Expression Of Glycosyltransferases Research Articles

Effect of β-1,4-GalTI on the biological function of astrocytes treated by LPS.

Inflammation of the central nervous system (CNS) is a common feature of neurological disorders and infections, playing a crucial role in the development of CNS-related conditions. CNS inflammation is primarily regulated by glial cells, with astrocytes being the most abundant type in the mammalian CNS. Numerous studies have demonstrated that astrocytes, as immunocompetent cells, perform diverse and complex functions in both health and disease. Glycosylation, a critical post-translational modification of proteins, regulates numerous biological functions. The expression and activity of glycosyltransferases, the enzymes responsible for glycosylation, are closely associated with the pathogenesis of various diseases. β-1,4-GalTI, a mammalian glycosyltransferase, plays a significant role in cell-cell interactions, adhesion, and migration. Although many studies have focused on β-1,4-GalTI, few have explored its effects on astrocyte function. In this study, we constructed lentiviral vectors for both interference and overexpression of β-1,4-GalTI and discovered that β-1,4-GalTI knockdown inhibited astrocyte migration and proliferation, while its overexpression promoted these processes. Concurrently, β-1,4-GalTI knockdown reduced the expression of TNF-α, IL-1β, and IL-6, whereas overexpression enhanced the expression of these cytokines. These findings suggest that modulating β-1,4-GalTI activity can influence the molecular functions of astrocytes and provide a theoretical foundation for further research into β-1,4-GalTI as a potential therapeutic target in astrocyte-mediated inflammation.

O-glycosylation of IgA1 and the pathogenesis of an autoimmune disease IgA nephropathy.

IgA nephropathy is a kidney disease characterized by deposition of immune complexes containing abnormally O-glycosylated IgA1 in the glomeruli. Specifically, some O-glycans are missing galactose that is normally β1,3-linked to N-acetylgalactosamine of the core 1 glycans. These galactose-deficient IgA1 glycoforms are produced by IgA1-secreting cells due to a dysregulated expression and activity of several glycosyltransferases. Galactose-deficient IgA1 in the circulation of patients with IgA nephropathy is bound by IgG autoantibodies and the resultant immune complexes can contain additional proteins, such as complement C3. These complexes, if not removed from the circulation, can enter the glomerular mesangium, activate the resident mesangial cells, and induce glomerular injury. In this review, we briefly summarize clinical and pathological features of IgA nephropathy, review normal and aberrant IgA1 O-glycosylation pathways, and discuss the origins and potential significance of natural anti-glycan antibodies, namely those recognizing N-acetylgalactosamine. We also discuss the features of autoantibodies specific for galactose-deficient IgA1 and the characteristics of pathogenic immune complexes containing IgA1 and IgG. In IgA nephropathy, kidneys are injured by IgA1-containing immune complexes as innocent bystanders. Most patients with IgA nephropathy progress to kidney failure and require dialysis or transplantation. Moreover, most patients after transplantation experience a recurrent disease. Thus, a better understanding of the pathogenetic mechanisms is needed to develop new disease-specific treatments.

Results from a real-world study: a novel glycosyltransferase risk score for prognosis, tumor microenvironment phenotypes and immunotherapy in bladder cancer

BackgroundAlthough immunotherapy shows tremendous potential in the treatment of bladder cancer (BLCA), the overall prognosis and response rates to immunotherapy in BLCA remain suboptimal.MethodsWe performed an extensive evaluation of glycosyltransferase expression patterns in BLCA patients by analyzing 210 glycosyltransferase-related genes. Subsequently, we established correlations between these glycosyltransferase patterns, prognosis, and tumor microenvironment (TME) phenotypes. To offer personalized patient assessments, we developed a glycosyltransferase risk score that accurately predicts prognosis, TME phenotypes, and molecular subtypes. Importantly, we developed a RNA-seq cohort, named Xiangya cohort, to validate our results.ResultsTwo distinct patterns of glycosyltransferase expression were identified, corresponding to inflamed and noninflamed TME phenotypes, and demonstrated the potential to predict prognosis. We developed and validated a comprehensive risk score that accurately predicted individual patient prognosis in the TCGA-BLCA cohort. Additionally, we constructed a nomogram that integrated the risk score with several key clinical factors. Importantly, this risk score was successfully validated in external cohorts, including the Xiangya cohort and GSE48075. Furthermore, we discovered a positive correlation between this risk score and tumor-infiltrating lymphocytes in both the TCGA-BLCA and Xiangya cohorts, suggesting that patients with a higher risk score exhibited an inflamed TME phenotype and were more responsive to immunotherapy. Finally, we observed that the high and low risk score groups were consistent with the luminal and basal subtypes of BLCA, respectively, providing further validation of the risk score’s role in the TME in terms of molecular subtypes.ConclusionsGlycosyltransferase patterns exhibit distinct TME phenotypes in BLCA. Our comprehensive risk score provides a promising approach for prognostic prediction and assessment of immunotherapy efficacy, offering valuable guidance for precision medicine.

Targeting sphingolipid metabolism in chronic lymphocytic leukemia

Elevated levels of circulating C16:0 glucosylceramides (GluCer) and increased mRNA expression of UDP-glucose ceramide glycosyltransferase (UGCG), the enzyme responsible for converting ceramides (Cer) to GluCer, represent unfavorable prognostic markers in chronic lymphocytic leukemia (CLL) patients. To evaluate the therapeutic potential of inhibiting GluCer synthesis, we genetically repressed the UGCG pathway using in vitro models of leukemic B cells, in addition to UGCG pharmacological inhibition with approved drugs such as eliglustat and ibiglustat, both individually and in combination with ibrutinib, assessed in cell models and primary CLL patient cells. Cell viability, apoptosis, and proliferation were evaluated in vitro, and survival and apoptosis were examined ex vivo. UGCG inhibition efficacy was confirmed by quantifying intracellular sphingolipid levels through targeted lipidomics using mass spectrometry. Other inhibitors of sphingolipid biosynthesis pathways were similarly assessed. Blocking UGCG significantly decreased cell viability and proliferation, highlighting the oncogenic role of UGCG in CLL. The efficient inhibition of UGCG was confirmed by a significant reduction in GluCer intracellular levels. The combination of UGCG inhibitors with ibrutinib demonstrated synergistic effect. Inhibitors that target alternative pathways within sphingolipid metabolism, like sphingosine kinases inhibitor SKI-II, also demonstrated promising therapeutic effects both alone and when used in combination with ibrutinib, reinforcing the oncogenic impact of sphingolipids in CLL cells. Targeting sphingolipid metabolism, especially the UGCG pathway, represents a promising therapeutic strategy and as a combination therapy for potential treatment of CLL patients, warranting further investigation.

Omics-based analysis of Akkermansia muciniphila cultivation in food-grade media

Background and Aim: Over the past years, the gut microbiota and its correlation to health and disease has been studied extensively. In terms of beneficial microbes, an increased interest in Akkermansia muciniphila (A. muciniphila ) has been observed since its discovery. Direct evidence for the role of A. muciniphila in host health has been provided in both mice and human studies. However, for human interventions with A. muciniphila cells, industrial-scale fermentations are needed, and hence, the used cultivation media should be free of animal-derived components, food-grade, non-allergenic and allow for efficient growth to high densities to provide cost-effective production platforms. In this study, we assessed the growth and performance of A. muciniphila in batch bioreactors using newly developed plant-based media. Methods: The bioreactors were supplemented with varying carbon sources, including different ratios of N-acetylglucosamine (GlcNAc) and glucose. We monitored the growth of A. muciniphila in the plant-based medium using optical density (OD600) measurements and microscopy. In addition, we used a combination of biochemical analysis as well as transcriptional and proteomics analysis to gain detailed insight into the physiology. Results: Comparisons between growth on these media and that on mucin revealed differences at both transcriptome and proteome levels, including differences in the expression of glycosyltransferases, signaling proteins, and stress response. Furthermore, elongated cells and higher OD600 values were observed using the plant-based media as compared to cultivation media containing mucin. Conclusion: These differences do not hamper growth, and therefore, our data suggest that the food-grade medium composition described here could be used to produce A. muciniphila with high yields for therapeutic purposes.

Rational Design for the Complete Synthesis of Stevioside in Saccharomyces cerevisiae.

Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a Saccharomyces cerevisiae strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in S. cerevisiae BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion.

#1261 A small-molecule inhibitor of Bruton's tyrosine kinase reduces production of galactose-deficient IgA1 in IgA nephropathy

Abstract Background and Aims IgA nephropathy (IgAN) is a chronic kidney disease characterized by glomerular immunodeposits that contain aberrantly glycosylated IgA1, i.e., IgA1 with some hinge-region O-glycans deficient in galactose (galactose-deficient IgA1; Gd-IgA1). These immunodeposits originate from circulating immune complexes that contain Gd-IgA1 bound by IgG autoantibodies. Consistent with this hypothesis, serum levels of Gd-IgA1 and IgG autoantibodies independently predict disease progression in IgAN patients. Biochemical studies using IgA1-secreting cell lines derived from peripheral blood of IgAN patients and healthy controls have revealed that Gd-IgA1 production is driven by altered biosynthetic pathways of IgA1 O-glycans. Specifically, cell lines from IgAN patients have altered expression and activity of several glycosyltransferases, C1GalT1 and ST6GalNAc2, and a C1GalT1-specific chaperone, C1GalT1C1. Genetic studies have shown that serum levels of Gd-IgA1 are genetically co-determined and genome-wide association studies (GWAS) identified genetic variations that impact the expression C1GALT1 and C1GALT1C1 genes. Moreover, macroscopic hematuria during an upper-respiratory-tract infection is common at the clinical onset of IgAN and during episodes of accentuated disease activity, indicating a connection between IgAN and mucosal inflammation. Notably, a pro-inflammatory cytokine interleukin-6 (IL-6), enhances production of Gd-IgA1 exclusively in the cell lines derived from IgAN patients. Leukemia inhibitory factor (LIF) has similar effects. LIF-induced effects are mediated by abnormal STAT1 signaling and pathways that include Lyn, a Src-family kinase. The corresponding gene, LYN, was recently identified in one of the 16 new IgAN-associated GWAS loci. Lyn, together with spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), is involved in B-cell-receptor (BCR)-signaling pathways that include activation and inhibitory signals. The aim of this study was to assess if Btk may be involved in Gd-IgA1 production in IgAN. Method IgA1-producing cell lines were cloned by limiting dilution of Epstein-Barr-virus-immortalized peripheral-blood B cells from patients with IgAN (n = 5) and healthy controls (n = 5). Cell signaling, induced by LIF or by BCR crosslinking with anti-IgA antibody, was assessed by flow cytometry or SDS-PAGE/immunoblotting using antibodies for specific phosphorylated sites of selected proteins. Data were normalized using antibodies for specific proteins. Total IgA and Gd-IgA1 were quantified in cell-culture medium using standard ELISA and lectin-ELISA, respectively. Gd-IgA1, normalized to IgA, was expressed in Units (U) of standard Gd-IgA1 protein. Results IgA1-producing cell lines from patients with IgAN and healthy controls secreted similar amounts of total IgA (17.2 ± 9.3 ug/ml vs. 14.6 ± 9.5 ug/ml), but the cells from IgAN patients secreted more Gd-IgA1 (24.2 ± 7.5 U vs. 15.5 ± 2.7 U; p < 0.05). Baseline phosphorylation of Btk (Y223), but not Syk (Y551), was higher in the cells from patients with IgAN than in those from healthy controls. We used an IgA1-secreting cell line from an IgAN patient to confirm that BCR crosslinking with F(ab’)2 anti-IgA antibody in IgA + CD19+ cells lines induced phosphorylation of Btk (Y223). Conversely, anti-IgM antibody, used as a negative control, did not activate Btk. LIF stimulation did not affect phosphorylation of Btk or Syk. A Btk inhibitor, PCI-32765 (ibrutinib), reduced Gd-IgA1 production in a dose-dependent fashion exclusively in the IgAN-derived cells. Notably, 1 uM PCI-32765 reduced Gd-IgA1 production to that for healthy controls (25.7 ± 1.2 U to 17.3 ± 1.8 U; p < 0.01). Conclusion Inhibition of Btk with a small-molecule inhibitor normalized Gd-IgA1 production in IgA1-secreting cells from patients with IgAN, revealing a previously unknown role of Btk in aberrant O-glycosylation of IgA1. Btk may thus represent a potential new target for disease-specific therapy of IgAN.

GROWTH REGULATING FACTOR 7-mediated arbutin metabolism enhances rice salt tolerance.

Salt stress is an environmental factor that limits plant growth and crop production. With the rapid expansion of salinized arable land worldwide, investigating the molecular mechanisms underlying the salt stress response in plants is urgently needed. Here, we report that GROWTH REGULATING FACTOR 7 (OsGRF7) promotes salt tolerance by regulating arbutin (hydroquinone-β-D-glucopyranoside) metabolism in rice (Oryza sativa). Overexpression of OsGRF7 increased arbutin content, and exogenous arbutin application rescued the salt-sensitive phenotype of OsGRF7 knockdown and knockout plants. OsGRF7 directly promoted the expression of the arbutin biosynthesis genes URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE 1 (OsUGT1) and OsUGT5, and knockout of OsUGT1 or OsUGT5 reduced rice arbutin content, salt tolerance, and grain size. Furthermore, OsGRF7 degradation through its interaction with F-BOX AND OTHER DOMAINS CONTAINING PROTEIN 13 reduced rice salinity tolerance and grain size. These findings highlight an underexplored role of OsGRF7 in modulating rice arbutin metabolism, salt stress response, and grain size, as well as its broad potential use in rice breeding.

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin

BackgroundIsoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards.ResultsHerein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three β-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor.ConclusionThe engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.

Mucosal affairs: glycosylation and expression changes of gill goblet cells and mucins in a fish-polyopisthocotylidan interaction.

Secreted mucins are highly O-glycosylated glycoproteins produced by goblet cells in mucosal epithelia. They constitute the protective viscous gel layer overlying the epithelia and are involved in pathogen recognition, adhesion and expulsion. The gill polyopisthocotylidan ectoparasite Sparicotyle chrysophrii, feeds on gilthead seabream (Sparus aurata) blood eliciting severe anemia. Control unexposed and recipient (R) gill samples of gilthead seabream experimentally infected with S. chrysophrii were obtained at six consecutive times (0, 11, 20, 32, 41, and 61 days post-exposure (dpe)). In histological samples, goblet cell numbers and their intensity of lectin labelling was registered. Expression of nine mucin genes (muc2, muc2a, muc2b, muc5a/c, muc4, muc13, muc18, muc19, imuc) and three regulatory factors involved in goblet cell differentiation (hes1, elf3, agr2) was studied by qPCR. In addition, differential expression of glycosyltransferases and glycosidases was analyzed in silico from previously obtained RNAseq datasets of S. chrysophrii-infected gilthead seabream gills with two different infection intensities. Increased goblet cell differentiation (up-regulated elf3 and agr2) leading to neutral goblet cell hyperplasia on gill lamellae of R fish gills was found from 32 dpe on, when adult parasite stages were first detected. At this time point, acute increased expression of both secreted (muc2a, muc2b, muc5a/c) and membrane-bound mucins (imuc, muc4, muc18) occurred in R gills. Mucins did not acidify during the course of infection, but their glycosylation pattern varied towards more complex glycoconjugates with sialylated, fucosylated and branched structures, according to lectin labelling and the shift of glycosyltransferase expression patterns. Gilthead seabream gill mucosal response against S. chrysophrii involved neutral mucus hypersecretion, which could contribute to worm expulsion and facilitate gas exchange to counterbalance parasite-induced hypoxia. Stress induced by the sparicotylosis condition seems to lead to changes in glycosylation characteristic of more structurally complex mucins.

The antimicrobial effect of different vitamin D compounds on Streptococcus mutans and their impact on glycosyltransferase expression

ABSTRACT Background Streptococcus mutans is a virulent microorganism associated with dental caries. This in vitro study aimed to investigate the antimicrobial effects of Cholecalciferol (D3) and Doxercalciferol (D2), against S. mutans and on glycosyltransferase gene expression. Methods Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of D3 and D2 for S. mutans were determined according to the Clinical Laboratory Standards Institute guidelines. The effect of the compounds on environmental pH in 1% w/v and 5% w/v sucrose broth cultures after 24 hours were assessed colorimetrically. Additionally, their impact on glycosyltransferases gene expression (GtfB, GtfC, GtfD) in 5% w/v sucrose culture was evaluated using quantitative real-time PCR. Results The MBCs of D3 and D2 were 83 µg/ml and 166 µg/ml respectively. Both compounds were effective in preventing the local pH drop <5.5 at ≥166 µg/ml in sucrose supplemented cultures. However, the compounds did not inhibit pH drop at MIC values. Notably, D2 upregulated GtfD expression significantly (p < 0.05) and downregulated GtfB and GtfC. Conclusion Vitamin D2 and D3 inhibited S. mutans mediated pH drop in sucrose supplemented cultures and altered glycosyltransferase expression, suggesting potential therapeutic roles in dental caries prevention. Further research is needed to assess their full impact on S. mutans survival under environmental stresses.

Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations.

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

Biosynthesis of apigenin glucosides in engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25℃, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin- 4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37℃. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-β-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-β-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-β-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-β-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Altered O-linked glycosylation in benign and malignant meningiomas.

Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 β1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 β-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Development and validation of glycosyltransferase related-gene for the diagnosis and prognosis of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous cancer characterized by difficulties in early diagnosis and outcome prediction. Aberrant glycosylated structures produced by the aberrant expression of glycosyltransferases are prevalent in HNSCC. In this study, we aim to construct glycosyltransferase-related gene signatures with diagnostic and prognostic value to better stratify patients with HNSCC and improve their diagnosis and prognosis. Bioinformatic tools were used to process data of patients with HNSCC from The Cancer Genome Atlas (TCGA) database. The prognostic model was formatted using univariate and multivariate Cox regression methods, while the diagnostic signature was constructed using support vector machine (SVM) and LASSO analysis. The results were verified using the Gene Expression Omnibus (GEO) cohort. The tumor microenvironment and benefits of immune checkpoint inhibitor (ICI) therapy in subgroups defined by glycosyltransferase-related genes were analyzed. Molecular biology experiments, including western blotting, cell counting kit (CCK)-8, colony formation, wound healing, and Transwell assays, were conducted to confirm the oncogenic function of beta-1,4-galactosyltransferase 3 (B4GALT3) in HNSCC. We established a five-gene prognostic signature and a 15-gene diagnostic model. Based on the median risk score, patients with low risk had longer overall survival than those in the high-risk group, which was consistent with the results of the GEO cohort. The concrete results suggested that high-risk samples were related to a high tumor protein (TP)53 mutation rate, high infiltration of resting memory cluster of differentiation (CD)4 T cells, resting natural killer (NK) cells, and M0 macrophages, and benefited from ICI therapy. In contrast, the low-risk subgroup was associated with a low TP53 mutation rate; and high infiltration of naive B cells, plasma cells, CD8 T cells, and resting mast cells; and benefited less from ICI therapy. In addition, the diagnostic model had an area under curve (AUC) value of 0.997 and 0.978 in the training dataset and validation cohort, respectively, indicating the high diagnostic potential of the model. Ultimately, the depletion of B4GALT3 significantly hindered the proliferation, migration, and invasion of HNSCC cells. We established two new biomarkers that could provide clinicians with diagnostic, prognostic, and treatment guidance for patients with HNSCC.

Abstract B099: Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations

Abstract Introduction: Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. Moreover, as glycans constitute the outermost layer of cells, diverse alterations during carcinogenesis affect the multiple processes of invasion and metastasis, making glycosylation a keystone mechanism in cancer progression. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells for comprehensive cell surface glycan expression profile using lectin microarray and transcriptomic analyses and then analyzed the relationship between altered glycans and glycan-related genes, with a particular focus on the status of KRAS mutations. Materials and methods: Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system containing extracellular matrix and specific niche factors. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. Results: We successfully established 16 3D-organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS mutations (7 G12V, 5 G12D, 1 Q61L) were found in 13 organoids, while wild-type KRAS without mutations was detected in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Pairings of a PDAC organoid with a non-PDAC counterpart were available from 2 patients; one was a PDAC-normal MPD pair and the other was PDAC-IPMN pair, eliminating the influence of individual variations in glycan expression status for those pairs. Lectin reactivity of AAL and AOL with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. And also, in the two cases with PDAC and non-PDAC organoids pairs, the reactivity of AAL were significantly elevated in the KRAS mutants, with FUT6 and FUT3 elevated. Conclusion: PDAC 3D-cultured organoids with KRAS mutations were covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers. Citation Format: Hiromitsu Nakahashi, Tatsuya Oda, Osamu Shimomura, Yoshimasa Akashi, Kazuhiro Takahashi, Yoshihiro Miyazaki, Tomoaki Furuta, Yukihito Kuroda, Pakavarin Louphrasitthiphol, Bryan J. Mathis, Hiroaki Tateno. Aberrant glycosylation in pancreatic ductal adenocarcinoma 3D organoids is mediated by KRAS mutations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B099.

IgM N-glycosylation correlates with COVID-19 severity and rate of complement deposition

The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID-19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease.

Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2

Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant Pq3-O-UGT2 was purified through Ni-NTA affinity purification, a two-step ion exchange chromatography, and subsequently size-exclusion chromatography (SEC). Notably, the purified Pq3-O-UGT2 showed substantial activity toward Rh2 and F2, catalyzing the formation of Rg3 and Rd, respectively. This activity was discernible within a pH range of 4.0–9.0 and temperature range of 30–55 °C, with optimal conditions observed at pH 7.0–8.0 and 37 °C. The catalytic efficiency of Pq3-O-UGT2 toward Rh2 and F2 was 31.43 s−1 mΜ−1 and 169.31 s−1 mΜ−1, respectively. We further crystalized Pq3-O-UGT2 in both its apo form and co-crystalized forms with UDPG, Rh2 and F2, respectively. High-quality crystals were obtained and X-ray diffraction data was collected for all co-crystalized samples. Analysis of the diffraction data revealed that the crystal of Pq3-O-UGT2 co-crystalized with UDP-Glc belonged to space group P1, while the other two crystals belonged to space group P212121. Together, this study has laid a robust foundation for subsequent structural analysis of Pq3-O-UGT2.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3′ untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.