Show legend Show as text

Structure type: structural motif or average structure
Aglycon: Asn-X-Ser/Thr
Trivial name: poly-LacNAc-containing N-glycan
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130655,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_158550,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• Article ID: 5913
  Damerow M, Graalfs F, Guther MLS, Mehlert A, Izquierdo L, Ferguson MA "A Gene of the beta3-Glycosyltransferase Family Encodes N-Acetylglucosaminyltransferase II Function in Trypanosoma brucei" - Journal of Biological Chemistry 291(26) (2016) 13834-13845
  

  The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose, and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manα1-3 and Manα1-6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the β3-glycosyltransferase superfamily (Damerow, M., Rodrigues, J. A., Wu, D., Güther, M. L., Mehlert, A., and Ferguson, M. A. (2014) J. Biol. Chem. 289, 9328-9339). Here, we demonstrate that TbGT15, another member of the same β3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manα1-6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains, whereas the Manα1-3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of β3-glycosyltransferase family members to catalyze β1-2 glycosidic linkages.

  glycosyltransferase, N-acetylglucosamine, glycobiology, Parasite, Trypanosoma brucei, post-translational modification (PTM)

  NCBI PubMed ID: 27189951
  Publication DOI: 10.1074/jbc.M116.733246
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: m.a.j.ferguson@dundee.ac.uk
  Institutions: From the Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
  Methods: methylation, GC-MS, DNA techniques, glycosyltransferase assays, ESI-MS, Western blotting, composition analysis, genetic methods, Southern blotting, RT-PCR, HPTLC, LC-MS, NaBD4 reduction, lectin blotting, RNA extraction, SEM
  
   
  
  Trypanosoma brucei 427 (variant 221)
  CSDB ID 4860 (all data & tools)

Show legend Show as text

Structure type: structural motif or average structure
Aglycon: Asn-X-Ser/Thr
Trivial name: poly-LacNAc-containing N-glycan
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130655,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_158550,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• Article ID: 5913
  Damerow M, Graalfs F, Guther MLS, Mehlert A, Izquierdo L, Ferguson MA "A Gene of the beta3-Glycosyltransferase Family Encodes N-Acetylglucosaminyltransferase II Function in Trypanosoma brucei" - Journal of Biological Chemistry 291(26) (2016) 13834-13845
  

  The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose, and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manα1-3 and Manα1-6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the β3-glycosyltransferase superfamily (Damerow, M., Rodrigues, J. A., Wu, D., Güther, M. L., Mehlert, A., and Ferguson, M. A. (2014) J. Biol. Chem. 289, 9328-9339). Here, we demonstrate that TbGT15, another member of the same β3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manα1-6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains, whereas the Manα1-3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of β3-glycosyltransferase family members to catalyze β1-2 glycosidic linkages.

  glycosyltransferase, N-acetylglucosamine, glycobiology, Parasite, Trypanosoma brucei, post-translational modification (PTM)

  NCBI PubMed ID: 27189951
  Publication DOI: 10.1074/jbc.M116.733246
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: m.a.j.ferguson@dundee.ac.uk
  Institutions: From the Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom
  Methods: methylation, GC-MS, DNA techniques, glycosyltransferase assays, ESI-MS, Western blotting, composition analysis, genetic methods, Southern blotting, RT-PCR, HPTLC, LC-MS, NaBD4 reduction, lectin blotting, RNA extraction, SEM
  
   
  
  Trypanosoma brucei TbGT15 null mutant
  CSDB ID 4861 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 992 [M+H+Na]2+
Aglycon: Asn263 N-terminal N-glycosylation site NET peptide
Trivial name: sVSG, soluble-form VSG, variant surface glycoprotein
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• 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 RNAi
  CSDB ID 4877 (all data & tools)

Show legend Show as text

Structure type: structural motif or average structure
Aglycon: -Asn-XSer/Thr-
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• 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 427 variant 221
  CSDB ID 4879 (all data & tools)

Show legend Show as text

Structure type: structural motif or average structure
Aglycon: -Asn-XSer/Thr-
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_158533,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• 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 null TbGT8
  CSDB ID 4880 (all data & tools)

Show legend Show as text

Structure type: structural motif or average structure
Aglycon: -Asn-XSer/Thr-
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130655,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_158550,IEDB_190606,IEDB_423128,IEDB_490033,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• 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 null TbGT10
  CSDB ID 4881 (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_115013,IEDB_123886,IEDB_130645,IEDB_130646,IEDB_130649,IEDB_130701,IEDB_135813,IEDB_135815,IEDB_136044,IEDB_136906,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140122,IEDB_141496,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_146694,IEDB_149558,IEDB_151528,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_190606,IEDB_418918,IEDB_423128,IEDB_490039,IEDB_540672,IEDB_548907,IEDB_689191,IEDB_918314,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_40,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_87,SB_88

• 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 4884 (all data & tools)

Show legend Show as text

Structure type: structural motif or average structure
Aglycon: -Asn-XSer/Thr-
Trivial name: VSG, variant surface glycoprotein
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• 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 4885 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: VSG complex N-glycan
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130655,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_150939,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_158548,IEDB_158550,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• Article ID: 5943
  Heng J, Naderer T, Ralph SA, McConville MJ "Glycosylated compounds of parasitic protozoa" - Book: Microbial Glycobiology (series: Structures, Relevance and Applications) (2010) 203-231
  

  This chapter describes the range of glycan structures and pathways that are found in different parasitic protozoa. All parasitic protists express a range of glycoconjugates that form protective protein-rich or carbohydrate-rich surface coats. Protein-rich coats are typically found on developmental stages that inhabit nonhydrolytic niches, such as the bloodstream and nonacidified intracellular vacuoles. These coats are commonly dominated by a limited repertoire of antigenically diverse proteins that are commonly, but not always, glycosylphosphatidylinositol- (GPI-) anchored and modified with N- or O-glycans. Carbohydrate-rich coats are commonly found on developmental stages that dwell within hydrolytic environments, such as vertebrate and arthropod digestive tracts and lysosomal vacuoles. These coats are dominated by GPI-anchored glycoproteins that are heavily modified with N-glycans, O-glycans, or phosphoglycans. Free GPI glycolipids (not attached to protein) can also be abundant or dominant components of these coats. Some parasitic protists can also form highly resistant cyst stages encased within polysaccharide-rich cell walls. Considerable progress has been made in defining the structures of the surface and intracellular glycans of the parasitic protists, their biosynthesis and the role that individual components play in parasite infectivity.

  O-glycosylation, Glycosylphosphatidylinositol, N-glycosylation, protozoan parasites, Phosphoglycosylation

  Publication DOI: 10.1016/B978-0-12-374546-0.00012-2
  Publisher: Amsterdam: Elsevier
  Correspondence: malcolmm@unimelb.edu.au
  Editors: Moran A
  Institutions: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Australia
  
   
  
  Trypanosoma brucei
  CSDB ID 4566 (all data & tools)

Show legend Show as text

Structure type: oligomer
Trivial name: VSG complex N-glycan
Compound class: N-glycan
Contained glycoepitopes: IEDB_115013,IEDB_123886,IEDB_130645,IEDB_130646,IEDB_130649,IEDB_130697,IEDB_130701,IEDB_135813,IEDB_135815,IEDB_136044,IEDB_136906,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_137776,IEDB_140108,IEDB_140122,IEDB_141496,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_146694,IEDB_149558,IEDB_150939,IEDB_151528,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153529,IEDB_153531,IEDB_153557,IEDB_158533,IEDB_190606,IEDB_418918,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_689191,IEDB_918314,IEDB_983930,SB_165,SB_166,SB_173,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_40,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_87,SB_88

• Article ID: 5943
  Heng J, Naderer T, Ralph SA, McConville MJ "Glycosylated compounds of parasitic protozoa" - Book: Microbial Glycobiology (series: Structures, Relevance and Applications) (2010) 203-231
  

  This chapter describes the range of glycan structures and pathways that are found in different parasitic protozoa. All parasitic protists express a range of glycoconjugates that form protective protein-rich or carbohydrate-rich surface coats. Protein-rich coats are typically found on developmental stages that inhabit nonhydrolytic niches, such as the bloodstream and nonacidified intracellular vacuoles. These coats are commonly dominated by a limited repertoire of antigenically diverse proteins that are commonly, but not always, glycosylphosphatidylinositol- (GPI-) anchored and modified with N- or O-glycans. Carbohydrate-rich coats are commonly found on developmental stages that dwell within hydrolytic environments, such as vertebrate and arthropod digestive tracts and lysosomal vacuoles. These coats are dominated by GPI-anchored glycoproteins that are heavily modified with N-glycans, O-glycans, or phosphoglycans. Free GPI glycolipids (not attached to protein) can also be abundant or dominant components of these coats. Some parasitic protists can also form highly resistant cyst stages encased within polysaccharide-rich cell walls. Considerable progress has been made in defining the structures of the surface and intracellular glycans of the parasitic protists, their biosynthesis and the role that individual components play in parasite infectivity.

  O-glycosylation, Glycosylphosphatidylinositol, N-glycosylation, protozoan parasites, Phosphoglycosylation

  Publication DOI: 10.1016/B978-0-12-374546-0.00012-2
  Publisher: Amsterdam: Elsevier
  Correspondence: malcolmm@unimelb.edu.au
  Editors: Moran A
  Institutions: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Australia
  
   
  
  Trypanosoma brucei
  CSDB ID 4567 (all data & tools)

Show legend Show as text

Structure type: oligomer
Aglycon: Asn58-protein
Trivial name: variant surface glycoprotein (VSG) MITat1.8
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_144983,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• Article ID: 5963
  Mehlert A, Sullivan L, Ferguson MAJ "Glycotyping of Trypanosoma brucei variant surface glycoprotein MITat1.8" - Molecular and Biochemical Parasitology 174(1) (2010) 74-77
  

  Following a switch from variant surface glycoprotein MITat1.4 to variant surface glycoprotein MITat1.8 expression by Lister strain 427 Trypanosoma brucei brucei parasites, the latter uncharacterized variant surface glycoprotein was analysed. Variant surface glycoprotein MITat1.8 was found to be a disulphide-linked homodimer, containing a complex N-linked glycan at Asn58 and a glycosylphosphatidylinositol membrane anchor attached to Asp419. Mass spectrometric analyses demonstrated that the N-glycan is exclusively Galβ1-4GlcNAcβ1-2Manα1-3(Galβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc and that the conserved Man3GlcN-myo-inositol glycosylphosphatidylinositol anchor glycan core is substituted with an average of 4 hexose, most likely galactose, residues. The presence of a complex N-glycan at Asn58 is consistent with the relatively acidic environment of the Asn58 N-glycosylation sequon, that predicts N-glycosylation by T. brucei oligosaccharyltransferase TbSTT3A with a Man5GlcNAc2 structure destined for processing to a paucimannose and/or complex N-glycan (Izquierdo L, Schulz B, Rodrigues JA et al. EMBO J 2009;28:2650–61 [12])

  mass spectrometry, Glycosylphosphatidylinositol, N-glycosylation, GPI, Trypanosoma brucei, N-linked oligosaccharides

  NCBI PubMed ID: 20558211
  Publication DOI: 10.1016/j.molbiopara.2010.06.007
  Journal NLM ID: 8006324
  Correspondence: M.A.J. Ferguson
  Institutions: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
  Methods: GC-MS, SDS-PAGE, ESI-MS, anion-exchange chromatography, Western blotting, MS/MS, MALDI-TOF MS, enzymatic digestion, HF treatment, permethylation, LC-MS
  
   
  
  Trypanosoma brucei 427, Trypanosoma brucei MITat1.8
  CSDB ID 7333 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 1622.7
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140116,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_141828,IEDB_144983,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_164174,IEDB_187201,IEDB_190606,IEDB_423128,IEDB_429156,IEDB_540672,IEDB_548907,IEDB_857734,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_77,SB_85,SB_88

• Article ID: 7480
  Hamilton BS, Wilson JD, Shumakovich MA, Fisher AC, Brooks JC, Pontes A, Naran R, Heiss C, Gao C, Kardish R, Heimburg-Molinaro J, Azadi P, Cummings RD, Merritt JH, DeLisa MP "A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors" - Scientific Reports 7(1) (2017) 15907
  

  Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, afnity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efciently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplifed purifcation scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases incding key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fuorescence-based screening of glycan-binding proteins. Taken together, these results confrm our combined synthesis strategy as a new, user-friendly route for supplying chemically defned human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

  NCBI PubMed ID: 29162910
  Publication DOI: 10.1038/s41598-017-15891-8
  Journal NLM ID: 101563288
  Publisher: London: Nature Publishing Group
  Correspondence: md255@cornell.edu
  Institutions: Complex Carbohydrate Research Center, The University of Georgia, Athens, USA, Glycobia, Inc., Ithaca, USA, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, USA
  Methods: 1H NMR, DNA techniques, MALDI-TOF MS, HPLC, extraction, enzymatic synthesis, glycan array analysis, hydrolysis
  
   
  
  Saccharomyces cerevisiae
  CSDB ID 43065 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 1987.9
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_140108,IEDB_140116,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_141828,IEDB_144983,IEDB_151079,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_153220,IEDB_164174,IEDB_187201,IEDB_190606,IEDB_423128,IEDB_429156,IEDB_540672,IEDB_548907,IEDB_857734,IEDB_983930,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_77,SB_85,SB_88

• Article ID: 7480
  Hamilton BS, Wilson JD, Shumakovich MA, Fisher AC, Brooks JC, Pontes A, Naran R, Heiss C, Gao C, Kardish R, Heimburg-Molinaro J, Azadi P, Cummings RD, Merritt JH, DeLisa MP "A library of chemically defined human N-glycans synthesized from microbial oligosaccharide precursors" - Scientific Reports 7(1) (2017) 15907
  

  Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, afnity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efciently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplifed purifcation scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases incding key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fuorescence-based screening of glycan-binding proteins. Taken together, these results confrm our combined synthesis strategy as a new, user-friendly route for supplying chemically defned human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

  NCBI PubMed ID: 29162910
  Publication DOI: 10.1038/s41598-017-15891-8
  Journal NLM ID: 101563288
  Publisher: London: Nature Publishing Group
  Correspondence: md255@cornell.edu
  Institutions: Complex Carbohydrate Research Center, The University of Georgia, Athens, USA, Glycobia, Inc., Ithaca, USA, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, USA
  Methods: 1H NMR, DNA techniques, MALDI-TOF MS, HPLC, extraction, enzymatic synthesis, glycan array analysis, hydrolysis
  
   
  
  Saccharomyces cerevisiae
  CSDB ID 43066 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2779,2921 [M+H]+
Aglycon: Pep2
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_1391962,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_142078,IEDB_143794,IEDB_144983,IEDB_150899,IEDB_151531,IEDB_152206,IEDB_153212,IEDB_190606,IEDB_423128,IEDB_540672,IEDB_548907,IEDB_983930,SB_137,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_29,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_88

• Article ID: 7877
  Cao L, Yu L, Guo Z, Shen A, Guo Y, Liang X "N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry" - Journal of Proteome Research 13(3) (2014) 1485-1493
  

  N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

  mass spectrometry, glycopeptide, Glycoproteomics, Saccharomyces cerevisiae, yeast, hydrophilic interaction liquid chromatography

  NCBI PubMed ID: 24527708
  Publication DOI: 10.1021/pr401049e
  Journal NLM ID: 101128775
  Publisher: Washington, DC: American Chemical Society
  Correspondence: Liang X
  Institutions: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
  Methods: enzymatic digestion, affinity chromatography, cell growth, HILIC, LC−MS, MALDI-QIT-TOF
  
   
  
  Saccharomyces cerevisiae
  CSDB ID 46351 (all data & tools)

Show legend Show as text

Structure type: oligomer ; 2925,2703 [M+H]+, 2957,2582 [M+H]+
Aglycon: Pep2; Pep1
Compound class: N-glycan
Contained glycoepitopes: IEDB_123886,IEDB_130646,IEDB_130701,IEDB_135813,IEDB_136044,IEDB_136045,IEDB_137340,IEDB_137472,IEDB_137485,IEDB_1391962,IEDB_140108,IEDB_140122,IEDB_141793,IEDB_141794,IEDB_141807,IEDB_142078,IEDB_142489,IEDB_143794,IEDB_144562,IEDB_144983,IEDB_150899,IEDB_151531,IEDB_152206,IEDB_152214,IEDB_153212,IEDB_174333,IEDB_190606,IEDB_423128,IEDB_490056,IEDB_540672,IEDB_548907,IEDB_983930,SB_137,SB_165,SB_166,SB_187,SB_195,SB_197,SB_198,SB_29,SB_30,SB_33,SB_44,SB_67,SB_7,SB_72,SB_73,SB_74,SB_85,SB_86,SB_88

• Article ID: 7877
  Cao L, Yu L, Guo Z, Shen A, Guo Y, Liang X "N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry" - Journal of Proteome Research 13(3) (2014) 1485-1493
  

  N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

  mass spectrometry, glycopeptide, Glycoproteomics, Saccharomyces cerevisiae, yeast, hydrophilic interaction liquid chromatography

  NCBI PubMed ID: 24527708
  Publication DOI: 10.1021/pr401049e
  Journal NLM ID: 101128775
  Publisher: Washington, DC: American Chemical Society
  Correspondence: Liang X
  Institutions: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
  Methods: enzymatic digestion, affinity chromatography, cell growth, HILIC, LC−MS, MALDI-QIT-TOF
  
   
  
  Saccharomyces cerevisiae
  CSDB ID 46353 (all data & tools)