RESUMO
The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe ß-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. ß-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. ß-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that ß-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that ß-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.
Assuntos
Cricetulus , Fucose , Fucose/metabolismo , Animais , Células CHO , Glicosilação , Humanos , Trastuzumab/farmacologia , Trastuzumab/metabolismo , Fucosiltransferases/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacosRESUMO
The O-glycoprotein Mucin-2 (MUC2) forms the protective colon mucus layer. While animal models have demonstrated the importance of Muc2, few studies have explored human MUC2 in similar depth. Recent studies have revealed that secreted MUC2 is bound to human feces. We hypothesized human fecal MUC2 (HF-MUC2) was accessible for purification and downstream structural and functional characterization. We tested this via histologic and quantitative imaging on human fecal sections; extraction from feces for proteomic and O-glycomic characterization; and functional studies via growth and metabolic assays in vitro. Quantitative imaging of solid fecal sections showed a continuous mucus layer of varying thickness along human fecal sections with barrier functions intact. Lectin profiling showed HF-MUC2 bound several lectins but was weak to absent for Ulex europaeus 1 (α1,2 fucose-binding) and Sambucus nigra agglutinin (α2,6 sialic acid-binding), and did not have obvious b1/b2 barrier layers. HF-MUC2 separated by electrophoresis showed high molecular weight glycoprotein bands (â¼1-2 MDa). Proteomics and Western analysis confirmed the enrichment of MUC2 and potential MUC2-associated proteins in HF-MUC2 extracts. MUC2 O-glycomics revealed diverse fucosylation, moderate sialylation, and little sulfation versus porcine colonic MUC2 and murine fecal Muc2. O-glycans were functional and supported the growth of Bacteroides thetaiotaomicron (B. theta) and short-chain fatty acid (SCFA) production in vitro. MUC2 could be similarly analyzed from inflammatory bowel disease stools, which displayed an altered glycomic profile and differential growth and SCFA production by B. theta versus healthy samples. These studies describe a new non-invasive platform for human MUC2 characterization in health and disease.
Assuntos
Colo , Fezes , Proteômica , Animais , Humanos , Camundongos , Colo/metabolismo , Glicoproteínas/metabolismo , Mucosa Intestinal/metabolismo , Mucina-2/genética , Mucina-2/metabolismo , Muco/metabolismo , Suínos , Masculino , Camundongos Endogâmicos C57BL , Microbioma GastrointestinalRESUMO
Primary familial brain calcification (PFBC) is characterised by abnormal deposits of calcium phosphate within various regions of the brain that are associated with severe cognitive impairments, psychiatric conditions, and movement disorders. Recent studies in diverse populations have shown a link between mutations in myogenesis-regulating glycosidase (MYORG) and the development of this disease. MYORG is a member of glycoside hydrolase (GH) family 31 (GH31) and, like the other mammalian GH31 enzyme α-glucosidase II, this enzyme is found in the lumen of the endoplasmic reticulum (ER). Though presumed to act as an α-glucosidase due to its localization and sequence relatedness to α-glucosidase II, MYORG has never been shown to exhibit catalytic activity. Here, we show that MYORG is an α-galactosidase and present the high-resolution crystal structure of MYORG in complex with substrate and inhibitor. Using these structures, we map detrimental mutations that are associated with MYORG-associated brain calcification and define how these mutations may drive disease progression through loss of enzymatic activity. Finally, we also detail the thermal stabilisation of MYORG afforded by a clinically approved small molecule ligand, opening the possibility of using pharmacological chaperones to enhance the activity of mutant forms of MYORG.
Assuntos
Encefalopatias , Glicosídeo Hidrolases , Animais , Encéfalo/metabolismo , Encefalopatias/genética , Encefalopatias/metabolismo , Glicosídeo Hidrolases/genética , Humanos , Ligantes , Mamíferos/metabolismo , Desenvolvimento Muscular , Linhagem , Especificidade por Substrato , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , alfa-Glucosidases/metabolismoRESUMO
Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.
Assuntos
Microbioma Gastrointestinal , Bactérias/metabolismo , Galactanos , Humanos , Polissacarídeos/metabolismo , SefaroseRESUMO
Cardiolipin is a mitochondrial phospholipid that is also detected in serum inferring its extracellular release; however, this process has not been directly demonstrated for any of the brain cell types. Nevertheless, extracellular cardiolipin has been shown to modulate several neuroimmune functions of microglia and astrocytes, including upregulation of their endocytic activity. Low cardiolipin levels are associated with brain aging, and may thus hinder uptake of amyloid-ß (Αß) in Alzheimer's disease. We hypothesized that glial cells are one of the sources of extracellular cardiolipin in the brain parenchyma where this phospholipid interacts with neighboring cells to upregulate the endocytosis of Αß. Liquid chromatography-mass spectrophotometry identified 31 different species of cardiolipin released from murine BV-2 microglial cells and revealed this process was accelerated by exposure to Aß42. Extracellular cardiolipin upregulated internalization of fluorescently-labeled Aß42 by primary murine astrocytes, human U118 MG astrocytic cells, and murine BV-2 microglia. Increased endocytic activity in the presence of extracellular cardiolipin was also demonstrated by studying uptake of Aß42 and pHrodo™ Bioparticles™ by human induced pluripotent stem cells (iPSCs)-derived microglia, as well as iPSC-derived human brain organoids containing microglia, astrocytes, oligodendrocytes and neurons. Our observations indicate that Aß42 augments the release of cardiolipin from microglia into the extracellular space, where it can act on microglia and astrocytes to enhance their endocytosis of Aß42. Our observations suggest that the reduced glial uptake of Aß due to the decreased levels of cardiolipin could be at least partially responsible for the extracellular accumulation of Aß in aging and Alzheimer's disease.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Microglia/metabolismo , Cardiolipinas/metabolismo , Doença de Alzheimer/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neuroglia/metabolismo , Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismoRESUMO
Vineyard exposure to wildfire smoke can taint grapes and wine. To understand the impact of this taint, it is imperative that the analytical methods used are accurate and precise. This study compared the variance across nine commercial and research laboratories following quantitative analysis of the same set of smoke-tainted wines. In parallel, correlations between the interlaboratory consensus values for smoke-taint markers and sensory analyses of the same smoke-tainted wines were evaluated. For free guaiacol, the mean accuracy was 94 ± 11% in model wine, while the free cresols and 4-methylguaiacol showed a negative bias and/or decreased precision relative to guaiacol. Similar trends were observed in smoke-tainted wines, with the cresols and glycosidically bound markers demonstrating high variance. Collectively, the interlaboratory results show that data from a single laboratory can be used quantitatively to understand smoke-taint. Results from different laboratories, however, should not be directly compared due to the high variance between study participants. Correlations between consensus compositional data and sensory evaluations suggest the risk of perceivable smoke-taint can be predicted from free cresol concentrations, overcoming limitations associated with the occurrence of some volatile phenols, guaiacol in particular, as natural constituents of some grape cultivars and of the oak used for barrel maturation.
Assuntos
Vitis , Compostos Orgânicos Voláteis , Vinho , Consenso , Cresóis/metabolismo , Guaiacol/análise , Humanos , Fenóis/análise , Fumaça/análise , Vitis/metabolismo , Compostos Orgânicos Voláteis/análise , Vinho/análiseRESUMO
Human milk oligosaccharides (HMOs) promote the development of the neonatal intestinal, immune, and nervous systems and has recently received considerable attention. Here we investigated how the maternal diet affects HMO biosynthesis and how any diet-induced HMO alterations influence the infant gut microbiome and immunity. Using capillary electrophoresis and MS-based analyses, we extracted and measured HMOs from breast milk samples and then correlated their levels with results from validated 24-h diet recall surveys and breast milk fatty acids. We found that fruit intake and unsaturated fatty acids in breast milk were positively correlated with an increased absolute abundance of numerous HMOs, including 16 sulfonated HMOs we identified here in humans for the first time. The diet-derived monosaccharide 5-N-glycolyl-neuraminic acid (Neu5Gc) was unambiguously detected in all samples. To gain insights into the potential impact of Neu5Gc on the infant microbiome, we used a constrained ordination approach and identified correlations between Neu5Gc levels and Bacteroides spp. in infant stool. However, Neu5Gc was not associated with marked changes in infant immune markers, in contrast with sulfonated HMOs, whose expression correlated with suppression of two major Th2 cytokines, IL-10 and IL-13. The findings of our work highlight the importance of maternal diet for HMO biosynthesis and provide as yet unexplored targets for future studies investigating interactions between HMOs and the intestinal microbiome and immunity in infants.
Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Leite Humano/metabolismo , Oligossacarídeos/farmacologia , Ácidos Sulfônicos/química , Bacteroides/efeitos dos fármacos , Bacteroides/isolamento & purificação , Sequência de Carboidratos , Dieta , Eletroforese Capilar , Ácidos Graxos Insaturados/metabolismo , Fezes/microbiologia , Humanos , Lactente , Recém-Nascido , Interleucina-10/metabolismo , Interleucina-13/metabolismo , Espectrometria de Massas , Ácidos Neuramínicos/química , Ácidos Neuramínicos/metabolismo , Ácidos Neuramínicos/farmacologia , Oligossacarídeos/análise , Ácidos Sulfônicos/metabolismo , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Smoke taint in wine is thought to be caused by smoke-derived volatile phenols (VPs) that are absorbed into grape tissues, trapped as conjugates that are imperceptible by smell, and subsequently released into wines as their free odor-active forms via metabolism by yeasts during fermentation. Blocking VP uptake into grapes would, therefore, be an effective way for vineyards to protect ripening grape crops exposed to smoke. Here, we re-evaluated a biofilm that had previously shown promise in pilot studies in reducing levels of smoke-derived VPs. A suite of nine free and acid-labile VPs were quantitated in Pinot Noir grapes that had been exposed to smoke after being coated with the biofilm one, seven or 14 days earlier. In contrast with earlier studies, our results demonstrated that in all cases, the biofilm treatments led to increased concentrations of both free and total VPs in smoke-exposed grapes, with earlier applications elevating concentrations of some VPs more than the later time points. Tracking VP concentrations through the grape ripening process demonstrated that some (phenol, p/m-cresol, and guaiacol) were not entirely sequestered in grapes as acid-labile conjugates, suggesting the presence of VP storage forms beyond simple glycosides. Free VPs in grapes, though a minor portion of the total, most clearly correlated with concentrations present in the resulting wines. Finally, red table grapes, available year round, were observed to replicate the effects of the biofilm treatments and were capable of transforming most VPs into acid-labile conjugates in under 24 h, indicating that they might be an effective model for rapidly assessing smoke-taint prophylactic products in the laboratory.
Assuntos
Fazendas , Fumaça , Vitis/crescimento & desenvolvimento , Compostos Orgânicos VoláteisRESUMO
When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing ß-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p-cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-ß-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae ß-glucosidase activity appear to be influenced by the VP moiety.
Assuntos
Fermentação , Frutas/metabolismo , Glicosídeos/metabolismo , Odorantes/análise , Fenol/metabolismo , Saccharomyces cerevisiae/enzimologia , Fumaça/efeitos adversos , Vitis/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Vinho/análise , Cresóis/metabolismo , Guaiacol/metabolismo , Fenóis/metabolismo , beta-Glucosidase/metabolismoRESUMO
MAIN CONCLUSION: The exposure of Vitis vinifera L. berries to forest fire smoke changes the concentration of phenylpropanoid metabolites in berries and the resulting wine. The exposure of Vitis vinifera L. berries (i.e., wine grapes) to forest fire smoke can lead to a wine defect known as smoke taint that is characterized by unpleasant "smoky" and "ashy" aromas and flavors. The intensity of smoke taint is associated with the concentration of organoleptic volatile phenols that are produced during the combustion-mediated oxidation of lignocellulosic biomass and subsequently concentrated in berries prior to fermentation. However, these same smoke-derived volatile phenols are also produced via metabolic pathways endogenous to berries. It follows then that an influx of exogenous volatile phenols (i.e., from forest fire smoke) could alter endogenous metabolism associated with volatile phenol synthesis, which occurs via the shikimic acid/phenylpropanoid pathways. The presence of ozone and karrikins in forest fire smoke, as well as changes to stomatal conductance that can occur from exposure to forest fire smoke also have the potential to influence phenylpropanoid metabolism. This study demonstrated changes in phenylpropanoid metabolites in Pinot noir berries and wine from three vineyards following the exposure of Vitis vinifera L. vines to simulated forest fire smoke. This included changes to metabolites associated with mouth feel and color in wine, both of which are important sensorial qualities to wine producers and consumers. The results reported are critical to understanding the chemical changes associated with smoke taint beyond volatile phenols, which in turn, may aid the development of preventative and remedial strategies.
Assuntos
Fenóis/metabolismo , Propanóis/metabolismo , Fumaça , Vitis/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Fermentação , Frutas/química , Frutas/metabolismo , Odorantes/análise , Polifenóis/metabolismo , Paladar , Vitis/química , Incêndios Florestais , Vinho/análiseRESUMO
INTRODUCTION: Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids. OBJECTIVE: To develop a rapid, quantitative and broadly applicable liquid chromatography-tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character. METHODOLOGY: Bulk diffusion coefficients were calculated using the Taylor-Aris open tubular method, with four reference compounds used to validate the experimental set-up. Three columns were quantitatively evaluated using van Deemter plots and fit-to-purpose performance metrics. Low (1.2 µL2 ) and standard (3.6 µL2 ) extra-column variance ultra-high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts. RESULTS: Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging Δ8 /Δ9 -tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids. CONCLUSIONS: The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra-column variance UPLC configurations.
Assuntos
Canabinoides/química , Cannabis/química , Cromatografia Líquida/métodos , Compostos Fitoquímicos/química , Espectrometria de Massas em Tandem/métodosRESUMO
O-Linked glycosylation of serine and threonine residues of nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) residues is catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc is conserved within mammals and is implicated in a wide range of physiological processes. Herein, we describe metabolic precursor inhibitors of OGT suitable for use both in cells and inâ vivo in mice. These 5-thiosugar analogues of N-acetylglucosamine are assimilated through a convergent metabolic pathway, most likely involving N-acetylglucosamine-6-phosphate de-N-acetylase (NAGA), to generate a common OGT inhibitor within cells. We show that of these inhibitors, 2-deoxy-2-N-hexanamide-5-thio-d-glucopyranoside (5SGlcNHex) acts inâ vivo to induce dose- and time-dependent decreases in O-GlcNAc levels in various tissues. Decreased O-GlcNAc correlates, both in vitro within adipocytes and inâ vivo within mice, with lower levels of the transcription factor Sp1 and the satiety-inducing hormone leptin, thus revealing a link between decreased O-GlcNAc levels and nutrient sensing in peripheral tissues of mammals.
Assuntos
Acetilglucosamina/metabolismo , Inibidores Enzimáticos/farmacologia , Leptina/metabolismo , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Adipócitos/metabolismo , Animais , Relação Dose-Resposta a Droga , Eletroforese Capilar , Ensaio de Imunoadsorção Enzimática , Glicosilação , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Especificidade por SubstratoRESUMO
Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.
Assuntos
Acetilglucosamina/química , Peptídeos/química , Animais , Glicosilação , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/metabolismo , Plasmídeos , Processamento de Proteína Pós-Traducional , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Fator de Transcrição Sp1/química , UbiquitinaçãoRESUMO
Pyrrolidine-based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O-GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O-GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O-GlcNAc, and should prove useful tools for studying the role of OGA in health and disease.
Assuntos
Encéfalo/metabolismo , Ciclitóis/farmacocinética , Inibidores Enzimáticos/farmacocinética , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Animais , Ciclitóis/química , Inibidores Enzimáticos/química , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , EstereoisomerismoRESUMO
Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked ß-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Catálise , Cristalografia por Raios X , Glicosilação , Humanos , Cinética , Modelos Moleculares , Mimetismo Molecular , N-Acetilglucosaminiltransferases/química , Conformação Proteica , Processamento de Proteína Pós-Traducional , Especificidade por SubstratoRESUMO
The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.
Assuntos
N-Acetilgalactosaminiltransferases/química , Conformação Proteica , Especificidade por Substrato , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
BACKGROUND & AIMS: Addition of sialic acids (sialylation) to glycoconjugates is a common capping step of glycosylation. Our study aims to determine the roles of the overall sialylation in intestinal mucosal homeostasis. METHODS: Mice with constitutive deletion of intestinal epithelial sialylation (IEC Slc35a1-/- mice) and mice with inducible deletion of sialylation in intestinal epithelium (TM-IEC Slc35a1-/- mice) were generated, which were used to determine the roles of overall sialylation in intestinal mucosal homeostasis by ex vivo and muti-omics studies. RESULTS: IEC Slc35a1-/- mice developed mild spontaneous microbiota-dependent colitis. Additionally, 30% of IEC Slc35a1-/- mice had spontaneous tumors in the rectum over the age of 12 months. TM-IEC Slc35a1-/- mice were highly susceptible to acute inflammation induced by 1% DSS vs controls. Loss of total sialylation was associated with reduced mucus thickness on fecal sections and within colon tissues. TM-IEC Slc35a1-/- mice showed altered microbiota with an increase in Clostridia disporicum, which is associated a global reduction in the abundance of at least 20 unique taxa; however, metabolomic analysis did not show any significant differences in short-chain fatty acid levels. Treatment with 5-fluorouracil (5-FU) led to more severe small intestine mucositis in the IEC Slc35a1-/- mice vs. WT littermates, which was associated with reduced Lgr5+ cell representation in small intestinal crypts in IEC Slc35a1-/-;Lgr5-GFP mice. CONCLUSIONS: Loss of overall sialylation impairs mucus stability and the stem cell niche leading to microbiota-dependent spontaneous colitis and tumorigenesis.
RESUMO
Sialyl-Lewis X (sLe(X)) is a tetrasaccharide that serves as a ligand for the set of cell adhesion proteins known as selectins. This interaction enables adhesion of leukocytes and cancer cells to endothelial cells within capillaries, resulting in their extravasation into tissues. The last step in sLe(X) biosynthesis is the α1,3-fucosyltrasferase (FUT)-catalyzed transfer of an L-fucose residue to carbohydrate acceptors. Impairing FUT activity compromises leukocyte homing to sites of inflammation and renders cancer cells less malignant. Inhibition of FUTs is, consequently, of great interest, but efforts to generate glycosyltransferase inhibitors, including FUT inhibitors, has proven challenging. Here we describe a metabolic engineering strategy to inhibit the biosynthesis of sLe(X) in cancer cells using peracetylated 5-thio-L-fucose (5T-Fuc). We show that 5T-Fuc is taken up by cancer cells and then converted into a sugar nucleotide analog, GDP-5T-Fuc, that blocks FUT activity and limits sLe(X) presentation on HepG2 cells with an EC(50) in the low micromolar range. GDP-5T-Fuc itself does not get transferred by either FUT3 or FUT7 at a measurable rate. We further demonstrate that treatment of cells with 5T-Fuc impaired their adhesive properties to immobilized adhesion molecules and human endothelial cells. 5T-Fuc, therefore, is a useful probe that can be used to modulate sLe(X) levels in cells to evaluate the consequences of inhibiting FUT-mediated sLe(X) formation. These data also reveal the utility of using sugar analogues that lead to formation of donor substrate analogues within cells as a general approach to blocking glycosyltransferases in cells.
Assuntos
Inibidores Enzimáticos/farmacologia , Fucose/análogos & derivados , Fucosiltransferases/antagonistas & inibidores , Oligossacarídeos/biossíntese , Selectinas/metabolismo , Animais , Células CHO , Adesão Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fucose/farmacologia , Fucosiltransferases/metabolismo , Glicosilação/efeitos dos fármacos , Células Hep G2 , Humanos , Engenharia Metabólica/métodos , Antígeno Sialil Lewis XRESUMO
The O-GlcNAc modification involves the attachment of single ß-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.
Assuntos
Acetilglucosaminidase/metabolismo , Amino Açúcares/metabolismo , Intestinos/enzimologia , Açúcares de Uridina Difosfato/metabolismo , Acetilglucosaminidase/genética , Amino Açúcares/genética , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Humanos , Intestinos/microbiologia , Simbiose/fisiologia , Açúcares de Uridina Difosfato/genéticaRESUMO
Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.