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1.  Identification and Functional Characterization of a Highly Divergent N-Acetylglucosaminyltransferase I (TbGnTI) in Trypanosoma brucei 
The Journal of Biological Chemistry  2014;289(13):9328-9339.
Background: Trypanosoma brucei expresses a highly glycosylated surface coat that is essential for parasite survival.
Results: The T. brucei gene TbGT11 encodes an N-acetylglucosaminyltransferase I, the key enzyme for initiating the biosynthesis of complex N-glycans.
Conclusion: T. brucei GnTI is not a homologue of metazoan GnTI, but a highly divergent enzyme belonging to the β3-glycosyltransferase family.
Significance: Deeper understanding of T. brucei N-glycosylation pathway.
Trypanosoma brucei expresses a diverse repertoire of N-glycans, ranging from oligomannose and paucimannose structures to exceptionally large complex N-glycans. Despite the presence of the latter, no obvious homologues of known β1–4-galactosyltransferase or β1–2- or β1–6-N-acetylglucosaminyltransferase genes have been found in the parasite genome. However, we previously reported a family of putative UDP-sugar-dependent glycosyltransferases with similarity to the mammalian β1–3-glycosyltransferase family. Here we characterize one of these genes, TbGT11, and show that it encodes a Golgi apparatus resident UDP-GlcNAc:α3-d-mannoside β1–2-N-acetylglucosaminyltransferase I activity (TbGnTI). The bloodstream-form TbGT11 null mutant exhibited significantly modified protein N-glycans but normal growth in vitro and infectivity to rodents. In contrast to multicellular organisms, where the GnTI reaction is essential for biosynthesis of both complex and hybrid N-glycans, T. brucei TbGT11 null mutants expressed atypical “pseudohybrid” glycans, indicating that TbGnTII activity is not dependent on prior TbGnTI action. Using a functional in vitro assay, we showed that TbGnTI transfers UDP-GlcNAc to biantennary Man3GlcNAc2, but not to triantennary Man5GlcNAc2, which is the preferred substrate for metazoan GnTIs. Sequence alignment reveals that the T. brucei enzyme is far removed from the metazoan GnTI family and suggests that the parasite has adapted the β3-glycosyltransferase family to catalyze β1–2 linkages.
doi:10.1074/jbc.M114.555029
PMCID: PMC3979372  PMID: 24550396
Glycobiology; Glycosyltransferases; Parasite; Post-translational Modification; Trypanosoma brucei; N-Acetylglucosamine
2.  Structure of a Complex Phosphoglycan Epitope from gp72 of Trypanosoma cruzi * 
The Journal of Biological Chemistry  2013;288(16):11093-11105.
Background: Trypanosoma cruzi expresses a highly immunogenic carbohydrate epitope in glycoprotein gp72.
Results: The epitope structure was solved using NMR and mass spectrometry and shown to be a phosphosaccharide, containing l-rhamnopyranose, l-fucopyranose, d-galactopyranose, d-galactofuranose, d-xylopyranose, and N-acetylglucosamine.
Conclusion: This is one of the most complex eukaryotic protein-linked carbohydrate structures yet described.
Significance: gp72 has been implicated in parasite differentiation, flagellar adhesion, and insect infectivity.
The parasitic protozoan organism Trypanosoma cruzi is the causative agent of Chagas disease. The insect vector-dwelling epimastigote form of the organism expresses a low abundance glycoprotein associated with the flagellum adhesion zone, called gp72. The gp72 glycoprotein was first identified with an anti-carbohydrate IgG3 monoclonal antibody called WIC29.26 and has been shown to have an unusual sugar composition. Here, we describe a new way to isolate the WIC29.26 carbohydrate epitope of gp72. Using 1H NMR and mass spectrometry before and after derivatization, we provide an almost complete primary chemical structure for the epitope, which is that of a complex phosphosaccharide: Galfβ1–4Rhapα1–2Fucpα1-4(Galpβ1–3)(Galpα1–2)Xylpβ1–4Xylpβ1–3(Xylpβ1–2Galpα1-4(Galpβ1–3)(Rhapα1–2)Fucpα1–4)GlcNAcp, with phosphate attached to one or other of the two Galp terminal residues and in which all residues are of the d-absolute configuration, except for fucose and rhamnose which are l. Combined with previous data (Haynes, P. A., Ferguson, M. A., and Cross, G. A. (1996) Glycobiology 6, 869–878), we postulate that this complex structure and its variants lacking one or more residues are linked to Thr and Ser residues in gp72 via a phosphodiester linkage (GlcNAcpα1-P-Thr/Ser) and that these units may form phosphosaccharide repeats through GlcNAcpα1-P-Galp linkages. The gp72 glycoprotein is associated with the flagellum adhesion zone on the parasite surface, and its ligation has been implicated in inhibiting parasite differentiation from the epimastigote to the metacyclic trypomastigote stage. The detailed structure of the unique phosphosaccharide component of gp72 reported here provides a template for future biosynthetic and functional studies.
doi:10.1074/jbc.M113.452763
PMCID: PMC3630849  PMID: 23436655
Carbohydrate Glycoprotein; Carbohydrate Structure; Glycoprotein Structure; Glycosylation; Parasitology; Trypanosoma cruzi; Carbohydrate Epitope; Galactofuranose; gp72; Phosphosaccharide
3.  Modeling of the N-Glycosylated Transferrin Receptor Suggests How Transferrin Binding Can Occur within the Surface Coat of Trypanosoma brucei 
PLoS Pathogens  2012;8(4):e1002618.
The transferrin receptor of bloodstream form Trypanosoma brucei is a heterodimer encoded by expression site associated genes 6 and 7. This low-abundance glycoprotein with a single glycosylphosphatidylinositol membrane anchor and eight potential N-glycosylation sites is located in the flagellar pocket. The receptor is essential for the parasite, providing its only source of iron by scavenging host transferrin from the bloodstream. Here, we demonstrate that both receptor subunits contain endoglycosidase H-sensitive and endoglycosidase H-resistant N-glycans. Lectin blotting of the purified receptor and structural analysis of the released N-glycans revealed oligomannose and paucimannose structures but, contrary to previous suggestions, no poly-N-acetyllactosamine structures were found. Overlay experiments suggest that the receptor can bind to other trypanosome glycoproteins, which may explain this discrepancy. Nevertheless, these data suggest that a current model, in which poly-N-acetyllactosamine glycans are directly involved in receptor-mediated endocytosis in bloodstream form Trypanosoma brucei, should be revised. Sequential endoglycosidase H and peptide-N-glycosidase F treatment, followed by tryptic peptide analysis, allowed the mapping of oligomannose and paucimannose structures to four of the receptor N-glycosylation sites. These results are discussed with respect to the current model for protein N-glycosylation in the parasite. Finally, the glycosylation data allowed the creation of a molecular model for the parasite transferrin receptor. This model, when placed in the context of a model for the dense variant surface glycoprotein coat in which it is embedded, suggests that receptor N-glycosylation may play an important role in providing sufficient space for the approach and binding of transferrin to the receptor, without significantly disrupting the continuity of the protective variant surface glycoprotein coat.
Author Summary
The tsetse fly transmitted parasite that causes human African trypanosomiasis, or sleeping sickness, scavenges iron from the bloodstream of the infected individual so that it can live, multiply and ultimately cause disease. To do this, it places a glycoprotein (a protein with carbohydrate chains attached) called the transferrin receptor on its surface to capture circulating human transferrin, an iron transport protein. It then internalizes transferrin receptor/transferrin complex and digests the transferrin part, releasing the iron for its own use. By analyzing the parasite transferrin receptor, we have been able to describe the carbohydrate chains of the transferrin receptor and thus complete a molecular model of this important glycoprotein. We have further built models of how we expect this low abundance glycoprotein will sit in the surface coat of the parasite, which is made of millions of copies of another glycoprotein. The results provide a ‘molecule's eye view’ of how the carbohydrate chains of the transferrin receptor provide the space necessary for the transferrin to bind to it without disrupting the protective coat.
doi:10.1371/journal.ppat.1002618
PMCID: PMC3320590  PMID: 22496646
4.  The lipid-linked oligosaccharide donor specificities of Trypanosoma brucei oligosaccharyltransferases 
Glycobiology  2012;22(5):696-703.
We recently presented a model for site-specific protein N-glycosylation in Trypanosoma brucei whereby the TbSTT3A oligosaccharyltransferase (OST) first selectively transfers biantennary Man5GlcNAc2 from the lipid-linked oligosaccharide (LLO) donor Man5GlcNAc2-PP-Dol to N-glycosylation sequons in acidic to neutral peptide sequences and TbSTT3B selectively transfers triantennary Man9GlcNAc2 to any remaining sequons. In this paper, we investigate the specificities of the two OSTs for their preferred LLO donors by glycotyping the variant surface glycoprotein (VSG) synthesized by bloodstream-form T. brucei TbALG12 null mutants. The TbALG12 gene encodes the α1-6-mannosyltransferase that converts Man7GlcNAc2-PP-Dol to Man8GlcNAc2-PP-Dol. The VSG synthesized by the TbALG12 null mutant in the presence and the absence of α-mannosidase inhibitors was characterized by electrospray mass spectrometry both intact and as pronase glycopetides. The results show that TbSTT3A is able to transfer Man7GlcNAc2 as well as Man5GlcNAc2 to its preferred acidic glycosylation site at Asn263 and that, in the absence of Man9GlcNAc2-PP-Dol, TbSTT3B transfers both Man7GlcNAc2 and Man5GlcNAc2 to the remaining site at Asn428, albeit with low efficiency. These data suggest that the preferences of TbSTT3A and TbSTT3B for their LLO donors are based on the c-branch of the Man9GlcNAc2 oligosaccharide, such that the presence of the c-branch prevents recognition and/or transfer by TbSTT3A, whereas the presence of the c-branch enhances recognition and/or transfer by TbSTT3B.
doi:10.1093/glycob/cws003
PMCID: PMC3311286  PMID: 22241825
N-glycosylation; oligosaccharyltransferase; STT3; Trypanosoma brucei
5.  Chemical Structure of Trichomonas vaginalis Surface Lipoglycan 
The Journal of Biological Chemistry  2011;286(47):40494-40508.
Background: Trichomonas vaginalis lipoglycan (TvLG) mediates interactions between the parasite and human host.
Results: TvLG is composed of a polyrhamnose backbone with branches of poly-N-acetyllactosamine that are involved in attachment to host epithelium.
Conclusion: TvLG has a unique structure among solved parasite glycans.
Significance: This work provides a template to analyze TvLG from T. vaginalis with different binding properties.
The extracellular parasite Trichomonas vaginalis contains a surface glycoconjugate that appears to mediate parasite-host cell interaction via binding to human galectin-1. This glycoconjugate also elicits cytokine production from human vaginal epithelial cells, implicating its role in modulation of host immune responses. We have analyzed the structure of this glycoconjugate, previously described to contain the sugars rhamnose (Rha), N-acetylglucosamine (GlcNAc), galactose (Gal), xylose (Xyl), N-acetylgalactosamine (GalNAc), and glucose (Glc), using gas chromatograph mass spectrometry (GC-MS), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF), electrospray MS/MS, and nuclear magnetic resonance (NMR), combined with chemical and enzymatic digestions. Our data reveal a complex structure, named T. vaginalis lipoglycan (TvLG), that differs markedly from Leishmania lipophosphoglycan and Entamoeba lipopeptidophosphoglycan and is devoid of phosphosaccharide repeats. TvLG is composed of an α1–3 linked polyrhamnose core, where Rha residues are substituted at the 2-position with either β-Xyl or chains of, on average, five N-acetyllactosamine (-3Galβ1–4GlcNAcβ1-) (LacNAc) units and occasionally lacto-N-biose (-3Galβ1-3GlcNAcβ1-) (LNB). These chains are themselves periodically substituted at the Gal residues with Xyl-Rha. These structural analyses led us to test the role of the poly-LacNAc/LNB chains in parasite binding to host cells. We found that reduction of poly-LacNAc/LNB chains decreased the ability of TvLG to compete parasite binding to host cells. In summary, our data provide a new model for the structure of TvLG, composed of a polyrhamnose backbone with branches of Xyl and poly-LacNAc/LNB. Furthermore, the poly-LacNAc side chains are shown to be involved in parasite-host cell interaction.
doi:10.1074/jbc.M111.280578
PMCID: PMC3220458  PMID: 21900246
Adhesion; Glycoconjugate; Glycolipid Structure; Host-Pathogen Interactions; Pathogenesis; Trichomonas; TvLG
6.  Glycotyping of Trypanosoma brucei variant surface glycoprotein MITat1.8 
Graphical abstract
VSG MITat1.8 was characterized with respect to its N-glycosylation, GPI anchor structure and found to be a disulfide-linked homodimer.
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]).
doi:10.1016/j.molbiopara.2010.06.007
PMCID: PMC2935967  PMID: 20558211
Trypanosoma brucei; N-linked oligosaccharides; N-glycosylation; Glycosylphosphatidylinositol; GPI; Mass spectrometry
7.  Application of electrospray mass spectrometry to the structural determination of glycosylphosphatidylinositol membrane anchors 
Glycobiology  2010;20(5):576-585.
The addition of glycosylphosphatidylinositol (GPI) anchors to proteins is an important posttranslational modification in eukaryotic cells. The complete structural elucidation of GPI anchors is a complex process that requires relatively large amounts of starting material. In this paper, we assess the degree of structural information that can be obtained by applying electrospray mass spectrometry and tandem mass spectrometry to permethylated GPI glycans prepared from a well-characterized GPI-anchored glycoprotein, the variant surface glycoprotein from Trypanosoma brucei. All GPI glycans contain a non-N-acetylated glucosamine residue, and permethylation leads to the formation of a fixed positive charge on the glycans, in the form of a quaternary amine. The permethylated glycans were detected as [M +- Na]2+- ions, and tandem mass spectrometry of these ions produced substantial, albeit incomplete, structural information on the branching patterns and linkage types for various GPI glycoforms of the variant surface glycoprotein.
doi:10.1093/glycob/cwq007
PMCID: PMC2850939  PMID: 20100693
glycosylphosphatidylinositol; GPI anchor; mass spectrometry; Trypanosoma brucei; variant surface glycoprotein
8.  Proteomic scale high-sensitivity analyses of GPI membrane anchors 
Glycoconjugate Journal  2008;26(8):915-921.
Glycosylphosphatidylinositol (GPI) anchored proteins are ubiquitous in eukaryotic cells. Earlier analysis methods required large amounts of purified protein to elucidate the structure of the GPI. This paper describes methods for analyzing GPIs on a ‘proteomic’ scale. Partially purified proteins may be run on sodium dodecyl sulphate polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride (PVDF) membrane. Following identification of the protein the piece of PVDF may be subjected to various chemical treatments, which are specific for GPI structures. The first method uses gas chromatography–mass spectrometry and it enables the presence of a GPI anchor to be confirmed. The second method depends on the cleavage of phosphate bonds and permits the carbohydrate structure to be elucidated by electrospray or matrix assisted laser desorption ionization-time of flight mass spectrometry. The final method described uses deamination of the glucosamine residue to release the lipid moiety for analysis by mass spectrometry.
doi:10.1007/s10719-008-9116-x
PMCID: PMC2791486  PMID: 18330699
Glycosylphosphatidylinositol (GPI); Mass spectrometry; Proteomic techniques
9.  The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness 
The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that phosphorylation-based signaling is a general and fundamental regulatory process that extends to this highly divergent lower eukaryote.
doi:10.1074/mcp.M800556-MCP200
PMCID: PMC2716717  PMID: 19346560
10.  Glycosylation Defects and Virulence Phenotypes of Leishmania mexicana Phosphomannomutase and Dolicholphosphate-Mannose Synthase Gene Deletion Mutants 
Molecular and Cellular Biology  2001;21(23):8168-8183.
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (ΔPMM and ΔDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana ΔDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana ΔPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
doi:10.1128/MCB.21.23.8168-8183.2001
PMCID: PMC99981  PMID: 11689705

Results 1-10 (10)