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.     

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.     

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.     

Degradation, recycling, and shedding of Trypanosoma brucei variant surface glycoprotein

Trypanosoma brucei bloodstream forms express a densely packed surface coat consisting of identical variant surface glycoprotein (VSG) molecules. This surface coat is subject to antigenic variation by sequential expression of different VSG genes and thus enables the cells to escape the mammalian host's specific immune response. VSG turnover was investigated and compared with the antigen switching rate. Living cells were radiochemically labeled with either 125I-Bolton-Hunter reagent or 35S-methionine, and immunogold-surface labeled for electron microscopy studies. The fate of labeled VSG was studied during subsequent incubation or cultivation of labeled trypanosomes. Our data show that living cells slowly released VSG into the medium with a shedding rate of 2.2 +/- 0.6% h-1 (t1/2 = 33 +/- 9 h). In contrast, VSG degradation accounted for only 0.3 +/- 0.06% h-1 (t1/2 = 237 +/- 45 h) and followed the classical lysosomal pathway as judged by electron microscopy. Since VSG uptake by endocytosis was rather high, our data suggest that most of the endocytosed VSG was recycled to the surface membrane. These results indicate that shedding of VSG at a regular turnover rate is sufficient to remove the old VSG coat within one week, and no increase of the VSG turnover rate seems to be necessary during antigenic variation.     

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.     

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.     

Trypanosoma brucei: frequent loss of a telomeric variant surface glycoprotein gene

We have observed the loss of an inactive telomeric variant surface glycoprotein (VSG) gene that is located on a minichromosome in Trypanosoma brucei. If this is due to gene conversion, it is the third "silent" gene conversion (i.e., one that does not produce an antigenic switch) detected in 19 antigenic switches of the IsTaR 1 serodeme. This is surprisingly frequent since the immune response cannot select against the inactive gene. We estimate that 10(-1) to 10(-3) telomeric VSG gene conversions occur per generation, which is at least 100 times more frequent than antigenic switching. Since all three "silent" gene conversions involved an IsTat 5 VSG gene, the frequency may vary among telomeric VSG genes. However, the high gene conversion frequency for the 5 VSG gene does not ensure a higher antigenic switch frequency than other telomeric VSG genes for which we have probes. These results suggest that gene conversion rapidly alters the repertoire of telomeric VSG genes, possibly including those on minichromosomes, producing a continual variation in the VSG genes that are more likely to be expressed.     

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.     

Molecular heterogeneity of the isolated surface glycoprotein from variant AnTat 1.1 of Trypanosoma brucei brucei

Variant surface glycoprotein (VSG) of Trypanosoma brucei brucei AnTat 1.1 was released by means of the procedure described by Baltz et al. ([1976], Ann. Immunol. [Inst. Pasteur] 127C, 761-774). The concanavalin-A chromatography yielded 3 VSG fractions according to the addition, in the elution buffer, of alpha-methyl-D-mannopyranoside, beta-mercaptoethanol, and sodium dodecyl sulfate. These VSG fractions showed heterogeneous behaviour on reverse-phase high performance liquid chromatography. The 3 VSG fractions as well as the myristylated VSG of AnTat 1.1 essentially consist of dimer VSG forms linked through a disulfide bridge, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and nonreducing conditions.     

The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat

The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the surface coat.     

Production of monoclonal antibodies against the purified glycosylphosphatidylinositol anchor of the variant surface glycoprotein from Trypanosoma brucei brucei.

The glycosylphosphatidylinositol anchor (GPI) from the membrane form variant surface glycoprotein (mfVSG) of Trypanosoma brucei brucei was isolated and identified after radioactive labeling with [3H]myristic acid, by immunostaining on HPTLC with a polyclonal antibody directed against mfVSG and by negative ion laser desorption and fast atom bombardment mass spectrometry of the GPI anchor before and after peracetylation. For the production of monoclonal antibodies the purified GPI molecule was incorporated into liposomes and injected intrasplenically in BALB/c mice. After fusion with the myeloma cell line X63-Ag 8.653 hybridoma cells were cloned by single cell cloning. The secreted antibodies were characterized by ELISA, Ouchterlony immunodiffusion, and Western blot and used in first immunofluorescent studies.