Journal articles on the topic 'Β-O-linked glycosylation'
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Claude, Patrick, Christian Lehmann, and Thomas Ziegler. "Dependency of the regio- and stereoselectivity of intramolecular, ring-closing glycosylations upon the ring size." Beilstein Journal of Organic Chemistry 7 (December 1, 2011): 1609–19. http://dx.doi.org/10.3762/bjoc.7.189.
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Phenyl 3,4,6-tri-O-benzyl-2-O-(3-carboxypropionyl)-1-thio-β-D-galactopyranoside (1) was condensed via its pentafluorophenyl ester 2 with 5-aminopentyl (4a), 4-aminobutyl (4b), 3-aminopropyl (4c) and 2-aminoethyl 4,6-O-benzylidene-β-D-glucopyranoside (4d), prepared from the corresponding N-Cbz protected glucosides 3a–d, to give the corresponding 2-[3-(alkylcarbamoyl)propionyl] tethered saccharides 5a–d. Intramolecular, ring closing glycosylation of the saccharides with NIS and TMSOTf afforded the tethered β(1→3) linked disaccharides 6a–c, the α(1→3) linked disaccharides 7a–d and the α(1→2) linked disaccharide 8d in ratios depending upon the ring size formed during glycosylation. No β(1→2) linked disaccharides were formed. Molecular modeling of saccharides 6–8 revealed that a strong aromatic stacking interaction between the aromatic parts of the benzyl and benzylidene protecting groups in the galactosyl and glucosyl moieties was mainly responsible for the observed regioselectivity and anomeric selectivity of the ring-closing glycosylation step.
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Fradin, Chantal, Marie Christine Slomianny, Céline Mille, Annick Masset, Raymond Robert, Boualem Sendid, Joachim F. Ernst, Jean Claude Michalski, and Daniel Poulain. "β-1,2 Oligomannose Adhesin Epitopes Are Widely Distributed over the Different Families of Candida albicans Cell Wall Mannoproteins and Are Associated through both N- and O-Glycosylation Processes." Infection and Immunity 76, no. 10 (July 21, 2008): 4509–17. http://dx.doi.org/10.1128/iai.00368-08.
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ABSTRACT β-1,2-Linked mannosides (β-Mans) are believed to contribute to Candida albicans virulence. The presence of β-Mans has been chemically established for two molecules (phosphopeptidomannan [PPM] and phospholipomannan) that are noncovalently linked to the cell wall, where they correspond to specific epitopes. However, a large number of cell wall mannoproteins (CWMPs) also express β-Man epitopes, although their nature and mode of β-mannosylation are unknown. We therefore used Western blotting to map β-Man epitopes for the different families of mannoproteins gradually released from the cell wall according to their mode of anchorage (soluble, released by dithiothreitol, β-1,3 glucan linked, and β-1,6 glucan linked). Reduction of β-Man epitope expression occurred after chemical and enzymatic deglycosylation of the different cell wall fractions, as well as in a secreted form of Hwp1, a representative of the CWMPs linked by glycosylphosphatidylinositol remnants. Enzyme-linked immunosorbent assay inhibition tests were performed to assess the presence of β-Man epitopes in released oligomannosides. A comparison of the results obtained with CWMPs to the results obtained with PPM and the use of mutants with mutations affecting O and N glycosylation demonstrated that both O glycosylation and N glycosylation participate in the association of β-Mans with the protein moieties of CWMPs. This process, which can alter the function of cell wall molecules and their recognition by the host, is therefore more important and more complex than originally thought, since it differs from the model established previously with PPM.
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Ryan, Philip, Andy Hsien Wei Koh, Anna Elizabeth Lohning, and Santosh Rudrawar. "Solid-Phase O-Glycosylation with a Glucosamine Derivative for the Synthesis of a Glycopeptide." Australian Journal of Chemistry 70, no. 10 (2017): 1151. http://dx.doi.org/10.1071/ch17201.
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An efficient synthesis of the O-linked glycosylamino acid Fmoc–l-Ser((Ac)3–β-d-GlcNAc)-OH building block is described. The utility of the method was demonstrated with direct solid-phase O-glycosylation of the hydroxyl group on the amino acid (Ser) side chain of a human α-A crystallin-derived peptide (AIPVSREEK) in nearly quantitative glycosylation yield.
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Schirm, M., M. Kalmokoff, A. Aubry, P. Thibault, M. Sandoz, and S. M. Logan. "Flagellin from Listeria monocytogenes Is Glycosylated with β-O-Linked N-Acetylglucosamine." Journal of Bacteriology 186, no. 20 (October 15, 2004): 6721–27. http://dx.doi.org/10.1128/jb.186.20.6721-6727.2004.
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ABSTRACT Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for β-O-linked GlcNAc confirmed that the linkage was in the β configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.
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KONRAD, Robert J., Irina MIKOLAENKO, Joseph F. TOLAR, Kan LIU, and Jeffrey E. KUDLOW. "The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic β-cell O-GlcNAc-selective N-acetyl-β-d-glucosaminidase." Biochemical Journal 356, no. 1 (May 8, 2001): 31–41. http://dx.doi.org/10.1042/bj3560031.
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Streptozotocin (STZ), an analogue of GlcNAc, inhibits purified rat spleen O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from protein. We have shown previously that STZ increases pancreatic islet O-linked protein glycosylation. In light of these data, we investigated the possibility further that STZ causes β-cell death by inhibiting O-GlcNAcase. In isolated islets, the time course and dose curve of STZ-induced O-glycosylation correlated with β-cell toxicity. STZ inhibition of rat islet O-GlcNAcase activity also paralleled that of its β-cell toxicity, with significant inhibition occurring at a concentration of 1mM. In contrast, STZ inhibition of rat brain O-GlcNAcase and β-TC3 insulinoma cell O-GlcNAcase was significantly right-shifted compared with islets, with STZ only significantly inhibiting activity at a concentration of 5mM, the same concentration required for β-TC3 cell toxicity. In comparison, N-methyl-N-nitrosourea, the nitric oxide-donating portion of STZ, did not cause increased islet O-glycosylation, β-cell toxicity or inhibition of β-cell O-GlcNAcase. Enhanced STZ sensitivity of islet O-GlcNAcase compared with O-GlcNAcase from other tissues or an insulinoma cell line suggests why actual islet β-cells are particularly sensitive to STZ. Confirming this idea, STZ-induced islet β-cell toxicity was completely blocked by GlcNAc, which also prevented STZ-induced O-GlcNAcase inhibition, but was not even partially blocked by glucose, glucosamine or GalNAc. Together, these data demonstrate that STZ's inhibition of β-cell O-GlcNAcase is the mechanism that accounts for its diabetogenic toxicity.
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Larsen, Ida Signe Bohse, Yoshiki Narimatsu, Hiren Jitendra Joshi, Lina Siukstaite, Oliver J. Harrison, Julia Brasch, Kerry M. Goodman, et al. "Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins." Proceedings of the National Academy of Sciences 114, no. 42 (October 2, 2017): 11163–68. http://dx.doi.org/10.1073/pnas.1708319114.
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The cadherin (cdh) superfamily of adhesion molecules carry O-linked mannose (O-Man) glycans at highly conserved sites localized to specific β-strands of their extracellular cdh (EC) domains. These O-Man glycans do not appear to be elongated like O-Man glycans found on α-dystroglycan (α-DG), and we recently demonstrated that initiation of cdh/protocadherin (pcdh) O-Man glycosylation is not dependent on the evolutionary conserved POMT1/POMT2 enzymes that initiate O-Man glycosylation on α-DG. Here, we used a CRISPR/Cas9 genetic dissection strategy combined with sensitive and quantitative O-Man glycoproteomics to identify a homologous family of four putative protein O-mannosyltransferases encoded by the TMTC1–4 genes, which were found to be imperative for cdh and pcdh O-Man glycosylation. KO of all four TMTC genes in HEK293 cells resulted in specific loss of cdh and pcdh O-Man glycosylation, whereas combined KO of TMTC1 and TMTC3 resulted in selective loss of O-Man glycans on specific β-strands of EC domains, suggesting that each isoenzyme serves a different function. In addition, O-Man glycosylation of IPT/TIG domains of plexins and hepatocyte growth factor receptor was not affected in TMTC KO cells, suggesting the existence of yet another O-Man glycosylation machinery. Our study demonstrates that regulation of O-mannosylation in higher eukaryotes is more complex than envisioned, and the discovery of the functions of TMTCs provide insight into cobblestone lissencephaly caused by deficiency in TMTC3.
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Pantaleon, M., H. Tan, and P. L. Kaye. "253. Toxic effects of hyperglycaemia arise from induced O-linked glycosylation in early mouse embryos." Reproduction, Fertility and Development 20, no. 9 (2008): 53. http://dx.doi.org/10.1071/srb08abs253.
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Glucose flux through the hexosamine biosynthetic pathway (HBP) which is essential for preimplantation development (1) produces uridine 5′-diphospho-N-acetylglucosamine, a donor substrate for multiple glycosylation reactions including O-linked glycosylation. This novel signalling arm of the HBP, known as the hexosamine signalling pathway (HSP) operates via reversible addition of an O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of proteins including transcription factors, cytoskeletal components, metabolic enzymes and cellular signalling components. O-linked glycosylation is functionally reciprocal to phosphorylation at the same residues, altering the activity and/or stability of targeted proteins, thus providing a mechanism for modulating cellular physiology in response to glucose availability. The enzymes regulating this O-GlcNAcylation are the β-linked-O-GlcNAc transferase (OGT) and an O-GlcNAc-selective β-N-acetylglucosaminidase (O-GlcNAcase). We hypothesised that the toxicity of hyperglycemia on early embryos arises from increased flux through HBP and increased O-GlcNAcylation of key proteins. Mouse zygotes (18 h post hCG) were cultured under conditions of modified flux through the HSP including hypoglycemia, hyperglycemia or supplemented with glucosamine which feeds exclusively into the HBP to increase downstream O-GlcNAcylation. BADGP was used to inhibit OGT and O-GlcNAcylation. Blastocyst formation, cell proliferation and apoptosis were assessed. Treatments that perturb levels of intracellular protein O-GlcNAcylation inhibited embryo development. Whilst some flux through HBP is required to activate embryonic differentiation (1), excess flux arising from a hyperglycemic environment or glucosamine supplementation reduced cell proliferation and blastocyst formation, confirming the criticality of this novel post-translational signalling pathway. Inhibition of OGT using 2 mM BADGP blocked the negative impact of hyperglycemia on blastocyst formation, cell number and apoptosis supporting our hypothesis that O-GlcNAcylation is a key mechanism used by the embryo to sense and respond to perturbations of glucose in its environment. (1) Pantaleon M, Scott J and Kaye PL (2008) Biol Reprod, 78(4):595–600
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Staudt, Konrad, Friederike Petra Maria Saxe, Heiko Schmied, Raphael Soeur, Wolfgang Böhme, and Werner Baumgartner. "Comparative Investigations of the Sandfish’s β-Keratin (Reptilia: Scincidae: Scincus scincus). Part 1: Surface and Molecular Examinations." Journal of Biomimetics, Biomaterials and Tissue Engineering 15 (October 2012): 1–16. http://dx.doi.org/10.4028/www.scientific.net/jbbte.15.1.
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The Sandfish (Scincidae: Scincus Scincus) Is a Lizard Capable of Moving through Desert Sand in a Swimming-Like Fashion. the Epidermis of this Lizard Shows a High Resistance against Abrasion Together with a Low Friction to Sand as an Adaption to a Subterranean Life below the Desert’s Surface, Outperforming even Steel. the Low Friction Is Mainly Caused by Chemical Composition of the Scales, which Consist of Glycosylated β-Keratins. in this Study, the Friction, the Micro-Structure, the Glycosylation of the β-Keratin Proteins and β-Keratin Coding DNA of the Sandfish in Comparison to other Reptilian Species Was Investigated, Mainly with the Closely Related Berber Skink (Scincidae: Eumeces Schneideri) and another Sand Swimming Species, the Not Closer Related Shovel-Snouted Lizard (Lacertidae: Meroles Anchietae). Glycosylated β-Keratins of the Sandfish, Visualized with Different Lectins Resulted in O-Linked Glycans through PNA Employed as Carbohydrate Marker. Furthermore, the Glycosylation of β-Keratins in Various Squamatean Species Was Investigated and All Species Tested Were Found Positive; however, it Seems Like both Sand Swimming Species Examined Have a much Stronger Glycosylation of their β-Keratins. in Order to Prove this Finding through a Genetic Foundation, DNA of a β-Keratin Coding Gene of the Sandfish Was Sequenced and Compared with a Homologue Gene of Eumeces Schneideri. by Comparison of the Protein Sequence, a Higher Abundance of O-Glycosylation Sites Was Found in the Sandfish (enabled through the Amino Acids Serine and Threonine), Giving Molecular Support for a Higher Glycosylation of the β-Keratins in this Species.
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Bimboese, Patricia, Craig J. Gibson, Stefan Schmidt, Wanqing Xiang, and Barbara E. Ehrlich. "Isoform-specific Regulation of the Inositol 1,4,5-Trisphosphate Receptor by O-Linked Glycosylation." Journal of Biological Chemistry 286, no. 18 (March 7, 2011): 15688–97. http://dx.doi.org/10.1074/jbc.m110.206482.
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The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium channel, has three isoforms with >65% sequence homology, yet the isoforms differ in their function and regulation by post-translational modifications. We showed previously that InsP3R-1 is functionally modified by O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) (Rengifo, J., Gibson, C. J., Winkler, E., Collin, T., and Ehrlich, B. E. (2007) J. Neurosci. 27, 13813–13821). We now report the effect of O-GlcNAcylation on InsP3R-2 and InsP3R-3. Analysis of AR4-2J cells, a rat pancreatoma cell line expressing predominantly InsP3R-2, showed no detectable O-GlcNAcylation of InsP3R-2 and no significant functional changes despite the presence of the enzymes for addition (O-β-N-acetylglucosaminyltransferase) and removal (O-β-N-acetylglucosaminidase) of the monosaccharide. In contrast, InsP3R-3 in Mz-ChA-1 cells, a human cholangiocarcinoma cell line expressing predominantly InsP3R-3, was functionally modified by O-GlcNAcylation. Interestingly, the functional impact of O-GlcNAcylation on the InsP3R-3 channel was opposite the effect measured with InsP3R-1. Addition of O-GlcNAc by O-β-N-acetylglucosaminyltransferase increased InsP3R-3 single channel open probability. Incubation of Mz-ChA-1 cells in hyperglycemic medium caused an increase in the InsP3-dependent calcium release from the endoplasmic reticulum. The dynamic and inducible nature of O-GlcNAcylation and the InsP3R isoform specificity suggest that this form of modification of InsP3R and subsequent changes in intracellular calcium transients are important in physiological and pathophysiological processes.
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Greer, Wenda L., Elizabeth Higgins, D. Robert Sutherland, Abraham Novogrodsky, Inka Brockhausen, Monica Peacocke, Laurence A. Rubin, Michael Baker, James W. Dennis, and Katherine A. Siminovitch. "Altered expression of leucocyte sialoglycoprotein in Wiskott-Aldrich syndrome is associated with a specific defect in O-glycosylation." Biochemistry and Cell Biology 67, no. 9 (September 1, 1989): 503–9. http://dx.doi.org/10.1139/o89-081.
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The Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency disorder characterized clinically by both lymphocyte and platelet dysfunction. Studies of WAS T lymphocytes have revealed deficient or defective cell surface expression of the highly O-glycosylated leucocyte sialoglycoprotein CD43. To further elucidate the basis for, and functional relevance of, CD43 modifications on WAS lymphocytes, we have studied lymphocytes from two WAS patients with regard to membrane glycoprotein profile and mitogen-induced proliferative responses. CD43 was found to be either absent or altered in size on peripheral blood lymphocytes and lectin-stimulated T cells from both patients. Compared with control cells, the WAS lymphocytes displayed reduced, but measurable proliferative responses to lectins and neuraminidase/galactose oxidase, and virtually no response to periodate, a mitogenic agent which targets sialic acid residues on membrane glycoproteins such as CD43. Analysis of activities of three glycosyltransferases involved in O-glycosylation revealed marked reduction in the level of activity of UDP-N-acetylglucosamine: Galβ1-3GalNAc-R β-1,6-N-acetylglucosamine (β-1,6-GlcNAc) transferase in one WAS patient and no detectable activity of this enzyme in a second. β-1,6-GlcNAc transferase activity has recently been shown to increase during T cell activation coincident with changes in the O-linked glycans on CD43. A selective reduction of this glycosyltransferase in WAS lymphocytes suggests that O-linked oligosaccharides may be important to the structure of membrane glycoproteins involved in lymphocyte activation.Key words: Wiskott-Aldrich syndrome, immune deficiency, O-glycosylation, glycosyltransferase, lymphocyte activation.
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Hull, Rebecca L., Sakeneh Zraika, Jayalakshmi Udayasankar, Robert Kisilevsky, Walter A. Szarek, Thomas N. Wight, and Steven E. Kahn. "Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and azaserine result in reduced islet amyloid formation in vitro." American Journal of Physiology-Cell Physiology 293, no. 5 (November 2007): C1586—C1593. http://dx.doi.org/10.1152/ajpcell.00208.2007.
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Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in ∼90% of individuals with Type 2 diabetes and is associated with decreased islet β-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet β-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri- O-acetyl-2,4-dideoxy-α-d- xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo. To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to 7 days in 16.7 mM glucose (conditions that result in amyloid deposition) plus increasing concentrations of the inhibitor. WAS-406 at doses of 0, 10, 100, and 1,000 μM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66 ± 0.14%, 0.10 ± 0.06%, 0.09 ± 0.07%, and 0.004 ± 0.003%, P < 0.001) and an increase in β-cell area in hIAPP transgenic islets (55.0 ± 2.6 vs. 60.6 ± 2.2% islet area for 0 vs. 100 μM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic islets. Azaserine, an inhibitor of the rate-limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.
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Garcia-Campayo, Vicenta, Tadashi Sugahara, and Irving Boime. "Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin β subunit." Molecular and Cellular Endocrinology 194, no. 1-2 (August 2002): 63–70. http://dx.doi.org/10.1016/s0303-7207(02)00189-2.
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Yoshida-Moriguchi, Takako, Tobias Willer, Mary E. Anderson, David Venzke, Tamieka Whyte, Francesco Muntoni, Hane Lee, Stanley F. Nelson, Liping Yu, and Kevin P. Campbell. "SGK196 Is a Glycosylation-Specific O-Mannose Kinase Required for Dystroglycan Function." Science 341, no. 6148 (August 8, 2013): 896–99. http://dx.doi.org/10.1126/science.1239951.
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Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine–β3-N-acetylglucosamine–β4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain–containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. We found that glycosyltransferase-like domain–containing 2 (GTDC2) is a protein O-linked mannose β 1,4-N-acetylglucosaminyltransferase whose product could be extended by β 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.
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GORDON, Kerry, Pierre REDELINGHUYS, Sylva L. U. SCHWAGER, Mario R. W. EHLERS, Anastassios C. PAPAGEORGIOU, Ramanathan NATESH, K. Ravi ACHARYA, and Edward D. STURROCK. "Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme." Biochemical Journal 371, no. 2 (April 15, 2003): 437–42. http://dx.doi.org/10.1042/bj20021842.
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Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEΔ36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0Å resolution respectively. Furthermore, a truncated, soluble tACE (tACEΔ36NJ), expressed in the presence of the glucosidase-I inhibitor N-butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P212121 space group (cell dimensions, a = 56.47Å, b = 84.90Å, c = 133.99Å, α = 90°, β = 90° and γ = 90°). These crystals diffracted to 2.0Å resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.
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Miguez, Jéssica S. G., Vanessa Dela Justina, Alecsander F. M. Bressan, Patrícia G. F. Marchi, Adenilda C. Honorio-França, Fernando S. Carneiro, R. Clinton Webb, Rita C. Tostes, Fernanda R. Giachini, and Victor V. Lima. "O-Glycosylation with O-linked β-N-acetylglucosamine increases vascular contraction: Possible modulatory role on Interleukin-10 signaling pathway." Life Sciences 209 (September 2018): 78–84. http://dx.doi.org/10.1016/j.lfs.2018.07.058.
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Shi, Wei-Wei, Yong-Liang Jiang, Fan Zhu, Hui Wu, Cong-Zhao Zhou, and Yuxing Chen. "Structure of pneumococcal GtfA reveals a novel prokaryotic O-GlcNAc transferase." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C303. http://dx.doi.org/10.1107/s205327331409696x.
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Protein glycosylation is increasingly recognized as an important process for bacterial physiology and pathophysiology. Glycosylation of serine-rich repeat (SRR) glycoprotein PsrP is essential for the pathogenesis of Streptococcus pneumoniae, one of the most common human pathogens. This glycosylation process is initiated by a glycosyltransferase complex comprising two components, a core enzyme GtfA and a co-activator GtfB. Here we report the 2.0-Å crystal structure of GtfA in complex with GlcNAc and UDP. The structure possesses a core domain of GT-B fold and an unprecedented "add-on" domain of DUF1975, which adopts a β-meander structure. This novel DUF1975 domain is critical for the intact glycosyltransferase activity of GtfA–GtfB complex via mediating their self-recognition and the binding to the acceptor protein PsrP. The glycoproteomic analysis revealed a novel pattern of protein O-linked glycosylation at the serine residue cluster. The findings suggest that GtfA is a new glycosyltransferase and provide a structural basis for the future design of inhibitors against the biogenesis of bacterial SRRPs.
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LEUENBERGER, Boris, Dagmar HAHN, Anastassios PISCHITZIS, Marianne K. HANSEN, and Erwin E. STERCHI. "Human meprinbeta: O-linked glycans in the intervening region of the type I membrane protein protect the C-terminal region from proteolytic cleavage and diminish its secretion." Biochemical Journal 369, no. 3 (February 1, 2003): 659–65. http://dx.doi.org/10.1042/bj20021398.
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Human meprin (hmeprin; N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase; EC 3.4.24.18) is a member of the astacin family of zinc metalloendopeptidases. The major site of expression is the brush border membrane of small intestinal and kidney epithelial cells. The enzyme is a type I integral membrane protein composed of two distinct subunits, α and β, which are linked by disulphide bridges. The enzyme complex is attached to the plasma membrane only via the β-subunit. The α-subunit is cleaved in the endoplasmic reticulum in a constitutive manner to remove the C-terminal membrane anchor which leads to secretion of the protein. While the β-subunit of hmeprin remains largely attached to the brush-border membrane some proteolytic processing occurs intracellularly as well as at the cell surface and results in the release of this subunit from the cell. In the present paper, we report that the β-subunit bears multiple O-linked sugar residues in the intervening domain. In contrast, the α-subunit does not contain O-linked oligosaccharides. Our results show that the O-linked carbohydrate side chains in hmeprinβ are clustered around a 13 amino acid sequence that contains the main cleavage site for proteolytic processing of the subunit. Prevention of O-glycosylation by specific inhibitors leads to enhanced proteolytic processing and the consequence is an increased release of hmeprinβ into the culture medium.
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Lenting, Peter J., Carina J. M. van Schooten, Ton Lisman, Marijke van den Berg, Jeroen C. J. Eikenboom, Frank W. G. Leebeek, Jenny Goudemand, Edith Fressinaud, Philip G. de Groot, and Cécile V. Denis. "O-Linked Glycosylation with Sialylated T-Antigen: A Novel Carbohydrate Determinant of von Willebrand Factor Antigen Levels." Blood 108, no. 11 (November 16, 2006): 178. http://dx.doi.org/10.1182/blood.v108.11.178.178.
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Abstract The glycosylation-profile of von Willebrand factor (VWF) is known to strongly influence its plasma levels. VWF contains several carbohydrate structures, including O-linked glycans that primarily consist of the sialylated T-antigen (NeuAc( α 2–3)Gal( β 1–3)[NeuAc( α 2–6)]GalNAc). It is unknown yet if O-linked carbohydrates affect VWF levels. We therefore developed an immunosorbent-based assay using the lectin Peanut Agglutinin (PNA) to determine O-linked T-antigen on VWF. Control experiments showed that PNA-binding was: (i) specific to T-antigen that was de-sialylated via neuramidase-treatment; (ii) proportional to the number of O-linked glycosylation sites and (iii) independent of the extent of multimerisation. Using this assay, we found a strong correlation between PNA binding and VWF-antigen levels in a series of randomly selected plasma samples (252±125% for VWF<0.5 U/ml (n=15); 131±36% for VWF between 0.5 and 1.5 U/ml (n=32); 92±40% for VWF>1.5 U/ml (n=19); p<0.003). Reduced or increased PNA binding was also observed for patients characterized by increased (liver cirrhosis; n=58) or reduced (von Willebrand disease (VWD)-type 1; n=32) VWF-antigen levels, respectively. VWD-type 1 patients further displayed increased ratios of propeptide over mature VWF-antigen levels (0.38±0.18 versus 0.17±0.03 for patients (n=32) and controls (n=20), respectively; p<0.0001), which is indicative for reduced VWF survival in these patients. Interestingly, a linear relation between PNA binding and propeptide/VWF ratio was observed (Spearman rank= 0.50; p=0.0039), suggesting a potential association between T-antigen glycosylation and VWF survival. Finally, we observed a marked decrease in PNA binding in post-DDAVP samples from various patients (131±45% versus 85±33% in pre- and post DDAVP, respectively; n=18; p=0.0013). Since these patients had distinct underlying pathologies (primary platelet function disorder, hemophilia A or VWD type 1, 2A or 2N), this seems to represent a general phenomenon that is not restricted to VWD. Moreover, it indicates that the O-linked glycosylation profile of VWF stored in endothelial storage-organelles may differ from that of constitutively secreted VWF.
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Jiménez-Martínez, María C., Ricardo Lascurain, Aniela Méndez-Reguera, Sergio Estrada-Parra, Iris Estrada-García, Patricia Gorocica, Salvador Martínez-Cairo, Edgar Zenteno, and Raúl Chávez. "O-Glycosylation of NnTreg Lymphocytes Recognized by theAmaranthus leucocarpusLectin." Clinical and Developmental Immunology 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/506807.
Full textAbstract:
O-glycosidically-linked glycans have been involved in development, maturation, homing, and immune regulation in T cells. Previous reports indicate thatAmaranthus leucocarpuslectin(ALL), specific for glycans containing galactose-N-acetylgalactosamine and N-acetylgalactosamine, recognizes human naïve CD27+CD25+CD4+T cells. Our aim was to evaluate the phenotype of CD4+T cells recognized byALLin peripheral blood mononuclear cells obtained from healthy volunteers. CD4+T cells were isolated by negative selection using magnetic beads-labeled monoclonal antibodies; the expression of T regulatory cell phenotypic markers was assessed onALL-recognized cells. In addition, IL-4, IL-10, IFN-γ, and TGF-βintracellular production inALL+cells was also evaluated. The analyses of phenotypic markers and intracellular cytokines were performed through flow cytometry.ALL-recognized CD4+T cells were mainly CD45RA+, CCR7+cells. Although52±10% CD25+Foxp3+cells were positive toALL, only34±4% ofALL+cells corresponded to CD25+Foxp3−cells. Intracellular cytokines in freshly obtainedALL+CD4+T cells exhibited 8% of IL-4, 15% of IL-10, 2% of IFN-γ, and 15% of TGF-β, whereasALL−CD4+T cells depicted 1% of IL-4, 2% of IL-10,<1% of IFN-γ, and 6% of TGF-β. Our results show that galactose-N-acetylgalactosamine and N-galactosamine-bearing CD4+T cells expressed phenotypic markers of NnTreg cells.
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Zhu, Qiang, Xuejun Cheng, Yaxian Cheng, Junchen Chen, Huan Xu, Yuntao Gao, Xiaotao Duan, Junfeng Ji, Xuekun Li, and Wen Yi. "O-GlcNAcylation regulates the methionine cycle to promote pluripotency of stem cells." Proceedings of the National Academy of Sciences 117, no. 14 (March 19, 2020): 7755–63. http://dx.doi.org/10.1073/pnas.1915582117.
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Methionine metabolism is critical for the maintenance of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) pluripotency. However, little is known about the regulation of the methionine cycle to sustain ESC pluripotency. Here, we show that adenosylhomocysteinase (AHCY), an important enzyme in the methionine cycle, is critical for the maintenance and differentiation of mouse embryonic stem cells (mESCs). We show that mESCs exhibit high levels of methionine metabolism, whereas decreasing methionine metabolism via depletion of AHCY promotes mESCs to differentiate into the three germ layers. AHCY is posttranslationally modified with an O-linked β-N-acetylglucosamine sugar (O-GlcNAcylation), which is rapidly removed upon differentiation. O-GlcNAcylation of threonine 136 on AHCY increases its activity and is important for the maintenance of trimethylation of histone H3 lysine 4 (H3K4me3) to sustain mESC pluripotency. Blocking glycosylation of AHCY decreases the ratio of S-adenosylmethionine versus S-adenosylhomocysteine (SAM/SAH), reduces the level of H3K4me3, and poises mESC for differentiation. In addition, blocking glycosylation of AHCY reduces somatic cell reprogramming. Thus, our findings reveal a critical role of AHCY and a mechanistic understanding of O-glycosylation in regulating ESC pluripotency and differentiation.
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Agirre, Jon, Antonio Ariza, Wendy A. Offen, Johan P. Turkenburg, Shirley M. Roberts, Stuart McNicholas, Paul V. Harris, et al. "Three-dimensional structures of two heavily N-glycosylatedAspergillussp. family GH3 β-D-glucosidases." Acta Crystallographica Section D Structural Biology 72, no. 2 (January 28, 2016): 254–65. http://dx.doi.org/10.1107/s2059798315024237.
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The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant β-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. β-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 β-D-glucosidases fromAspergillus fumigatusandA. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley β-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy4C1chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. TheAspergillusGH3 structures, in light of other recent three-dimensional structures, provide insight into fungal β-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application.
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Srivastava, Om P., and Ole Hindsgaul. "Synthesis of the subterminally 6-O-phosphorylated trimannosides found on carbohydrate chains of lysosomal enzymes." Canadian Journal of Chemistry 64, no. 12 (December 1, 1986): 2324–30. http://dx.doi.org/10.1139/v86-382.
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The three possible subterminally 6-O-phosphorylated trimannosides (2–4) found on the asparagine-linked carbohydrate chains of lysosomal enzymes have been chemically synthesized as their 8-methoxycarbonyloctyl glycosides, R = (CH2)8COOCH3. The key step in the syntheses involves glycosylation of suitably protected α-D-mannopyranosides 5–7 with the phosphorylated mannobiosyl donor 2-O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-3,4-di-O-benzyl-6-O-di-phenylphosphoryl-α-D-mannopyranosyl chloride (14), which produced mixtures of α- and β-linked trisaccharidic products. Compounds 2 (αDMan(1 → 2) αDMan 6-phosphate(1 → 2)αDManOR), 3 (αDMan(1 → 2)αDMan 6-phosphate(1 → 3)αDManOR), and 4 (αDMan(1 → 2)αDMan 6-phosphate(1 → 6)αDManOR) are being used in a study of the targeting of enzymes to the lysosomes by phosphomannosyl receptors.
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Souza-Silva, Leonardo, Rheure Alves-Lopes, Jéssica Silva Miguez, Vanessa Dela Justina, Karla Bianca Neves, Fabíola Leslie Mestriner, Rita de Cassia Tostes, Fernanda Regina Giachini, and Victor Vitorino Lima. "Glycosylation with O-linked β-N-acetylglucosamine induces vascular dysfunction via production of superoxide anion/reactive oxygen species." Canadian Journal of Physiology and Pharmacology 96, no. 3 (March 2018): 232–40. http://dx.doi.org/10.1139/cjpp-2017-0225.
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Overproduction of superoxide anion (•O2−) and O-linked β-N-acetylglucosamine (O-GlcNAc) modification in the vascular system are contributors to endothelial dysfunction. This study tested the hypothesis that increased levels of O-GlcNAc-modified proteins contribute to •O2− production via activation of NADPH oxidase, resulting in impaired vasodilation. Rat aortic segments and vascular smooth muscle cells (VSMCs) were incubated with vehicle (methanol) or O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc) (100 μM). PUGNAc produced a time-dependent increase in O-GlcNAc levels in VSMC and decreased endothelium-dependent relaxation, which was prevented by apocynin and tiron, suggesting that •O2− contributes to endothelial dysfunction under augmented O-GlcNAc levels. Aortic segments incubated with PUGNAc also exhibited increased levels of reactive oxygen species, assessed by dihydroethidium fluorescence, and augmented •O2− production, determined by lucigenin-enhanced chemiluminescence. Additionally, PUGNAc treatment increased Nox-1 and Nox-4 protein expression in aortas and VSMCs. Translocation of the p47phox subunit from the cytosol to the membrane was greater in aortas incubated with PUGNAc. VSMCs displayed increased p22phox protein expression after PUGNAc incubation, suggesting that NADPH oxidase is activated in conditions where O-GlcNAc protein levels are increased. In conclusion, O-GlcNAc levels reduce endothelium-dependent relaxation by overproduction of •O2− via activation of NADPH oxidase. This may represent an additional mechanism by which augmented O-GlcNAc levels impair vascular function.
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Zarschler, Kristof, Bettina Janesch, Sonja Zayni, Christina Sch�ffer, and Paul Messner. "Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T, Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP." Applied and Environmental Microbiology 75, no. 10 (March 20, 2009): 3077–85. http://dx.doi.org/10.1128/aem.00087-09.
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ABSTRACT The gram-positive bacterium Paenibacillus alvei CCM 2051T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO2→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051T.
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Goldberg, Howard J., Catharine I. Whiteside, Gerald W. Hart, and I. George Fantus. "Posttranslational, Reversible O-Glycosylation Is Stimulated by High Glucose and Mediates Plasminogen Activator Inhibitor-1 Gene Expression and Sp1 Transcriptional Activity in Glomerular Mesangial Cells." Endocrinology 147, no. 1 (January 1, 2006): 222–31. http://dx.doi.org/10.1210/en.2005-0523.
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Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG-stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG-induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral β-N-acetylglucosaminidase, and by knockdown of O-linked β-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG-induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells.
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Haurum, John S., Ingelise Bjerring Høier, Gemma Arsequell, Anne Neisig, Gregorio Valencia, Jesper Zeuthen, Jacques Neefjes, and Tim Elliott. "Presentation of Cytosolic Glycosylated Peptides by Human Class I Major Histocompatibility Complex Molecules in Vivo." Journal of Experimental Medicine 190, no. 1 (July 1, 1999): 145–50. http://dx.doi.org/10.1084/jem.190.1.145.
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Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8–10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-β-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-β-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-β-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-β-linked GlcNAc can be presented by class I MHC molecules to the immune system.
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López-Ramírez, Luz A., Iván Martínez-Duncker, Anayeli Márquez-Márquez, Ana P. Vargas-Macías, and Héctor M. Mora-Montes. "Silencing of ROT2, the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii–Host Interaction." Journal of Fungi 8, no. 11 (November 18, 2022): 1220. http://dx.doi.org/10.3390/jof8111220.
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Sporothrix schenckii is a member of the Sporothrix pathogenic clade and one of the most common etiological agents of sporotrichosis, a subcutaneous fungal infection that affects both animal and human beings. Like other fungal pathogens, the Sporothrix cell wall is composed of structural polysaccharides and glycoproteins that are covalently modified with both N-linked and O-linked glycans. Thus far, little is known about the N-linked glycosylation pathway in this organism or its contribution to cell wall composition and interaction with the host. Here, we silenced ROT2, which encodes the catalytic subunit of the endoplasmic reticulum α-glucosidase II, a processing enzyme key for the N-linked glycan core processing. Silencing of ROT2 led to the accumulation of the Glc2Man9GlcNAC2 glycan core at the cell wall and a reduction in the total content of N-linked glycans found in the wall. However, the highly silenced mutants showed a compensatory mechanism with increased content of cell wall O-linked glycans. The phenotype of mutants with intermediate levels of ROT2 silencing was more informative, as they showed changes in the cell wall composition and exposure of β-1.3-glucans and chitin at the cell surface. Furthermore, the ability to stimulate cytokine production by human mononuclear cells was affected, along with the phagocytosis by human monocyte-derived macrophages, in a mannose receptor-, complement receptor 3-, and TLR4-dependent stimulation. In an insect model of experimental sporotrichosis, these mutant cells showed virulence attenuation. In conclusion, S. schenckii ROT2 is required for proper N-linked glycosylation, cell wall organization and composition, and interaction with the host.
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Kim, Eun. "Chemical Reporters and Their Bioorthogonal Reactions for Labeling Protein O-GlcNAcylation." Molecules 23, no. 10 (September 20, 2018): 2411. http://dx.doi.org/10.3390/molecules23102411.
Full textAbstract:
Protein O-GlcNAcylation is a non-canonical glycosylation of nuclear, mitochondrial, and cytoplasmic proteins with the attachment of a single O-linked β-N-acetyl-glucosamine (O-GlcNAc) moiety. Advances in labeling and identifying O-GlcNAcylated proteins have helped improve the understanding of O-GlcNAcylation at levels that range from basic molecular biology to cell signaling and gene regulation to physiology and disease. This review describes these advances in chemistry involving chemical reporters and their bioorthogonal reactions utilized for detection and construction of O-GlcNAc proteomes in a molecular mechanistic view. This detailed view will help better understand the principles of the chemistries utilized for biology discovery and promote continued efforts in developing new molecular tools and new strategies to further explore protein O-GlcNAcylation.
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Hjalmarsson, Anna, Eric Carlemalm, and Einar Everitt. "Infectious Pancreatic Necrosis Virus: Identification of a VP3-Containing Ribonucleoprotein Core Structure and Evidence for O-Linked Glycosylation of the Capsid Protein VP2." Journal of Virology 73, no. 4 (April 1, 1999): 3484–90. http://dx.doi.org/10.1128/jvi.73.4.3484-3490.1999.
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ABSTRACT Virions of infectious pancreatic necrosis virus (IPNV) were completely disintegrated upon dialysis against salt-free buffers. Direct visualization of such preparations by electron microscopy revealed 5.0- to 6.5-nm-thick entangled filaments. By using a specific colloidal gold immunolabeling technique, these structures were shown to contain the viral protein VP3. Isolation by sucrose gradient centrifugation of the filaments, followed by serological analysis, demonstrated that the entire VP3 content of the virion was recovered together with the radiolabeled genomic material forming the unique threadlike ribonucleoprotein complexes. In a sensitive blotting assay, the outer capsid component of IPNV, i.e., the major structural protein VP2, was shown to specifically bind lectins recognizing sugar moieties of N-acetylgalactosamine, mannose, and fucose. Three established metabolic inhibitors of N-linked glycosylation did not prevent addition of sugar residues to virions, and enzymatic deglycosylation of isolated virions using N-glycosidase failed to remove sugar residues of VP2 recognized by lectins. However, gentle alkaline β elimination clearly reduced the ability of lectins to recognize VP2. These results suggest that the glycosylation of VP2 is of the O-linked type when IPNV is propagated in RTG-2 cells.
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Qutyan, Mohammed, Matthew Henkel, Joseph Horzempa, Michael Quinn, and Peter Castric. "Glycosylation of Pilin and Nonpilin Protein Constructs by Pseudomonas aeruginosa 1244." Journal of Bacteriology 192, no. 22 (September 10, 2010): 5972–81. http://dx.doi.org/10.1128/jb.00007-10.
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ABSTRACT PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the β carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.
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Gómez-Gaviria, Manuela, Nancy E. Lozoya-Pérez, Monika Staniszewska, Bernardo Franco, Gustavo A. Niño-Vega, and Hector M. Mora-Montes. "Loss of Kex2 Affects the Candida albicans Cell Wall and Interaction with Innate Immune Cells." Journal of Fungi 6, no. 2 (April 29, 2020): 57. http://dx.doi.org/10.3390/jof6020057.
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The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host–fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and β-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.
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Zeituni, Amir E., William McCaig, Elizabeth Scisci, David G. Thanassi, and Christopher W. Cutler. "The Native 67-Kilodalton Minor Fimbria of Porphyromonas gingivalis Is a Novel Glycoprotein with DC-SIGN-Targeting Motifs." Journal of Bacteriology 192, no. 16 (June 18, 2010): 4103–10. http://dx.doi.org/10.1128/jb.00275-10.
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ABSTRACT We recently reported that the oral mucosal pathogen Porphyromonas gingivalis, through its 67-kDa Mfa1 (minor) fimbria, targets the C-type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased CD4+ T-cell response. We have now purified the native minor fimbria by ion-exchange chromatography and sequenced the fimbria by tandem mass spectrometry (MS/MS), confirming its identity and revealing two putative N-glycosylation motifs as well as numerous putative O-glycosylation sites. We further show that the minor fimbria is glycosylated by ProQ staining and that glycosylation is partially removed by treatment with β(1-4)-galactosidase, but not by classic N- and O-linked deglycosidases. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that the minor fimbria contains the DC-SIGN-targeting carbohydrates fucose (1.35 nmol/mg), mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg), and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy revealed that the minor fimbria forms fibers approximately 200 nm in length that could be involved in targeting or cross-linking DC-SIGN. These findings shed further light on molecular mechanisms of invasion and immunosuppression by this unique mucosal pathogen.
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Johnsen, Virginia L., Darrell D. Belke, Curtis C. Hughey, Dustin S. Hittel, Russell T. Hepple, Lauren G. Koch, Steven L. Britton, and Jane Shearer. "Enhanced cardiac protein glycosylation (O-GlcNAc) of selected mitochondrial proteins in rats artificially selected for low running capacity." Physiological Genomics 45, no. 1 (January 2013): 17–25. http://dx.doi.org/10.1152/physiolgenomics.00111.2012.
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O-linked β-N-acetyl glucosamine (O-GlcNAc) is a posttranslational modification consisting of a single N-acetylglucosamine moiety attached by an O-β-glycosidic linkage to serine and threonine residues of both nuclear and cytosolic proteins. Analogous to phosphorylation, the modification is reversible and dynamic, changing in response to stress, nutrients, hormones, and exercise. Aims of this study were to examine differences in O-GlcNAc protein modification in the cardiac tissue of rats artificially selected for low (LCR) or high (HCR) running capacity. Hyperinsulinemic-euglycemic clamps in conscious animals assessed insulin sensitivity while 2-[14C] deoxyglucose tracked both whole body and tissue-specific glucose disposal. Immunoblots of cardiac muscle examined global O-GlcNAc modification, enzymes that control its regulation (OGT, OGA), and specific proteins involved in mitochondrial oxidative phosphorylation. LCR rats were insulin resistant disposing of 65% less glucose than HCR. Global tissue O-GlcNAc, OGT, OGA, and citrate synthase were similar between groups. Analysis of cardiac proteins revealed enhanced O-GlcNAcylation of mitochondrial Complex I, Complex IV, VDAC, and SERCA in LCR compared with HCR. These results are the first to establish an increase in specific protein O-GlcNAcylation in LCR animals that may contribute to progressive mitochondrial dysfunction and the pathogenesis of insulin resistance observed in the LCR phenotype.
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Davies, Gideon J., and Carlos Martinez-Fleites. "The O-GlcNAc modification: three-dimensional structure, enzymology and the development of selective inhibitors to probe disease." Biochemical Society Transactions 38, no. 5 (September 24, 2010): 1179–88. http://dx.doi.org/10.1042/bst0381179.
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Carbohydrates, their structures and the enzymes responsible for their synthesis and degradation, offer numerous possibilities for the design and application of probes with which to study and treat disease. The intracellular dynamic O-GlcNAc (O-linked β-N-acetylglucosamine) modification is one such glycosylation with considerable medical interest, reflecting its implication in diseases such as Type 2 diabetes and neurodegeneration. In the present paper, we review recent structural and mechanistic studies into the enzymes responsible for this modification, highlighting how mechanism-inspired small-molecule probes may be applied to study potential disease processes. Such studies have questioned a causal link between O-GlcNAc and Type 2 diabetes, but do offer potential for the study, and perhaps the treatment, of tauopathies.
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Boulard, Mathieu, Sofia Rucli, John R. Edwards, and Timothy H. Bestor. "Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters." Proceedings of the National Academy of Sciences 117, no. 25 (June 10, 2020): 14292–98. http://dx.doi.org/10.1073/pnas.1912074117.
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The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1β), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed withO-linked β-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer ofN-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed thatO-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss ofO-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal ofO-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed thatO-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.
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Hu, Jiachang, Yimei Wang, Shuan Zhao, Jing Chen, Shi Jin, Ping Jia, and Xiaoqiang Ding. "Remote Ischemic Preconditioning Ameliorates Acute Kidney Injury due to Contrast Exposure in Rats through Augmented O-GlcNAcylation." Oxidative Medicine and Cellular Longevity 2018 (August 13, 2018): 1–15. http://dx.doi.org/10.1155/2018/4895913.
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Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues or organs. O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of intracellular proteins denotes a type of posttranslational modification that influences multiple cytoplasmic and nuclear protein functions. Growing evidence indicates that stress can induce an acute increase in O-GlcNAc levels, which can be cytoprotective. The current study aimed to determine whether RIPC can provide renoprotection against contrast-induced acute kidney injury (CI-AKI) by augmenting O-GlcNAc signaling. We established a stable model of CI-AKI using 5/6 nephrectomized rats exposed to dehydration followed by iohexol injection via the tail vein. We found that RIPC increased UDP-GlcNAc levels through the hexosamine biosynthetic pathway as well as global renal O-GlcNAcylation. RIPC-induced elevation of O-GlcNAc signaling ameliorated CI-AKI based on the presence of less tubular damage and apoptosis and the amount of reactive oxygen species. In addition, the use of alloxan, an O-GlcNAc transferase inhibitor, and azaserine, a glutamine fructose-6-phosphate amidotransferase inhibitor, neutralized the protective effect of RIPC against oxidative stress and tubular apoptosis. In conclusion, RIPC attenuates local oxidative stress and tubular apoptosis induced by contrast exposure by enhancing O-GlcNAc glycosylation levels; this can be a potentially useful approach for lowering the risk of CI-AKI.
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Kindahl, Lill, Corine Sandström, A. Grey Craig, Thomas Norberg, and Lennart Kenne. "1H NMR studies on the solution conformation of contulakin-G and analogues." Canadian Journal of Chemistry 80, no. 8 (August 1, 2002): 1022–31. http://dx.doi.org/10.1139/v02-115.
Full textAbstract:
The conformation of contulakin-G, a bioactive 16 amino acid O-linked glycopeptide (ZSEEGGSNAT*KKPYIL) with the disaccharide β-D-Gal(1[Formula: see text]3)α-D-GalNAc attached to the threonine residue in position 10, has been investigated by 1H NMR spectroscopy. The 1H NMR data for the non-glycosylated peptide and for two glycopeptide analogues, one with the monosaccharide α-D-GalNAc at Thr10 and one with the disaccharide β-D-Gal(1–>3)α-D-GalNAc at Ser7, all of lower bioactivity than contulakin-G, have also been collected. The chemical shifts, NOEs, temperature coefficients of amide protons, and 3JNH,αH-values suggest that all four compounds exist mainly in random coil conformations. Some transient populations of folded conformations are also present in the glycopeptides and turns, probably induced by the sugars, are present in the peptide chain around the site of glycosylation. In the two peptides O-glycosylated at Thr10, the rotation of α-D-GalNAc around the linkage between the sugar and the peptide is restricted. There is evidence for a hydrogen bond between the amide proton of α-D-GalNAc and the peptide chain that could contribute to this torsional rigidity. An intramolecular hydrogen bond between the carbohydrate and the peptide chain does not exist in the peptide O-glycosylated at the Ser7 residue. Key words: conformation, contulakin-G, NMR, O-linked glycopeptide.
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Chen, Yabing, Xinyang Zhao, and Hui Wu. "Metabolic Stress and Cardiovascular Disease in Diabetes Mellitus." Arteriosclerosis, Thrombosis, and Vascular Biology 39, no. 10 (October 2019): 1911–24. http://dx.doi.org/10.1161/atvbaha.119.312192.
Full textAbstract:
Mammalian cells metabolize glucose primarily for energy production, biomass synthesis, and posttranslational glycosylation; and maintaining glucose metabolic homeostasis is essential for normal physiology of cells. Impaired glucose homeostasis leads to hyperglycemia, a hallmark of diabetes mellitus. Chronically increased glucose in diabetes mellitus promotes pathological changes accompanied by impaired cellular function and tissue damage, which facilitates the development of cardiovascular complications, the major cause of morbidity and mortality of patients with diabetes mellitus. Emerging roles of glucose metabolism via the hexosamine biosynthesis pathway (HBP) and increased protein modification via O -linked β- N -acetylglucosamine ( O -GlcNAcylation) have been demonstrated in diabetes mellitus and implicated in the development of diabetic cardiovascular complications. This review will discuss the biological outcomes of the glucose metabolism via the hexosamine biogenesis pathway and protein O -GlcNAcylation in regulating cellular homeostasis, and highlight the regulations and contributions of elevated O -GlcNAcylation to the pathogenesis of diabetic cardiovascular disease.
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Nagelberg, S. B., L. A. Cole, and S. W. Rosen. "A novel form of ectopic human chorionic gonadotrophin β-subunit in the serum of a woman with epidermoid cancer." Journal of Endocrinology 107, no. 3 (December 1985): 403–8. http://dx.doi.org/10.1677/joe.0.1070403.
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ABSTRACT A novel form of free human chorionic gonadotrophin β-subunit (hCGβ) was found in serum from ElBre, a woman with epidermoid carcinoma of unknown origin. ElBre hCGβ was larger than standard (pregnancy urine) hCGβ when analysed by gel chromatography (apparent molecular weight 54 000 vs 44000). This size difference appeared to be due to a larger carboxyterminal extension (CTE) of ElBre hCGβ since thermolysin cleavage of the CTE from standard hCGβ and Elbre hCGβ yielded core products of the same size. Oligosaccharides, O-linked to serine or threonine, were present in ElBre hCGβ, presumably on its CTE as judged by the complete binding of desialylated ElBre hCGβ to immobilized peanut agglutinin (this lectin is specific for terminal galactose linked β1 → 3 to N-acetylgalactosamine, a disaccharide exposed after desialylation of the O-linked oligosaccharides of standard hCGβ). ElBre hCGβ, however, was incompletely recognized by antisera specific for the CTE of standard hCGβ, especially the carbohydrate-sensitive antiserum R141. The O-linked oligosaccharides of standard hCGβ are heterogeneous in size; 13% are of the largest (hexasaccharide) form. In contrast, over 50% of the O-linked oligosaccharides in hCGβ from the JAr choriocarcinoma cell line are hexasaccharides. Like desialylated ElBre hCGβ, desialylated JAr hCGβ bound completely to peanut agglutinin, but was incompletely recognized by antisera to the hCGβ-CTE. Furthermore, JAr hCGβ was intermediate in size between standard hCGβ and ElBre hCGβ when analysed by gel chromatography (apparent molecular weight 49 000). Thus, we propose that ElBre hCGβ had an even higher proportion of large O-linked oligosaccharides than had JAr hCGβ. Moreover, the N-linked oligosaccharides of ElBre hCGβ differed from standard hCGβ; only 55% of ElBre hCGβ bound to Concanavalin A versus 89% of standard hCGβ. These data further support the concepts of aberrant glycosylation by neoplastic tissues and carbohydrate heterogeneity of hCGβ produced by various tissues. J. Endocr. (1985) 107, 403–408
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Lima, Victor V., Kathryn Spitler, Hyehun Choi, R. Clinton Webb, and Rita C. Tostes. "O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?" Clinical Science 123, no. 8 (June 27, 2012): 473–86. http://dx.doi.org/10.1042/cs20110638.
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O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury.
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Moll, Tobias, Pamela J. Shaw, and Johnathan Cooper-Knock. "Disrupted glycosylation of lipids and proteins is a cause of neurodegeneration." Brain 143, no. 5 (November 14, 2019): 1332–40. http://dx.doi.org/10.1093/brain/awz358.
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Abstract Glycosyltransferases represent a large family of enzymes that catalyse the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates. A number of studies have implicated glycosyltransferases in the pathogenesis of neurodegenerative diseases but differentiating cause from effect has been difficult. We have recently discovered that mutations proximal to the substrate binding site of glycosyltransferase 8 domain containing 1 (GLT8D1) are associated with familial amyotrophic lateral sclerosis (ALS). We demonstrated that ALS-associated mutations reduce activity of the enzyme suggesting a loss-of-function mechanism that is an attractive therapeutic target. Our work is the first evidence that isolated dysfunction of a glycosyltransferase is sufficient to cause a neurodegenerative disease, but connection between neurodegeneration and genetic variation within glycosyltransferases is not new. Previous studies have identified associations between mutations in UGT8 and sporadic ALS, and between ST6GAL1 mutations and conversion of mild cognitive impairment into clinical Alzheimer’s disease. In this review we consider potential mechanisms connecting glycosyltransferase dysfunction to neurodegeneration. The most prominent candidates are ganglioside synthesis and impaired addition of O-linked β-N-acetylglucosamine (O-GlcNAc) groups to proteins important for axonal and synaptic function. Special consideration is given to examples where genetic mutations within glycosyltransferases are associated with neurodegeneration in recognition of the fact that these changes are likely to be upstream causes present from birth.
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McColgan, Nicole M., Marissa N. Feeley, Ashley M. Woodward, Damien Guindolet, and Pablo Argüeso. "The O-GlcNAc modification promotes terminal differentiation of human corneal epithelial cells." Glycobiology 30, no. 11 (April 13, 2020): 872–80. http://dx.doi.org/10.1093/glycob/cwaa033.
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Abstract Dynamic modification of nuclear and cytoplasmic proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) plays an important role in orchestrating the transcriptional activity of eukaryotic cells. Here, we report that the O-GlcNAc modification contributes to maintaining ocular surface epithelial homeostasis by promoting mucin biosynthesis and barrier function. We found that induction of human corneal epithelial cell differentiation stimulated the global transfer of O-GlcNAc to both nuclear and cytosolic proteins. Inflammatory conditions, on the other hand, were associated with a reduction in the expression of O-GlcNAc transferase at the ocular surface epithelia. Loss- and gain-of-function studies using small interfering RNA targeting O-GlcNAc transferase, or Thiamet G, a selective inhibitor of O-GlcNAc hydrolase, respectively, revealed that the presence of O-GlcNAc was necessary to promote glycocalyx barrier function. Moreover, we found that Thiamet G triggered a correlative increase in both surface expression of MUC16 and apical epithelial cell area while reducing paracellular permeability. Collectively, these results identify intracellular protein O-glycosylation as a novel pathway responsible for promoting the terminal differentiation of human corneal epithelial cells.
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Halfinger, Bernhard, Angelika Hammerer-Lercher, Benno Amplatz, Bettina Sarg, Leopold Kremser, and Herbert H. Lindner. "Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro–B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure." Clinical Chemistry 63, no. 1 (January 1, 2017): 359–68. http://dx.doi.org/10.1373/clinchem.2016.265397.
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Abstract BACKGROUND Currently, N-terminal pro–B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. METHODS The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. RESULTS We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. CONCLUSIONS Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms.
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Gellai, Renata, Judit Hodrea, Lilla Lenart, Adam Hosszu, Sandor Koszegi, Dora Balogh, Agota Ver, et al. "Role of O-linked N-acetylglucosamine modification in diabetic nephropathy." American Journal of Physiology-Renal Physiology 311, no. 6 (December 1, 2016): F1172—F1181. http://dx.doi.org/10.1152/ajprenal.00545.2015.
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Increased O-linked β- N-acetylglucosamine glycosylation ( O-GlcNAcylation) is a known contributor to diabetes; however, its relevance in diabetic nephropathy (DN) is poorly elucidated. Here, we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein (HSP)72 signaling. Since tubular injury is the prominent site of DN, the effect of hyperglycemia was first measured in proximal tubular (HK2) cells cultured in high glucose. In vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), phosphorylated (p)Akt/Akt, peNOS/eNOS, and HSP72 were assessed in the kidney cortex of streptozotocin-induced diabetic rats. The effects of various renin-angiotensin-aldosterone system (RAAS) inhibitors were also evaluated. In proximal tubular cells, hyperglycemia-induced OGT expression led to increased O-GlcNAcylation, which was followed by a compensatory increase of OGA. In parallel, peNOS and pAkt levels decreased, whereas HSP72 increased. In diabetic rats, elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS inhibitors ameliorated diabetes-induced kidney damage and prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS, and HSP72. In conclusion, hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS blockers successfully inhibit this process, suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.
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Wong, Siew L., Linda L. Wu, Rebecca L. Robker, Jeremy G. Thompson, and Melanie L. Sutton McDowall. "Hyperglycaemia and lipid differentially impair mouse oocyte developmental competence." Reproduction, Fertility and Development 27, no. 4 (2015): 583. http://dx.doi.org/10.1071/rd14328.
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Maternal diabetes and obesity are characterised by elevated blood glucose, insulin and lipids, resulting in upregulation of specific fuel-sensing and stress signalling pathways. Previously, we demonstrated that, separately, upregulation of the hexosamine biosynthetic pathway (HBP; under hyperglycaemic conditions) and endoplasmic reticulum (ER) stress (due to hyperlipidaemia) pathways reduce blastocyst development and alter oocyte metabolism. In order to begin to understand how both glucose and lipid metabolic disruptions influence oocyte developmental competence, in the present study we exposed mouse cumulus–oocyte complexes to hyperglycaemia (30 mM) and/or lipid (40 μM) and examined the effects on embryo development. The presence of glucosamine (GlcN; a hyperglycaemic mimetic) or increased lipid during in vitro maturation severely perturbed blastocyst development (P < 0.05). Hyperglycaemia, GlcN and hyperglycaemia + lipid treatments significantly increased HBP activity, increasing total O-linked glycosylation (O-GlcNAcylation) of proteins (P < 0.0001). All treatments also induced ER stress pathways, indicated by the expression of specific ER stress genes. The expression of genes encoding the HBP enzymes glutamine:fructose-6-phosphate amidotransferase 2 (Gfpt2) and O-linked β-N-acetylglucosaminyltransferase (Ogt) was repressed following lipid treatment (P < 0.001). These findings partially implicate the mechanism of O-GlcNAcylation and ER stress as likely contributors to compromised fertility of obese women.
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Pantaleon, M., and P. L. Kaye. "034. Nutrient sensing by the early mouse embryo: hexosamine biosynthesis and glucose signalling during preimplantation development." Reproduction, Fertility and Development 17, no. 9 (2005): 70. http://dx.doi.org/10.1071/srb05abs034.
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Adequate nutrient supply prior to implantation is not only essential to early embryonic growth and development but has also been implicated in metabolic programming events that influence later stage development and the onset of adult disease. The molecular mechanisms involved in early embryonic nutrient sensing and subsequent programming however have not yet been determined. Glucose can act as an essential molecular signal for metabolic differentiation and blastocyst formation.1,2 Our work demonstrates that propagation of this nutrient signal involves glucose metabolism through the hexosamine biosynthetic pathway, whose end-product, uridine 5′-diphospho-N-acetylglucosamine (UDP-Glc-NAc) acts as a donor substrate adding a single O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of nucleocytoplasmic proteins. The number of proteins modified by this O-linked glycosylation is large and includes transcription factors, cytoskeletal components, metabolic enzymes and other cellular signaling components. This tightly regulated and dynamic modification operates in a functionally reciprocal relationship to the more familiar phosphorylation at the same sites hence altering the activity and/or stability of targeted proteins and providing a mechanism for modulating cellular physiology in response to nutrient availability. We show that early embryonic glucose exposure, whilst not essential for energy generation during cleavage development, is nonetheless critical for the maintenance of cellular homeostasis with perturbations in glucose levels during early development leading to decreased levels of cell survival. Furthermore, using antisera specific for O-GlcNAc we have examined levels of O-glycosylated proteins in early mouse embryos in response to the presence or absence of glucose and find dramatically reduced global levels of O-linked glycosylation as well as altered nuclear levels of key transcription factors in embryos deprived of glucose. We believe that this is the first demonstration of a nutrient effect on levels of transcriptional regulators in early development. Elucidation of the mechanisms by which the nutrient environment influences embryonic development is of fundamental importance to our understanding of the origins of adult disease. (1)Pantaleon et al. (2001). Proc 32nd Annual SRB conference, Gold Coast, Qld. Abstract #42.(2)Martin and Leese (1995). Mol. Reprod. Dev. 40, 436–443.
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Lee, Young Ah, Kyeong Ah Kim, and Myeong Heon Shin. "Naegleria fowleri Induces Jurkat T Cell Death via O-deGlcNAcylation." Korean Journal of Parasitology 59, no. 5 (October 22, 2021): 501–5. http://dx.doi.org/10.3347/kjp.2021.59.5.501.
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The pathogenic free-living amoeba Naegleria fowleri causes primary amoebic meningoencephalitis, a fatal infection, by penetrating the nasal mucosa and migrating to the brain via the olfactory nerves. N. fowleri can induce host cell death via lytic necrosis. Similar to phosphorylation, O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is involved in various cell-signaling processes, including apoptosis and proliferation, with O-GlcNAc addition and removal regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), respectively. However, the detailed mechanism of host cell death induced by N. fowleri is unknown. In this study, we investigated whether N. fowleri can induce the modulation of O-GlcNAcylated proteins during cell death in Jurkat T cells. Co-incubation with live N. fowleri trophozoites increased DNA fragmentation. In addition, incubation with N. fowleri induced a dramatic reduction in O-GlcNAcylated protein levels in 30 min. Moreover, pretreatment of Jurkat T cells with the OGA inhibitor PUGNAc prevented N. fowleri–induced O-deGlcNAcylation and DNA fragmentation. These results suggest that O-deGlcNAcylation is an important signaling process that occurs during Jurkat T cell death induced by N. fowleri.
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Kubicek, C. P., T. Panda, G. Schreferl-kunar, F. Gruber, and R. Messner. "O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei." Canadian Journal of Microbiology 33, no. 8 (August 1, 1987): 698–703. http://dx.doi.org/10.1139/m87-122.
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The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.
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Paunola, Eija, Taina Suntio, Eija Jämsä, and Marja Makarow. "Folding of Active β-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum." Molecular Biology of the Cell 9, no. 4 (April 1998): 817–27. http://dx.doi.org/10.1091/mbc.9.4.817.
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Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli β-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. β-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150Δ, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150Δ-β-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar β-lactamase activity andK m values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.
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Rumbold, Karl, Peter Biely, Maria Mastihubová, Marinka Gudelj, Georg Gübitz, Karl-Heinz Robra, and Bernard A. Prior. "Purification and Properties of a Feruloyl Esterase Involved in Lignocellulose Degradation by Aureobasidium pullulans." Applied and Environmental Microbiology 69, no. 9 (September 2003): 5622–26. http://dx.doi.org/10.1128/aem.69.9.5622-5626.2003.
Full textAbstract:
ABSTRACT The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k cat/Km ) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.