Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Targeting the variable surface of African trypanosomes with variant surface glycoprotein-specific,serum-stable RNA aptamers

African trypanosomes cause sleeping sickness in humans and Nagana in cattle. The parasites multiply in the blood and escape the immune response of the infected host by antigenic variation. Antigenic variation is characterized by a periodic change of the parasite protein surface, which consists of a variant glycoprotein known as variant surface glycoprotein (VSG). Using a SELEX (systematic evolution of ligands by exponential enrichment) approach, we report the selection of small, serum-stable RNAs, so-called aptamers, that bind to VSGs with subnanomolar affinity. The RNAs are able to recognize different VSG variants and bind to the surface of live trypanosomes. Aptamers tethered to an antigenic side group are capable of directing antibodies to the surface of the parasite in vitro. In this manner, the RNAs might provide a new strategy for a therapeutic intervention to fight sleeping sickness.     

The surface of the African trypanosomes

The African trypanosomes bear on the outside of their cell membrane a single 10-15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000-63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy ; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement “the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier” appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.     

Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation.

A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites.     

The Surface of the African Trypanosomes1,2

The African trypanosomes bear on the outside of their cell membrane a single 10–15 nm thick coat of a glycoprotein. This glycoprotein may differ in structure in the predominant populations of parasitemic waves found in relapsing infections. Variant Specific Glycoprotein (VSG) range in MW between 53,000–63,000 d and may have variable amounts of carbohydrate attached at one, two, or several loci. Such differences in carbohydrate content may account in part for their range in molecular size. Approximately 30 C-terminal residues demonstrate isotypy; i.e. these regions fall into classes having similar amino acid sequence. Modest homology has been demonstrated in two VSGs of T. congolense arising in relapsing infections although comparison of many VSG show little or no obvious homology. More recently, lipid-associated forms of VSG have been described and it is believed that these forms may be transmembrane proteins. Different VSGs appear to have different amounts of the primary sequence which have alpha-helix-forming potential. In some VSG, in excess of 80% of the structure is helical as judged by both Chou-Fasman calculations and by circular dichroism. This raises the possibility that different VSG may have different folding patterns. The arrangement of VSG on the trypanosome surface probably places the basic amino acid-rich carbohydrate-bearing C-terminus of the polypeptide chain close to the membrane. There is some protein-protein association between VSGs for which (in T. evansi) the C-terminal tail is not required. The importance of VSG structure lies not only in the fact that the molecule mediates the phenomenon of antigenic variation but also in the recent observation that VSG may act on the cellular immune system to suppress the humoral immune responses of the host.     

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.     

Functional analysis of the Enterococcus faecalis plasmid pAD1-encoded stability determinant par

Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.     

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.     

Purification of major variable-surface glycoproteins from African trypanosomes by reverse-phase high-performance liquid chromatography

Reverse-phase high-performance liquid chromatography (RP-HPLC) was used in a one-step procedure to purify and analyze several different major variable-surface glycoproteins (VSGs) from lysates of African trypanosomes. RP-HPLC was used to fractionate lysates of trypanosomes and the VSG localized to the major peak of the elution profile using a rabbit antiserum to the cross-reacting determinant of the VSG. Polyacrylamide gel electrophoresis of HPLC fractions showed that the purity of isolated VSGs was equivalent to or better than that attained using conventional purification procedures. The elution positions of purified VSGs from a variety of cloned trypanosomes were identical, indicating the presence of a common hydrophobic feature on the surface of these highly polymorphic antigens. Preliminary experiments have shown that purification of VSG from trypanosome lysates may be scaled up to preparative levels. The results show that RP-HPLC is a useful procedure for rapid preparation of highly purified trypanosome VSGs and that analysis of their various molecular forms will be facilitated by the application of HPLC methods.     

Molecular basis of antigenic variation in African trypanosomes

African trypanosomes, which cause sleeping sickness in man and other mammals, are able to evade immune destruction in their hosts by altering the expression of a major cell surface molecule, the variant surface glycoprotein (VSG). The VSGs are encoded by a multigene family, and antigenic variation occurs when the trypanosome switches from expression of one VSG gene to another. This switching process involves changes in the arrangement of the trypanosome genomic DNA.     

Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short,identical exon at their 5′ end

The nucleotide sequence of the 5′ end of the mRNA for variant surface glycoprotein (VSG) 117 has been determined and compared with the sequence of the unexpressed basic copy (BC) of the VSG 117 gene. This shows the existence of an exon 35 nucleotides long at the 5′ end of the mRNA. The evidence suggests that this ‘mini-exon’ is derived from the expression site into which the VSG 117 BC is transposed during activation. The nucleotide sequence of this mini-exon is indistinguishable from that recently found for a different VSG, 118 (Van der Ploeg et al., Nucl. Acids Res. 10 (1982) 3591–3604). Analysis of the 5′ end of the mRNA for another VSG (221) whose gene is thought to be activated by a different mechanism to that of VSGs 117 and 118 indicates that the 5′- most 35 nucleotides of the VSG 221 mRNA are identical to the 117/118 mini-exon sequence. The implications of these results for the mechanism of VSG gene expression are discussed.     

Trypanosoma evansi: Identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain

African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts’ antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.     

Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5

At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.     

Structure of a glycosylphosphatidylinositol-anchored domain from a trypanosome variant surface glycoprotein

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.     

Antigenic variation during the developmental cycle of Trypanosoma brucei

During the complex life cycle of Trypanosoma brucei, changes in the exposed surface antigens occur in both the mammalian host and the insect vector (Glossina spp.). These antigenic changes are associated with alterations of the variant surface glycoprotein (VSG) composition or with the loss of the VSG. In the bloodstream of the mammalian host, trypanosomes successfully evade destruction by the host's immune response by continuously expressing alternative VSGs, at low frequency, which are not destroyed by host antibodies. When ingested by the tsetse fly, the bloodstream trypanosomes rapidly lose their surface coat and surface membrane antigens are exposed which are normally covered in the bloodstream. In the salivary glands of the tsetse fly, the trypanosomes differentiate to the metacyclic stage, which reacquires a surface coat. The antigenic composition of the metacyclics is heterogeneous. The same metacyclic types are expressed regardless of the bloodstream antigenic type ingested by the tsetse fly. In the mammal the metacyclics differentiate to long-slender bloodstream forms but continue to express the metacyclic VSG for at least three days. The next VSGs expressed in the mammalian host appear to be influenced by the antigenic type ingested by the tsetse. The ingested antigenic type is often expressed in the first parasitemia following expression of the metacyclic antigenic types.