Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.     

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

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

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.     

Release and purification of Trypanosoma brucei variant surface glycoprotein

Conditions affecting the solubilization of variant surface glycoprotein (VSG) from Trypanosoma brucei have been investigated. The results obtained form the basis for a convenient and efficient method for VSG purification. VSG release from the cell surface was temperature-dependent, following osmotic lysis at 0 degree C, and was inhibited by low concentrations of Zn2+ but not by tosyl-lysine chloromethyl-ketone (TLCK), phenylmethylsulfonylfluoride (PMSF), or iodoacetamide. These and other results eliminated the possibility that release was due to proteolytic cleavage of the C-terminal hydrophobic tail present on newly synthesized VSG. Bolton and Hunter reagent reacted with several components on living cells.     

Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein

The expression of the Trypanosoma brucei variant surface glycoprotein AnTat 1.1 proceeds by a mechanism that transfers a duplicated gene copy into a new genomic environment, the so-called expression site, where it will be expressed. We have isolated a genomic fragment containing the region spanning the expression site-transposon junction, and the 5' half of the coding sequence. Comparing this DNA segment with its template copy (basic copy) allowed us to identify the exact breaking point and indicated a base sequence which could be involved in initiating the transposition event. Sequencing data also indicated that the co-transposed segment 5' to the coding sequence is 430 bp in length. The extreme 5' end of the mRNA is derived from a region in the expression site not immediately adjacent to the transposed DNA segment. This particular sequence exists in multiple copies in the genome and is common to the mRNA of all variant surface glycoproteins so far analysed.     

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.     

Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein

The variant surface glycoprotein (VSG) of Trypanosoma brucei has a glycolipid covalently attached to its C terminus. This glycolipid, which anchors the protein to the cell membrane, is attached to the VSG polypeptide within 1 min after translation (Bangs, J. D. Hereld, D., Krakow, J.L., Hart, G. W., and Englund, P. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3207-3211). This rapid processing suggests that, prior to incorporation, the glycolipid may exist in the cell as a preformed precursor which is transferred to the VSG polypeptide en bloc. We have isolated a molecule which has properties consistent with it being a VSG glycolipid precursor. It is highly polar and can be labeled by [3H] myristate but not by [3H]palmitate. It reaches steady state during continuous labeling with [3H]myristate and shows rapid turnover in pulse-chase experiments, suggesting that it is a metabolic intermediate rather than an end product. When treated with HNO2 it liberates phosphatidylinositol, as does VSG (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Also, like VSG, it releases a compound which co-migrates on thin layer chromatography with dimyristylglycerol when treated with the purified endogenous phospholipase C from trypanosomes. After treatment with this lipase, the putative precursor can be immunoprecipitated by antibodies directed against the C-terminal cross-reactive antigenic determinant of the VSG. These data provide strong evidence that this glycolipid is a VSG precursor.     

Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei

Endocytosis and intracellular transport has been studied in the bloodstream forms of Trypanosoma brucei by light and electron microscopy, using colloidal gold coupled to bovine transferrin (transferrin-gold). The endocytosed transferrin-gold, visualized by silver intensification for light microscopy, was present in vesicular structures between the cell nucleus and flagellar pocket of the organism. At the ultrastructural level, transferrin-gold was present after a 10-min incubation in the flagellar pocket, coated vesicles, cisternal networks, and lysosomelike structures. Endocytosis and intracellular processing of T. brucei variable surface glycoprotein (VSG) was studied using two preparations of affinity-purified rabbit IgG directed against different parts of the VSG. One preparation of IgG was directed against the cross-reacting determinant (CRD): a complex glycolipid side chain covalently linked to the COOH-terminus of the VSG molecule. The other was directed against determinants on the rest of the VSG molecule. When the two IgG preparations were used on thawed, thin cryosections of trypanosomes that had been incubated in transferrin-gold before fixation, the organelles involved with transferrin-gold endocytosis labeled with both antibodies, as well as many vesicular, tubular, and vacuolar structures that did not contain endocytosed transferrin-gold. Both antibodies also labeled the cell surface. In double-labeling experiments both antibodies were closely associated except that IgG directed against the VSG molecule labeled all the cisternae of the Golgi apparatus, whereas anti-CRD IgG was shown to label only half of the Golgi apparatus. Evidence for sorting of VSG molecules from endocytosed transferrin-gold was found. Double-labeling experiments also showed some tubular profiles which labeled on one side with anti-CRD IgG and on the other side with anti-VSG IgG, suggesting a possible segregation of parts of the VSG molecule.     

Topologic analysis of the epitopes of a variant surface glycoprotein of Trypanosoma brucei

A panel of variant-specific mAb has been raised against the Trypanosoma brucei variant MITat 1.2. The binding characteristics of these mAb have been determined by a combination of immunofluorescence assays, using living or fixed trypanosomes, and solid phase assays, using purified variant surface glycoprotein. In addition, these mAb have been tested for their ability to neutralize MITat 1.2 infections in mice. Finally, the epitopes recognized by the mAb have been defined by competitive binding assays. These results are discussed with respect to the structural organization of the surface coat of T. brucei.     

Beyond immune escape: a variant surface glycoprotein causes suramin resistance in Trypanosoma brucei

Suramin is one of the first drugs developed in a medicinal chemistry program (Bayer, 1916), and it is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense. Cellular uptake of suramin occurs by endocytosis, and reverse genetic studies with T. b. brucei have linked downregulation of the endocytic pathway to suramin resistance. Here we show that forward selection for suramin resistance in T. brucei spp. cultures is fast, highly reproducible and linked to antigenic variation. Bloodstream‐form trypanosomes are covered by a dense coat of variant surface glycoprotein (VSG), which protects them from their mammalian hosts' immune defenses. Each T. brucei genome contains over 2000 different VSG genes, but only one is expressed at a time. An expression switch to one particular VSG, termed VSG^Sur, correlated with suramin resistance. Reintroduction of the originally expressed VSG gene in resistant T. brucei restored suramin susceptibility. This is the first report of a link between antigenic variation and drug resistance in African trypanosomes.     

Trypanosoma brucei: release of variant surface glycoprotein during the parasite life cycle

African trypanosome variant surface antigen, which was released from the Trypanosoma brucei parasite at two stages in its life cycle, has been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. Variant surface antigen released as exoantigen into the bloodstream of infected rats resembled the soluble form of the surface antigen. Variant surface antigen released from parasites undergoing transformation to the uncoated procyclic stage was detected as two molecular species: soluble variant surface antigen and a cleavage product of variant surface antigen. The data presented are consistent with enzymatic cleavage of the variant surface antigen C-terminal hydrophobic moiety operating to release parasite surface coat from living parasites.     

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

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