Show legend Show as text

Structure type: oligomer
Trivial name: high mannose
Compound class: N-glycan
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: 2758
  Mendelzon DH, Previato JO, Parodi AJ "Characterization of protein-linked oligosaccharides in trypanosomatid flagellates" - Molecular and Biochemical Parasitology 18 (1986) 355-367
  

  Protein-linked, endo-β-N-acetylglucosaminidase H-sensitive oligosaccharides were isolated from several trypanosomatids incubated with [U-14C]glucose. Structural analysis of the compounds revealed that Man9GlcNAc2 was the oligosaccharide transferred from dolichol-P-P derivatives to proteins in Trypanosoma dionisii, Trypanosoma conorhini, Leptomonas samueli and Herpetomonas samuelpessoai and Man6GlcNAc2 in Blastocrithidia culicis and Leishmania adleri. In all cases, transiently glucosylated compounds were detected: Glc1Man7-9GlcNAc2 in T. dionisii, T. conorhini, L. samueli; Glc1Man9GlcNAc2 in H. samuelpessoai, Glc1Man6GlcNAc2 in B. culicis and Glc1Man6GlcNAc2 and Glc1Man5GlcNAc2 in L. adleri. The mechanism of protein glycosylation in T. dionisii and T. conorhini appeared to be similar to that described before for Trypanosoma cruzi epimastigotes, although some differences were found between the structures of the main isomers of Man7GlcNAc2 and Man8GlcNAc2 present in T. conorhini and T. cruzi. Differences between the mechanisms of glycosylation occurring in Leishmania mexicana and L. adleri were also found: Man6GlcNAc2 in the latter microorganism was demannosylated to Man5GlcNAc2, a step not detected in the former parasite. A novel substituent in N-linked high mannose-type oligosaccharides was found in L. samueli and H. samuelpessoai: galactose in the furanose configuration. In the latter trypanosomatid, Man9GlcNAc2 was demannosylated only to Man8GlcNAc2, whereas in all other parasites in which the same oligosaccharide was transferred to proteins, Man5-7GlcNAc2 were also detected

  glycoproteins, Dolichol, Trypanosomatid flagellates

  NCBI PubMed ID: 3083255
  Publication DOI: 10.1016/0166-6851(86)90092-7
  Journal NLM ID: 8006324
  Institutions: Instituto de Investigaciones Bioquímicas ‘Fundación Campomar’, Antonio Machado 151, 1405 Buenos Aires, Argentina, Departamento de Microbiologia Geral, Universidade Federal de Rio de Janeiro, Bloco I, Ilha do Fundão, Rio de Janeiro, RJ 21941, Brazil
  
   
  
  Blastocrithidia culicis, Herpetomonas samuelpessoai, Leishmania adleri, Leishmania mexicana, Leptomonas samueli, Trypanosoma conorhini, Trypanosoma dionisii
  CSDB ID 143735 (all data & tools)

Show legend Show as text

Structure type: oligomer
Compound class: N-glycan
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_142488,IEDB_143632,IEDB_144983,IEDB_144998,IEDB_146664,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,IEDB_983931,SB_136,SB_191,SB_192,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: 2758
  Mendelzon DH, Previato JO, Parodi AJ "Characterization of protein-linked oligosaccharides in trypanosomatid flagellates" - Molecular and Biochemical Parasitology 18 (1986) 355-367
  

  Protein-linked, endo-β-N-acetylglucosaminidase H-sensitive oligosaccharides were isolated from several trypanosomatids incubated with [U-14C]glucose. Structural analysis of the compounds revealed that Man9GlcNAc2 was the oligosaccharide transferred from dolichol-P-P derivatives to proteins in Trypanosoma dionisii, Trypanosoma conorhini, Leptomonas samueli and Herpetomonas samuelpessoai and Man6GlcNAc2 in Blastocrithidia culicis and Leishmania adleri. In all cases, transiently glucosylated compounds were detected: Glc1Man7-9GlcNAc2 in T. dionisii, T. conorhini, L. samueli; Glc1Man9GlcNAc2 in H. samuelpessoai, Glc1Man6GlcNAc2 in B. culicis and Glc1Man6GlcNAc2 and Glc1Man5GlcNAc2 in L. adleri. The mechanism of protein glycosylation in T. dionisii and T. conorhini appeared to be similar to that described before for Trypanosoma cruzi epimastigotes, although some differences were found between the structures of the main isomers of Man7GlcNAc2 and Man8GlcNAc2 present in T. conorhini and T. cruzi. Differences between the mechanisms of glycosylation occurring in Leishmania mexicana and L. adleri were also found: Man6GlcNAc2 in the latter microorganism was demannosylated to Man5GlcNAc2, a step not detected in the former parasite. A novel substituent in N-linked high mannose-type oligosaccharides was found in L. samueli and H. samuelpessoai: galactose in the furanose configuration. In the latter trypanosomatid, Man9GlcNAc2 was demannosylated only to Man8GlcNAc2, whereas in all other parasites in which the same oligosaccharide was transferred to proteins, Man5-7GlcNAc2 were also detected

  glycoproteins, Dolichol, Trypanosomatid flagellates

  NCBI PubMed ID: 3083255
  Publication DOI: 10.1016/0166-6851(86)90092-7
  Journal NLM ID: 8006324
  Institutions: Instituto de Investigaciones Bioquímicas ‘Fundación Campomar’, Antonio Machado 151, 1405 Buenos Aires, Argentina, Departamento de Microbiologia Geral, Universidade Federal de Rio de Janeiro, Bloco I, Ilha do Fundão, Rio de Janeiro, RJ 21941, Brazil
  
   
  
  Blastocrithidia culicis, Herpetomonas samuelpessoai, Leishmania adleri, Leishmania mexicana, Leptomonas samueli, Trypanosoma conorhini, Trypanosoma dionisii
  CSDB ID 143736 (all data & tools)

Show legend Show as text

Structure type: oligomer
Aglycon: dolichol-PP
Trivial name: Man9GlcNAc2
Compound class: glycoprotein
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: 2891
  Previato JO, Mendelzon DH, Parodi AJ "Characterization of dolichol monophosphate- and dolichol diphosphate-linked saccharides in trypanosomatid flagellates" - Molecular and Biochemical Parasitology 18 (1986) 343-353
  

  Dolichol-P- and dolichol-P-P-linked saccharides were isolated from several trypanosomatid flagellates incubated with [U-14C]glucose. Formation of Glc-P-dolichol and Man-P-dolichol was observed in Herpetomonas muscarum and Leishmania adleri, whereas only the latter derivative was synthesized in Trypanosoma dionisii and Leptomonas samueli. The main and largest dolichol-P-P-linked oligosaccharide formed in Trypanosoma conorhini, T. dionisii, L. samueli, Herpetomonas samuelpessoai and H. muscarum appeared to be Man9GlcNAc2, whereas in Blastocrithidia culicis it was Man6GlcNAc2. In L. adleri there were two main oligosaccharides linked to dolichol-P-P, Man6GlcNAc2 and Man5GlcNAc2. The structures of the oligosaccharides were identical with those of the intermediates in the formation of Glc3Man9GlcNAc2-P-P-dolichol in higher eucaryotes. It was concluded that similarly to Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana, no glucosylated derivatives of dolichol-P-P were formed in the additional seven trypanosomatids studied here. The results obtained suggest that the defective step could be in some cases the formation of Glc-P-dolichol and in others, the transfer of glucose residues from the latter compound to dolichol-P-P-linked oligosaccharides.

  Publication DOI: 10.1016/0166-6851(86)90091-5
  Journal NLM ID: 8006324
  Institutions: Departamento de Microbiologia Geral, Universidade Federal de Rio de Janeiro, Bloco 1, llha do Fundao, Rio de Janeiro, RJ 21941, Brazil, Instituto de lnvestigaciones Bioqulmicas ''Fundacion Campomar'', Antonio Machado 151, (1405) Buenos Aires, Argentina
  Methods: acid hydrolysis, paper chromatography, isotopic labeling
  
   
  
  Herpetomonas muscarum, Herpetomonas samuelpessoai, Leptomonas samueli, Trypanosoma conorhini, Trypanosoma dionisii, Trypanosoma cruzi
  CSDB ID 143731 (all data & tools)

Show legend Show as text

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)

Show legend Show as text

Structure type: oligomer
Trivial name: Man9GlcNAc2
Compound class: N-glycan, mannan
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: 5895
  Bushkin GG, Ratner DM, Cui J, Banerjee S, Duraisingh MT, Jennings CV, Dvorin JD, Gubbels M, Robertson SD, Steffen M, O'Keefe BR, Robbins PW, Samuelson J "Suggestive evidence for Darwinian Selection against asparagine-linked glycans of Plasmodium falciparum and Toxoplasma gondii" - Eukaryotic Cell 9(2) (2010) 248-241
  

  We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc(3)Man(5)GlcNAc(2)) and the host 14-sugar precursor (Glc(3)Man(9)GlcNAc(2)) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.

  N-glycans, Toxoplasma gondii, Plasmodium falciparum

  NCBI PubMed ID: 19783771
  Publication DOI: 10.1128/EC.00197-09
  Journal NLM ID: 101130731
  Publisher: American Society for Microbiology
  Correspondence: jsamuels@bu.edu
  Institutions: Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA
  Methods: biochemical methods, bioinformatic analysis, LC-MS/MS, fluorescence microscopy, lectin blotting, morphological methods
  
   
  
  Toxoplasma gondii RH
  CSDB ID 7265 (all data & tools)
• Article ID: 6685
  Archer DB, Peberdy JF "The molecular biology of secreted enzyme production by fungi" - Critical Reviews in Biotechnology 17(4) (1997) 273-306
  

  Enzymes from filamentous fungi are already widely exploited, but new applications for known enzymes and new enzymic activities continue to be found. In addition, enzymes from less amenable non-fungal sources require heterologous production and fungi are being used as the production hosts. In each case there is a need to improve production and to ensure quality of product. While conventional, mutagenesis-based, strain improvement methods will continue to be applied to enzyme production from filamentous fungi the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis of fungal enzyme production and this knowledge is now being applied both in the laboratory and commercially. We review the current state of knowledge on the molecular basis of enzyme production by filamentous fungi. We focus on transcriptional and post-transcriptional regulation of protein production, the transit of proteins through the secretory pathway and the structure of the proteins produced including glycosylation.

  enzyme, glycosylation, Aspergillus, fungus, protein secretion, Trichoderma, heterologous gene expression

  NCBI PubMed ID: 9397531
  Publication DOI: 10.3109/07388559709146616
  Journal NLM ID: 8505177
  Publisher: CRC Press
  Institutions: Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich, UK
  
   
  
  Aspergillus niger
  CSDB ID 40033 (all data & tools)
• Article ID: 8598
  Harada Y, Huang C, Yamaki S, Dohmae N, Suzuki T "Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-β-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc.In vitroENGase assays revealed that the presence of an α1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man4GlcNAc2-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs." - Journal of Biological Chemistry 291(15) (2016) 8048-8058
  

  Phosphorylated oligosaccharides (POSs) are produced by the degradation of dolichol-linked oligosaccharides (DLOs) by an unclarified mechanism in mammalian cells. Although POSs are exclusively found in the cytosol, their intracellular fates remain unclear. Our findings indicate that POSs are catabolized via a non-lysosomal glycan degradation pathway that involves a cytosolic endo-β-N-acetylglucosaminidase (ENGase). Quantitative and structural analyses of POSs revealed that ablation of the ENGase results in the significant accumulation of POSs with a hexasaccharide structure composed of Manα1,2Manα1,3(Manα1,6)Manβ1,4GlcNAcβ1,4GlcNAc.In vitroENGase assays revealed that the presence of an α1,2-linked mannose residue facilitates the hydrolysis of POSs by the ENGase. Liquid chromatography-mass spectrometric analyses and fluorescent labeling experiments show that such POSs contain one phosphate group at the reducing end. These results indicate that ENGase efficiently hydrolyzes POSs that are larger than Man4GlcNAc2-P, generating GlcNAc-1-P and neutral Gn1-type free oligosaccharides. These results provide insight into important aspects of the generation and degradation of POSs.

  oligosaccharide, N-linked glycosylation, glycobiology, Carbohydrate Metabolism, endoplasmic reticulum (ER), endo-β-N-acetylglucosaminidase, dolichol-linked oligosaccharides, non-lysosomal glycan degradation, phosphorylated oligosaccharides

  NCBI PubMed ID: 26858256
  Publication DOI: 10.1074/jbc.M115.685313
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: Suzuki T
  Institutions: Glycometabolome Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, Global Research Cluster, RIKEN, Wako, Saitama, Japan, Global Application Development Center, Analytical and Measuring Instruments Division, Shimadzu Corp., Hadano, Kanagawa, Japan, Collaboration Promotion Unit, RIKEN Global Research Cluster, Wako, Saitama, Japan
  Methods: HPLC, LC-MS, fluorescent labeling with 2-aminopyridine
  
   
  
  Saccharomyces cerevisiae alg6Δ
  CSDB ID 49427 (all data & tools)
• Article ID: 10575
  Hayashi M, Tsuru A, Mitsui T, Takahashi N, Hanzawa H, Arata Y, Akazawa T "Structure and biosynthesis of the xylose-containing carbohydrate moiety of rice α-amylase" - European Journal of Biochemistry 191 (1990) 287-295
  

  Suspension-cultured cells of rice secrete α-amylase into the culture medium. It has been shown that the mature form of the α-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete α-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, α-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.

  NCBI PubMed ID: 2143471
  Publication DOI: 10.1111/j.1432-1033.1990.tb19122.x
  Journal NLM ID: 0107600
  Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies
  Institutions: Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Japan, Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, Japan, Nagoya City University, College of Nursing, Nagoya, Japan, Faculty of Pharmaceutical Science, University of Tokyo, Japan
  Methods: 1H NMR, SDS-PAGE, HPLC, enzymatic digestion, pulse-labeling assay
  
   
  
  Oryza sativa
  CSDB ID 123443 (all data & tools)

Show legend Show as text

Structure type: oligomer
Aglycon: Asn428
Trivial name: variant surface glycoproteins (VSG)
Compound class: N-glycan
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: 5902
  Castillo-Acosta VM, Vidal AE, Ruiz-Pérez LM, Van Damme EJ, Igarashi Y, Balzarini J, González-Pacanowska D "Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei" - Molecular Microbiology 90(4) (2013) 665-679
  

  The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

  virulence, glycosylation, Trypanosoma brucei, variant surface glycoprotein

  NCBI PubMed ID: 23926900
  Publication DOI: 10.1111/mmi.12359
  Journal NLM ID: 8712028
  Publisher: Blackwell Publishing
  Correspondence: dgonzalez@ipb.csic.es
  Institutions: Instituto de Parasitología y Biomedicina 'López-Neyra'. Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, s/n 18016, Armilla, Granada, Spain
  Methods: RT-PCR, binding assays, fluorescence microscopy, lectin blotting, FACS assay
  
   
  
  Trypanosoma brucei
  CSDB ID 7272 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 1236 [M+H+Na]2+
Aglycon: Asn428 C-terminal N-glycosylation site GNTNTT peptide
Trivial name: sVSG, soluble-form VSG, variant surface glycoprotein
Compound class: N-glycan
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: 5917
  Denton H, Fyffe S, Smith TK "GDP-mannose pyrophosphorylase is essential in the bloodstream form of Trypanosoma brucei" - Biochemical Journal 425(3) (2010) 603-614
  

  A putative GDP-Man PP (guanidine diphosphomannose pyrophosphorylase) gene from Trypanosoma brucei (TbGDP-Man PP) was identified in the genome and subsequently cloned, sequenced and recombinantly expressed, and shown to be a catalytically active dimer. Kinetic analysis revealed a Vmax of 0.34 μmol/min per mg of protein and Km values of 67 μM and 12 μM for GTP and mannose 1-phosphate respectively. Further kinetic studies showed GDP-Man was a potent product feedback inhibitor. RNAi (RNA interference) of the cytosolic TbGDP-Man PP showed that mRNA levels were reduced to ~20% of wild-type levels, causing the cells to die after 3-4 days, demonstrating that TbGDP-Man PP is essential in the bloodstream form of T. brucei and thus a potential drug target. The RNAi-induced parasites have a greatly reduced capability to form GDP-Man, leading ultimately to a reduction in their ability to synthesize their essential GPI (glycosylphosphatidylinositol) anchors. The RNAi-induced parasites also showed aberrant N-glycosylation of their major cell-surface glycoprotein, variant surface glycoprotein, with loss of the high-mannose Man9GlcNAc2 N-glycosylation at Asn428 and formation of complex N-glycans at Asn263.

  Glycosylphosphatidylinositol, N-glycosylation, Trypanosoma brucei, variant surface glycoprotein, essentiality, guanidine diphosphomannose pyrophosphorylase

  NCBI PubMed ID: 19919534
  Publication DOI: 10.1042/BJ20090896
  Journal NLM ID: 2984726R
  Publisher: London, UK : Published by Portland Press on behalf of the Biochemical Society
  Correspondence: tks1@st-andrews.ac.uk
  Institutions: Biomolecular Sciences Research Complex, The North Haugh, The University, St Andrews, Fife KY16 9ST, Scotland, UK
  Methods: gel filtration, ESI-MS, ESI-MS/MS, genetic methods, HPLC, enzymatic digestion, Southern blotting, RT-PCR, HPTLC, cloning, enzymatic assay
  
   
  
  Trypanosoma brucei 427 variant 221, Trypanosoma brucei RNAi
  CSDB ID 4869 (all data & tools)

Show legend Show as text

Structure type: oligomer
Aglycon: -Asn-XSer/Thr-
Trivial name: VSG, variant surface glycoprotein
Compound class: N-glycan
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: 5919
  Duncan SM, Nagar R, Damerow M, Yashunsky DV, Bruzzi B, Nikolaev AV, Ferguson MAJ "A Trypanosoma brucei β3 glycosyltransferase superfamily gene encodes a β1-6 GlcNAc-transferase mediating N-glycan and GPI anchor modification" - Journal of Biological Chemistry 294(4) (2021) 101153
  

  The parasite Trypanosoma brucei exists in both a bloodstream form (BSF) and a procyclic form (PCF), which exhibit large carbohydrate extensions on the N-linked glycans and glycosylphosphatidylinositol (GPI) anchors, respectively. The parasite's glycoconjugate repertoire suggests at least 38 glycosyltransferase (GT) activities, 16 of which are currently uncharacterized. Here, we probe the function(s) of the uncharacterized GT67 glycosyltransferase family and a β3 glycosyltransferase (β3GT) superfamily gene, TbGT10. A BSF-null mutant, created by applying the diCre/loxP method in T. brucei for the first time, showed a fitness cost but was viable in vitro and in vivo and could differentiate into the PCF, demonstrating nonessentiality of TbGT10. The absence of TbGT10 impaired the elaboration of N-glycans and GPI anchor side chains in BSF and PCF parasites, respectively. Glycosylation defects included reduced BSF glycoprotein binding to the lectin ricin and monoclonal antibodies mAb139 and mAbCB1. The latter bind a carbohydrate epitope present on lysosomal glycoprotein p67 that we show here consists of (-6Galβ1-4GlcNAcβ1-)≥4 poly-N-acetyllactosamine repeats. Methylation linkage analysis of Pronase-digested glycopeptides isolated from BSF wild-type and TbGT10 null parasites showed a reduction in 6-O-substituted- and 3,6-di-O-substituted-Gal residues. These data define TbGT10 as a UDP-GlcNAc:βGal β1-6 GlcNAc-transferase. The dual role of TbGT10 in BSF N-glycan and PCF GPI-glycan elaboration is notable, and the β1-6 specificity of a β3GT superfamily gene product is unprecedented. The similar activities of trypanosome TbGT10 and higher-eukaryote I-branching enzyme (EC 2.4.1.150), which belong to glycosyltransferase families GT67 and GT14, respectively, in elaborating N-linked glycans, are a novel example of convergent evolution.

  N-acetylglucosaminyltransferase, Glycosylphosphatidylinositol, N-glycosylation, GPI, N-glycan, Trypanosoma brucei glycosyltransferase

  NCBI PubMed ID: 34478712
  Publication DOI: 10.1016/j.jbc.2021.101153
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: m.a.j.ferguson@dundee.ac.uk
  Institutions: Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom
  Methods: methylation, PCR, GC-MS, DNA techniques, Western blotting, genetic methods, enzymatic digestion, conjugation, lectin blotting, biolayer interferometry (BLI) measurements
  
   
  
  Trypanosoma brucei
  CSDB ID 4882 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2108.7 [M+Na]+
Aglycon: s-gp130 peptide
Compound class: N-glycan
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: 5922
  Feasley C, Johnson JM, West CM, Chia CP "Glycopeptidome of a Heavily N-Glycosylated Cell Surface Glycoprotein of Dictyostelium Implicated in Cell Adhesion" - Journal of Proteome Research 9(7) (2010) 3495-3510
  

  Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man(8)GlcNAc(4) species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core alpha 3-fucose. Site mapping confirmed the occupancy of 15 sequons, some variably, and glycopeptide analysis confirmed 14 sites and revealed extensive heterogeneity at most sites. Glycopeptide glycoforms ranged from Man(6) to Man(9), GlcNAc(0-2) (peripheral), Fuc(0-2) (including core alpha 3 and peripheral), (SO(4))(0-1), and (MePO(4))(0-1), which represented elements of virtually the entire known cellular N-glycome as inferred from prior metabolic labeling and mass spectrometry studies. gp130, and a family of 14 related predicted glycoproteins whose polypeptide sequences are rapidly diverging in the Dictyostelium lineage, may contribute a functionally important shroud of high-mannose N-glycans at the interface of the amoebae with each other, their predators and prey, and the soil environment.

  mass spectrometry, glycobiology, N-glycan, Dictyostelium, glycopeptidome, gp130

  NCBI PubMed ID: 20443635
  Publication DOI: 10.1021/pr901195c
  Journal NLM ID: 101128775
  Publisher: Washington, DC: American Chemical Society
  Correspondence: Cwest2@ouhsc.edu
  Institutions: Department of Biochemistry & Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA
  Methods: gel filtration, SDS-PAGE, sugar analysis, Western blotting, amino acid analysis, MALDI-TOF MS, genetic methods, enzymatic digestion, HPAEC-PAD, permethylation, cloning, RP-HPLC, MALDI-TOF/TOF MS, reductive amination, nanoLC-MS/MS
  
   
  
  Dictyostelium discoideum HL250, Dictyostelium discoideum HW11
  CSDB ID 4895 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2391.8 [M+Na]+
Compound class: N-glycan
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_142488,IEDB_143632,IEDB_144983,IEDB_144998,IEDB_146664,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_164046,IEDB_164174,IEDB_187201,IEDB_232584,IEDB_429156,IEDB_540671,IEDB_548907,IEDB_857734,IEDB_983930,IEDB_983931,SB_136,SB_191,SB_192,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: 5923
  Feasley C, van der Wel H, West CM "Evolutionary diversity of social amoebae N-glycomes may support interspecific autonomy" - Glycoconjugate Journal 32(6) (2015) 345-359
  

  Multiple species of cellular slime mold (CSM) amoebae share overlapping subterranean environments near the soil surface. Despite similar life-styles, individual species form independent starvation-induced fruiting bodies whose spores can renew the life cycle. N-glycans associated with the cell surface glycocalyx have been predicted to contribute to interspecific avoidance, resistance to pathogens, and prey preference. N-glycans from five CSM species that diverged 300-600 million years ago and whose genomes have been sequenced were fractionated into neutral and acidic pools and profiled by MALDI-TOF-MS. Glycan structure models were refined using linkage specific antibodies, exoglycosidase digestions, MALDI-MS/MS, and chromatographic studies. Amoebae of the type species Dictyostelium discoideum express modestly trimmed high mannose N-glycans variably modified with core α3-linked Fuc and peripherally decorated with 0-2 residues each of β-GlcNAc, Fuc, methylphosphate and/or sulfate, as reported previously. Comparative analyses of D. purpureum, D. fasciculatum, Polysphondylium pallidum, and Actyostelium subglobosum revealed that each displays a distinctive spectrum of high-mannose species with quantitative variations in the extent of these modifications, and qualitative differences including retention of Glc, mannose methylation, and absence of a peripheral GlcNAc, fucosylation, or sulfation. Starvation-induced development modifies the pattern in all species but, except for universally observed increased mannose-trimming, the N-glycans do not converge to a common profile. Correlations with glycogene repertoires will enable future reverse genetic studies to eliminate N-glycomic differences to test their functions in interspecific relations and pathogen evasion.

  evolution, N-glycan, Dictyostelium, Cellular slime molds, Social amoebae

  NCBI PubMed ID: 25987342
  Publication DOI: 10.1007/s10719-015-9592-8
  Journal NLM ID: 8603310
  Publisher: Kluwer Academic Publishers
  Correspondence: Christopher M. West ; Christopher M. West
  Institutions: Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC-415, OUHSC, Oklahoma City, OK, 73104, USA
  Methods: SDS-PAGE, Western blotting, MALDI-TOF MS, enzymatic digestion, permethylation, column chromatography, MALDI-TOF/TOF MS, bioinformatic analysis (BLASTp)
  
   
  
  Dictyostelium purpureum
  CSDB ID 7293 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2067.7 [M+Na]+
Compound class: N-glycan
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_142488,IEDB_143632,IEDB_144983,IEDB_144998,IEDB_146664,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,IEDB_983931,SB_136,SB_191,SB_192,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: 5923
  Feasley C, van der Wel H, West CM "Evolutionary diversity of social amoebae N-glycomes may support interspecific autonomy" - Glycoconjugate Journal 32(6) (2015) 345-359
  

  Multiple species of cellular slime mold (CSM) amoebae share overlapping subterranean environments near the soil surface. Despite similar life-styles, individual species form independent starvation-induced fruiting bodies whose spores can renew the life cycle. N-glycans associated with the cell surface glycocalyx have been predicted to contribute to interspecific avoidance, resistance to pathogens, and prey preference. N-glycans from five CSM species that diverged 300-600 million years ago and whose genomes have been sequenced were fractionated into neutral and acidic pools and profiled by MALDI-TOF-MS. Glycan structure models were refined using linkage specific antibodies, exoglycosidase digestions, MALDI-MS/MS, and chromatographic studies. Amoebae of the type species Dictyostelium discoideum express modestly trimmed high mannose N-glycans variably modified with core α3-linked Fuc and peripherally decorated with 0-2 residues each of β-GlcNAc, Fuc, methylphosphate and/or sulfate, as reported previously. Comparative analyses of D. purpureum, D. fasciculatum, Polysphondylium pallidum, and Actyostelium subglobosum revealed that each displays a distinctive spectrum of high-mannose species with quantitative variations in the extent of these modifications, and qualitative differences including retention of Glc, mannose methylation, and absence of a peripheral GlcNAc, fucosylation, or sulfation. Starvation-induced development modifies the pattern in all species but, except for universally observed increased mannose-trimming, the N-glycans do not converge to a common profile. Correlations with glycogene repertoires will enable future reverse genetic studies to eliminate N-glycomic differences to test their functions in interspecific relations and pathogen evasion.

  evolution, N-glycan, Dictyostelium, Cellular slime molds, Social amoebae

  NCBI PubMed ID: 25987342
  Publication DOI: 10.1007/s10719-015-9592-8
  Journal NLM ID: 8603310
  Publisher: Kluwer Academic Publishers
  Correspondence: Christopher M. West ; Christopher M. West
  Institutions: Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC-415, OUHSC, Oklahoma City, OK, 73104, USA
  Methods: SDS-PAGE, Western blotting, MALDI-TOF MS, enzymatic digestion, permethylation, column chromatography, MALDI-TOF/TOF MS, bioinformatic analysis (BLASTp)
  
   
  
  Dictyostelium purpureum
  CSDB ID 4486 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 1905.7 [M+Na]+
Compound class: N-glycan
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: 5923
  Feasley C, van der Wel H, West CM "Evolutionary diversity of social amoebae N-glycomes may support interspecific autonomy" - Glycoconjugate Journal 32(6) (2015) 345-359
  

  Multiple species of cellular slime mold (CSM) amoebae share overlapping subterranean environments near the soil surface. Despite similar life-styles, individual species form independent starvation-induced fruiting bodies whose spores can renew the life cycle. N-glycans associated with the cell surface glycocalyx have been predicted to contribute to interspecific avoidance, resistance to pathogens, and prey preference. N-glycans from five CSM species that diverged 300-600 million years ago and whose genomes have been sequenced were fractionated into neutral and acidic pools and profiled by MALDI-TOF-MS. Glycan structure models were refined using linkage specific antibodies, exoglycosidase digestions, MALDI-MS/MS, and chromatographic studies. Amoebae of the type species Dictyostelium discoideum express modestly trimmed high mannose N-glycans variably modified with core α3-linked Fuc and peripherally decorated with 0-2 residues each of β-GlcNAc, Fuc, methylphosphate and/or sulfate, as reported previously. Comparative analyses of D. purpureum, D. fasciculatum, Polysphondylium pallidum, and Actyostelium subglobosum revealed that each displays a distinctive spectrum of high-mannose species with quantitative variations in the extent of these modifications, and qualitative differences including retention of Glc, mannose methylation, and absence of a peripheral GlcNAc, fucosylation, or sulfation. Starvation-induced development modifies the pattern in all species but, except for universally observed increased mannose-trimming, the N-glycans do not converge to a common profile. Correlations with glycogene repertoires will enable future reverse genetic studies to eliminate N-glycomic differences to test their functions in interspecific relations and pathogen evasion.

  evolution, N-glycan, Dictyostelium, Cellular slime molds, Social amoebae

  NCBI PubMed ID: 25987342
  Publication DOI: 10.1007/s10719-015-9592-8
  Journal NLM ID: 8603310
  Publisher: Kluwer Academic Publishers
  Correspondence: Christopher M. West ; Christopher M. West
  Institutions: Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC-415, OUHSC, Oklahoma City, OK, 73104, USA
  Methods: SDS-PAGE, Western blotting, MALDI-TOF MS, enzymatic digestion, permethylation, column chromatography, MALDI-TOF/TOF MS, bioinformatic analysis (BLASTp)
  
   
  
  Dictyostelium discoideum AX3, Dictyostelium purpureum, Dictyostelium fasciculatum, Polysphondylium pallidum
  CSDB ID 4487 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2470 [M+H]+
Aglycon: 2-aminopyridine (PA)
Compound class: N-glycan
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_142488,IEDB_143632,IEDB_144983,IEDB_144998,IEDB_146664,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_164046,IEDB_164174,IEDB_187201,IEDB_232584,IEDB_429156,IEDB_540671,IEDB_548907,IEDB_857734,IEDB_983930,IEDB_983931,SB_136,SB_191,SB_192,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: 5945
  Hykollari A, Balog CIA, Rendic D, Braulke W, Wilson IBH "Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL" - Journal of Proteome Research 12(3) (2013) 1173-1187
  

  The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.

  glycan, mass spectrometry, fucose, sulfate, Mannosyltransferase, N-glycans, Dictyostelium, methylphosphate

  NCBI PubMed ID: 23320427
  Publication DOI: 10.1021/pr300806b
  Journal NLM ID: 101128775
  Publisher: Washington, DC: American Chemical Society
  Correspondence: iain.wilson@boku.ac.at
  Institutions: Department für Chemie, Universität für Bodenkultur, A-1190 Wien, Austria, Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands, Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany
  Methods: gel filtration, SDS-PAGE, sugar analysis, Western blotting, MALDI-TOF MS, enzymatic digestion, HF treatment, RT-PCR, cloning, RP-HPLC, RNA sequencing, HIAX-HPLC, MALDI-TOF MS/MS
  
   
  
  Dictyostelium discoideum AX3
  CSDB ID 8136 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2146 [M+H]+
Aglycon: 2-aminopyridine (PA)
Compound class: N-glycan
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_142488,IEDB_143632,IEDB_144983,IEDB_144998,IEDB_146664,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,IEDB_983931,SB_136,SB_191,SB_192,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: 5945
  Hykollari A, Balog CIA, Rendic D, Braulke W, Wilson IBH "Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL" - Journal of Proteome Research 12(3) (2013) 1173-1187
  

  The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism.

  glycan, mass spectrometry, fucose, sulfate, Mannosyltransferase, N-glycans, Dictyostelium, methylphosphate

  NCBI PubMed ID: 23320427
  Publication DOI: 10.1021/pr300806b
  Journal NLM ID: 101128775
  Publisher: Washington, DC: American Chemical Society
  Correspondence: iain.wilson@boku.ac.at
  Institutions: Department für Chemie, Universität für Bodenkultur, A-1190 Wien, Austria, Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands, Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany
  Methods: gel filtration, SDS-PAGE, sugar analysis, Western blotting, MALDI-TOF MS, enzymatic digestion, HF treatment, RT-PCR, cloning, RP-HPLC, RNA sequencing, HIAX-HPLC, MALDI-TOF MS/MS
  
   
  
  Dictyostelium discoideum AX3
  CSDB ID 8137 (all data & tools)

Show legend Show as text

Structure type: oligomer
Aglycon: Asn
Trivial name: Man9GlcNAc2
Compound class: N-glycan
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: 5954
  Koeller CM, Tiengwe C, Schwartz KJ, Bangs JD "Steric constraints control processing of glycosylphosphatidylinositol anchors in Trypanosoma brucei" - Journal of Biological Chemistry 295(8) (2020) 2227-2238
  

  The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site-associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4-6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ∼5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (aa 4-7) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL-reactive, suggesting the addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

  Trypanosome, N-linked glycosylation, glycobiology, Trypanosoma brucei, glycosylphosphatidylinositol (GPI anchor), glycosylphosphatidylinositol processing, kinetoplastid protozoa, N-glycan processing, transferrin, transferrin receptor

  NCBI PubMed ID: 31932305
  Publication DOI: 10.1074/jbc.RA119.010847
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: jdbangs@buffalo.edu
  Institutions: Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo (SUNY), Buffalo, New York 14214, Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, 53706
  Methods: radiolabeling, serological methods, genetic methods, enzymatic digestion, RT-PCR, immunoprecipitation assay
  
   
  
  Trypanosoma brucei
  CSDB ID 8156 (all data & tools)