axis as a potential target for HCC therapy.

B3GALT4 remodels the tumor microenvironment through GD2-mediated lipid raft formation and the c-met/AKT/mTOR/IRF-1 axis in neuroblastoma.

BACKGROUND: Beta-1,3-galactosyltransferase-4 (B3GALT4) plays a critical regulatory role in tumor biology. However, the role of B3GALT4 in modulating the tumor microenvironment (TME) of neuroblastoma (NB) remains unknown. METHODS: Public datasets and clinical NB samples were collected to evaluate the expression and clinical significance of GD2 and B3GALT4 in NB patients. CCK-8, colony formation, and transwell assays and experiments in tumor-bearing mouse models were conducted to investigate the function of B3GALT4. Flow cytometry, ELISA, immunohistochemistry, immunofluorescence, western blotting, and chemotaxis assays were conducted to ascertain the immunomodulatory mechanism of B3GALT4. The combined therapeutic effect of the lipid raft inhibitor MßCD and anti-GD2 mAb was validated in a murine model of NB. RESULTS: GD2 was overexpressed in NB tissues and high expression of GD2 was associated with poor prognosis in NB patients. B3GALT4 was downregulated in NB tissues, and low expression of B3GALT4 indicated poor prognosis in NB patients. Silencing B3GALT4 significantly enhanced tumor progression both in vitro and in vivo. Meanwhile, the overexpression of B3GALT4 increased the recruitment of CD8+ T lymphocytes via the chemokines CXCL9 and CXCL10. Additionally, B3GALT4 regulated NB-cell GD2 expression and lipid raft formation. Mechanistically, B3GALT4 regulated the expression of CXCL9 and CXCL10 via the c-Met signaling in the lipid rafts and the downstream AKT/mTOR/IRF-1 pathway. The lipid raft inhibitor, MßCD, attenuated B3GALT4 deficiency-induced tumor progression and immune evasion. Last, MßCD combined with anti-GD2 mAb treatment significantly enhanced the antitumor effect and the infiltration of CD8+ T cells. CONCLUSIONS: Upregulation of B3GALT4 promotes the secretion of CXCL9 and CXCL10 to recruit CD8+ T lymphocytes via the GD2-mediated lipid rafts and the c-Met/AKT/mTOR/IRF-1 pathway. Moreover, lipid raft inhibitors may enhance the efficacy of anti-GD2 immunotherapy for NB.

Comprehensive analysis of the GALACTINOL SYNTHASE (GolS) gene family in citrus and the function of CsGolS6 in stress tolerance.

Galactinol synthase (GolS) catalyzes the first and rate-limiting step in the synthesis of raffinose family of oligosaccharides (RFOs), which serve as storage and transport sugars, signal transducers, compatible solutes and antioxidants in higher plants. The present work aimed to assess the potential functions of citrus GolS in mechanisms of stress response and tolerance. By homology searches, eight GolS genes were found in the genomes of Citrus sinensis and C. clementina. Phylogenetic analysis showed that there is a GolS ortholog in C. clementina for each C. sinensis GolS, which have evolved differently from those of Arabidopsis thaliana. Transcriptional analysis indicated that most C. sinensis GolS (CsGolS) genes show a low-level tissue-specific and stress-inducible expression in response to drought and salt stress treatments, as well as to 'Candidatus Liberibacter asiaticus' infection. CsGolS6 overexpression resulted in improved tobacco tolerance to drought and salt stresses, contributing to an increased mesophyll cell expansion, photosynthesis and plant growth. Primary metabolite profiling revealed no significant changes in endogenous galactinol, but different extents of reduction of raffinose in the transgenic plants. On the other hand, a significant increase in the levels of metabolites with antioxidant properties, such as ascorbate, dehydroascorbate, alfa-tocopherol and spermidine, was observed in the transgenic plants. These results bring evidence that CsGolS6 is a potential candidate for improving stress tolerance in citrus and other plants.

Dysregulation and prometastatic function of glycosyltransferase C1GALT1 modulated by cHP1BP3/ miR-1-3p axis in bladder cancer.

BACKGROUND: Abnormal glycosylation in a variety of cancer types is involved in tumor progression and chemoresistance. Glycosyltransferase C1GALT1, the key enzyme in conversion of Tn antigen to T antigen, is involved in both physiological and pathological conditions. However, the mechanisms of C1GALT1 in enhancing oncogenic phenotypes and its regulatory effects via non-coding RNA are unclear. METHODS: Abnormal expression of C1GALT1 and its products T antigen in human bladder cancer (BLCA) were evaluated with BLCA tissue, plasma samples and cell lines. Effects of C1GALT1 on migratory ability and proliferation were assessed in YTS-1 cells by transwell, CCK8 and colony formation assay in vitro and by mouse subcutaneous xenograft and trans-splenic metastasis models in vivo. Dysregulated circular RNAs (circRNAs) and microRNAs (miRNAs) were profiled in 3 pairs of bladder cancer tissues by RNA-seq. Effects of miR-1-3p and cHP1BP3 (circRNA derived from HP1BP3) on modulating C1GALT1 expression were investigated by target prediction program, correlation analysis and luciferase reporter assay. Functional roles of miR-1-3p and cHP1BP3 on migratory ability and proliferation in BLCA were also investigated by in vitro and in vivo experiments. Additionally, glycoproteomic analysis was employed to identify the target glycoproteins of C1GALT1. RESULTS: In this study, we demonstrated upregulation of C1GALT1 and its product T antigen in BLCA. C1GALT1 silencing suppressed migratory ability and proliferation of BLCA YTS-1 cells in vitro and in vivo. Subsets of circRNAs and miRNAs were dysregulated in BLCA tissues. miR-1-3p, which is reduced in BLCA tissues, inhibited transcription of C1GALT1 by binding directly to its 3'-untranslated region (3'-UTR). miR-1-3p overexpression resulted in decreased migratory ability and proliferation of YTS-1 cells. cHP1BP3 was upregulated in BLCA tissues, and served as an miR-1-3p "sponge". cHP1BP3 was shown to modulate migratory ability, proliferation, and colony formation of YTS-1 cells, and displayed tumor-suppressing activity in BLCA. Target glycoproteins of C1GALT1, including integrins and MUC16, were identified. CONCLUSIONS: This study reveals the pro-metastatic and proliferative function of upregulated glycosyltransferase C1GLAT1, and provides preliminary data on mechanisms underlying dysregulation of C1GALT1 via miR-1-3p / cHP1BP3 axis in BLCA.

O-fucosylation stabilizes the TSR3 motif in thrombospondin-1 by interacting with nearby amino acids and protecting a disulfide bond.

Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by ß3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.

Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV-B irradiation.

Flavonol glycosides are bioactive compounds important for plant defence and human nutrition. Glycosylation and methylation play an important role in enriching the diversity of flavonols in response to the environment. Peach flowers and fruit are rich in flavonol diglycosides such as isorhamnetin 3-O-rutinoside (I3Rut), kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside, and flavonol monoglycosides such as I 3-O-glucoside and Q 3-O-galactoside. UV-B irradiation of fruit significantly induced accumulation of all these flavonol glycosides. Candidate biosynthetic genes induced by UV-B were identified by genome homology searches and the in vitro catalytic activities of purified recombinant proteins determined. PpUGT78T3 and PpUGT78A2 were identified as flavonol 3-O-glucosyltransferase and 3-O-galactosyltransferase, respectively. PpUGT91AK6 was identified as flavonol 1,6-rhamnosyl trasferase catalysing the formation of flavonol rutinosides and PpFOMT1 was identified as a flavonol O-methyltransferase that methylated Q at the 3'-OH-OH to form isorhamnetin derivatives. Transient expression in Nicotiana benthamiana confirmed the specificity of PpUGT78T3 as a flavonol 3-O-glucosyltransferase, PpUGT78A2 as a 3-O-galactosyltransferase, PpUGT91AK6 as a 1,6-rhamnosyltrasferase and PpFOMT1 as an O-methyltransferase. This study provides new insights into the mechanisms of glycosylation and methylation of flavonols, especially the formation of flavonol diglycosides such as I3Rut, and will also be useful for future potential metabolic engineering of complex flavonols.

A unique miR775-GALT9 module regulates leaf senescence in Arabidopsis during post-submergence recovery by modulating ethylene and the abscisic acid pathway.

The submergence-induced hypoxic condition negatively affects the plant growth and development, and causes early onset of senescence. Hypoxia alters the expression of a number of microRNAs (miRNAs). However, the molecular function of submergence stress-induced miRNAs in physiological or developmental changes and recovery remains poorly understood. Here, we show that miR775 is an Arabidopsis thaliana-specific young and unique miRNA that possibly evolved non-canonically. miR775 post-transcriptionally regulates GALACTOSYLTRANSFERASE 9 (GALT9) and their expression is inversely affected at 24 h of complete submergence stress. The overexpression of miR775 (miR775-Oe) confers enhanced recovery from submergence stress and reduced accumulation of RBOHD and ROS, in contrast to wild-type and MIM775 Arabidopsis shoot. A similar recovery phenotype in the galt9 mutant indicates the role of the miR775-GALT9 module in post-submergence recovery. We predicted that Golgi-localized GALT9 is potentially involved in protein glycosylation. The altered expression of senescence-associated genes (SAG12, SAG29 and ORE1), ethylene signalling (EIN2 and EIN3) and abscisic acid (ABA) biosynthesis (NCED3) pathway genes occurs in miR775-Oe, galt9 and MIM775 plants. Thus, our results indicate the role for the miR775-GALT9 module in post-submergence recovery through a crosstalk between the ethylene signalling and ABA biosynthesis pathways.

Circ_0009910 sponges miR-491-5p to promote acute myeloid leukemia progression through modulating B4GALT5 expression and PI3K/AKT signaling pathway.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous group of leukemias with an overall poor prognosis. Circular RNAs (circRNAs) have been verified to play important regulatory roles in AML progression. However, the role and molecular mechanism of circ_0009910 in AML development have not be completely clarified. METHODS: The expression levels of circ_0009910, microRNA-491-5p (miR-491-5p), and ß-1, 4-galactosyltransferase 5 (B4GALT5) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Cell proliferation and self-renewal ability were assessed via Cell Counting Kit-8 (CCK-8) and sphere formation assay. Cell cycle distribution and cell apoptosis were evaluated by flow cytometry. Caspase-3 activity was tested by Caspase-3 Activity Assay Kit. Western blot was used to examine the protein levels of autophagy-related markers and PI3K/AKT pathway-related markers. The interaction between miR-491-5p and circ_0009910 or B4GALT5 was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay, or RNA pull-down assay. RESULTS: Circ_0009910 was highly expressed in AML tissues and cells. Silenced circ_0009910 could significantly inhibit the proliferation, sphere formation, and autophagy and promoted the apoptosis of AML cells. Circ_0009910 bound to miR-491-5p in AML cells, and circ_0009910 promoted AML progression partly through sponging miR-491-5p in vitro. B4GALT5 was a target of miR-491-5p, and miR-491-5p overexpression-mediated influences in AML cells were effectually overturned by the addition of B4GALT5 overexpression plasmid. Furthermore, circ_0009910 could regulate the expression of B4GALT5 by downregulating miR-491-5p in AML cells. Additionally, circ_0009910 could activate the PI3K/AKT signaling pathway by sponging miR-491-5p. CONCLUSION: Circ_0009910 could suppress the proliferation, sphere formation, and autophagy and accelerated apoptosis by modulating B4GALT5 expression and activating the PI3K/AKT signaling pathway via sponging miR-491-5p in AML cells, suggesting that circ_0009910 might be a potential biomarker for the treatment of AML.

Validation of a Novel Multitarget Blood Test Shows High Sensitivity to Detect Early Stage Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although biannual ultrasound surveillance with or without α-fetoprotein (AFP) testing is recommended for at-risk patients, sensitivity for early stage HCC, for which potentially curative treatments exist, is suboptimal. We conducted studies to establish the multitarget HCC blood test (mt-HBT) algorithm and cut-off values and to validate test performance in patients with chronic liver disease. METHODS: Algorithm development and clinical validation studies were conducted with participants in an international, multicenter, case-control study. Study subjects had underlying cirrhosis or chronic hepatitis B virus; HCC cases were diagnosed per the American Association for the Study of Liver Diseases criteria and controls were matched for age and liver disease etiology. Whole blood and serum were shipped to a central laboratory and processed while blinded to case/control status. An algorithm was developed for the mt-HBT, which incorporates methylation biomarkers (HOXA1, TSPYL5, and B3GALT6), AFP, and sex. RESULTS: In algorithm development, with 136 HCC cases (60% early stage) and 404 controls, the mt-HBT showed 72% sensitivity for early stage HCC at 88% specificity. Test performance was validated in an independent cohort of 156 HCC cases (50% early stage) and 245 controls, showing 88% overall sensitivity, 82% early stage sensitivity, and 87% specificity. Early stage sensitivity in clinical validation was significantly higher than AFP at 20 ng/mL or greater (40%; P < .0001) and GALAD (gender, age, Lens culinaris agglutinin-reactive AFP, AFP, and des-γ-carboxy-prothrombin score) of -0.63 or greater (71%; P = .03), although AFP and GALAD at these cut-off values had higher specificities (100% and 93%, respectively). CONCLUSIONS: The mt-HBT may significantly improve early stage HCC detection for patients undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality. ClinicalTrials.gov number NCT03628651.