Cooperativity of catalytic and lectin-like domain of Trypanosoma congolense trans-sialidase modulates its catalytic activity.

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-ß-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-ß-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.

Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase.

BACKGROUND: Animal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay's high specificity. METHODOLOGY/PRINCIPAL FINDINGS: The structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay. CONCLUSIONS/SIGNIFICANCE: The results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays.

Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA-2 and Trypanosoma congolense ISA.

African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector-borne diseases of humans and livestock in sub-Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic-Specific Surface Antigen-2 (TbPSSA-2). To investigate the underlying molecular mechanism of TbPSSA-2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter-lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector.

Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense.

BACKGROUND: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.

Structural characterization and epitope mapping of the glutamic acid/alanine-rich protein from Trypanosoma congolense: defining assembly on the parasite cell surface.

Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal.

Differential protein expression throughout the life cycle of Trypanosoma congolense, a major parasite of cattle in Africa.

Trypanosoma congolense is an important pathogen of livestock in Africa. To study protein expression throughout the T. congolense life cycle, we used culture-derived parasites of each of the three main insect stages and bloodstream stage parasites isolated from infected mice, to perform differential protein expression analysis. Three complete biological replicates of all four life cycle stages were produced from T. congolense IL3000, a cloned parasite that is amenable to culture of major life cycle stages in vitro. Cellular proteins from each life cycle stage were trypsin digested and the resulting peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ). The peptides were then analyzed by tandem mass spectrometry (MS/MS). This method was used to identify and relatively quantify proteins from the different life cycle stages in the same experiment. A search of the Wellcome Trust's Sanger Institute's semi-annotated T. congolense database was performed using the MS/MS fragmentation data to identify the corresponding source proteins. A total of 2088 unique protein sequences were identified, representing 23% of the ∼9000 proteins predicted for the T. congolense proteome. The 1291 most confidently identified proteins were prioritized for further study. Of these, 784 yielded annotated hits while 501 were described as "hypothetical proteins". Six proteins showed no significant sequence similarity to any known proteins (from any species) and thus represent new, previously uncharacterized T. congolense proteins. Of particular interest among the remainder are several membrane molecules that showed drastic differential expression, including, not surprisingly, the well-studied variant surface glycoproteins (VSGs), invariant surface glycoproteins (ISGs) 65 and 75, congolense epimastigote specific protein (CESP), the surface protease GP63, an amino acid transporter, a pteridine transporter and a haptoglobin-hemoglobin receptor. Several of these surface disposed proteins are of functional interest as they are necessary for survival of the parasites.

Production of congopain, the major cysteine protease of Trypanosoma (Nannomonas) congolense, in Pichia pastoris reveals unexpected dimerisation at physiological pH.

African animal trypanosomosis (nagana) is arguably the most important parasitic disease affecting livestock in sub-Saharan Africa. Since none of the existing control measures are entirely satisfactory, vaccine development is being actively pursued. However, due to antigenic variation, the quest for a conventional vaccine has proven elusive. As a result, we have sought an alternative 'anti-disease vaccine approach', based on congopain, a cysteine protease of Trypanosoma congolense, which was shown to have pathogenic effects in vivo. Congopain was initially expressed as a recombinant protein in bacterial and baculovirus expression systems, but both the folding and yield obtained proved inadequate. Hence alternative expression systems were investigated, amongst which Pichia pastoris proved to be the most suitable. We report here the expression of full length, and C-terminal domain-truncated congopain in the methylotrophic yeast P. pastoris. Differences in yield were observed between full length and truncated proteins, the full length producing 2-4 mg of protein per litre of culture, while the truncated form produced 20-30 mg/l. The protease was produced as a proenzyme, but underwent spontaneous activation when acidified (pH <5). To investigate whether this activation was due to autolysis, we produced an inactive mutant (active site Cys→Ala) by site-directed mutagenesis. The mutant form was produced at a much higher rate, up to 100mg/l culture, as a proenzyme. It did not undergo spontaneous cleavage of the propeptide when subjected to acidic pH suggesting an autocatalytic process of activation for congopain. These recombinant proteins displayed a very unusual feature for cathepsin L-like proteinases, i.e. complete dimerisation at pH >6, and by reversibly monomerising at acidic pH <5. This attribute is of utmost importance in the context of an anti-disease vaccine, given that the epitopes recognised by the sera of trypanosome-infected trypanotolerant cattle appear dimer-specific.

The protease resistant surface (PRS) glycoconjugate from Trypanosoma congolense has an inositol-acylated glycosylphosphatidylinositol anchor, containing a significant proportion of myristate at the sn-2 position.

In the tsetse fly, the surface of Trypanosoma congolense parasites is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include EPGENGT procyclin and protease resistant surface molecule (PRS), as well as congolense epimastigote-specific protein, CESP, and glutamic acid- and alanine-rich protein (GARP). The GPI structures of EPGENGT and GARP have been partially elucidated, but very little is known about PRS. We now purified PRS and analyzed its GPI lipid structure and carbohydrate composition using mass spectrometry. We found that unlike EPGENGT and GARP, the GPI anchor of PRS is unusually composed of inositol-acylated diacyl-phosphatidylinositols, including species containing either myristic or oleic acid at the sn-2 position of the glycerol backbone. This is the first identification of a tri-acylated GPI anchor containing myristate in procyclic form trypanosomes. In addition, we found that PRS is highly rich in galactose and sialic acid residues, suggesting that it may represent a major acceptor of the parasite trans-sialidase.

Identification and molecular characterization of a novel stage-specific surface protein of Trypanosoma congolense epimastigotes.

The cattle pathogen Trypanosoma congolense expresses life cycle stage-specific surface molecules involved in adaptation to different host and vector environments. Here we report the discovery and molecular characterization of a novel stage-specific GPI-anchored surface glycoprotein that is selectively expressed in the epimastigote (EMF) life cycle stage of T. congolense. Culture supernatants of EMF but not of procyclic culture forms (PCFs) promoted adhesion of PCF parasites in an in vitro assay. Biosynthetic labeling experiments showed that these EMF culture supernatants contained a 100kDa trypanosome-derived protein that was not present in supernatants from PCF. We named this molecule "congolense epimastigote-specific protein" (CESP). The gene encoding CESP was isolated from an EMF cDNA library after immunoscreening. The multicopy gene had a 2070-bp open reading frame that encodes a polypeptide of 689 amino acids with a predicted mass of 72.9kDa. The discrepancy between the predicted (72.9kDa) and observed (100kDa) masses may be explained partially by glycosylation of the molecule which has six potential N-glycosylation sites and a predicted GPI anchor. Indeed, metabolic labeling of CESP with [(3)H] ethanolamine revealed that CESP was a GPI-anchored protein. Confocal laser scanning microscopy showed that CESP was expressed only on the surface of the EMF stage of the parasite. The identification of CESP as a unique component of culture supernatants from EMF and that such supernatants can confer plastic-adhesive ability on PCF suggest that CESP is worth further investigation as an adhesion molecule that perhaps allows T. congolense EMF to adhere to the tsetse proboscis.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms.

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Partial structure of glutamic acid and alanine-rich protein, a major surface glycoprotein of the insect stages of Trypanosoma congolense.

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the major surface glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing an sn-1-stearoyl-2-l-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growing in vitro.

Trypanosoma simiae and Trypanosoma congolense: surface glycoconjugates of procyclic forms-the same coats on different hangers?

Organic solvent extraction, reverse-phase high performance liquid chromatography and enzyme-linked immunosorbent assay with surface binding monoclonal antibodies were used to isolate membrane molecules of procyclic culture forms of Trypanosoma simiae and Trypanosoma congolense. Gel electrophoresis of the purified molecules revealed two predominant molecular species from each parasite that were broadly similar yet showed different apparent molecular masses and staining characteristics. The molecules were shown to be glycosylphosphatidylinositol-lipid anchored glycoconjugates, rich in carbohydrates. Each moiety displayed surface-disposed carbohydrate epitopes that were recognized on the surface of both species of trypanosomes by monoclonal antibodies specific for procyclic parasites of the subgenus Nannomonas. The epitopes were previously shown to be displayed on the glutamic acid-alanine rich protein of T. congolense yet neither this protein, nor its encoding gene is present in T. simiae. The results indicate that although T. congolense and T. simiae share common carbohydrate surface epitopes, these are displayed on biochemically different molecules. We speculate that the surface disposed carbohydrate structures are involved in parasite-tsetse interactions since these species have the same developmental cycles in the insect vector.

Unravelling the phylogenetic relationships of African trypanosomes of suids.

African trypanosomes of the subgenera Nannomonas and Pycnomonas have been recorded from both wild and domestic suids. However, complete descriptions of some of these trypanosomes with regard to host range, pathogenicity, transmission and distribution are still lacking. Neither the recently described Trypanosoma (Nannomonas) godfreyi nor Trypanosoma (Nannomonas) congolense Tsavo have been isolated from mammalian hosts, while Trypanosoma (Pycnomonas) suis remains the rarest of the Salivarian trypanosomes. The only isolate presumed to be of the latter species is maintained at the Kenya Trypanosomiasis Research Institute, Nairobi. We present here the results of characterization of this isolate by morphology, tsetse transmission, the use of species-specific DNA probes and DNA sequence analysis. Morphology in stained blood smears revealed a small trypanosome with a free flagellum. Experimental transmission through Glossina morsitans morsitans showed a developmental cycle typical of subgenus Nannomonas A positive identification was obtained with species-specific PCR primers for T. congolense Tsavo; moreover, the sequence of the SSU rRNA gene was almost identical to that of T. congolense Tsavo on database. In phylogenetic analysis of the SSU rRNA genes of Salivarian trypanosomes, T. congolense Tsavo grouped with T. simiae rather than T. congolense, suggesting that the name T. simiae Tsavo is more appropriate.

The structure-function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense.

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Km D-glucose = 41 microM versus 64 microM). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Ki D-glucose = 0.8 mM). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for D-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.

Study of the effect of gamma-irradiation on bovine serum samples on the ability of monoclonal antibodies to detect invariant antigens of Trypanosoma congolense, T. vivax and T. brucei in enzyme-linked immunosorbent assays.

Samples of bovine serum from uninfected and African trypanosomes-infected animals were tested before and after gamma-irradiation, using three sandwich enzyme-linked immunosorbent assays (ELISA). Each test system utilized a different monoclonal antibody, reputedly allowing the specific detection of conserved-invariant cytoplasmic antigens of trypanonosomes, T. congolense, T. vivax, and T. brucei, respectively. Results have identified two groups of samples. The first contained samples where there were unequivocal ELISA results indicating positivity and negativity, for non-irradiated samples. In this group, irradiation had no effect on the diagnostic sensitivity of the assays. All samples shown to be positive before irradiation remained positive and those shown to be negative, remained negative. There was, however, a statistically significant reduction in signal in each of the ELISAs following irradiation. The second group contained samples identified before irradiation as flanking the diagnostic negative/positive threshold of OD > or =0.05. These showed a negative bias after irradiation of the order of OD -0.01, which was shown to be statistically significant by paired t-statistics. Without correction of the given diagnostic negative/positive threshold, bovine sera with OD values around the threshold were expected to deliver more false negative test results upon irradiation. This was confirmed when serological data were compared with parasitological findings; where three times more false negative test results were found from irradiated serum samples. Consequently, for this group of irradiated bovine samples tested by ELISA, the re-adjustment of the diagnostic negative/positive threshold of the ELISAs using defined irradiated serum samples is recommended; otherwise, the frequency of false negative results might be increased.

Cytoskeletal architecture and components involved in the attachment of Trypanosoma congolense epimastigotes.

Scanning and transmission electron microscopy of Trypanosoma congolense epimastigotes attached to a plastic substratum shows them to elaborate a complex flagellum filament system and plaque with a highly organized structure. Non-ionic detergent extraction of these cells shows that the resulting cytoskeletons remain attached to the plaque. The subpellicular corset of microtubules can be removed by salt or Ca2+ treatment leaving the axoneme, paraflagellar rod, associated filaments and the plaque. Neither of these treatments therefore removed the plaque-associated material from the substratum. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis reveals an abundant 70 kDa protein that is highly enriched in the salt extracted 'minimal plaque' structures and appears likely to be a major constituent of this structure. These studies reveal that the complex filament and microtubule systems of the cytoskeleton involved the attachment of trypanosomes to substrata and have established a method of biochemical fractionation of the structures and components involved.

The primary structure of Trypanosoma (Nannomonas) congolese variant surface glycoproteins.

The complete nucleotide sequences were determined for three transcripts each encoding a different variant surface glycoprotein (VSG) of Trypanosoma (Nannomonas) congolense. The nucleotide sequence was determined also for a transcript encoding a fourth VSG, but this was truncated. The data obtained confirm absence of the canonical polyadenylation signal, lack of conserved sequence elements in the 3' untranslated region, and heterogeneity in the spliced-leader acceptor site in the T. congolense VSG transcripts examined. A comparison of the amino acids deduced from the nucleotide sequences of the four VSGs and those of other VSGs published previously reveals a strong conservation of several structural domains, particularly cysteine residues located throughout most of the molecules. The majority of T. congolense VSGs analyzed in this study resemble most the N-terminal cysteine residue domain type B of T. brucei, characterized by a cysteine residue located toward the N-terminal end, a cluster of cysteine residues in the central region, and at least three cysteine residues between positions 250 and 300 of the molecules. One of the VSGs analyzed, ILNat3.3, did not fit into any of the classification schemes proposed for the VSGs so far studied, and thus may represent a different class of these surface molecules. Unlike VSGs of T. brucei, the T. congolense VSGs have no cysteine residues at the carboxy-terminal end. These data now make it possible to predict general primary structural features of T. congolense VSGs.

Glycosyl-phosphatidylinositols of Trypanosoma congolense: two common precursors but a new protein-anchor.

The parasitic protozoan Trypanosoma congolense exhibits a dense surface coat which is pivotal for immunoevasion of the parasite. This dense surface coat is made of a single protein species, the variant surface glycoprotein, which is present in a high copy number. The protein is anchored to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor. A detailed study of the structure of T. congolense strain 423 (clone BENat 1.3) variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor was performed. Radioactively labelled core-glycan prepared by dephosphorylation, deamination and reduction was analysed by high-pH anion-exchange chromatography, size-exclusion and lectin affinity chromatography. Additionally the glycosyl-phosphatidylinositol membrane anchor core-glycan was purified from a bulk preparation of variant surface glycoprotein and subjected to mass spectrometry and methylation analysis. Using these methods we could identify a novel galactose-beta 1,6-N-acetyl-glucosamine-beta 1,4-branch modifying the mannose adjacent to the glucosamine of the mannose-alpha 1,2-mannose-alpha 1,6-mannose-alpha 1,4-glucosamine core-glycan of the variant surface glycoprotein glycosyl-phosphatidylinositol membrane anchor. Furthermore the biosynthetic pathway leading to this novel structure was investigated. Two putative glycosyl-phosphatidylinositol anchor precursors were identified having structures identical to the previously characterized Trypanosoma brucei brucei glycolipids P2 and P3 (also designated glycolipid A and C) consistent with a trimannosyl core and a dimyristoyl-glycerol. Both glycosyl-phosphatidylinositol anchor precursors of T. congolense do not possess the side-branch modification found on the mature protein membrane anchor, implying that the sugar side-chain is added to the anchor during its passage through the Golgi-apparatus.

Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker.

Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

Characterization of concanavalin A-binding glycoproteins from procyclic culture forms of Trypanosoma congolense, T. simiae and T. brucei brucei.

Concanavalin A-binding glycoproteins were obtained from procyclic culture forms (PCFs) of Trypanosoma congolense, T. simiae, and T. b. brucei strains. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that glycoproteins of 38.5, 30.5, and 27 kDa were conserved between the different species and strains of the procyclic parasites. There were few similarities in the profiles of the high-molecular-weight glycoconjugates between the parasites. Monoclonal antibody analysis revealed that the 38.5- and 27-kDa glycoproteins were intracellular molecules and that they contained cross-reactive antigenic determinants. Surface biotinylation of PCF T. congolense K45/1 identified surface-accessible glycoproteins of 81.5, 59, and 38-42 kDa. By use of lectin blots and enzymatic deglycosylation studies, we demonstrated that the 81.5-, 59-, 38.5-, and 27-kDa glycoproteins contained N-linked oligosaccharide chains with both high-mannose-type and complex-type oligosaccharides, and the 81.5- and 59-kDa surface glycoproteins contained sialic acid residues. The glycoproteins identified in this study provide a starting point for further structure and function studies.