Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants.

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.

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.

The mucin-like glycoprotein super-family of Trypanosoma cruzi: structure and biological roles.

Trypanosoma cruzi expresses at its surface large amounts of mucin-like glycoproteins. The T. cruzi mucins (TcMUC), a group of highly glycosylated GPI-anchored proteins rich in Thr, Ser, and Pro residues, are expressed in high copy numbers in both insect and mammalian stages of the parasite. These molecules are encoded by a multigene family and contain a unique type of glycosylation consisting of several sialylated O-glycans linked to the protein backbone via N-acetylglucosamine residues. The TcMUC are important because of their role in host cell invasion and the ability to induce secretion of proinflammatory cytokines and nitric oxide in activated macrophages. The TcMUC are also significant in being the major substrate for the cell surface trans-sialidase. In this review, we summarize the recent knowledge on the molecular structure and function of this family of T. cruzi glycoproteins.

Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite-host cell interaction.

The 35/50 kDa mucin-like surface glycoprotein (gp35/50) of Trypanosoma cruzi metacyclic trypomastigotes has been implicated in mammalian cell invasion. In this study we investigated whether the sialyl residues of gp35/50 are required for interaction of parasites with target cells. After treatment with bacterial neuraminidase, the metacyclic forms (G strain) remained reactive with the monoclonal antibody (mAb) 10D8 but lost their reactivity with mAb 3C9, that recognizes sialic acid-containing epitopes on gp35/50, and entered HeLa cells in significantly higher numbers as compared to untreated controls. Resialylation of gp35/50, by incubation of parasites with T. cruzi trans-sialidase and sialyl lactose, restored the reactivity with mAb 3C9 as well as the affinity for sialic acid specific lectin. Accordingly, the rate of invasion of resialylated parasites was reduced to levels similar to those observed before desialylation. Purified G strain gp35/50, desialylated by neuraminidase treatment, bound to HeLa cells more than its sialylated counterpart. The Ca2+ signaling activity, which has been associated with cell invasion, was also determined by measuring the cytosolic Ca2+ concentration ([Ca2+]i), in HeLa cells upon interaction with sonicated extracts from untreated or neuraminidase-treated parasites, or with purified gp35/50 in its sialylated or desialylated form. Consistent with the results of cell invasion assay, the desialylated parasite preparations, as well as the sialic acid free gp35/50, induced an average elevation in [Ca2+]i significantly higher than that triggered by untreated controls. None of these effects, namely the increase in infectivity and Ca2+ signaling activity, was observed with neuraminidase-treated CL strain metacyclic trypomastigotes, which express a variant form of sialic acid gp35/50 molecule that is not recognized by mAb 10D8 and apparently is not involved in target cell invasion.

Glycosyl phosphatidylinositol myristoylation in African trypanosomes. New intermediates in the pathway for fatty acid remodeling.

Glycosyl phosphatidylinositol (GPI) anchors in the bloodstream form of Trypanosoma brucei are unusual in that their two fatty acids are myristate. The myristates are added in the final stages of GPI biosynthesis in a remodeling reaction. Remodeling occurs first at the sn-2 position of glycerol, involving removal of a longer fatty acid and subsequent attachment of myristate. The second myristate is then incorporated into the sn-1 position, but the mechanism has been unclear due to the unavailability of a reliable cell-free system supporting complete remodeling. Here, we first refined the cell-free system (by removing Mn(2+) ions), thereby allowing efficient production of the dimyristoylated GPI precursor. Using this improved system, we made three new discoveries concerning the pathway for fatty acid remodeling. First, we discovered a monomyristoylated GPI (known as glycolipid theta') as an intermediate involved in remodeling at the sn-1 position. Second, we found an alternative pathway for production of glycolipid theta, the first lyso intermediate in remodeling. The alternative pathway involves an inositol-acylated GPI known as glycolipid lyso-C'. Finally, we found that there is significant breakdown of GPIs during remodeling in the cell-free system, and we speculate that this breakdown has a regulatory role in GPI biosynthesis.

Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway.

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine-linked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N- glycan of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants is predominantly a hybrid type with a terminal N- acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.

The procyclin repertoire of Trypanosoma brucei. Identification and structural characterization of the Glu-Pro-rich polypeptides.

The surface of the insect stages of the protozoan parasite Trypanosoma brucei is covered by abundant glycosyl phosphatidylinositol (GPI)-anchored glycoproteins known as procyclins. One type of procyclin, the EP isoform, is predicted to have 22-30 Glu-Pro (EP) repeats in its C-terminal domain and is encoded by multiple genes. Because of the similarity of the EP isoform sequences and the heterogeneity of their GPI anchors, it has been impossible to separate and characterize these polypeptides by standard protein fractionation techniques. To facilitate their structural and functional characterization, we used a combination of matrix-assisted laser desorption ionization and electrospray mass spectrometry to analyze the entire procyclin repertoire expressed on the trypanosome cell. This analysis, which required removal of the GPI anchors by aqueous hydrofluoric acid treatment and cleavage at aspartate-proline bonds by mild acid hydrolysis, provided precise information about the glycosylation state and the number of Glu-Pro repeats in these proteins. Using this methodology we detected in a T. brucei clone the glycosylated products of the EP3 gene and two different products of the EP1 gene (EP1-1 and EP1-2). Furthermore, only low amounts of the nonglycosylated products of the GPEET and EP2 genes were detected. Because all procyclin genes are transcribed polycistronically, the latter finding indicates that the expression of the GPEET and EP2 genes is post-transcriptionaly regulated. This is the first time that the whole procyclin repertoire from procyclic trypanosomes has been characterized at the protein level.

The surface coat of procyclic Trypanosoma brucei: programmed expression and proteolytic cleavage of procyclin in the tsetse fly.

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 10(7) molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

Mucin-like molecules form a negatively charged coat that protects Trypanosoma cruzi trypomastigotes from killing by human anti-alpha-galactosyl antibodies.

In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.