Variant-specific Surface Glycoprotein Research Articles

Affinity Is an Important Determinant of the Anti-Trypanosome Activity of Nanobodies

BackgroundThe discovery of Nanobodies (Nbs) with a direct toxic activity against African trypanosomes is a recent advancement towards a new strategy against these extracellular parasites. The anti-trypanosomal activity relies on perturbing the highly active recycling of the Variant-specific Surface Glycoprotein (VSG) that occurs in the parasite's flagellar pocket.Methodology/Principal FindingsHere we expand the existing panel of Nbs with anti-Trypanosoma brucei potential and identify four categories based on their epitope specificity. We modified the binding properties of previously identified Nanobodies Nb_An05 and Nb_An33 by site-directed mutagenesis in the paratope and found this to strongly affect trypanotoxicity despite retention of antigen-targeting properties. Affinity measurements for all identified anti-trypanosomal Nbs reveal a strong correlation between trypanotoxicity and affinity (KD), suggesting that it is a crucial determinant for this activity. Half maximal effective (50%) affinity of 57 nM was calculated from the non-linear dose-response curves. In line with these observations, Nb humanizing mutations only preserved the trypanotoxic activity if the KD remained unaffected.Conclusions/SignificanceThis study reveals that the binding properties of Nanobodies need to be compatible with achieving an occupancy of >95% saturation of the parasite surface VSG in order to exert an anti-trypanosomal activity. As such, Nb-based approaches directed against the VSG target would require binding to an accessible, conserved epitope with high affinity.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

High Affinity Nanobodies against the Trypanosome brucei VSG Are Potent Trypanolytic Agents that Block Endocytosis

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

Both Type-I and Type-II Responses Contribute to Murine Trypanotolerance

The host immune system has been documented to influence the course and outcome of infection with the phospholipase-C-deficient (PLC(-/-)) Trypanosoma brucei brucei. We addressed the resistant mechanisms during trypanosomosis by comparing the immune response to variant-specific surface glycoprotein (VSG) in relatively susceptible C3H mice and trypanotolerant (C57BL/6 x BALB/c)-F1 (B6B-F1) mice infected with PLC(-/-) parasites. During the early stage of infection, lymphoid cells from both PLC(-/-)-susceptible C3H and -tolerant B6B-F1 mice mainly secreted VSG-specific IFN-gamma. Although C3H mice remained locked in a type-I cytokine environment (IFN-gamma, TNF-alpha) during late stage of infection, B6B-F1 mice switched to production of type-II cytokines (IL-4, IL-10) from late stage of infection onwards. It seems that VSG-specific cytokine responses associated with resistance to murine African trypanosomosis are infection-stage dependent, with type-I cytokine responses being critical during the early stage of infection while type-II cytokine responses appear to be more important during the late and chronic phases of the disease. Because of the striking similarities in the course of the PLC(-/-)infection in B6B-F1 mice with that of the trypanotolerant N'dama cattle naturally-infected with T. congolense, the PLC(-/-)-infected B6B-F1 mice represents a suitable model to study the course of infection and immune responses during bovine trypanosomosis.

Differential production of immune parameters by mouse strains that differ in their susceptibility to <i>Trypanosoma brucei brucei</i>

African trypanosomes are extracellular protozoan haemoparasites causing sleeping sickness to man or nagana to livestock in sub-Saharan Africa. Efforts to control trypanosomiasis have been hampered mainly by the parasites\' ability to constantly undergo antigenic variation. Characterization of immune mechanisms responsible for resistance during trypanosomosis may unravel novel strategies to control the pathogenic consequences of the disease. In the present study, we analyse the severity of infection and immune responses during the acute phase of Trypanosoma b. brucei infection in mouse stains (Balb/c, C57B1/6, C3H/HeNand {Balb/c×C57B1/6} F1 i.e. CB6-F1), that differ in their susceptibility to infection. Mice were intraperitoneally inoculated with 2 Differential productions of immune parameters by mouse strains that differ in their susceptibility to Trypanosoma brucei brucei. Morbidity was quantified by rough coat, reduced activity/movement and anaemia. The kinetics production of plasma level tumor necrosis factor (TNF) and interleukin-4 (IL-4) were evaluated by cytokine-specific sandwich ELISA. Further more, plasma from T.b. brucei-infected mice were tested in antibody-specific ELISAs for variant-specific surface glycoprotein (VSG) reactivity to determine the levels of VSG-specific antibodies. Results showed that T.b. brucei-infected mice differentially produce TNF. Mice (C57B1/6, CB6-FI) that efficiently controlled parasitaemia exhibited significantly higher plasma TNF levels during and after the first peak of parasitaemia than those showing reduced ability to control parasitaemia (C3H/HeN, Balb/c). Furthermore, the production of IL-4 was observed in all mouse strains except for C3H/HeN which exhibited the most susceptible phenotype. Interestingly, infected C3H/HeN mice showed a greatly reduced antibody-searching ability as compared to other strains. Data from this study suggest that resistant in T.b. brucei-infected mice is associated with the production of increased levels of TNF, coupled with low but sufficient le veils of IL-4 and increased antibody levels during the acute phase of infection. The Kenya Veterinarian Vol. 27 2004: pp. 109-112

The Induction of a Type 1 Immune Response following a Trypanosoma brucei Infection Is MyD88 Dependent

The initial host response toward the extracellular parasite Trypanosoma brucei is characterized by the early release of inflammatory mediators associated with a type 1 immune response. In this study, we show that this inflammatory response is dependent on activation of the innate immune system mediated by the adaptor molecule MyD88. In the present study, MyD88-deficient macrophages are nonresponsive toward both soluble variant-specific surface glycoprotein (VSG), as well as membrane-bound VSG purified from T. brucei. Infection of MyD88-deficient mice with either clonal or nonclonal stocks of T. brucei resulted in elevated levels of parasitemia. This was accompanied by reduced plasma IFN-gamma and TNF levels during the initial stage of infection, followed by moderately lower VSG-specific IgG2a Ab titers during the chronic stages of infection. Analysis of several TLR-deficient mice revealed a partial requirement for TLR9 in the production of IFN-gamma and VSG-specific IgG2a Ab levels during T. brucei infections. These results implicate the mammalian TLR family and MyD88 signaling in the innate immune recognition of T. brucei.

Variant Surface Glycoprotein from Trypanosoma evansi Is Partially Responsible for the Cross-Reaction between Trypanosoma evansi and Trypanosoma vivax

Salivarian trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as a defense against the host immune system. Although about 1000 VSG and pseudo-VSG genes are scattered throughout the trypanosome genome, each trypanosome expresses only one VSG, while the rest of the genes are transcriptionally silent. A 64-kDa glycosylated cross-reacting antigen between Trypanosoma evansi and Trypanosoma vivax (p64), which was purified from the TEVA1 T. evansi Venezuelan isolate, was proven here to represent the soluble form of a VSG. Initially, a biochemical characterization of p64 was carried out. Gel filtration chromatography, sedimentation, and chemical cross-linking provided evidences of the dimeric nature of p64. The hydrodynamic parameters indicated that p64 is asymmetrical with a frictional ratio f/fo = 1.57. Isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis revealed that p64 contained two isoforms with isoelectric points of 6.8-6.9 and 7.1-7.2. When p64 and three p64 Staphylococcus aureus V8 proteolytic fragments were sequenced, the same N-termini sequence was obtained: Ala-Pro-Ile-Thr-Asp-Ala-Asp-Leu-Gly-Pro-Ala-Gln-Ile-Ala-Asp, which displayed a significant homology with a putative Trypanosoma brucei VSG gene located on chromosome 4. Additionally, immunofluorescence microscopy on T. evansi and T. vivax established that p64 and its T. vivax homologue were confined to the surface of both parasites. An immunological characterization of this antigen was also carried out using several Venezuelan T. evansi isolates expressing different VSGs, which were obtained from naturally infected animals. Although sera from animals infected with the various T. evansi isolates recognized p64, only one isolate, besides TEVA1, contained polypeptides that were recognized by anti-p64 antibodies. All these results together with prior evidences [Uzcanga, G. et al. (2002) Parasitology 124, 287-299] confirmed that p64 is the soluble form of a T. evansi VSG, containing common epitopes recognized by sera from animals infected with T. evansi or T. vivax. Despite the huge repertoire of VSG genes existing on bloodstream trypanosomes, our data also demonstrated the potential use of a VSG variant from the TEVA1 T. evansi isolate as a diagnostic reagent.

A Conserved Flagellar Pocket Exposed High Mannose Moiety Is Used by African Trypanosomes as a Host Cytokine Binding Molecule

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.

The Glycosyl-Inositol-Phosphate and Dimyristoylglycerol Moieties of the Glycosylphosphatidylinositol Anchor of the Trypanosome Variant-Specific Surface Glycoprotein Are Distinct Macrophage-Activating Factors

The TNF-alpha-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C3H/HeJ mice and LPS-sensitive C3H/HeN mice. The variant-specific surface glycoprotein (VSG) was identified as the major TNF-alpha-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-alpha-inducing capacities, indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-alpha triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-alpha-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-alpha production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-alpha-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-gamma preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages toward IL-1alpha secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-alpha induction, the DMG compound of the mfVSG anchor contributes to IL-1 alpha induction and LPS sensitization.

A developmentally regulated position effect at a telomeric locus in Trypanosoma brucei

Trypanosoma brucei undergoes antigenic variation in the mammalian host. This can be achieved by activation and inactivation of telomeric variant-specific surface glycoprotein genes (vsg). In procyclic (insect midgut stage) cells, Vsg is not expressed. The mechanisms that regulate transcription of vsg expression sites (ESs) are unknown. Here we demonstrate that transcription from three different promoters was repressed when they were inserted at a transcriptionally silent telomere-proximal locus in bloodstream-form cells. This position effect was stable and heritable. Only transcription from an ES promoter was repressed in procyclic cells. The observed position effect and the promoter-specific developmental regulation suggest that these phenomena reflect the mechanisms that regulate vsg expression.

Insertion of the promoter for a variant surface glycoprotein gene expression site in an RNA polymerase II transcription unit of procyclic Trypanosoma brucei

The variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei are invariably expressed near the ends of chromosomes (telomeres). We have targeted a VSG gene expression site (ES) promoter driving a selectable marker gene (neomycin phosphotransferase) into a chromosome-internal transcription unit, the tubulin gene array of procyclic trypanosomes. To avoid read through transcription of the marker gene from the tubulin promoter, we targeted the ES promoter in inverse orientation relative to tubulin gene transcription. The only correctly targeted transformant obtained contained the marker gene close to the border of the tubulin gene array, and expression of this gene was relatively low. Possible reasons for the low targeting efficiency and expression level are discussed.

Synthesis of a peptidyldimannosyl phosphate: fragment of the variant-specific surface glycoprotein membrane anchor from trypanosoma brucei

Condensation of protected β-benzyl-aspartate 4 with 2-amino-ethanol, subsequent deprotection of the amino function and coupling with N, N′-di-benzyloxycarbonyl-lysine ( 7) afforded the peptidyl alcohol 8. Glycosylation of methyl 3,4,6 tri- O-benzyl-α- d-mannopyranoside ( 11) by ethyl 2,6-di- O-benzoyl-3,4-di- O-benzyl-1-thio-α- d-mannopyranoside ( 14) gave, after debenzoylation and protection of the 2′-hydroxyl function, the suitably protected dimannoside 21. Phosphitylation of 21 with benzyloxy- bis-( N, N-diisopropylamino)phosphine ( 18) followed by the addition of 8 gave, after oxidation and subsequent hydrogenolysis of the benzyl groups, the title compound in a good yield.

Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes

Transcription of the predominant surface antigen genes in Trypanosoma brucei is unusual in its resistance to the RNA polymerase inhibitor alpha-amanitin, a property typical for rDNA transcription in eukaryotes. Transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. To investigate whether RNA polymerase I, the polymerase that transcribes rRNA genes, can give rise to functional mRNAs in trypanosomes, we have fused the putative promoter of the T.brucei rRNA genes to the chloramphenicol acetyl transferase (CAT) gene and determined CAT activity after transient expression of chimeric constructs in procyclic trypanosomes. We show here that the rRNA promoter yields the same high CAT activity as the promoters for the two predominant surface antigen genes of trypanosomes, the Variant-specific Surface Glycoprotein (VSG) gene of bloodstream trypanosomes and the procyclin gene of insect-form trypanosomes, both of which are also transcribed by an alpha-amanitin-insensitive RNA polymerase. RNA polymerase I of trypanosomes seems therefore able to synthesize pre-mRNAs that are effectively processed into translatable mRNAs. Dissection of the promoter segments showed the minimal elements for a VSG gene expression site promoter to be confined to a segment of -60 to +77 bp, overlapping the most 5' putative transcription start sites as determined in vivo by RNase protection experiments. For the ribosomal promoter region a segment of -258 to +200 bp relative to the putative transcription start site was sufficient for maximal CAT activity. There is a precise requirement for specific nucleotides at the rRNA transcription start site. We detect no homology between the sequences required for promoter function of the three alpha-amanitin-resistant transcription units, rRNA, VSG and procyclin (parp) genes. This suggests that the sequence-specific recognition of these promoters either occurs by common factors detecting sequence homologies that escape us, or by separate factors that bind to different DNA sequences but interact with a common alpha-amanitin-resistant RNA polymerase.

A novel DNA nucleotide inTrypanosoma bruceionly present in the mammalian phase of the life-cycle

The existence of an unusual form of DNA modification in the bloodstream form of the African trypanosome Trypanosoma brucei has been inferred from partial resistance to cleavage of nuclear DNA with PstI and PvuII (Bernards et al, 1984; Pays et al, 1984). This putative modification is correlated with the shut-off of telomeric Variant-specific Surface Glycoprotein (VSG) gene expression sites (ESs). The modification only affects inactive VSG genes with a telomeric location, and it is absent in procyclic (insect form) trypanosomes in which no VSG is made at all. Previous attempts to detect unusual nucleosides in T.brucei DNA were unsuccessful, but we now report the detection of two unusual nucleotides, called pdJ and pdV, in T.brucei DNA, using the 32P-postlabeling technique. Nucleotide pdV was present in both bloodstream form and procyclic T.brucei DNA and co-migrated in two different two-dimensional thin layer chromatography (2D-TLC) systems with hydroxymethyldeoxyuridine 5'-monophosphate (pHOMedU). In contrast, nucleotide pdJ was exclusively present in bloodstream form trypanosomal DNA. Levels of pdJ were higher in DNA enriched for telomeric sequences than in total genomic DNA and pdJ was also detected in other Kinetoplastida species exhibiting antigenic variation. Postlabeling and 2D-TLC analyses showed base J to be different from the known eukaryotic unusual DNA bases 5-methylcytosine, N6-methyladenine and hydroxymethyluracil, and also from (glucosylated) hydroxymethylcytosine, uracil, alpha-putrescinylthymine, 5-dihydroxypentyluracil and N6-carbamoylmethyladenine. We conclude that pdJ is a novel eukaryotic DNA nucleotide and that it is probably responsible for the partial resistance to cleavage by PvuII and PstI of inactive telomeric VSG genes. It may therefore be involved in the regulation of ES activity in bloodstream form trypanosomes.

Antigenic variation inTrypanosoma brucei: a telomeric expression site for variant-specific surface glycoprotein genes with novel features

African trypanosomes evade the immune response of their host by periodically changing their variant surface glycoprotein (VSG) coat. Each coat is encoded by a separate VSG gene. Expressed genes are in a telomeric expression site (ES) and there are several sites in each trypanosome. To study the transcription control of VSG genes in Trypanosoma brucei we have analyzed an ES, called the dominant ES (DES), that readily switches off and on. The promoter area of the DES is very similar to that of the 221 ES (Zomerdijk et al., 1990). It can be switched off and on in vivo without detectable DNA alterations in the vicinity of the transcription start and it can drive high transient expression of a reporter gene in transfection experiments. However, there are also two major differences between the DES and the 221 ES. First, one version of the DES contains an additional upstream transcription unit overlapping the VSG gene ES promoter. The presence of this upstram transcription is dispensable, however, for the VSG gene ES promoter is active, even if transcription through this start from the upstream promoter is blocked using UV light. Moreover, a second version of the DES present in another trypanosome variant does not produce these upstream transcripts. Secondly, we find that the inactivation of DES transcription in one trypanosome variant is accompanied by DNA alterations in the DES upstream (greater than 2 kb) of the transcription start; reactivation of DES transcription is accompanied by another alteration far upstream. Although we cannot exclude that these DNA rearrangements are incidental, our results raise the possibility that the activity of ES promoters is negatively controlled in cis by far upstream sequences not included in transfection constructs and that alterations in these sequences may lead to (in)activation of the promoter.

Control of surface antigen genes in African trypanosomes

Conference Article| October 01 1990 Control of surface antigen genes in African trypanosomes PIET BORST; PIET BORST *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JANET H. GOMMERS-AMPT; JANET H. GOMMERS-AMPT *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JAN LUTGERINK; JAN LUTGERINK †Department of Human Genetics, Free University Amsterdam, 1081 HV Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar MARC OUELLETTE; MARC OUELLETTE *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar ANNELOOR L. M. A. TEN ASBROEK; ANNELOOR L. M. A. TEN ASBROEK *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar JOOST C. B. M. ZOMERDIJK JOOST C. B. M. ZOMERDIJK *Division of Molecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1990) 18 (5): 741–742. https://doi.org/10.1042/bst0180741 Article history Received: April 30 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation PIET BORST, JANET H. GOMMERS-AMPT, JAN LUTGERINK, MARC OUELLETTE, ANNELOOR L. M. A. TEN ASBROEK, JOOST C. B. M. ZOMERDIJK; Control of surface antigen genes in African trypanosomes. Biochem Soc Trans 1 October 1990; 18 (5): 741–742. doi: https://doi.org/10.1042/bst0180741 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: VSG, variant-specific surface glycoprotein, ES, expression site, CAT, chloramphenicol acetyl transferase, PGK, phosphoglycerate kinase This content is only available as a PDF. © 1990 Biochemical Society1990 Article PDF first page preview Close Modal You do not currently have access to this content.

The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region map at nucleotide sequences that do not strongly resemble rRNA transcriptional starts even though the transcripts are synthesized by an RNA polymerase highly resistant to alpha-amanitin. The cloned VSG gene 221 ES transcription initiation region promotes high CAT gene expression, when reintroduced by electroporation into T. brucei. We show that the activity of this expression site is controlled at or near transcription initiation in bloodstream trypanosomes. The 221 ES is inactivated without any sequence alteration within 1.4 kb of the transcription start site. This excludes mechanisms of promoter inactivation involving DNA rearrangements in the vicinity of the transcription start site, e.g. promoter inversion or conversion.

Structure of a telomeric expression site for variant specific surface antigens in Trypanosoma brucei

We have studied the organization of the expression site, in which most chromosome-internal variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei strain 427 are expressed (the dominant expression site) and compared it to the previously characterized VSG 221 expression site [1]. With the exception of a 500 bp segment and a VSG pseudogene, which are absent from the dominant expression site, overall all major sequence elements of the two sites are organized similarly, as judged from their relative mapping positions by UV inactivation of transcription. Transcription is insensitive to 1 mg α-amanitin per ml, a characteristic property of VSG gene expression sites analyzed thus far. The sequence elements of the dominant expression site include at least one other expressed gene of unknown function [2] and homologues of at least two other open reading frames. The large internal duplications of the 60-kb 221 expression site appear to be missing from the dominant site, resulting in a shorter, 40-kb transcription unit. As judged from its relative sensitivity to UV inactivation of transcription, a subsidiary promoter, identified by other methods in the dominant expression site [3] appears fully dependent for its activity on the promoter located 40 kb upstream of the VSG gene. We conclude that all VSG gene expression sites may be similarly organized as large polygenic transcription units.

Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum

We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Superlcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.

Trypanosoma brucei gambiense and T. b. rhodesiense: concanavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms.

We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3-16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed greater than 95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.