Β-linked N-acetylglucosamine Research Articles

Abstract P1138: Transcriptomic Insights Into How Previously Increased Cardiac Protein O-GlcNAcylation Enhances Susceptibility To Cardiac Dysfunction In A Response To Pressure-Overload

Increased levels of cardiac protein posttranslational β-linked N-acetylglucosamine (O-GlcNAc) modification are implicated in adverse remodeling seen in hypertrophy and diabetes. Our previous work found elevated O-GlcNAc levels in hearts from diabetic mice, and a chronic increase in O-GlcNAc caused adverse cardiac remodeling. In diabetes, increased cardiac disease risk persists even after patients return to tight glycemic control. Thus, we hypothesize that a transient increase in O-GlcNAc is sufficient to exacerbate adverse cardiac remodeling under subsequent pressure-overload, and we propose that this is due to epigenetic modifications causing persistent changes in gene expression. We used an inducible, cardiomyocyte specific, dominant negative O-GlcNAcase (dnOGA) mouse. After a 2-wk induction (which leads to a 1.8-fold increase in O-GlcNAc levels) and subsequent 2-wk washout, Control (Con) and dnOGA mice undergo transverse-aortic constriction (TAC) or Sham surgery. After 8-wks, cardiac function is measured via echocardiography, gravimetric measures at harvest, and RNA-seq analysis is done on cardiac tissue. In both Con+TAC and dnOGA+TAC vs. Sham groups, systolic function is decreased ( -12%, p < 0.001, -11%, p < 0.01 ), and ventricular weight is increased ( +43%, +69%, both p < 0.0001 ), consistent with cardiac hypertrophy. Interestingly, we see further exacerbation of cardiac hypertrophy ( +29%, p < 0.0001 ) and pulmonary edema ( +36%, p < 0.01 ) between dnOGA+TAC vs. Con+TAC groups. Also, RNA-seq results show distinct transcriptomic profiles between the experimental groups via hierarchical clustering ( |FC| > 1.50, FDR < 0.1 ). A candidate gene approach shows enhanced and exacerbated changes in genes associated with cardiac disease states like heart failure and fibrosis (e.g., Atp2a2, Col1a1, & Nox4 ), consistent with cardiac remodeling. Our results support the hypothesis that transient increase in cardiac O-GlcNAc levels increases susceptibility to subsequent cardiac pathology. Moreover, the exacerbation of pathology is consistent with epigenetic mediated effects of transient O-GlcNAc induction; and pertaining to diabetes, provide a possible mechanism into the continued increased of cardiac disease risk.

Is The Hexosamine Biosynthesis Pathway Affected By Inflammation In Endothelial Cells?

Excessive addition of β-linked N-acetylglucosamine (O-GlcNAc) via the hexosamine biosynthesis pathway (HBP) occurs with diabetes and decreases in muscles with exercise. HBP flux increases inflammation and may be specifically important in the activation phase of the inflammatory response. Inflammation plays a key role in insulin resistance and hyperglycemia (HG), with the process contributing to the development of diabetes and associated complications. β-cell development and differentiation are affected by the HBP, and O-GlcNAc has been indicated in insulin secretion from the pancreas (1). PURPOSE: We explored whether in the absence of HG, an inflammatory insult, can affect key factors in this pathway and total O-GlcNAcylation within the endothelium. Because African Americans are overrepresented in conditions involving chronic inflammation, potential racial differences were also examined. METHODS: Human umbilical vein endothelial cells (HUVECs), n=6, were stimulated with 10ng/mL of tumor necrosis factor alpha (TNFα) for 4 hours, or treated as a control. Western blot procedures were used to measure expression of the HBP rate-limiting enzyme (GFAT) and the transferase that adds the end-product onto proteins (OGT). Total protein O-GlcNAcylation (O-GlcNAc) was also measured. RESULTS: With TNFα stimulation, there was no difference in GFAT (0.63 + .09 vs 0.62 + .08, p=.89), OGT (0.49 + .09 vs 0.41 + .08, p=.56), nor total O-GlcNAc (1.56 + .38 vs 1.77 + .42, p=.71) expression compared to controls. There were no racial differences found among the various conditions (p>.05) with either of the proteins examined. CONCLUSIONS: The HBP is not altered with acute inflammation in endothelial cells (ECs), with no racial differences additionally found. Low-grade inflammation vitally contributes to diabetes, atherosclerosis, and their complications, with the HBP playing a role in each. We conclude that HG may be necessary to work in conjunction with inflammation to increase protein O-GlcNAcylation in ECs. Although increased flux though the HBP has been shown to upregulate inflammation, the reverse relationship cannot be confirmed. Future research is needed to examine links between this pathway, various conditions, and the ability of exercise to alter it within the endothelium.

F199. O-GLCNAC DYSREGULATION IN SCHIZOPHRENIA CORTEX

BackgroundO-linked β-linked N-acetylglucosamine (O-GlcNAc) is a post-translational glycosylation modification with ubiquitous functions in cell biology. The attachment of GlcNAc to serine or threonine (S/T) residues transiently adorns thousands of nuclear, cytosolic, and mitochondrial proteins and modulates protein function and localization via dynamic cooperation with kinase and phosphatase enzymes. O-GlcNAc transferase (OGT) exists in complex with S/T phosphatase subunits PP1β and PP1γ and, along with the activity of O-GlcNAcase (OGA), facilitates rapid cycling between O-GlcNAcylation and phosphorylation states to serve as an “on/off switch” for substrate activation. Altered levels of OGT, OGA, and/or O-GlcNAc have been shown to influence many pathways pertinent to schizophrenia (SZ) pathophysiology. Notably, elevated O-GlcNAc and enhanced O-GlcNAcylation mediate glucose tolerance and insulin resistance which can lead to diabetes, an illness often found comorbid with SZ. Elevated O-GlcNAcylation can also produce mitochondrial abnormalities consistent with those identified in SZ. In an exploratory study of glycosylation enzyme transcript expression, our lab found OGT mRNA levels 253% higher in SZ than non-psychiatrically ill comparison (COMP) subjects (p < 0.0001). Based on this evidence, we hypothesized that OGT protein levels or the ratio of OGT:OGA enzymes are elevated in SZ brain.MethodsExpression of OGT and OGA were measured using western blots of superior temporal gyrus (STG; Brodmann Area 22) homogenates from sex- and age-matched pairs of SZ and COMP subjects (N = 17). Standard immunoblotting methods and commercially available antibodies were used to detect the targets of interest and protein levels were normalized to intralane valosin containing protein (VCP) expression; VCP expression has previously been found to be unchanged in SZ STG.ResultsIn the current study, we found OGA protein levels reduced 18% in SZ (p < 0.01) and SZ subjects demonstrate a trend toward increased ratios of OGT:OGA (p = 0.05). OGT was not different between groups (p = 0.77).DiscussionOur current results partially support our original hypothesis that an altered ratio of OGT:OGA may contribute to abnormalities of O-GlcNAcylation and consequent cellular metabolic abnormalities in SZ. A trend toward increased OGT:OGA along with decreased expression of OGA would produce the same functional outcome as the originally predicted OGT increase: upregulation of protein O-GlcNAcylation. Given that the mRNA study used samples of dorsolateral prefrontal cortex (DLPFC) while our protein-level measures were from STG, it is not inconceivable that potential O-GlcNAc dysregulation could arise from upregulated OGT in one brain region, but downregulated OGA in another. To elaborate on these findings, we will investigate OGT and OGA expression in the DLPFC and will assess total O-GlcNAcylation in both brain regions to determine functional consequences of altered enzyme expression.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

Silencing β-linked N-acetylglucosamine transferase induces apoptosis in human gastric cancer cells through PUMA and caspase-3 pathways.

β-linked N-acetylglucosamine (GlcNAc) is a monosaccharide that is catalyzed by O-GlcNAcylation transferase (OGT) to bind serine or threonine hydroxyl moieties of numerous nuclear and cytoplasmic proteins. Recent studies have shown that O-GlcNAcylation is elevated in various cancer types, which is associated with oncogenesis and tumor progression. However, whether OGT is expressed and/or plays a role in gastric cancer is unknown. In the present study, we used qPCR to determine that OGT mRNA levels are significantly elevated in gastric cancer tissues compared with that in corresponding adjacent tissues. In addition, in vivo silencing of OGT in nude mice suppressed tumor proliferation and decreased tumor burden. Furthermore, in vitro OGT knockdown induced more cell apoptosis through increasing PUMA and caspase-3 expression. We used a glycan-binding protein gene microarray to identify potential downstream target genes of OGT, and found that apoptosis-related genes such as galectin and HBEGF were decreased after OGT suppression, suggesting that OGT silencing induces apoptosis in gastric cancer tissues. We concluded that OGT plays a key role in gastric cancer proliferation and survival, and could be a potential target for therapy.

Rational design of a glycosynthase by the crystal structure of β-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures

The crystal structure of β-galactosidase from Bacillus circulans (BgaC) was determined at 1.8Å resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-β domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae β(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from α-d-galactopyranosyl fluoride (αGalF) to different β-linked N-acetylglucosamine acceptor substrates in good yield (40–90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of β1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.

Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus

The thick peptidoglycan layers of Gram-positive bacteria are connected to polyanionic glycopolymers called wall teichoic acids (WTA). Pathogens such as Staphylococcus aureus, Listeria monocytogenes, or Enterococcus faecalis produce WTA with diverse, usually strain-specific structure. Extensive studies on S. aureus WTA mutants revealed important functions of WTA in cell division, growth, morphogenesis, resistance to antimicrobials, and interaction with host or phages. While most of the S. aureus WTA-biosynthetic genes have been identified it remained unclear for long how and why S. aureus glycosylates WTA with α- or β-linked N-acetylglucosamine (GlcNAc). Only recently the discovery of two WTA glycosyltransferases, TarM and TarS, yielded fundamental insights into the roles of S. aureus WTA glycosylation. Mutants lacking WTA GlcNAc are resistant towards most of the S. aureus phages and, surprisingly, TarS-mediated WTA β-O-GlcNAc modification is essential for β-lactam resistance in methicillin-resistant S. aureus. Notably, S. aureus WTA GlcNAc residues are major antigens and activate the complement system contributing to opsonophagocytosis. WTA glycosylation with a variety of sugars and corresponding glycosyltransferases were also identified in other Gram-positive bacteria, which paves the way for detailed investigations on the diverse roles of WTA modification with sugar residues.

Structural, Dynamic, and Chemical Characterization of a Novel S-Glycosylated Bacteriocin

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two β-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two α-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.

Glycosidic intermediates identified in1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways

The glycosylation process, through the addition of carbohydrates, is a major post-translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N-linked or O-linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β-linked N-acetylglucosamine. In both cases, the key precursors are the uridine diphospho-N-acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho-N-acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho-N-acetylhexosamines visible in high-resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho-N-acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes.

Deciphering the roles of O‐GlcNAcylation during CD4+ T‐cells activation

O-GlcNAcylation is a dynamic post-translational modification of serine/threonine with β-linked N-acetylglucosamine. This cyto-nuclear modification is found in all multi-cellular organisms. Previous observations during chemical activation of CD4+ T-cells showed decreasing O-GlcNAc levels on cytosolic proteins while increasing on nuclear proteins. In addition, transcription factor Elf-1 involved in T-cell activation requires O-GlcNAcylation for proper translocation into nucleus. These data indicate that O-GlcNAcylation is an integral part of signal transduction during T-cell activation. To investigate this, we examined O-GlcNAcylation changes resulting from PMA/ionomycin activation of CD4+ T-cells. O-GlcNAc levels increased for nuclear and cytosolic proteins through the first 2 hrs then decreased through 5hrs. Further more, individual bands showed unique O-GlcNAcylation level changes. The cellular localization and amount of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGase) were unchanged. In vitro measured enzymatic activity of O-GlcNAcase also did not change. These data suggest that O-GlcNAc level changes during T-cell activation are due to alteration in OGT or OGase specific interactions. (Supported by NIH grants CA42486 and HD13563. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU.)

804 Expression of a RNK-TRYP-2-homologue serinprotease mRNA in the mouse brain

Two genes, HEXA and HEXB, encode the α- and β-subunits, respectively, of human β-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, β-hexosaminidase A (αβ), B (ββ), and S (αα), that have the capacity to degrade a variety of substrates containing β-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a β-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse β-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5′ flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G + C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs. The widespread tissue distribution of the Hexa and Hexb transcripts conformed to the housekeeping role of the enzyme. However, the expression pattern of the genes was not coincident, implying tissue-specific differences in promoter function.

Characterization of the parasitic interface between Erysiphe pisi and Pisum sativum using fluorescent probes

The constituents of the interface between a strain of Erysiphe pisiand two susceptible and five resistant lines of Pisum sativum were investigated by fluorescence microscopy after treatment with specific reagents. Fragments of epidermes were detached so that the haustoria and extrahaustorial membranes were exposed directly to the reagents. The specificities of the 23 reagents employed included α- and β-linkages in polysaccharides; constituent sugars; aldehyde, amino, sulphydryl and disulphide groups; proteins, lysine, arginine, lipid, calcium and anions; and anion transport sites. The main conclusions are that in the susceptible cultivars the extrahaustorial membrane includes polysaccharides (β1–4 linked) with small amounts of α-glucose, α-mannose and galactose, β-linked N-acetylglucosamine (haustorial face only), protein, arginine, amino and sulphydryl groups and calcium. The extrahaustorial matrix includes varying quantities of β-linked polysaccharides. Haustorial walls contain β-linked polysaccharides including α-glucose, α-mannose, galactose and N-acetyl glucosamine (β-linked). The haustorial cytoplasm contains protein and releases fluorescein from fluorescein diacetate and 3- o-methyl fluorescein phosphate. The main differences between the susceptible lines and the resistant lines is that the resistant line, show enhanced reactivity of terminal glucose, mannose and N-acetylglucosamine groups in the extrahaustorial membranes of some lines. The last is probably due to greater accessibility of lectin effected by rupture of extrahaustorial membranes. Constituents of extrahaustorial membranes fluorescing with 4-acetamido-4′- iso-thiocyanato-stilbene-2,2′-disulphonic acid developed ore slowly and included less cross-linked protein. Calcium was rare in the extrahaustorial membranes and host cytoplasm in resistant lines. The results are discussed in relation to the molecular and functional properties of the interface.