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Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and β-1,4-galactosyltransferase-7 (β-GALT-7)-the first and second enzymes of GAG biosynthesis-when microinjected into a cell. In another study, 4-deoxy-4-fluoro-β-xylosides inhibited β-GALT-7 at 1 mM concentration in vitro . In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or β-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and β-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug-drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC 50 = 380 nM (prodrug), 122 μM (drug), and U87 cells at IC 50 = 10.57 μM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus β-nitro xyloside conformer to XYLT-1 and β-GALT-7 enzymes.

O-GlcNAcylation is a post-translational modification catalysed by O-GlcNAc transferase (OGT). Missense mutations in OGT have been associated with developmental disorders, OGT-linked congenital disorder of glycosylation (OGT-CDG), which are characterized by intellectual disability. OGT relies on the hexosamine biosynthetic pathway (HBP) for provision of its UDP-GlcNAc donor. We considered whether mutations in UDP-N-acetylhexosamine pyrophosphorylase (UAP1), which catalyses the final step in the HBP, would phenocopy OGT-CDG mutations. A de novo mutation in UAP1 (NM_001324114:c.G685A:p.A229T) was reported in a patient with intellectual disability. We show that this mutation is pathogenic and decreases the stability and activity of the UAP1 isoform AGX1 in vitro. X-ray crystallography reveals a structural shift proximal to the mutation, leading to a conformational change of the N-terminal domain. These data suggest that the UAP1 A229T missense mutation could be a contributory factor to the patient phenotype., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P k ) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Automated chemical oligosaccharide synthesis is an attractive concept that has been successfully applied to a large number of target structures, but requires excess quantities of suitably protected and activated building blocks. Herein we demonstrate the use of biocatalysis to supply such reagents for automated synthesis. By using the promiscuous NmLgtB-B β1-4 galactosyltransferase from Neisseria meningitidis we demonstrate fast and robust access to the LacNAc motif, common to many cell-surface glycans, starting from either lactose or sucrose as glycosyl donors. The enzymatic product was shown to be successfully incorporated as a complete unit into a tetrasaccharide target by automated assembly., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

β1,4-GalT1 is a type II membrane glycosyltransferase. It catalyzes the production of lactose in the lactating mammary gland and is supposedly also involved in the galactosylation of terminal GlcNAc of complex-type N-glycans. In-vitro studies of the bovine β4Gal-T1 homolog showed that replacing a single residue of tyrosine with leucine at position 289 alters the donor substrate specificity from UDP-Gal to UDP-N-acetyl-galactosamine (UDP-GalNAc). The effect of this peculiar change in β1,4GalT1 specificity was investigated in-vivo, by generating biallelic Tyr286Leu β1,4GalT1 mice using CRISPR/Cas9 and crossbreeding. Mice bearing this mutation showed no appreciable defects when compared to wild-type mice, with the exception of biallelic female B4GALT1 mutant mice, which were unable to produce milk. The detailed comparison of wild-type and mutant mice derived from liver, kidney, spleen, and intestinal tissues showed only small differences in their N-glycan pattern. Comparable N-glycosylation was also observed in HEK 293 wild-type and knock-out B4GALT1 cells. Remarkably and in contrast to the other analyzed tissue samples, sialylation and galactosylation of serum N-glycans of biallelic Tyr286Leu GalT1 mice almost disappeared completely. These results suggest that β1,4GalT1 plays a special role in the synthesis of serum N-glycans. The herein described Tyr286Leu β1,4GalT1 mutant mouse model may, therefore, prove useful in the investigation of the mechanism which regulates tissue-dependent galactosylation.

Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5- knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca 2+ , reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5- KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5 -KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca 2+ , and conducive for transition to naïve pluripotency., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin-type O-glycosylation. We evaluated alterations in the colon mucus layer in 2-, 16-, and 24-month-old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR-124-3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T-synthase, the rate-limiting enzyme in O-glycosylation, displayed an age-related decline in protein expression. Further experiments indicated that miR-124-3p modulated O-glycosylation by directly targeting T-synthase. Moreover, young mice overexpressing miR-124-3p exhibited abnormal glycosylation, early-onset, and more severe colitis. These data suggest that miR-124-3p predisposes to senile colitis by reducing T-synthase, and the miR-124-3p/T-synthase/O-glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Rhizobium leguminosarum bv. trifolii produces exopolysaccharide (EPS) composed of glucose, glucuronic acid, and galactose residues at a molar ratio 5:2:1. A majority of genes involved in the synthesis, modification, and export of exopolysaccharide are located in the chromosomal Pss-I region. In the present study, a Δ pssJ deletion mutant was constructed and shown to produce EPS lacking terminal galactose in the side chain of the octasaccharide subunit. The lack of galactose did not block EPS subunit translocation and polymerization. The in trans delivery of the pssJ gene restored the production of galactose-containing exopolysaccharide. The mutant was compromised in several physiological traits, e.g., motility and biofilm production. An impact of the pssJ mutation and changed EPS structure on the symbiotic performance was observed as improper signaling at the stage of molecular recognition, leading to formation of a significant number of non-infected empty nodules. Terminal galactosyltransferase PssJ was shown to display a structure typical for the GT-A class of glycosyltransferases and interact with other GTs and Wzx/Wzy system proteins. The latter, together with PssJ presence in soluble and membrane protein fractions indicated that the protein plays its role at the inner membrane interface and as a component of a larger complex.

Human milk oligosaccharides (HMOs) are unique components of human breast milk. Their large-scale production by fermentation allows infant formulas to be fortified with HMOs, but current fermentation processes require lactose as a starting material, increasing the costs, bioburden, and environmental impact of manufacturing. Here we report the development of an Escherichia coli strain that produces 2'-fucosyllactose (2'-FL), the most abundant HMO, de novo using sucrose as the sole carbon source. Strain engineering required the expression of a novel glucose-accepting galactosyltransferase, overexpression of the de novo UDP-d-galactose and GDP-l-fucose pathways, the engineering of an intracellular pool of free glucose, and overexpression of a suitable α(1,2)-fucosyltransferase. The export of 2'-FL was facilitated using a sugar efflux transporter. The final production strain achieved 2'-FL yields exceeding 60 g/L after fermentation for 84 h. This efficient strategy facilitates the lactose-independent production of HMOs by fermentation, which will improve product quality and reduce the costs of manufacturing.

Aluminium (Al) is a key element that plays a major role in inhibiting plant growth and productivity under acidic soils. While lipids may be involved in plant tolerance/sensitivity to Al, the role of monogalactosyldiacylglycerol (MGDG) in Al response remains unknown. In this study, Arabidopsis MGDG synthase (AtMGD) mutants (mgd1, mgd2 and mgd3) and wild-type (Col-0) plants were treated with AlCl 3 ; the effect of aluminium on root growth, aluminium distribution, plasma membrane integrity, lipid peroxidation, hydrogen peroxide content and membrane lipid compositions were analysed. Under Al stress, mgd mutants exhibited a more severe root growth inhibition, plasma membrane integrity damage and lipid peroxidation compared to Col-0. Al accumulation in root tips showed no difference between Col-0 and mutants under Al stress. Lipid analysis demonstrated that under Al treatment the MGDG content in all plants and MGDG/DGDG (digalactosyldiacylglycerol) remarkably reduced, especially in mutants impairing the stability and permeability of the plasma membrane. These results indicate that the Arabidopsis mgd mutants are hypersensitive to Al stress due to the reduction in MGDG content, and this is of great significance in the discovery of effective measures for plants to inhibit aluminium toxicity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The catalysis of inverting glycosyltransferases consists of several biophysical and biochemical processes during which the transfer of a sugar residue from the purine phosphate donor substrate to an acceptor substrate occurs with stereo-inversion of the anomeric C1 center at a product. During catalysis a highly charged phosphate by-product (UDP3-) is formed and a mechanism of how the enzyme stabilizes it back to the UDP2- form is not known. Using methods of molecular modeling (hybrid DFT-QM/MM calculations) we proposed and validated a catalytic mechanism of bovine inverting β-1,4-galactosyltransferase-1 (β4Gal-T1) with native (UDP-galactose) and thio donor substrates (UDP-5' thio galactose). We focused on three aspects of the mechanism not yet investigated: (i) the formation of an oxocarbenium ion intermediate, which was only found for the retaining glycosyltransferases for the time being; (ii) the mechanism of stabilization of a highly charged phosphate by-product (UDP3-) back to its standard in vivo form (UDP2-); (iii) explanation for why in experimental measurements the rate of catalysis with the thio donor substrate is only 8% of the rate of that with the natural substrate. To understand the differences in the interaction patterns between the complexes enzyme : UDP-Gal and enzyme : UDP-5S-Gal, fragmented molecular orbital (FMO) decomposition energy analysis was carried out at the DFT level.

Purpose: Breast cancer stem cells (BCSCs) contribute to the initiation, development, and recurrence of breast carcinomas. β1,4-Galactosyltransferase V (B4GalT5), which catalyzes the addition of galactose to GlcNAcβ1-4Man of N-glycans, is involved in embryogenesis. However, its role in the modulation of BCSCs remains unknown., Materials and Methods: The relationship between B4GalT5 and breast cancer stemness was investigated by online clinical databases and immunohistochemistry analysis. Mammosphere formation, fluorescence-activated cell sorting (FACS), and in-vivo assays were used to evaluate B4GalT5 expression in BCSCs and its effect on BCSCs. B4GalT5 regulation of Wnt/β-catenin signaling was examined by immunofluorescence and Ricinus communis agglutinin I pull-down assays. Cell surface biotinylation and FACS assays were performed to assess the association of cell surface B4GalT5 and BCSCs., Results: B4GalT5, but not other B4GalTs, was highly correlated with BCSC markers and poor prognosis. B4GalT5 significantly increased the stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and promoted the production of CD44+CD24-/low cells and the formation of mammospheres. Furthermore, B4GalT5 overexpression resulted in dramatic tumor growth in vivo. Mechanistically, B4GalT5 modified and protected Frizzled-1 from degradation via the lysosomal pathway, promoting Wnt/β-catenin signaling which was hyperactivated in BCSCs. B4GalT5, located on the surface of a small subset of breast carcinoma cells, was not responsible for the stemness of BCSCs., Conclusion: B4GalT5 modulates the stemness of breast cancer through glycosylation modification to stabilize Frizzled-1 and activate Wnt/β-catenin signaling independent of its cell surface location. Our studies highlight a previously unknown role of B4GalT5 in regulating the stemness of breast cancer and provide a potential drug target for anticancer drug development.

Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5- to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys 261 lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H 2 O 2 . The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Multiple mechanisms contribute to the survival and growth of metastatic castration-resistant prostate cancer (mCRPC) cells without androgen, including androgen receptor splice variants (AR-V) and de novo intratumoral androgen synthesis. AKR1C3 is a critical androgenic enzyme that plays different roles in mCRPC, such as an EMT driver or AR coactivator. However, the relationship and regulatory mechanisms between AKR1C3 and AR-V remain largely unknown. In this study, we observed a positive correlation between AKR1C3 and AR-V7 staining in tissues from prostate rebiopsy at mCRPC. Mechanistically, AKR1C3 interacts with AR-V7 protein in CRPC cells, which can reciprocally inhibit AR-V7 and AKR1C3 protein degradation. Biologically, this complex is essential for in vitro and in vivo tumour growth of CRPC cells after androgen deprivation as it represses B4GALT1, a unique tumour suppressor gene in PCa. Together, this study reveals AKR1C3/AR-V7 complex as a potential therapeutic target in mCRPC., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The glycans on enveloped viruses are synthesized by host-cell machinery. Some of these glycans on zoonotic viruses of mammalian reservoirs are recognized by human natural antibodies that may protect against such viruses. These antibodies are produced mostly against carbohydrate antigens on gastrointestinal bacteria and fortuitously, they bind to carbohydrate antigens synthesized in other mammals, neutralize and destroy viruses presenting these antigens. Two such antibodies are: anti-Gal binding to α -gal epitopes synthesized in non-primate mammals, lemurs, and New World monkeys, and anti-N-glycolyl neuraminic acid (anti-Neu5Gc) binding to N-glycolyl-neuraminic acid (Neu5Gc) synthesized in apes, Old World monkeys, and many non-primate mammals. Anti-Gal appeared in Old World primates following accidental inactivation of the α 1,3galactosyltransferase gene 20-30 million years ago. Anti-Neu5Gc appeared in hominins following the inactivation of the cytidine-monophosphate-N-acetyl-neuraminic acid hydroxylase gene, which led to the loss of Neu5Gc <6 million-years-ago. It is suggested that an epidemic of a lethal virus eliminated ancestral Old World-primates synthesizing α -gal epitopes, whereas few mutated offspring lacking α -gal epitopes and producing anti-Gal survived because anti-Gal destroyed viruses presenting α -gal epitopes, following replication in parental populations. Similarly, anti-Neu5Gc protected few mutated hominins lacking Neu5Gc in lethal virus epidemics that eliminated parental hominins synthesizing Neu5Gc. Since α -gal epitopes are presented on many zoonotic viruses it is suggested that vaccines elevating anti-Gal titers may be of protective significance in areas endemic for such zoonotic viruses. This protection would be during the non-primate mammal to human virus transmission, but not in subsequent human to human transmission where the virus presents human glycans. In addition, production of viral vaccines presenting multiple α -gal epitopes increases their immunogenicity because of effective anti-Gal-mediated targeting of vaccines to antigen presenting cells for extensive uptake of the vaccine by these cells.

Galactose-deficient IgA1 (Gd-IgA1) plays a crucial role in the development of Immunoglobulin A nephropathy (IgAN), however, the underlying pathogenic mechanisms driving Gd-IgA1 production in B cells are not well understood. In this study, RNA-seq analysis identified 337 down-regulated and 405 up-regulated genes in B cells from 17 patients with IgAN and 6 healthy controls. Among them, ST6Gal1, which was associated with IgAN in a previous genome-wide association study (GWAS), was up-regulated in IgAN and significantly positive correlated with elevated Gd-IgA1. In addition, we identified increased plasma ST6Gal1 levels in 100 patients with IgAN, which were associated with higher levels of proteinuria, plasma IgA, Gd-IgA1 levels, greater degrees of systemic complement activation including C3a, Bb, C4d, MAC and a lower proportion classified as C2 grade (crescent proportion ≥25%). Interesting, in vitro, recombinant ST6Gal1 (rST6Gal1) exposure reduced the production of Gd-IgA1 in cultured peripheral blood mononuclear cells from IgAN patients. rST6Gal1 stimuli also increased expression of C1GALT1, which were well-known proportional to the decrease in galactose deficiency of IgA1. In conclusions, we identified increased plasma ST6Gal1 levels and the association of ST6Gal1 with disease severity of IgAN. Additionally, rST6Gal1 administration in vitro increased expression of C1GALT1 and reduced the production of Gd-IgA1., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Conclusions: This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25-33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

Chloroplast membranes have a high content of the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). These galactolipids are essential for the biogenesis of plastids and functioning of the photosynthetic machinery. A monotopic glycosyltransferase, monogalactosyldiacylglycerol synthase synthesizes the bulk of MGDG. It is embedded in the outer leaflet of the inner envelope membrane of chloroplasts. The protein transfers a galactose residue from UDP-galactose to diacylglycerol (DAG); it needs anionic lipids such as phosphatidylglycerol (PG) to be active. The intricacy of the organization and the process of active complex assembly and synthesis have been investigated at the Coarse-Grained and All-Atom of computer simulation levels to cover large spatial and temporal scales. The following self-assembly process and catalytic events can be drawn; (1) in the membrane, in the absence of protein, there is a spontaneous formation of PG clusters to which DAG molecules associate, (2) a reorganization of the clusters occurs in the vicinity of the protein once inserted in the membrane, (3) an accompanying motion of the catalytic domain of the protein brings DAG in the proper position for the formation of the active complex MGD1/UDP-Gal/DAG/PG for which an atomistic model of interaction is proposed.

Bacterial glycosphingolipids such as glucuronosylceramide and galactosylceramide have been identified as ligands for invariant natural killer T cells and play important roles in host defense. However, the glycosphingolipid synthases required for production of these ceramides have not been well-characterized. Here, we report the identification and characterization of glucuronosylceramide synthase (ceramide UDP-glucuronosyltransferase [Cer-GlcAT]) in Z ymomonas mobilis , a Gram-negative bacterium whose cellular membranes contain glucuronosylceramide. On comparing the gene sequences that encode the diacylglycerol GlcAT in bacteria and plants, we found a homologous gene that is widely distributed in the order Sphingomonadales in the Z. mobilis genome. We first cloned the gene and expressed it in Escherichia coli , followed by protein purification using nickel-Sepharose affinity and gel filtration chromatography. Using the highly enriched enzyme, we observed that it has high glycosyltransferase activity with UDP-glucuronic acid and ceramide as sugar donor and acceptor substrate, respectively. Cer-GlcAT deletion resulted in a loss of glucuronosylceramide and increased the levels of ceramide phosphoglycerol, which was expressed in WT cells only at very low levels. Furthermore, we found sequences homologous to Cer-GlcAT in Sphingobium yanoikuyae and Bacteroides fragilis , which have been reported to produce glucuronosylceramide and α-galactosylceramide, respectively. We expressed the two homologs of the cer-glcat gene in E. coli and found that each gene encodes Cer-GlcAT and Cer-galactosyltransferase, respectively. These results contribute to the understanding of the roles of bacterial glycosphingolipids in host-bacteria interactions and the function of bacterial glycosphingolipids in bacterial physiology., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Okino et al.)

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media. This root phenotype was associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Characterization of recombinant GH43 variants revealed that the exo-β-1,3-galactosidase activity of GH43 enzymes is hindered by β-1,6 branches on β-1,3-galactans. In line with this steric hindrance, the recombinant GH43 variants did not release galactose from cell wall-extracted glycoproteins or AGP-rich gum arabic. These results indicate that the lack of exo-β-1,3-galactosidase activity alters cell wall extensibility in roots, a phenotype that could be explained by the involvement of galactosidases in AGP glycan biosynthesis., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nibbering et al.)

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N -glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Morimoto et al.)

Tn-antigen (Tn), a single N -acetylgalactosamine (GalNAc) monosaccharide attached to protein Ser/Thr residues, is found on most cancer yet rarely detected in adult normal tissues as reported in previous studies, featuring it as one of the most distinctive signatures of cancer. Although it is important in cancer, Tn modified glycoproteins are not entirely clear owing to the lack of a suitable method. Knowing the Tn-glycosylated proteins and glycosylation sites are essential to the prevention, diagnosis, and therapy of cancer associated with the expression of Tn. Here, we introduce a method named EXoO-Tn for large-scale mapping of Tn-glycosylated proteins and glycosylation sites. EXoO-Tn utilizes solid-phase immobilization of proteolytic peptides of proteins, which modifies Tn by glycosyltransferase C1GalT1 with isotopically labeled UDP-Gal( 13 C 6 ), to tag and convert Tn to Gal( 13 C 6 )-Tn, which gives rise to a unique glycan mass. The exquisite Gal( 13 C 6 ) modified Tn are then recognized by a human-gut-bacterial enzyme, OpeRATOR, and released at the N-termini of the Gal( 13 C 6 )-Tn-occupied Ser/Thr residues from immobilized peptides to yield site-containing glycopeptides. The effectiveness of EXoO-Tn was benchmarked by analyzing Jurkat cells, where 947 Tn-glycosylation sites from 480 glycoproteins were mapped. The EXoO-Tn was further applied to the analysis of pancreatic cancer sera, where Tn-glycoproteins were identified. Given the significance of Tn in cancer, EXoO-Tn is anticipated to have broad translational and clinical utilities.

Objective: β-1,4 galactosyltransferase-V (β-1,4 GalT-V) is an enzyme that synthesises a glycosphingolipid known as lactosylceramide, which has been implicated in general inflammation and atherosclerosis. We asked if β-1,4 GalT-V was present at elevated levels in patients with SLE, a disease which is associated with increased risk of atherosclerosis., Methods: In this case-control observational study, serum samples were obtained from patients with SLE who are part of the Johns Hopkins Lupus Cohort. Control serum samples were obtained from healthy adult community members recruited from the Baltimore area. All serum samples (n=50 in the SLE group and n=50 in the healthy control group) were analysed with enzyme-linked immunoassays. These assays used antibodies raised against antigens that enabled us to measure the absorbance of oxidised phosphocholines per apolipoprotein B-100 (ox-PC/apoB) and the concentration of lipoprotein(a) (Lp(a)) and β-1,4 GalT-V., Results: Absorbance of ox-PC/apoB and concentrations of Lp(a) and β-1,4 GalT-V were significantly higher in the SLE serum samples as compared with the control serum (p<0.0001)., Conclusions: We conclude that patients with SLE have elevated levels of β-1,4 GalT-V and ox-PC, which have previously been recognised as risk factors for atherosclerosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Raffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of RAFS from maize (or corn, Zea mays ) ( ZmRAFS ) is induced by drought, heat, cold, and salinity stresses. We found that zmrafs mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants. This indicated that ZmRAFS and its product raffinose contribute to plant drought tolerance. ZmRAFS overexpression in Arabidopsis enhanced drought stress tolerance by increasing myo -inositol levels via ZmRAFS-mediated galactinol hydrolysis in the leaves due to sucrose insufficiency in leaf cells and also enhanced raffinose synthesis in the seeds. Supplementation of sucrose to detached leaves converted ZmRAFS from hydrolyzing galactinol to synthesizing raffinose. Taken together, we demonstrate that ZmRAFS enhances plant drought tolerance through either raffinose synthesis or galactinol hydrolysis, depending on sucrose availability in plant cells. These results provide new avenues to improve plant drought stress tolerance through manipulation of the raffinose anabolic pathway., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Li et al.)

Background: Animal tissues and tissue-derived biomaterials are widely used in the field of xenotransplantation and regenerative medicine. A potential immunogenic risk that affects the safety and effectiveness of xenografts is the presence of remnant α -Gal antigen (synthesized by GGTA1 or/and iGb3S ). GGTA1 knockout mice have been developed as a suitable model for the analysis of anti-Gal antibody-mediated immunogenicity. However, we are yet to establish whether GGTA1/iGb3S double knockout (G/i DKO) mice are sensitive to Gal antigen-positive xenoimplants., Methods: α -Gal antigen expression in the main organs of G/i DKO mice or bovine bone substitutes was detected via a standardized ELISA inhibition assay. Serum anti- α -Gal antibody titers of G/i DKO mice after immunization with rabbit red blood cells (RRBC) and implantation of raw lyophilized bone substitutes (Gal antigen content was 8.14 ± 3.17 × 10 12 /mg) or Guanhao Biotech bone substitutes (50% decrease in Gal antigen relative to the raw material) were assessed. The evaluation of total serum antibody, inflammatory cytokine, and splenic lymphocyte subtype populations and the histological analysis of implants and thymus were performed to systematically assess the immune response caused by bovine bone substitutes and bone substitute grafts in G/i DKO mice., Results: α -Gal epitope expression was reduced by 100% in the main organs of G/i DKO mice, compared with their wild-type counterparts. Following immunization with RRBC, serum anti-Gal antibody titers of G/i DKO mice increased from 80- to 180-fold. After subcutaneous implantation of raw lyophilized bone substitutes and Guanhao Biotech bone substitutes into G/i DKO mice, specific anti- α -Gal IgG, anti- α -Gal IgM, and related inflammatory factors (IFN- γ and IL-6) were significantly increased in the raw lyophilized bone substitute group but showed limited changes in the Guanhao Biotech bone substitute group, compared with the control., Conclusion: G/i DKO mice are sensitive to Gal antigen-positive xenogeneic grafts and can be effectively utilized for evaluating the α -Gal-mediated immunogenic risk of xenogeneic grafts., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 Anliang Shao et al.)

A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacterial glycosyltransferases are potential targets for the development of novel antibiotics and anti-virulence agents. We report a novel inhibitor design for the retaining α-1,4-galactosyltransferase LgtC from Neisseria meningitidis. Our design is based on the installation of an electrophilic warhead on the LgtC acceptor substrate and targeted at a non-catalytic cysteine residue in the LgtC active site. We have successfully synthesised two prototype inhibitors in four steps from lactulose. The key step in our synthesis is a Heyns rearrangement, during which we observed the formation of a hitherto unknown side product. While both lactosamine derivatives behaved as moderate inhibitors of LgtC, they also retained residual substrate activity. These results suggest that in contrast to our original design, these inhibitors do not act via a covalent mode of action, but are most likely non-covalent inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background : β-1, 4-galactosyltransferase III (B4GALT3) plays an important role in development tumourigenesis. But its role in human glioblastoma (GBM) is still unknown. Materials and methods : To perform gain-of-function and loss-of-function experiment, we transfected siRNA or vector into GBM cells. CCK-8, EdU staining, transwell, western blot, immunofluorescence staining were applied to perform phenotype and mechanism experiment. GlioVis platform was used to analyze B4GALT3 expression from TCGA datasets. Results : B4GALT3 is upregulated in glioblastoma and predicts poor prognosis. Upregulation of B4GALT3 promotes GBM cell proliferation and invasion while the downregulation of B4GALT3 reduces GBM cell proliferation and invasion. B4GALT3 regulates cell proliferation and invasion via β-catenin and vimentin. B4GALT3 correlates with cell invasion markers in clinical samples. Conclusion : B4GALT3 might be a potential molecular therapeutic target of glioblastoma.

The β4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) transfers GalNAc in a β1,4-linkage to GlcNAc forming the LacdiNAc (LDN) determinant on oligosaccharides. The LacdiNAc-binding adhesin (LabA) has been suggested to mediate attachment of Helicobacter pylori to the gastric mucosa via binding to the LDN determinant. The O-glycan core chain specificity of B4GALNT3 is poorly defined. We investigated the specificity of B4GALNT3 on GlcNAc residues carried by O-glycan core 2, core 3 and extended core 1 precursors using transient transfection of CHO-K1 cells and a mucin-type immunoglobulin fusion protein as reporter protein. Binding of the LabA-positive H. pylori J99 and 26695 strains to mucin fusion proteins carrying the LDN determinant on different O-glycan core chains and human gastric mucins with and without LDN was assessed in a microtiter well-based binding assay, while the binding of 125 I-LDN-BSA to various clinical H. pylori isolates was assessed in solution. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blotting confirmed the requirement of a terminal GlcNAc for B4GALNT3 activity. B4GALNT3 added a β1,4-linked GalNAc to GlcNAc irrespective of whether the latter was carried by a core 2, core 3 or extended core 1 chain. No LDN-mediated adhesion of H. pylori strains 26 695 and J99 to LDN determinants on gastric mucins or a mucin-type fusion protein carrying core 2, 3 and extended core 1 O-glycans were detected in a microtiter well-based adhesion assay and no binding of a 125 I-labelled LDN-BSA neoglycoconjugate to clinical H. pylori isolates was identified.

Mono- and digalactosyldiacylglycerol are essential galactolipids for the biogenesis of plastids and functioning of the photosynthetic machinery. In Arabidopsis, the first step of galactolipid synthesis is catalyzed by monogalactosyldiacylglycerol synthase 1 (MGD1), a monotopic protein located in the inner envelope membrane of chloroplasts, which transfers a galactose residue from UDP-galactose to diacylglycerol (DAG). MGD1 needs anionic lipids such as phosphatidylglycerol (PG) to be active, but the mechanism by which PG activates MGD1 is still unknown. Recent studies shed light on the catalytic mechanism of MGD1 and on the possible PG binding site. Particularly, Pro189 was identified as a potential residue implicated in PG binding and His155 as the putative catalytic residue. In the present study, using a multifaceted approach (Langmuir membrane models, atomic force microscopy, molecular dynamics; MD), we investigated the membrane binding properties of native MGD1 and mutants (P189A and H115A). We demonstrated that both residues are involved in PG binding, thus suggesting the existence of a PG-His catalytic dyad that should facilitate deprotonation of the nucleophile hydroxyl group of DAG acceptor. Interestingly, MD simulations showed that MGD1 induces a reorganization of lipids by attracting DAG molecules to create an optimal platform for binding., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of the present study was to investigate the effect of microRNAs (miRNAs) on the expression level of core1β3‑galactosyltransferase‑specific molecular chaperone (COSMC) in immunoglobulin A nephropathy (IgAN). miRNA expression levels were determined in pediatric patients with IgAN (IgAN group), in patients with other renal diseases (control group) and healthy pediatrics (control group). The target miRNAs of COSMC were investigated in the present study. Western blot analysis was performed to examine the effects of miRNAs on COSMC expression levels. In addition, galactose‑deficient IgA1 (Gd‑IgA1) expression levels were detected following the addition of miRNA‑196b. The present results suggested that the expression levels of 205 miRNAs significantly differed between the IgAN and healthy control groups. The present results also suggested that miRNA‑196b and miRNA‑33a‑3p targeted COSMC, and that miRNA‑196b expression in B lymphocytes was significantly higher in the IgAN group compared with the control group (P<0.0001). However, COSMC expression level was significantly downregulated in isolated B lymphocytes transfected with miRNA‑196b mimics, but Gd‑IgA1 expression levels were increased. Therefore, miRNA‑196b may play a role in the formation of Gd‑IgA1 and IgAN pathogenesis via COSMC regulation.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG., (© 2020 Shen et al.)

Objective: To discuss the role and mechanism of β4GalT1 both in vivo and in vitro glioma, observe whether pathophysiological processes of glioma can be improved after β4GalT1 is knocked down, and study whether β4GalT1 plays a role in malignant biological processes of glioma by regulating the apoptosis and immune processes., Patients and Methods: Firstly, the distribution difference of β4GalT1 in tumor tissues and normal tissues was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) tumor analysis system to deduce the possible role of β4GalT1 in glioma. Secondly, whether the malignant degree of glioma was related to the expression of β4GalT1 and its immunity using human tumor tissues and blood lymphocyte subsets was analyzed. Thirdly, interfere lentivirus vector with β4GalT1 and knockdown β4GalT1 was analyzed to observe whether the malignant degree of glioma has changed. Fourthly, interfere lentivirus vector with recombinant β4GalT protein and β4GalT1 was analyzed to verify the effect of β4GalT in vitro test. Fifth, interfere lentivirus vector with recombinant β4GalT protein and β4GalT1 was analyzed to verify effect of β4GalT in vivo test. Finally, we discuss whether β4GalT is involved in the biological process of glioma through inflammatory reaction., Results: In the GEPIA tumor analysis system, the expression in tumor was significantly higher than that in normal tissues. The expression of β4GalT1 in glioma tissues was higher than that in normal tissues, and the higher the malignancy of the tumor, the higher the expression of β4GalT1 in the glioma tissues, and the lower the immune level was. The expression of IDH1, MGMT, and ki-67 was reduced, and the survival rate of the mice with glioma was improved after β4GalT1 was knocked down. In vitro tests, the activity of tumor cells and their reproductive ability can be reduced after β4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group. In vivo tests, gross tumor volume can be reduced after β4GalT1 was knocked down, the immune level of the body can be improved, and the level of tissue apoptosis can be reduced. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group. After knocking down β4GalT1, the expression of inflammatory factors can be reduced both in vivo and in vitro, and the inflammatory microenvironment of tumors can be improved. After recombinant β4GalT1 was given alone, the result was opposite to that of β4GalT1 knocked down group., Conclusions: The level of β4GalT1 expression in tumor tissues was increased. The malignant degree of glioma is related to the expression of β4GalT1 and its immunity. The level of tumor marker can be decreased, and the survival rate of glioma model mice can be increased after β4GalT1 is knocked down. Apoptosis and immune injury caused by tumor can be improved and gross tumor volume can be deduced after β4GalT1 is knocked down. During the development of glioma, β4GalT1 may play a malignant biological role through inflammatory response.

Our previous study demonstrated that downregulation of transcription factor Specificity protein (Sp) 1 suppresses the malignant potentials of A549 human lung cancer cell line with the reduced β4-galactosylation of highly branched N-glycans on cell surface glycoproteins. The reduced β4-galactosylation was brought about by the decreased expression of the β4-galactosyltransferase 1 (β4GalT1) gene. Herein, we examined whether the reduced β4-galactosylation by decreasing the β4GalT1 gene expression suppresses the malignant potentials of A549 cells. In the β4GalT1-downregulated cells, the β4-galactosylation of highly branched N-glycans was reduced in several glycoproteins such as lysosome-associated membrane protein-1 and E-cadherin. The anchorage-independent growth and migratory ability of the β4GalT1-downregulated cells decreased when compared with the control cells. Furthermore, the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) decreased in the β4GalT1-downregulated cells. These results indicate that downregulation of the β4GalT1 gene decreases the β4-galactosylation of highly branched N-glycans and the phosphorylation of p44/42 MAPK, and suppresses the malignant potentials of A549 cells.

The successful enzymatic synthesis of various ganglioside-related oligosaccharides requires many available glycan-processing enzymes. However, the number of available glycan-processing enzymes remains limited. In this study, the full-length CgtA43456 (β-(1→4)-N-acetylgalactosaminyltransferase) and CgtB11168 (β-(1→3)-galactosyltransferase) were successfully produced from Escherichia coli through the optimization of E. coli-preferable codon usage, selection of E. coli strain, and use of the molecular chaperone GroEL-GroES (GroEL/ES). The CgtA43456 enzyme was produced as a soluble form in E. coli C41(DE3) co-expressed with codon-optimized CgtA43456 and GroEL/ES. However, soluble CgtB11168 was well expressed in E. coli C41(DE3) with only the codon-optimized CgtB11168. Rather, when co-expressed with GroEL/ES, total production of CgtB11168 was reduced. Using immobilized-metal affinity chromatography, the CgtA43456 and CgtB11168 proteins were obtained with approximately 75-78 % purity. The purified CgtA43456 showed a specific activity of 21 mU/mg using UDP-N-acetylgalactosamine and GM3 trisaccharide as donor and acceptor, respectively. The purified CgtB11168 catalyzed the transfer of galactose from UDP-Gal to GM2 tetrasaccharide with a specific activity of 16 mU/mg. We propose that they could be used as catalysts for enzymatic synthesis of GM1 ganglioside-related oligosaccharides., Competing Interests: Declaration of Competing Interest The authors declare no financial or commercial conflicts of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Previous studies have demonstrated that the freezing tolerance (FT) of grapevine was enhanced by foliar application of exogenous abscisic acid (exo-ABA), a treatment which might be incorporated into cultural practices to mitigate cold damage in vineyards. To investigate the underlying mechanisms of this response, a two-year (2017 and 2018) study was conducted to characterize the effects of exo-ABA on greenhouse-grown 'Cabernet franc' grapevine. In control grapevines, both physiological (deeper dormancy) and biochemical (sugar accumulation in buds) changes occurred, indicating that grapevines initiated cold acclimation in the greenhouse. Compared to control, exo-ABA decreased stomatal conductance 2 h after application. Two weeks post application, exo-ABA treated grapevines showed accelerated transition of grapevine physiology during cold acclimation (increased depth of dormancy, decreased bud water content and enhanced bud FT), relative to control. Exo-ABA induced the accumulation of several sugars in buds including the raffinose family oligosaccharides (RFOs), and the RFO precursor, galactinol. The expression of raffinose and galactinol synthase genes was higher in exo-ABA treated grapevine buds, compared to control. The new findings from this study have advanced our understanding of the role of ABA in grapevine FT, which will be useful to develop future strategies to protect grapevines from cold damage., Competing Interests: Declaration of Competing Interest Authors have no competing interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest associated with the content of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Collagen β (1-O) galactosyltransferase 1 (GLT25D1) has been reported to transfer galactose to hydroxylysine residues via β (1-O) linkages in collagen. However, the role of Glt25d1 in liver fibrogenesis is still unknow. Recently, we generated a Glt25d1 knockout mouse to elucidate the role of Glt25d1 in vivo. However, we found that complete deletion of the Glt25d1 gene resulted in embryonic lethality at E11.5. Histopathological analysis revealed that dysplasia in Glt25d1 -/- labyrinth with defects of the vascular network. Immunohistochemical showed that the decrease in proliferation of Glt25d1 -/- liver and the developing central nervous system (CNS). The role of Glt25d1 in liver fibrogenesis was explored by Glt25d1 +/- mice. Glt25d1 +/- mice and wild-type (WT) mice were injected intraperitoneally with the same dose of CCl 4 . The higher level of serum alanine aminotransferase was observed in Glt25d1 +/- mice. Reverse transcription-quantitative polymerase chainreaction demonstrated that the mRNA expression levels of the inflammatory cytokines such as, Tnf-α, Cxcl-1 and Mcp-1, showed a significantly increase in CCl 4 -treated Glt25d1 +/- mice. Collagen-I, collagen-III and α-SMA transcripts accumulation was markedly increased in the Glt25d1 +/- mice. However, Masson's trichrome staining revealed a trend to decrease in the ECM proteins deposition of Glt25d1 +/- liver. Immunohistochemistry and Western blots revealed that the protein expression of Collagen-III was reduced and a trend to a decrease in collagen-I was observed in the Glt25d1 +/- liver compared with those of WT mice. Our results demonstrate that Glt25d1 knockout results in embryonic lethality and down-regulation of Glt25d1 may inhibit collagen secretion during liver fibrogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

C1GALT1 controls the crucial step of GalNAc-type O-glycosylation and is associated with both physiologic and pathologic conditions, including cancers. EPH receptors comprise the largest family of receptor tyrosine kinases (RTKs) and modulate a diverse range of developmental processes and human diseases. However, the role of C1GALT1 in the signaling of EPH receptors remains largely overlooked. Here, we showed that C1GALT1 high expression in gastric adenocarcinomas correlated with adverse clinicopathologic features and is an independent prognostic factor for poor overall survival. Silencing or loss of C1GALT1 inhibited cell viability, migration, invasion, tumor growth and metastasis, as well as increased apoptosis and cytotoxicity of 5-fluorouracil in AGS and MKN45 cells. Phospho-RTK array and western blot analysis showed that C1GALT1 depletion suppressed tyrosine phosphorylation of EPHA2 induced by soluble Ephrin A1-Fc. O-glycans on EPHA2 were modified by C1GALT1 and both S277A and T429A mutants, which are O-glycosites on EPHA2, dramatically enhanced phosphorylation of Y588, suggesting that not only overall O-glycan structures but also site-specific O-glycosylation can regulate EPHA2 activity. Furthermore, depletion of C1GALT1 decreased Ephrin A1-Fc induced migration and reduced Ephrin A1 binding to cell surfaces. The effects of C1GALT1 knockdown or knockout on cell invasiveness in vitro and in vivo were phenocopied by EPHA2 knockdown in gastric cancer cells. These results suggest that C1GALT1 promotes phosphorylation of EPHA2 and enhances soluble Ephrin A1-mediated migration primarily by modifying EPHA2 O-glycosylation. Our study highlights the importance of GalNAc-type O-glycosylation in EPH receptor-regulated diseases and identifies C1GALT1 as a potential therapeutic target for gastric cancer.

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.

The emerging evidence has shown that plant serine/arginine-rich (SR) proteins play a crucial role in abiotic stress responses by regulating the alternative splicing (AS) of key genes. Recently, we have shown that drought stress enhances the expression of SR45a (also known as SR-like 3 ) in Brassica rapa. Herein, we unraveled the hitherto unknown functions of BrSR45a in drought stress response by comparing the phenotypes, chlorophyll a fluorescence and splicing patterns of the drought-responsive genes of Arabidopsis BrSR45a overexpressors (OEs), homozygous mutants (SALK&#95;052345), and controls (Col-0). Overexpression and loss of function did not result in aberrant phenotypes; however, the overexpression of BrSR45a was positively correlated with drought tolerance and the stress recovery rate in an expression-dependent manner. Moreover, OEs showed a higher drought tolerance index during seed germination (38.16%) than the control lines. Additionally, the overexpression of BrSR45a induced the expression of the drought stress-inducible genes RD29A, NCED3 , and DREB2A under normal conditions. To further illustrate the molecular linkages between BrSR45a and drought tolerance, we investigated the AS patterns of key drought-tolerance and BrSR45a interacting genes in OEs, mutants, and controls under both normal and drought conditions. The splicing patterns of DCP5, RD29A, GOLS1, AKR, U2AF, and SDR were different between overexpressors and mutants under normal conditions. Furthermore, drought stress altered the splicing patterns of NCED2, SQE, UPF1 , U4/U6-U5 tri-snRNP - associated protein , and UPF1 between OEs and mutants, indicating that both overexpression and loss of function differently influenced the splicing patterns of target genes. This study revealed that BrSR45a regulates the drought stress response via the alternative splicing of target genes in a concentration-dependent manner., Competing Interests: The authors declare that they have no conflicts of interest.

Objective: To investigate the effect of bone marrow stromal cell glycosyltransferase B4GALT1 expression on hematopoietic cell proliferation and its upstream regulation mechanism., Methods: B4GALT1 was overexpressed in human bone marrow stromal cell line HS5, which was then co-cultured with acute myeloid leukemia cell line KG1a. And its effect on hematopoietic cell proliferation was detected by flow cytometry. Dual luciferase reporter assay, real-time PCR and Western blot were used to predict and validate upstream transcription factors that regulate stromal cell B4GALT1 expression., Results: Overexpression of B4GALT1 in HS5 significantly promoted the proliferation of KG1a in the co-culture system. B4GALT1 expression in stromal cells positively correlated with upstream c-Jun expression, which was verified by JNK/c-Jun inhibitors., Conclusion: The differential expression of glycosyltransferases and their corresponding glycosylation in the hematopoietic microenvironment play an important role.

Alternative polyadenylation (APA) is a pervasive mechanism for gene regulation in eukaryotes. Stachyose is the main assimilate translocated in the cucumber phloem. Stachyose synthase (CsSTS) catalyzes the last step of stachyose biosynthesis in cucumber leaves and plays a key role in the regulation of assimilate partitioning between source and sink. In this study, three CsSTS mRNAs with the same open reading frame and the 5`untranslated region (UTR), but differing in their 3`UTRs, named CsSTS1 (short), CsSTS2 (medium), and CsSTS3 (long), were identified. Southern blot and sequence analysis of the cucumber genome confirmed that these transcripts are regulated through APA from a single gene. No significant difference of in vitro translation efficiency was found among three mRNAs. However, the relative stabilities of three transcripts varied among different tissues and different leaf development stages of cucumber. CsSTS1 expression in cucumber calli was up-regulated by the raffinose (substrate of CsSTS) and down-regulated by stachyose (product of CsSTS), respectively. In cucumber plants, all three isoforms have considerable expression in non-fruit node leaves. However, in fruit-carrying node leaves, the expression of CsSTS2 and CsSTS3 was severely inhibited and only CsSTS1 was highly expressed, indicating fruit setting has a remarkable effect on the relative expression level of three transcripts. This "fruit setting" effect could be observed until at least 36 h after the fruit was removed from the node. Our results suggest that abundant expression of CsSTS1 is beneficial for stachyose loading in source leaves, and APA is a delicate mechanism for CsSTS to regulate cucumber source-sink balance., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. In this study, we report that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation was significantly decreased compared with controls after chilling stress. A similar characterization of the maize dehydration responsive element (DRE)-binding protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts, which consequently upregulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc), which was controlled by the ZmRAFS promoter. Deletion of the single dehydration-responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression, while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative PCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance., (© The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glycosylation is critical to megakaryocyte (MK) and thrombopoiesis in the context of gene mutations that affect sialylation and galactosylation. Here, we identify the conserved B4galt1 gene as a critical regulator of thrombopoiesis in MKs. β4GalT1 deficiency increases the number of fully differentiated MKs. However, the resulting lack of glycosylation enhances β1 integrin signaling leading to dysplastic MKs with severely impaired demarcation system formation and thrombopoiesis. Platelets lacking β4GalT1 adhere avidly to β1 integrin ligands laminin, fibronectin, and collagen, while other platelet functions are normal. Impaired thrombopoiesis leads to increased plasma thrombopoietin (TPO) levels and perturbed hematopoietic stem cells (HSCs). Remarkably, β1 integrin deletion, specifically in MKs, restores thrombopoiesis. TPO and CXCL12 regulate β4GalT1 in the MK lineage. Thus, our findings establish a non-redundant role for β4GalT1 in the regulation of β1 integrin function and signaling during thrombopoiesis. Defective thrombopoiesis and lack of β4GalT1 further affect HSC homeostasis.

Metabolites are small intermediate products of cellular metabolism perturbed in a variety of complex disorders. Identifying genetic markers associated with metabolite concentrations could delineate disease-related metabolic pathways in humans. We tested genetic variants for associations with 136 metabolites in 1954 Chinese from Singapore. At a conservative genome-wide threshold (3.7 × 10-10), we detected 1899 variant-metabolite associations at 16 genetic loci. Three loci (ABCA7, A4GALT, GSTM2) represented novel associations with metabolites, with the strongest association observed between ABCA7 and d18:1/24:1 dihexosylceramide. Among 13 replicated loci, we identified six new variants independent of previously reported metabolite or lipid signals. We observed variant-metabolite associations at two loci (ABCA7, CHCHD2) that have been linked to neurodegenerative diseases. At SGPP1 and SPTLC3 loci, genetic variants showed preferential selectivity for sphingolipids with d16 (rather than d18) sphingosine backbone, including sphingosine-1-phosphate (S1P). Our results provide new genetic associations for metabolites and highlight the role of metabolites as intermediate modulators in disease metabolic pathways., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: More and more studies demonstrated that genetic variation at C1GALT1 influences Gd-IgA1 level in IgAN. However, whether the expression of β1, 3-galactosyltransferase (β1, 3Gal-T) was influenced may provide insights into how Gd-IgA1 levels are controlled in IgAN., Methods: Thirty IgAN patients diagnosed in Tianjin Medical University General Hospital from April to September 2018 and 30 healthy volunteers whose age and gender matched with patients were enrolled in this study. Total Gd-IgA1 levels in plasma were determined by ELISA and C1GALT1 levels were determined by RT-PCR. Four databases (PubMed, EMBASE, CNKI, WanFang Medical Network) were searched to identify eligible studies that evaluated a difference in the expression of C1GALT1 in IgAN patients compared with total controls (non-IgAN and health controls). The C1GALT1C1 expression levels, which was indispensable to β1, 3Gal-T of IgA1, was also been compared., Results: Gd-IgA1 levels were remarkable higher in IgAN patients compared with healthy control. The expression levels of C1GALT1 gene were remarkably down-regulated in IgAN patients compared with healthy control. And the mRNA level of C1GALT1 was inversely correlated to Gd-IgA1 levels. In meta-analysis, six articles including 316 participants that analyzed the expression of β1, 3Gal-T were met inclusion criteria. There was no significant difference in the expression of C1GALT1 between IgAN patients compared with controls. And we found patients with IgAN had lower levels of C1GALT1 gene expression in the B cells compared to controls. The C1GALT1C1 levels in the IgAN patients were not different from the levels in the control group, which were unchanged no matter according to different ethnic population, different control group and different cell source. Two studies including 46 persons compared enzymatic activity of β1, 3Gal-T in B cells, and the result showed the β1, 3Gal-T activity was decreased in B cells., Conclusions: We found expression levels of C1GALT1 were remarkably downregulated in IgAN patients and negatively correlated with higher levels of Gd-IgA1. Subsequent meta-analysis validated the low expression and activity of β1, 3Gal-T in B cells in patients with IgAN. However, there was no apparent disparity in the aspect of C1GALT1C1 expression between IgAN and control groups.

Pectin is a major polysaccharide component that promotes plant growth and fiber elongation in cotton. In previous studies, the galacturonosyltransferase-like (GATL) gene family has been shown to be involved in pectin synthesis. However, few studies have been performed on cotton GATL genes. Here, a total of 33, 17, and 16 GATL genes were respectively identified in Gossypium hirsutum , Gossypium raimondii , and Gossypium arboreum . In multiple plant species, phylogenetic analysis divided GATL genes into five groups named GATL-a to GATL-e, and the number of groups was found to gradually change over evolution. Whole genome duplication (WGD) and segmental duplication played a significant role in the expansion of the GATL gene family in G. hirsutum . Selection pressure analyses revealed that GATL-a and GATL-b groups underwent a great positive selection pressure during evolution. Moreover, the expression patterns revealed that most of highly expressed GhGATL genes belong to GATL-a and GATL-b groups, which have more segmental duplications and larger positive selection value, suggesting that these genes may play an important role in the evolution of cotton plants. We overexpressed GhGATL2 , GhGATL9 , GhGATL12, and GhGATL15 in Arabidopsis and silenced the GhGATL15 gene in cotton through a virus induced gene silencing assay (VIGS). The transgenic and VIGS lines showed significant differences in stem diameter, epidermal hair length, stamen length, seed size, and fiber length than the control plant. In addition, the pectin content test proved that the pectin was significantly increased in the transgenic lines and reduced in VIGS plants, demonstrating that GhGATL genes have similar functions and act on the pectin synthesis to regulate plant growth and fiber elongation. In summary, we performed a comprehensive analysis of GhGATL genes in G. hirsutum including evolution, structure and function, in order to better understand GhGATL genes in cotton for further studies.