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.     

Expression of procyclin mRNAs during cyclical transmission of Trypanosoma brucei

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3' untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised.     

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man9GlcNAc2 from Man9GlcNAc2-PP-Dol while others receive Man5GlcNA(2 from Man5GlcNAc2-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the alpha-mannosyltransferase that converts Man5GlcNAc2-PP-Dol to Man6GlcNAc2-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man9GlcNAc2 and processed no further than Man5GlcNAc2 in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man5GlcNAc2-PP-Dol is the only route to complex N-glycans in T. brucei and that Man9GlcNAc2-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man5GlcNAc2 or Man9GlcNAc2 transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.     

Multiple procyclin isoforms are expressed differentially during the development of insect forms of Trypanosoma brucei

Transmission of Trypanosoma brucei by the tsetse fly entails several rounds of differentiation as the parasite migrates through the digestive tract to the salivary glands of its vector. Differentiation of the bloodstream to the procyclic form in the fly midgut is accompanied by the synthesis of a new coat consisting of EP and GPEET procyclins. There are three closely related EP isoforms, two of which (EP1 and EP3) contain N-glycans. To identify the individual EP isoforms that are expressed early during synchronous differentiation in vitro, we exploited the selective extraction of GPI-anchored proteins and mass spectrometry. Unexpectedly, we found that GPEET and all isoforms of EP were coexpressed for a few hours at the onset of differentiation. At this time, the majority of EP1 and EP3 molecules were already glycosylated. Within 24 hours, GPEET became the major surface component, to be replaced in turn by glycosylated forms of EP, principally EP1, at a later phase of development. Transient transfection experiments using reporter genes revealed that each procyclin 3' untranslated region contributes to differential expression as the procyclic form develops. We postulate that programmed expression of other procyclin species will accompany further rounds of differentiation, enabling the parasite to progress through the fly.     

Removal or maintenance of inositol-linked acyl chain in glycosylphosphatidylinositol is critical in trypanosome life cycle

The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.     

Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes

In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.     

Procyclin null mutants of Trypanosoma brucei express free glycosylphosphatidylinositols on their surface

Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.     

GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat

The procyclic form of Trypanosoma brucei exists in the midgut of the tsetse fly. The current model of its surface glycocalyx is an array of rod-like procyclin glycoproteins with glycosylphosphatidylinositol (GPI) anchors carrying sialylated poly-N-acetyllactosamine side chains interspersed with smaller sialylated poly-N-acetyllactosamine-containing free GPI glycolipids. Mutants for TbGPI12, deficient in the second step of GPI biosynthesis, were devoid of cell surface procyclins and poly-N-acetyllactosamine-containing free GPI glycolipids. This major disruption to their surface architecture severely impaired their ability to colonize tsetse fly midguts but, surprisingly, had no effect on their morphology and growth characteristics in vitro. Transmission electron microscopy showed that the mutants retained a cell surface glycocalyx. This structure, and the viability of the mutants in vitro, prompted us to look for non-GPI-anchored parasite molecules and/or the adsorption of serum components. Neither were apparent from cell surface biotinylation experiments but [3H]glucosamine biosynthetic labeling revealed a group of previously unidentified high apparent molecular weight glycoconjugates that might contribute to the surface coat. While characterizing GlcNAc-PI that accumulates in the TbGPI12 mutant, we observed inositolphosphoceramides for the first time in this organism.     

Cleavage of trypanosome surface glycoproteins by alkaline trypsin-like enzyme(s) in the midgut of Glossina morsitans

EP and GPEET procyclin, the major surface glycoproteins of procyclic forms of Trypanosoma brucei, are truncated by proteases in the midgut of the tsetse fly Glossina morsitans morsitans. We show that soluble extracts from the midguts of teneral flies contain trypsin-like enzymes that cleave the N-terminal domains from living culture-derived parasites. The same extract shows little activity against a variant surface glycoprotein on living bloodstream form T. brucei (MITat 1.2) and none against glutamic acid/alanine-rich protein, a major surface glycoprotein of Trypanosoma congolense insect forms although both these proteins contain potential trypsin cleavage sites. Gel filtration of tsetse midgut extract revealed three peaks of tryptic activity against procyclins. Trypsin alone would be sufficient to account for the cleavage of GPEET at a single arginine residue in the fly. In contrast, the processing of EP at multiple sites would require additional enzymes that might only be induced or activated during feeding or infection. Unexpectedly, the pH optima for both the procyclin cleavage reaction and digestion of the trypsin-specific synthetic substrate Chromozym-TRY were extremely alkaline (pH 10). Direct measurements were made of the pH within different compartments of the tsetse digestive tract. We conclude that the gut pH of teneral flies, from the proventriculus to the hindgut, is alkaline, in contradiction to previous measurements indicating that it was mildly acidic. When tsetse flies were analysed 48 h after their first bloodmeal, a pH gradient from the proventriculus (pH 10.6+/-0.6) to the posterior midgut (pH 7.9+/-0.4) was observed.     

The regulation of procyclin expression in Trypanosoma bruceli: making or breaking the rules?

The identification of procyclins as stage-specific coat proteins of procyclic forms of Trypanosoma brucei has not only provided a convenient molecular marker for the differentiation of bloodstream-form trypanosomes into procyclic forms, but has also allowed some important insights into gene regulation in trypanosomes. Here, Adrian Hehl and Isabel Roditi summarize what has been learnt in the past few years about the control mechanisms that may contribute to the stage-specific expression of procyclins.     

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.     

Major Surface Glycoproteins of Insect Forms of Trypanosoma brucei Are Not Essential for Cyclical Transmission by Tsetse

Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the null mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the null mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the null mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.     

Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.     

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris

Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).     

Identification of a second catalytically active trans-sialidase in Trypanosoma brucei

The procyclic stage of Trypanosoma brucei is covered by glycosylphosphatidylinositol (GPI)-anchored surface proteins called procyclins. The procyclin GPI anchor contains a side chain of N-acetyllactosamine repeats terminated by sialic acids. Sialic acid modification is mediated by trans-sialidases expressed on the parasite’s cell surface. Previous studies suggested the presence of more than one active trans-sialidases, but only one has so far been reported. Here we cloned and examined enzyme activities of four additional trans-sialidase homologs, and show that one of them, Tb927.8.7350, encodes another active trans-sialidase, designated as TbSA C2. In an in vitro assay, TbSA C2 utilized α2-3 sialyllactose as a donor, and produced an α2-3-sialylated product, suggesting that it is an α2-3 trans-sialidase. We suggest that TbSA C2 plays a role in the sialic acid modification of the trypanosome cell surface.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.     

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.     

The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.