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Structure type: oligomer
Trivial name: N-linked oligosaccharide
Compound class: N-glycan, glycoprotein, polysaccharide, mannan, oligosaccharide, N-polysaccharide
Contained glycoepitopes: IEDB_123886,IEDB_130701,IEDB_135813,IEDB_136104,IEDB_137340,IEDB_137485,IEDB_140116,IEDB_140942,IEDB_141793,IEDB_141807,IEDB_141828,IEDB_141829,IEDB_141830,IEDB_141831,IEDB_143632,IEDB_144983,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_164046,IEDB_164174,IEDB_187201,IEDB_429156,IEDB_540671,IEDB_548907,IEDB_857734,IEDB_983930,SB_136,SB_191,SB_196,SB_197,SB_198,SB_33,SB_44,SB_53,SB_67,SB_72,SB_73,SB_74,SB_77,SB_85

• Article ID: 5255
  Qu Y, Feng J, Deng S, Cao L, Zhang Q, Zhao R, Zhang Z, Jiang Y, Zink EM, Baker SE, Lipton MS, Paša-Tolić L, Hu JZ, Wu S "Structural analysis of N- and O-glycans using ZIC-HILIC/dialysis coupled to NMR detection" - Fungal Genetics and Biology 72 (2014) 207-215
  

  Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes, including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we developed a glycan enrichment strategy that couples Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) with dialysis to enrich the glycans from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is a simple, fast, and efficient sample preparation approach. The approach was thus applied to analysis of a biological complex sample, the pronase E digest of the secreted proteins from the fungus Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core similar to the structure of the glycan from RNase B, and O-linked glycans bearing mannose and glucose with 1→3 and 1→6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled with dialysis is very effective and accessible in preparing glycans for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.

  NMR, glycan, A. niger, dialysis, secretome, ZIC-HILIC

  NCBI PubMed ID: 25117693
  Publication DOI: 10.1016/j.fgb.2014.08.001
  Journal NLM ID: 9607601
  Publisher: Orlando, FL : Academic Press / Elsevier
  Correspondence: Wu S
  Institutions: Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, USA, Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, USA, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, USA
  Methods: 13C NMR, 1H NMR, NMR-2D, HPSEC, enzymatic digestion, extraction, ZIC-HILIC
  
   
  
  Streptomyces griseus
  CSDB ID 46597 (all data & tools)
• Article ID: 7342
  Akao T, Yahara A, Sakamoto K, Yamada O, Akita O, Yoshida T "Lack of endoplasmic reticulum 1,2-α-mannosidase activity that trims N-glycan Man9GlcNAc2 to Man8GlcNAc2 isomer B in a manE gene disruptant of Aspergillus oryzae" - Journal of Bioscience and Bioengineering 113(4) (2012) 438-441
  

  The gene manE, encoding a probable class I endoplasmic reticulum 1,2-α-mannosidases (ER-Man), was identified from the filamentous fungus Aspergillus oryzae due to similarity to orthologs. It removes a single mannose residue from Man9GlcNAc2, generating Man8GlcNAc2 isomer B. Disruption of manE caused drastic decreases in ER-Man activity in A. oryzae microsomes.

  glycoprotein, 1, N-glycan processing, endoplasmic reticulum, Aspergillus oryzae, 2-α-mannosidase

  NCBI PubMed ID: 22169093
  Publication DOI: 10.1016/j.jbiosc.2011.11.015
  Journal NLM ID: 100888800
  Publisher: Osaka, Japan, Amsterdam, The Netherlands: Society for Bioscience and Bioengineering
  Correspondence: akao_t@nrib.go.jp
  Institutions: National Research Institute of Brewing, Hiroshima, Japan, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Japan
  Methods: DNA sequencing, TLC, HPLC, fluorescence spectroscopy, cell growth
  
   
  
  Aspergillus oryzae NS4, Aspergillus oryzae NS4 (ΔmanE mutant)
  CSDB ID 44190 (all data & tools)
• Article ID: 7800
  Yamaguchi T, Kamiya Y, Choo YM, Yamamoto S, Kato K "Terminal spin labeling of a high-mannose-type oligosaccharide for quantitative NMR analysis of its dynamic conformation" - Chemistry Letters 42(5) (2013) 544-546
  

  To achieve quantitative conformational characterization of oligosaccharides possessing branched structures with internal mobility, we developed a paramagnetic tagging protocol for NMR relaxation analysis. A nitroxide spin label was attached chemically to the reducing terminus of a specific high-mannose-type undecasaccharide that was overexpressed in genetically engineered yeast cells. This hybrid approach provides a source of long-distance atomic information, enabling detailed characterization of the conformational dynamics of the triantennary sugar chain that is involved in protein-fate-determination processes in cells.

  NMR, oligosaccharide

  Publication DOI: 10.1246/cl.130040
  Journal NLM ID: 0320606
  Publisher: Chemical Society of Japan
  Correspondence: kkatonmr@ims.ac.jp
  Institutions: The Glycoscience Institute, Ochanomizu University, Tokyo, Japan, Institute for Molecular Science, National Institutes of Natural Sciences, Aichi, Japan, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Aichi, Japan, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan, Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia, GLYENCE Co., Ltd., Aichi, Japan
  Methods: 1H NMR, NMR-2D, HPLC, extraction, hydrazinolysis
  
   
  
  Saccharomyces cerevisiae (Δoch1 Δmnn1 Δmnn4 Δmns1 mutant)
  CSDB ID 45433 (all data & tools)
• Article ID: 8758
  Flores RJD, Ohashi T, Sakai K, Gonoi T, Kawasaki H, Fujiyama K "The neutral N-linked glycans of the Basidiomycetous yeasts Pseudozyma antarctica and Malassezia furfur (Subphylum Ustilaginomycotina)" - Journal of General and Applied Microbiology 65(2) (2019) 53-63
  

  Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from Man3GlcNAc2-PA to Man9GlcNAc2-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.

  N-linked glycan, Pseudozyma, basidiomycetous yeasts, Malassezia

  NCBI PubMed ID: 30305477
  Publication DOI: 10.2323/jgam.2018.05.003
  Journal NLM ID: 0165543
  Publisher: Tokyo: Microbiology Research Foundation
  Correspondence: fujiyama@icb.osaka-u.ac.jp
  Institutions: International Center for Biotechnology, Osaka University, Osaka, Japan, NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), Chiba, Japan, Medical Mycology Research Center, Chiba University, Chiba, Japan
  Methods: HPLC, enzymatic digestion, extraction, hydrazinolysis, RP-HPLC, cell growth, LC-MS/MS, precipitation, derivatization, centrifugation
  
   
  
  Pseudozyma antarctica NBRC 10260, Malassezia furfur IFM 48685
  CSDB ID 49825 (all data & tools)
• Article ID: 8761
  Kar B, Patel P, Ao J, Free SJ "Neurospora crassa family GH72 glucanosyltransferases function to crosslink cell wall glycoprotein N-linked galactomannan to cell wall lichenin" - Fungal Genetics and Biology 123 (2019) 60-69
  

  The formation of a glucan/chitin/glycoprotein cell wall matrix is vital for fungal survival, growth, and morphogenesis. The cell wall proteins are important cell wall components and function in adhesion, signal transduction, and as cell wall structural elements. In this report we demonstrate that Neurospora crassa GH72 glucan transferases function to crosslink cell wall glycoproteins into the cell wall. With an in vitro assay, we show that the glucan transferases are able to attach lichenin, a cell wall glucan with a repeating β-1,4-glucose-β-1,4-glucose-β-1,3-glucose structure, to cell wall glycoproteins. We propose that the pathway for attachment of lichenin to the glycoprotein has four steps. First, N-linked oligosaccharides present on the glycoproteins are modified by the addition of a galactomannan. As part of our report we have characterized the structure of the galactomannan, which consists of an α-1,6-mannose backbone with galactofuranose side chains. In the second step, the galactomannan is processed by members of the GH76 α-1,6-mannanases. In the third step, the glucan transferases cleave the lichenin and create substrate-enzyme intermediates. In the final step, the transferases transfer the lichenin to the processed galactomannan. We demonstrate that the N. crassa glucan transferases have demonstrate specificity for the processed galactomannan and for lichenin. The energy from the cleaved glycosidic bond in lichenin is retained in the substrate-enzyme intermediate and used to create a new glycosidic bond between the lichenin and the processed galactomannan. The pathway effectively crosslinks glycoproteins into the fungal cell wall.

  oligosaccharide, cell wall, glycosyltransferase, N-linked glycosylation, fungi, Neurospora, protein cross-linking

  NCBI PubMed ID: 30503329
  Publication DOI: 10.1016/j.fgb.2018.11.007
  Journal NLM ID: 9607601
  Publisher: Orlando, FL : Academic Press / Elsevier
  Correspondence: free@buffalo.edu
  Institutions: Department of Biological Sciences, SUNY University at Buffalo, Buffalo, NY, USA
  Methods: GC-MS, SDS-PAGE, Western blotting, enzymatic digestion, gel immunoprecipitation, cell growth, MALDI-TOF/TOF MS, enzymatic assay, precipitation, centrifugation
  
   
  
  Neurospora crassa (Δdfg-5 mutant), Neurospora crassa (Δdcw-1 mutant), Neurospora crassa (Δgel-1 mutant), Neurospora crassa (Δgel-2 mutant), Neurospora crassa (Δgel-5 mutant), Neurospora crassa (Δoch-1 mutant)
  CSDB ID 49841 (all data & tools)
• Article ID: 8770
  Patel PK, Free SJ "The genetics and biochemistry of cell wall structure and synthesis in Neurospora crassa, a model filamentous fungus" - Frontiers in Microbiology 10 (2019) ID 2294
  

  This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a "processed" galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.

  cell wall, Galactomannan, glucan, filamentous fungi, melanin, Neurospora, glucanosyltransferase, mannanase

  NCBI PubMed ID: 31649638
  Publication DOI: 10.3389/fmicb.2019.02294
  Journal NLM ID: 101548977
  Publisher: Lausanne: Frontiers Research Foundation
  Correspondence: free@buffalo.edu
  Institutions: Department of Biological Sciences, SUNY University at Buffalo, Buffalo, NY, USA
  
   
  
  Neurospora crassa (Δoch-1 mutant)
  CSDB ID 49876 (all data & tools)
• Article ID: 9082
  Andjelković U, Gudelj I, Klarić T, Hinneburg H, Vinković M, Wittine K, Dovezenski N, Vikić-Topić D, Lauc G, Vujčić Z, Josić D "Increased yield of enzymatic synthesis by chromatographic selection of different N-glycoforms of yeast invertase" - Electrophoresis 2020 (2020) ID 202000092
  

  Invertases are glycosidases applied for synthesis of alkyl glycosides that are important and effective surfactants. Stability of invertases in the environment with increased content of organic solvent is crucial for increase of productivity of glycosidases. Their stability is significantly influenced by N-glycosylation. However, yeast N-glycosylation pathways may synthesize plethora of N-glycan structures. A total natural crude mixture of invertase glycoforms (EINV) extracted from Saccharomyces cerevisiae was subfractionated by anion-exchange chromatography on industrial monolithic supports to obtain different glycoforms (EINV1-EINV3). Separated glycoforms exhibited different stabilities in water-alcohol solutions that are in direct correlation with the amount of phosphate bound to N-glycans. Observed differences in stability of different invertase glycoforms were used to improve productivity of methyl β-D-fructofuranoside (MF) synthesis. The efficiency and yield of MF synthesis were improved more than 50% when the most stabile glycoform bearing the lowest amount of phosphorylated N-glycans is selected and utilized. These data underline the importance of analysis of glycan structures attached to glycoproteins, demonstrate different impact of N-glycans on the surface charge and enzyme stability in regard to particular reaction environment, and provide a platform for improvement of yield of industrial enzymatic synthesis by chromatographic selection of glycoforms on monolithic supports.

  N-glycosylation, organic solvent, enzyme stability, glycoform separation, monolithic supports

  NCBI PubMed ID: 33026663
  Publication DOI: 10.1002/elps.202000092
  Journal NLM ID: 8204476
  Publisher: Wiley-VCH
  Correspondence: Andjelković U
  Institutions: Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, Faculty of Chemistry, University of Belgrade, Belgrade, Serbia, University of Belgrade, Institute of Chemistry, Technology and Metallurgy, National Institute of the Republic of Serbia, Belgrade, Serbia, Department of Biotechnology, University of Rijeka, Rijeka, Croatia, Genos Glycoscience Research Laboratory, Zagreb, Croatia, NMR Centre, Ruđer Bošković Institute, Zagreb, Croatia, Institute for Medical Research, University of Belgrade, Belgrade, Serbia, Department of Natural and Health Sciences, Juraj Dobrila University of Pula, Pula, Croatia, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
  Methods: 13C NMR, 1H NMR, NMR-2D, enzymatic digestion, ion-exchange chromatography, fluorescence spectroscopy, column chromatography, MALDI-TOF/TOF MS, enzymatic assay, spectrophotometry, HILIC, fluorescent labeling, UHPLC, filtration, DNS method, denaturation
  
   
  
  Saccharomyces cerevisiae
  CSDB ID 50690 (all data & tools)
• Article ID: 9375
  Patel PK, Tung SK, Porfirio S, Sonon R, Azadi P, Free SJ "Extracellular targeting of Neurospora crassa cell wall and secreted glycoproteins by DFG-5" - Fungal Genetics and Biology 160 (2022) 103686
  

  The formation of a cell wall is vital for the survival and growth of a fungal cell. Fungi express members of the GH76 family of α-1,6-mannanases which play an important role in cell wall biogenesis. In this report we characterize the Neurospora crassa DFG-5 α-1,6-mannanase and demonstrate that it binds to the α-1,6-mannose backbone of an N-linked galactomannan found on cell wall glycoproteins. We show that DFG-5 has an enzymatic activity and provide evidence that it processes the α-1,6-mannose backbone of the N-linked galactomannan. Site-directed mutagenesis and complementation experiments show that D116 and D117 are located at the DFG-5 active site. D76 and E130, which are located in a groove on the opposite side of the protein, are also important for enzyme function. Cell wall glycoproteins co-purify with DFG-5 demonstrating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 is able to discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans present on secreted glycoproteins. DFG-5 plays a key role in targeting extracellular glycoproteins to their final destinations. By processing the galactomannans on cell wall proteins, DFG-5 targets them for cell wall incorporation by lichenin transferases. The N-linked galactomannans on secreted proteins are not processed by DFG-5, which targets these proteins for release into the extracellular medium.

  Galactomannan, protein secretion, cell wall biosynthesis, DFG5, Extracellular protein targeting, GH76 α-1, 6-mannanase

  NCBI PubMed ID: 35306147
  Publication DOI: 10.1016/j.fgb.2022.103686
  Journal NLM ID: 9607601
  Publisher: Orlando, FL : Academic Press / Elsevier
  Correspondence: S.J. Free
  Institutions: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA, Department of Biological Sciences, SUNY University at Buffalo, Buffalo, NY, USA
  Methods: PCR, Western blotting, MALDI-TOF MS, enzymatic digestion, cloning, enzymatic assay, complementation, site-directed mutagenesis, co-purification experiments
  
   
  
  Neurospora crassa A Δoch-1
  CSDB ID 51652 (all data & tools)
• Article ID: 11711
  Bardor M, Cabanes-Macheteau M, Faye L, Lerouge P "Monitoring of N-glycosylation of plant glycoproteins by fluorophore-assisted carbohydrate electrophoresis" - Electrophoresis 21(12) (2000) 2550-2556
  

  We have evaluated the efficiency of a fast, simple and efficient method, fluorophore-assisted carbohydrate electrophoresis (FACE), for the characterization of plant N-linked glycans. After their enzymatic release from plant glycoproteins, N-glycans were reductively aminated to the charged fluorophore 8-aminonaphthalene-1, 3, 6-trisulfonic acid (ANTS) and separated using high resolution polyacrylamide gel electrophoresis. In addition, an affinity purification procedure using concanavalin A was developed for separation of ANTS-labeled high-mannose-type N-glycans from other plant oligosaccharides.

  3, plant glycoproteins, 8-aminonaphthalene-1, 6-trisulfonic acid labeled plant N-glycans, fluorophore-assisted carbohydrate electrophoresis

  NCBI PubMed ID: 10939471
  Publication DOI: 10.1002/1522-2683(20000701)21:12<2550::AID-ELPS2550>3.0.CO;2-G
  Journal NLM ID: 8204476
  Publisher: Wiley-VCH
  Correspondence: plerouge@crihan.fr
  Institutions: Laboratoire des Transports Intracellulaires, CNRS ESA, Université de Rouen, Faculté des Sciences, Mont Saint Aignan, France
  Methods: MALDI-TOF MS, electrophoresis, FACE, PAGE, binding assays, fluorescence microscopy, fluorescence labeling, centrifugation
  
   
  
  Arabidopsis thaliana
  CSDB ID 64835 (all data & tools)