Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei

The genome of Trypanosoma brucei carries over a hundred genes coding for different variants of the major surface glycoprotein. Activation of some of these genes is accompanied by a duplication and transposition of the gene (the basic copy) to another region in the genome where it is transcribed. We present here physical maps of the basic and transposition-activated genes for two surface glycoproteins of Trypanosoma brucei, stock 427. In both cases the transposed segment starts 1-2 kb in front of the coding region and ends within the 3'-terminal region of the gene. The DNA segments flanking both transposed genes are indistinguishable and share a 6-kb stretch upstream and a 8-kb stretch downstream of the transposed segment not cut by several restriction endonucleases. The 5' borders of the two transposed segments are homologous and contain sequences present in many copies in the genome. A different repeated sequence has previously been found at the 3' edge of the transposed segment. The replicative transposition may, therefore, involve a unidirectional gene conversion initiated by base pairing between the edges of the transposed sequence and a single expression site elsewhere in the genome.     

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.     

Trypanosoma brucei brucei: variability in the association of some variant surface glycoproteins

In our isolation procedure, the surface antigens of the variants AnTat 1.1 and 1.10 (Trypanosoma brucei brucei) are essentially obtained as a disulfide-linked dimer while the AnTat 1.8 surface antigen is found as a mixture of monomer and disulfide-linked dimer. This observation may be related to the localization of the cysteine residues in the protein sequences. In the purification procedure using concanavalin-A Sepharose chromatography, besides the VSG elution by methyl-alpha-D-mannopyranoside, a quantitative elution of still bound VSG may be obtained by the addition of beta-mercaptoethanol to methyl-alpha-D-mannopyrannoside in the elution buffer. The surface antigen of the variant AnTat 1.1 was examined for molecular form at several different times during the release procedure. The disulfide-linked dimer could be observed within 30 min of the surface coat release, indicating its presence within the parasite.     

Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins

The complete primary structures of the Asn-linked oligosaccharides from the conserved glycosylation site of the type-I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose-type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.     

Crystallization of amino-terminal domains and domain fragments of variant surface glycoproteins from Trypanosoma brucei brucei

Crystals were produced from variant surface glycoproteins (VSG) of Trypanosoma brucei brucei antigenic variants MITat 1.2, 1.6, and ILTat 1.22, 1.23, 1.24, 1.25, and 1.26. Purified VSGs had molecular weights from 60,000 to 68,000 on sodium dodecyl sulfate-polyacrylamide gels, whereas the crystals obtained were composed of polypeptides of approximate Mr 40,000-50,000. Amino-terminal amino acid sequences determined from the crystallized VSGs were identical to sequences obtained from the respective intact proteins, indicating that the crystals contained VSG amino-terminal fragments. Crystallization conditions and lattice dimensions of the crystals are given.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins

Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.     

Physical studies of Trypanosoma brucei variant surface glycoproteins and their antigenic determinants

Secondary structure determinations have been carried out on two antigenically related variant surface glycoproteins (VSG's) from Trypanosoma brucei, WaTat 1.1 and WaTat 1.12. The two molecules, which bear highly homologous amino-terminal sequences, showed subtle differences in their circular dichroism (CD). Computer analysis revealed that the contribution of alpha helix to the secondary structure of the VSG's was 49% for WaTat 1.1 and 52% for WaTat 1.12. Unfolding studies using guanidine hydrochloride suggested that the WaTat 1.12 VSG was slightly more resistant than WaTat 1.1 VSG to the effect of this reagent. The membrane form of WaTat 1.1 VSG purified by reverse-phase high-performance liquid chromatography gave CD and fluorescence spectra indicative of a partially unfolded or denatured molecule. It was also shown that the antigenic differences between the VSG's were due to surface-oriented topographically assembled epitopes which were highly sensitive to structural perturbations. Monoclonal antibodies specific for these epitopes bound to four discreet determinants on WaTat 1.1, one of which was absent from WaTat 1.12.     

Trypanosoma brucei: radioimmunoassay of variant surface glycoproteins from organisms grown in vitro and in vivo

Bloodstream forms of Trypanosoma brucei that were infective for mammals, when grown in vitro at 37 C for 29 days or 25 months had amounts of variant surface glycoprotein similar to the amounts from bloodstream forms isolated from infected rat blood. The amounts were measured by competition radioimmunoassays for both unique and cross-reacting determinants and the results were the same, providing evidence that a single type of variant surface glycoprotein was measured. Neither radioimmunoassay detected determinants of variant surface glycoproteins in trypanosomes transformed by culturing at 27 C to insect forms not infective for mammals.     

The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma brucei

Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei.     

Nuclear genes that encode mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome

The plant mitochondrial genome is complex in structure, owing to a high degree of recombination activity that subdivides the genome and increases genetic variation. The replication activity of various portions of the mitochondrial genome appears to be nonuniform, providing the plant with an ability to modulate its mitochondrial genotype during development. These and other interesting features of the plant mitochondrial genome suggest that adaptive changes have occurred in DNA maintenance and transmission that will provide insight into unique aspects of plant mitochondrial biology and mitochondrial-chloroplast coevolution. A search in the Arabidopsis genome for genes involved in the regulation of mitochondrial DNA metabolism revealed a region of chromosome III that is unusually rich in genes for mitochondrial DNA and RNA maintenance. An apparently similar genetic linkage was observed in the rice genome. Several of the genes identified within the chromosome III interval appear to target the plastid or to be targeted dually to the mitochondria and the plastid, suggesting that the process of endosymbiosis likely is accompanied by an intimate coevolution of these two organelles for their genome maintenance functions.     

Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.     

Purification and properties of the membrane form of variant surface glycoproteins (VSGs) from Trypanosoma brucei

Variant surface glycoproteins (VSG) of Trypanosoma brucei are released in a water soluble form on impairment of membrane integrity. We have previously shown that this release is the result of an enzyme-mediated event which converts the hydrophobic membrane form VSG into the hydrophilic water-soluble form. We now present further details of the methods by which membrane form VSG ( mfVSG ) may be isolated, uncontaminated by water-soluble VSG ( sVSG ). The sensitivity to different metal ions of the enzyme that mediated the conversion event is discussed, and some biochemical characteristics of different mfVSG preparations are presented.     

Carbohydrate composition of variant-specific surface antigen glycoproteins from Trypanosoma brucei

The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetylglucosamine). The glycoprotein from variant 048, strain 427 contained (+20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an intergral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120, 000).     

Biosynthesis and processing of a variant surface glycoprotein from Trypanosoma brucei brucei

Iowa trypanosome antigen type (IaTat) 1.2 variant surface glycoprotein (VSG) is synthesized in vitro as a Mr 54,000 preprotein that contains a 31-amino-acid signal peptide. Translation of mRNA in the presence of either dog pancreas or trypanosome microsomal membranes results in cotranslational cleavage of the signal peptide and addition of core oligosaccharide side chains to the protein. Analysis of these products on sodium dodecyl sulfate (SDS)-gels indicates that removal of the signal peptide (Mr 3200) is almost exactly compensated for by an increase in molecular weight due to carbohydrate addition. Pulse-chase experiments in cultures of isolated trypanosomes indicate that two IaTat 1.2 VSG species (Mr 58,000 and 60,000) occur in vivo. When glycosylation is inhibited by incubation of trypanosomes with tunicamycin, a single Mr 50,000 polypeptide is immunoprecipitated. The multiple protein species, therefore, arise from heterogeneity in carbohydrate side chains whose synthesis and transfer to the protein are tunicamycin sensitive. Sequence analysis verified that both species of VSG contain identical amino-terminal sequences. Further post-translational processing of IaTat 1.2 VSG includes addition of phosphate and myristic acid residues, both of which have been shown to be located in the immunologically cross-reactive determinant at the carboxyl terminus of the protein. Exposure of this attachment site requires post-translational proteolytic removal of a 17-amino-acid peptide from the carboxyl terminus of an intermediate form of VSG.     

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.     

Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes

The variant specific surface glycoprotein from Trypanosoma brucei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.