Purification and Properties of the Membrane Form of Variant Surface Glycoproteins (VSGs) from Trypanosoma brucei1,2,3

Variant surface glycoproteins (VSG) of Trypanosoma brucei are released in a water soluble form on impairment of membrane integrity. We have previously shown that this release is the result of an enzyme-mediated event which converts the hydrophobic membrane form VSG into the hydrophilic water-soluble form. We now present further details of the methods by which membrane form VSG (mfVSG) may be isolated, uncontaminated by water-soluble VSG (sVSG). The sensitivity to different metal ions of the enzyme that mediated the conversion event is discussed, and some biochemical characteristics of different mfVSG preparations are presented.     

Trypanosoma brucei variant surface glycoprotein has a sn-1,2-dimyristyl glycerol membrane anchor at its COOH terminus

The membrane form of Trypanosoma brucei variant surface glycoprotein (mfVSG) is acylated with ester-linked tetradecanoic (myristic) acid (Ferguson, M. A. J., and Cross, G. A. M. (1984) J. Biol. Chem. 259, 3011-3015). Comparative analysis of Pronase peptides from mfVSG and soluble VSG localizes the site of mfVSG acylation to a COOH-terminal oligosaccharide structure. Chemical and enzymatic treatment of the acylated Pronase mfVSG fragment revealed that the myristic acid is present as a diglyceride (sn-1,2-dimyristin) that is probably linked to the COOH-terminal oligosaccharide via a phosphodiester bond between the sn-3-glycerol hydroxyl and a sugar hydroxyl group. The endogenous membrane-associated enzyme, which quantitatively cleaves myristic acid from mfVSG to produce soluble VSG, releases diglyceride, as would be expected of a phospholipase C.     

Intermolecular interactions involved in the association of the variant surface glycoprotein of Trypanosoma brucei

Trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (VSG). Depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. VSG is accessible as soluble globular protein (sVSG), or as insoluble membrane form (mfVSG). In order to get insight into the two-dimensional association of VSG within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. No self-assembly of sVSG could be detected even at protein concentrations close to the local packing in the surface layer. The absence of preferential interactions with soybean phospholipid or lysolecithin monolayers (spread on a Langmuir trough) suggests that the soluble form of the protein is not integrated into a model lipid-water interface. Thus, the two-dimensional arrangement of the protein in situ seems to be determined by hydrophobic interactions of the lipid components rather than protein-lipid interactions. In contrast to sVSG, the membrane form (mfVSG) undergoes aggregation and shows a strong tendency to absorb to surfaces and chromatographic matrices, thus interfering with standard techniques of protein purification.     

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.     

Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.     

Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei.

Surface proteins of the mammalian stage of the parasitic protozoan, Trypanosoma brucei, were biotinylated with sulfosuccinimidyl 6-(biotinamido) hexanoate. Since the predominant protein labeled by this reagent is the membrane form of the variant surface glycoprotein (mfVSG), a procedure was developed to convert mfVSG to its soluble form by the endogenous glycosylphosphatidylinositol-specific phospholipase C while retaining other biotinylated surface proteins in a membrane-bound state. From these membranes, three novel glycoproteins of 60, 65, and 75 kDa could be isolated by a combination of Triton X-114 phase separation and precipitations by streptavidin and concanavalin A coupled to solid supports. These polypeptides were detected in trypanosomes expressing different mfVSGs and are thus considered to be invariant. In a variant clone in which the mfVSG is trypsin-sensitive, the invariant surface glycoproteins of 65 and 75 kDa, designated ISG65 and ISG75, respectively, were proteolytically degraded with similar kinetics as the mfVSG. Neither ISG65 nor ISG75 could be detected in procyclic trypanosomes, the stage of the parasite characteristic for the insect midgut. Gene cloning reported in the accompanying paper (Ziegelbauer, K., Multhaup, G., and Overath, P. (1992) J. Biol. Chem. 267, 10797-10803) suggests that ISG65 and ISG75 are transmembrane proteins.     

Evidence of myristylated disulfide-linked dimer of variant surface glycoprotein of Trypanosoma brucei-brucei

1. Variant surface glycoprotein (VSGs) of Trypanosoma brucei-brucei may exist as a disulfide-linked dimer in both forms: myristylated (mfVSG) and non-myristylated (sVSG), as judge by fluorography and immunoblotting of SDS-PAGE under non-reducing conditions. 2. The dimeric VSG form is labeled with [3H]-myristic acid in our incorporation conditions. 3. AnTat 1.1 trypanosomes preincubated with tunicamycin and incubated with [3H]-myristic acid synthesized a labeled molecule that has an apparent molecular weight slightly smaller than the native form, and that also corresponds to a disulfide-linked dimer.     

Rapid lateral diffusion of the variant surface glycoprotein in the coat of Trypanosoma brucei

The membrane form of the variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of Trypanosoma brucei by a dimyristoylphosphatidylinositol residue connected via a glycan to the COOH-terminal amino acid. The glycoprotein molecules are tightly packed, forming a coat that is impenetrable to lytic serum components. Lateral diffusion of mfVSG was measured by the fluorescence recovery after photobleaching technique. mfVSG labeled on the cell surface with rhodamine-conjugated anti-VSG Fab fragments showed a diffusion coefficient of 1 X 10(-10) cm2/s at 37 degrees C and of 0.7 X 10(-10) cm2/s at 27 degrees C. About 80% of the molecules were mobile. Affinity-purified mfVSG molecules implanted into the plasma membrane of baby hamster kidney cells exhibited a similar mobility to that found in the trypanosome coat [D = (0.4-0.7) X 10(-10) cm2/s at 4 degrees C]. Phospholipid mobility in the plasma membrane of trypanosomes was characterized by a diffusion coefficient of 2.2 X 10(-9) cm2/s at 37 degrees C. It is concluded that mfVSG mobility in the surface coat of the parasite is rapid and comparable to that of other membrane-bound glycoproteins but slower than that of phospholipids.     

Purification and characterization of the membrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei

The conversion of the membrane-form variant surface glycoprotein (mfVSG) of the unicellular parasitic flagellate Trypanosoma brucei to soluble variant surface glycoprotein and sn-1,2-dimyristoyl glycerol is catalyzed by an endogeneous, membrane bound phospholipase C-like hydrolase. Using a monoclonal antibody against the enzyme the hydrolase was purified 3,000-fold with a yield of 32%. The enzyme has a molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The rate with which mfVSG hydrolase cleaves phosphatidylinositol is 170 times lower than the cleavage rate for mfVSG, whereas phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine cannot serve as substrates. Reconstitution experiments into phospholipid vesicles show that the enzyme can hydrolyze mfVSG when present in the same phospholipid bilayer but not when present in separate bilayers.     

Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.     

Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles,sorting by default in endosomes,and exocytosis via RAB11-positive carriers

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endo- and exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50-60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11- as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.     

How Does the VSG Coat of Bloodstream Form African Trypanosomes Interact with External Proteins?

Variations on the statement “the variant surface glycoprotein (VSG) coat that covers the external face of the mammalian bloodstream form of Trypanosoma brucei acts a physical barrier” appear regularly in research articles and reviews. The concept of the impenetrable VSG coat is an attractive one, as it provides a clear model for understanding how a trypanosome population persists; each successive VSG protects the plasma membrane and is immunologically distinct from previous VSGs. What is the evidence that the VSG coat is an impenetrable barrier, and how do antibodies and other extracellular proteins interact with it? In this review, the nature of the extracellular surface of the bloodstream form trypanosome is described, and past experiments that investigated binding of antibodies and lectins to trypanosomes are analysed using knowledge of VSG sequence and structure that was unavailable when the experiments were performed. Epitopes for some VSG monoclonal antibodies are mapped as far as possible from previous experimental data, onto models of VSG structures. The binding of lectins to some, but not to other, VSGs is revisited with more recent knowledge of the location and nature of N-linked oligosaccharides. The conclusions are: (i) Much of the variation observed in earlier experiments can be explained by the identity of the individual VSGs. (ii) Much of an individual VSG is accessible to antibodies, and the barrier that prevents access to the cell surface is probably at the base of the VSG N-terminal domain, approximately 5 nm from the plasma membrane. This second conclusion highlights a gap in our understanding of how the VSG coat works, as several plasma membrane proteins with large extracellular domains are very unlikely to be hidden from host antibodies by VSG.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.     

Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion.

Using transformed procyclic trypanosomes, the synthesis, intracellular transport and secretion of wild-type and mutant variant surface glycoprotein (VSG) is characterized. We find no impediment to the expression of this bloodstream stage protein in insect stage cells. VSG receives a procyclic-type phosphatidylinositol-specific phospholipase C-resistant glycosyl phosphatidylinositol (GPI) anchor, dimerizes and is N-glycosylated. It is transported to the plasma membrane with rapid kinetics (t(1/2) approximately 1 h) and then released by a cell surface zinc-dependent metalloendoprotease activity, a possible homolog of leishmanial gp63. Deletion of the C-terminal GPI addition signal generates a soluble form of VSG that is exported with greatly reduced kinetics (t(1/2) approximately 5 h). Fusion of the procyclic acidic repetitive protein (PARP) GPI anchor signal to the C-terminus of the truncated VSG reporter restores both GPI addition and transport competence, suggesting that GPI anchors play a critical role in the folding and/or forward transport of newly synthesized VSG. The VSG-PARP fusion is also processed near the C-terminus by events that do not involve N-linked oligosaccharides and which are consistent with GPI side chain modification. This unexpected result suggests that GPI processing may be influenced by adjacent peptide sequence or conformation.     

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.     

Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides

The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.     

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.     

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein.

The major surface antigen of the mammalian bloodstream form of Trypanosoma brucei, the variant surface glycoprotein (VSG), is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The VSG anchor is susceptible to phosphatidylinositol-specific phospholipase C (PI-PLC). Candidate precursor glycolipids, P2 and P3, which are PI-PLC-sensitive and -resistant respectively, have been characterized in the bloodstream stage. In the insect midgut stage, the major surface glycoprotein, procyclic acidic repetitive glycoprotein, is also GPI-anchored but is resistant to PI-PLC. To determine how the structure of the GPI anchor is altered at different life stages, we characterized candidate GPI molecules in procyclic T. brucei. The structure of a major procyclic GPI, PP1, is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6 Man alpha 1-GlcN-acylinositol, linked to lysophosphatidic acid. The inositol can be labeled with [3H]palmitic acid, and the glyceride with [3H]stearic acid. We have also found that all detectable ethanolamine-containing GPIs from procyclic cells contain acylinositol and are resistant to cleavage by PI-PLC. This suggests that the procyclic acidic repetitive glycoprotein GPI anchor structure differs from that of the VSG by virtue of the structures of the GPIs available for transfer.