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Gloria Rudenko - One of the best experts on this subject based on the ideXlab platform.
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tbsap is a novel chromatin protein repressing metacyclic Variant Surface Glycoprotein expression sites in bloodstream form trypanosoma brucei
Nucleic Acids Research, 2021Co-Authors: Carys Davies, Bill Wickstead, Cherpheng Ooi, Georgios Sioutas, Belinda S Hall, Haneesh Sidhu, Falk Butter, Sam Alsford, Gloria RudenkoAbstract:The African trypanosome Trypanosoma brucei is a unicellular eukaryote, which relies on a protective Variant Surface Glycoprotein (VSG) coat for survival in the mammalian host. A single trypanosome has >2000 VSG genes and pseudogenes of which only one is expressed from one of ∼15 telomeric bloodstream form expression sites (BESs). Infectious metacyclic trypanosomes present within the tsetse fly vector also express VSG from a separate set of telomeric metacyclic ESs (MESs). All MESs are silenced in bloodstream form T. brucei. As very little is known about how this is mediated, we performed a whole genome RNAi library screen to identify MES repressors. This allowed us to identify a novel SAP domain containing DNA binding protein which we called TbSAP. TbSAP is enriched at the nuclear periphery and binds both MESs and BESs. Knockdown of TbSAP in bloodstream form trypanosomes did not result in cells becoming more 'metacyclic-like'. Instead, there was extensive global upregulation of transcripts including MES VSGs, VSGs within the silent VSG arrays as well as genes immediately downstream of BES promoters. TbSAP therefore appears to be a novel chromatin protein playing an important role in silencing the extensive VSG repertoire of bloodstream form T. brucei.
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Mechanistic and Functional Studies of Proteins Maintaining the protective Variant Surface Glycoprotein coat of African trypanosomes
2020Co-Authors: Gloria RudenkoAbstract:Abstract The African trypanosome Trypanosoma brucei has a precarious existence as an extracellular parasite of the mammalian bloodstream, where it is faced with continuous immune attack. Key to survival is a dense VSG (Variant Surface Glycoprotein) coat, which is repeatedly switched during the course of a chronic infection. New data demonstrate a link between VSG synthesis and cell cycle progression, indicating that VSG is monitored during the trypanosome cell cycle
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blocking Variant Surface Glycoprotein synthesis alters endoplasmic reticulum exit sites golgi homeostasis in trypanosoma brucei
Traffic, 2018Co-Authors: Cherpheng Ooi, Terry K Smith, Eva Gluenz, Nadina Vasileva Wand, Sue Vaughan, Gloria RudenkoAbstract:The predominant secretory cargo of bloodstream form Trypanosoma brucei is Variant Surface Glycoprotein (VSG), comprising ~10% total protein and forming a dense protective layer. Blocking VSG translation using Morpholino oligonucleotides triggered a precise pre-cytokinesis arrest. We investigated the effect of blocking VSG synthesis on the secretory pathway. The number of Golgi decreased, particularly in post-mitotic cells, from 3.5 ± 0.6 to 2.0 ± 0.04 per cell. Similarly, the number of endoplasmic reticulum exit sites (ERES) in post-mitotic cells dropped from 3.9 ± 0.6 to 2.7 ± 0.1 eight hours after blocking VSG synthesis. The secretory pathway was still functional in these stalled cells, as monitored using Cathepsin L. Rates of phospholipid and glycosylphosphatidylinositol-anchor biosynthesis remained relatively unaffected, except for the level of sphingomyelin which increased. However, both endoplasmic reticulum and Golgi morphology became distorted, with the Golgi cisternae becoming significantly dilated, particularly at the trans-face. Membrane accumulation in these structures is possibly caused by reduced budding of nascent vesicles due to the drastic reduction in the total amount of secretory cargo, that is, VSG. These data argue that the total flux of secretory cargo impacts upon the biogenesis and maintenance of secretory structures and organelles in T. brucei, including the ERES and Golgi.
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the role of genomic location and flanking 3 utr in the generation of functional levels of Variant Surface Glycoprotein in trypanosoma brucei
Molecular Microbiology, 2017Co-Authors: Sophie Ridewood, Cherpheng Ooi, Nadina Vasileva Wand, Georgios Sioutas, Belinda S Hall, Anna Trenaman, Iris Scherwitzl, Gloria RudenkoAbstract:Summary Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I-transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16-mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I-transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell-cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell-cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I-transcribed ES, as well as conserved VSG 3′UTR 16-mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei.
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blocking Variant Surface Glycoprotein synthesis in trypanosoma brucei triggers a general arrest in translation initiation
PLOS ONE, 2009Co-Authors: Terry K Smith, Mark Carrington, Eva Gluenz, Keith Gull, Nadina Vasileva, Stephen J Terry, Neil Portman, Susanne Kramer, Shulamit Michaeli, Gloria RudenkoAbstract:Background: The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. Methodology/Principal Findings: Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. Conclusions: The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell Surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.
Etienne Pays - One of the best experts on this subject based on the ideXlab platform.
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transcription is initiated on silent Variant Surface Glycoprotein expression sites despite monoallelic expression in trypanosoma brucei
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Ali Kassem, Etienne Pays, Luc VanhammeAbstract:African trypanosomes survive the immune defense of their hosts by regularly changing their antigenic coat made of Variant Surface Glycoprotein (VSG). The Trypanosoma brucei genome contains more than 1,000 VSG genes. To be expressed, a given VSG gene must be located in one of 15 telomeric regions termed "VSG expression sites" (ESs), each of which contains a polycistronic transcription unit that includes ES-associated genes. Only one ES is fully active at a time, so only one VSG gene is transcribed per cell. Although this monoallelic expression is controlled at the transcriptional level, the precise molecular mechanism for this control is not understood. Here we report that in single cells transcription is initiated on several ESs simultaneously, indicating that the monoallelic control is not determined only at transcription initiation, but also at further control steps such as transcription elongation or RNA processing.
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the Variant Surface Glycoprotein as a tool for adaptation in african trypanosomes
Microbes and Infection, 2006Co-Authors: Etienne PaysAbstract:African trypanosomes (prototype: Trypanosoma brucei) are flagellated protozoan parasites that infect a wide variety of mammals, causing nagana in cattle and sleeping sickness in humans. These organisms can cause prolonged chronic infections due to their ability to successively expose different antigenic Variants of the Variant Surface Glycoprotein (VSG). The genomic loci where the VSG genes are expressed are telomeric and contain polycistronic transcription units with several genes that are involved in adaptation of the parasite to the host. At least three of these genes, which respectively encode the two subunits of the heterodimeric receptor for transferrin and a protein conferring resistance to the human trypanolytic factor apolipoprotein L-I, share the same origin as the VSG. The high recombination potential of the telomeric VSG expression sites, coupled to their dynamic mono-allelic expression control, provides trypanosomes with a powerful capacity for adaptation to their hosts.
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control and function of the bloodstream Variant Surface Glycoprotein expression sites in trypanosoma brucei
International Journal for Parasitology, 2001Co-Authors: Luc Vanhamme, Laurence Lecordier, Etienne PaysAbstract:African trypanosomes escape the host immune response through a periodical change of their Surface coat made of one major type of protein, the Variant Surface Glycoprotein. From a repertoire of a thousand Variant Surface Glycoprotein genes available, only one is expressed at a time, and this takes place in a specialised expression site itself selected from a collection of an estimated 20-30 sites. As the specialised expression sites are long polycistronic transcription units, the Variant Surface Glycoprotein is co-transcribed with several other genes termed expression site-associated genes. How do the trypanosomes only use a single specialised expression site at a time? Why are there two dozen specialised expression sites? What are the functions of the other genes of these transcription units? We review the currently available answers to these questions.
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differential rna elongation controls the Variant Surface Glycoprotein gene expression sites of trypanosoma brucei
Molecular Microbiology, 2000Co-Authors: Luc Vanhamme, Annette Pays, Patricia Tebabi, Philippe Poelvoorde, Hoang Van Xong, Etienne PaysAbstract:The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of Variant Surface Glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different Variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.
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a single stranded dna binding protein shared by telomeric repeats the Variant Surface Glycoprotein transcription promoter and the procyclin transcription terminator of trypanosoma brucei
Nucleic Acids Research, 2000Co-Authors: Magali Berberof, Patricia Tebabi, Stéphane Lips, Luc Vanhamme, Sylvie Alexandre, Etienne PaysAbstract:In Trypanosoma brucei the genes are organised into long polycistronic transcription units and only three promoters for protein-encoding genes and a single terminator have been characterised. These promoters recruit a polI-like RNA polymerase for the transcription units encoding the two major stage-specific antigens of the parasite, the Variant Surface Glycoprotein (VSG) of the bloodstream form and procyclin of the insect-specific procyclic form, while the terminator is that of a procyclin transcription unit. By deletional and mutational analysis we defined the two DNA sequences essential for the activity of the VSG promoter from a bloodstream form transcription unit and one of the functional elements of the procyclin terminator. These three short sequences are similar, and their C-rich strand binds the same protein of 40 kDa. In addition, this factor also binds to the C-rich strand of the telomeric repeats, the consensus target sequence being 5′-CCCTNN-3′. The factor-binding sequences are functionally interchangeable in chimeric promoter or terminator constructs, although additional elements are required for full activity.
Mark Carrington - One of the best experts on this subject based on the ideXlab platform.
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structural basis for the shielding function of the dynamic trypanosome Variant Surface Glycoprotein coat
Nature microbiology, 2017Co-Authors: Thomas Bartossek, Nicola G Jones, Christin Schafer, Mislav Cvitkovic, Marius Glogger, Helen R Mott, Jochen Kuper, Martha Brennich, Mark Carrington, Anasuncana SmithAbstract:The most prominent defence of the unicellular parasite Trypanosoma brucei against the host immune system is a dense coat that comprises a Variant Surface Glycoprotein (VSG). Despite the importance of the VSG family, no complete structure of a VSG has been reported. Making use of high-resolution structures of individual VSG domains, we employed small-angle X-ray scattering to elucidate the first two complete VSG structures. The resulting models imply that the linker regions confer great flexibility between domains, which suggests that VSGs can adopt two main conformations to respond to obstacles and changes of protein density, while maintaining a protective barrier at all times. Single-molecule diffusion measurements of VSG in supported lipid bilayers substantiate this possibility, as two freely diffusing populations could be detected. This translates into a highly flexible overall topology of the Surface VSG coat, which displays both lateral movement in the plane of the membrane and variation in the overall thickness of the coat.
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chaperone requirements for biosynthesis of the trypanosome Variant Surface Glycoprotein
PLOS ONE, 2010Co-Authors: Mark C Field, Yanan Wang, Tatiana Sergeenko, Susanne Bohm, Mark CarringtonAbstract:BACKGROUND Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER) stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC) of ER protein folding and export may be present in trypanosomes. As the Variant Surface Glycoprotein (VSG) represents approximately 90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden. METHODOLOGY/PRINCIPAL FINDINGS We demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as approximately 2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation. CONCLUSIONS/SIGNIFICANCE The retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only approximately 30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient evolutionary origin, and was present in the last common eukaryotic ancestor.
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blocking Variant Surface Glycoprotein synthesis in trypanosoma brucei triggers a general arrest in translation initiation
PLOS ONE, 2009Co-Authors: Terry K Smith, Mark Carrington, Eva Gluenz, Keith Gull, Nadina Vasileva, Stephen J Terry, Neil Portman, Susanne Kramer, Shulamit Michaeli, Gloria RudenkoAbstract:Background: The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. Methodology/Principal Findings: Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. Conclusions: The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell Surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.
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structure of a glycosylphosphatidylinositol anchored domain from a trypanosome Variant Surface Glycoprotein
Journal of Biological Chemistry, 2007Co-Authors: Nicola G Jones, Helen R Mott, Daniel Nietlispach, Reuben Sunil Kumar Sharma, David F Burke, Isobel Eyres, Marsilius Mues, Mark CarringtonAbstract:The cell Surface of African trypanosomes is covered by a densely packed monolayer of a single protein, the Variant Surface Glycoprotein (VSG). The VSG protects the trypanosome cell Surface from effector molecules of the host immune system and is the mediator of antigenic variation. The sequence divergence between VSGs that is necessary for antigenic variation can only occur within the constraints imposed by the structural features necessary to form the monolayer barrier. Here, the structures of the two domains that together comprise the C-terminal di-domain of VSG ILTat1.24 have been determined. The first domain has a structure similar to the single C-terminal domain of VSG MITat1.2 and provides proof of structural conservation in VSG C-terminal domains complementing the conservation of structure present in the N-terminal domain. The second domain, although based on the same fold, is a minimized version missing several structural features. The structure of the second domain contains the C-terminal residue that in the native VSG is attached to a glycosylphosphatidylinositol (GPI) anchor that retains the VSG on the external face of the plasma membrane. The solution structures of this domain and a VSG GPI glycan have been combined to produce the first structure-based model of a GPI-anchored protein. The model suggests that the core glycan of the GPI anchor lies in a groove on the Surface of the domain and that there is a close association between the GPI glycan and protein. More widely, the GPI glycan may be an integral part of the structure of other GPI-anchored proteins.
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Variant Surface Glycoprotein gene repertoires in trypanosoma brucei have diverged to become strain specific
BMC Genomics, 2007Co-Authors: Clyde O Hutchinson, Nicola G Jones, Helen R Mott, Reuben Sunil Kumar Sharma, Kim Picozzi, Susan C Welburn, Mark CarringtonAbstract:In a mammalian host, the cell Surface of African trypanosomes is protected by a monolayer of a single Variant Surface Glycoprotein (VSG). The VSG is central to antigenic variation; one VSG gene is expressed at any one time and there is a low frequency stochastic switch to expression of a different VSG gene. The genome of Trypanosoma brucei contains a repertoire of > 1000 VSG sequences. The degree of conservation of the genomic VSG repertoire in different strains has not been investigated in detail. Eighteen expressed VSGs from Ugandan isolates were compared with homologues (> 40 % sequence identity) in the two available T. brucei genome sequences. Fourteen homologues were present in the genome of Trypanosoma brucei brucei TREU927 from Kenya and fourteen in the genome of T. b. gambiense Dal972 from Cote d'Ivoire. The Ugandan VSGs averaged 71% and 73 % identity to homologues in T. b. brucei and T. b. gambiense respectively. The sequence divergence between homologous VSGs from the three different strains was not random but was more prevalent in the parts of the VSG believed to interact with the host immune system on the living trypanosome. It is probable that the VSG repertoires in the different isolates contain many common VSG genes. The location of divergence between VSGs is consistent with selection for strain-specific VSG repertoires, possibly to allow superinfection of an animal by a second strain. A consequence of strain-specific VSG repertoires is that any vaccine based on large numbers of VSGs from a single strain will only provide partial protection against other strains.
Michael A. J. Ferguson - One of the best experts on this subject based on the ideXlab platform.
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glycotyping of trypanosoma brucei Variant Surface Glycoprotein mitat1 8
Molecular and Biochemical Parasitology, 2010Co-Authors: Angela Mehlert, Lauren Sullivan, Michael A. J. FergusonAbstract: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]).
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deletion of the glucosidase ii gene in trypanosoma brucei reveals novel n glycosylation mechanisms in the biosynthesis of Variant Surface Glycoprotein
Journal of Biological Chemistry, 2005Co-Authors: Deuan C Jones, Angela Mehlert, Lucia M S Guther, Michael A. J. FergusonAbstract:The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the Variant Surface Glycoprotein, the main Glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcα1–3Manα1–2Manα1–2Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc, Glcα1–3Manα1–2Manα1–2Manα1–3(GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc, and Glcα1–3Manα1–2Manα1–2Manα1–3(Galβ1–4GlcNAcβ1–2Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid α-mannosidase processing was ruled out using a mixture of α-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the Variant Surface Glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: Glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man4–3GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.
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the glycoforms of a trypanosoma brucei Variant Surface Glycoprotein and molecular modeling of a glycosylated Surface coat
Glycobiology, 2002Co-Authors: Angela Mehlert, Charles S Bond, Michael A. J. FergusonAbstract:The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective Surface coat. This Surface coat is a monolayer of five million Variant Surface Glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The Surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode Glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the Glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the Surface coat.
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structure of the glycosylphosphatidylinositol membrane anchor glycan of a class 2 Variant Surface Glycoprotein from trypanosoma brucei
Journal of Molecular Biology, 1998Co-Authors: Angela Mehlert, Julia M Richardson, Michael A. J. FergusonAbstract:Abstract The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 Variant Surface Glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen fluoride dephosphorylation and nitrous acid deamination of the purified Glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-filtration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Manα1-2Manα1-6Man and a complex α-galactose branch of up to Galα1-2Galα1-6(Galα1-2)Gal, but most also contained an additional galactose residue attached α1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached β1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone.
Piet Borst - One of the best experts on this subject based on the ideXlab platform.
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delineation of the regulated Variant Surface Glycoprotein gene expression site domain of trypanosoma brucei
Molecular and Biochemical Parasitology, 2003Co-Authors: Karen Sheader, Magali Berberof, Piet Borst, Tomoko Isobe, Gloria RudenkoAbstract:The African trypanosome Trypanosoma brucei is protected in the bloodstream of the mammalian host by a dense Variant Surface Glycoprotein (VSG) coat. Although an individual cell has hundreds of VSG genes, the active VSG is transcribed in a mutually exclusive fashion from one of about twenty telomeric VSG expression sites. Expression sites are regulated domains flanked by 50 bp repeat arrays and extensive tracts of repetitive elements. We have integrated exogenous rDNA and expression site promoters upstream of the 50 bp repeats of the VO2 VSG expression site. Transcription from both types of exogenous promoter is downregulated and comparable to promoters targeted into the VSG Basic Copy arrays. We show that the upstream exogenous rDNA promoter escapes VSG expression site control, as switching the downstream VO2 VSG expression site on and off does not affect its activity. Therefore, the 50 bp repeat arrays appear to be the boundary of the regulated expression site domain.
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expression site activation in trypanosoma brucei with three marked Variant Surface Glycoprotein gene expression sites
Molecular and Biochemical Parasitology, 2002Co-Authors: Sebastian Ulbert, Ines Chaves, Piet BorstAbstract:The genes for the Variant Surface Glycoprotein (VSG) of Trypanosoma brucei are transcribed in telomeric expression sites (ESs). There are about 20 different ESs per trypanosome nucleus. Usually, only one is active at a time, but trypanosomes can switch the ES that is active at a low rate ( 10−1 per cell per generation). Unstable triple-resistant trypanosomes were not obtained. We conclude that the unstable rapid-switching state is a natural intermediate in ES switching. It only involves two ESs, whereas the other ESs are not expressed. Furthermore, we show that ‘inactive’ ESs can exist at several different stable levels of activation. Whereas, a ‘silent’ ES shows a low level of expression of promoter proximal sequences, the level of activation can be reversibly increased, leading to partially activated ESs.
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control of Variant Surface Glycoprotein gene expression sites in trypanosoma brucei
The EMBO Journal, 1999Co-Authors: Ines Chaves, Gloria Rudenko, Anita Dirksmulder, Mike Cross, Piet BorstAbstract:Trypanosoma brucei has 20 similar telomeric‐expression sites for Variant Surface Glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug‐resistance genes and selected these on agarose plates containing both drugs. Double‐resistant clones arose at a low frequency of 10 −7 per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double‐resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a ‘pre‐active’ silent state from which they can be readily activated.
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selection for activation of a new Variant Surface Glycoprotein gene expression site in trypanosoma brucei can result in deletion of the old one
Molecular and Biochemical Parasitology, 1998Co-Authors: Gloria Rudenko, Ines Chaves, Anita Dirksmulder, Piet BorstAbstract:The African trypanosome Trypanosoma brucei expresses the active Variant Surface Glycoprotein (VSG) gene in a telomeric VSG gene expression site. We have generated trypanosomes with a neomycin resistance gene inserted behind an active VSG gene expression site promoter, and a hygromycin resistance gene behind a silent one. By alternating drug selection, we could select for trypanosomes that had switched between the two marked VSG gene expression sites. Surprisingly, trypanosomes that had activated a new VSG gene expression site had often lost the old one. Using polymerase chain reaction (PCR), we screened large numbers of switched trypanosomes and found that sequences lost invariably included the drug marker near the promoter, as well as the telomeric VSG gene many tens of kilobases away. We postulate that stable activation of a new expression site requires silencing of the old one. If silencing does not occur at a sufficient rate by normal switch-off, stable activation of the new site can only occur if the old site is lost in random deletion events. The fact that we pick up these normally infrequent deletions, indicates that inactivation of the old VSG expression site could be rate limiting during switching in our strain of T. brucei.
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analysis of a Variant Surface Glycoprotein gene expression site promoter of trypanosoma brucei by remodelling the promoter region
Molecular and Biochemical Parasitology, 1998Co-Authors: Patricia A Blundell, Piet BorstAbstract:Abstract Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its Variant Surface Glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3′ of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5′ of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5′ ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5′ of an ES promoter is required for optimal functioning of an ES.
