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.     

Release of the surface coat from the plasma membrane of intact bloodstream forms of Trypanosoma brucei requires Ca2+

A-antigenicity; Glycocalyx; Hydrolases; Membrane glycoproteins     

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.     

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.     

Trypanosoma brucei: immunogenicity of the variant surface coat glycoprotein of virulent and avirulent subspecies

Comparative analyses were made to define the immunogenic role in mice of the variant surface coat glycoprotein (VSG) of African trypanosomes. Less than 10 micrograms of the glycoprotein fixed to trypanosomes or covalently linked to sheep erythrocytes were 100 times more immunogenic than soluble VSG. Therefore, although VSG is present on the parasites and in the blood of infected hosts, the cell-bound form most likely elicits immunity. Intravenous administration of soluble or cell-bound VSG was a better route of immunization than the subcutaneous route. Therefore, although parasites grow at the site of infection, in tissue spaces, and in the blood, control of blood parasitemia is best developed if the antigen is introduced to the vascular bed. Full protection against homologous challenge occurred by 4 days and was maintained through 30 days. Trypanosome-agglutinating antibody titers could be measured at 3 days, peaked at 5 days, and remained high through 14 days after immunization. Therefore, mice immunized with an optimal dosage of VSG, 2 days before challenge, should have had ample time to elicit a protective response. Most of these mice, however, developed patent infections, and one-third died during the first peak of parasitemia at about the same time as untreated control mice. This indicates that active infection inhibits the early phases of induction of immunity. Mice, suboptimally immunized against and challenged with an avirulent isolate of Trypanosoma brucei gambiense, survived at higher rates than mice immunized and challenged with a virulent clone of T. b. rhodesiense. Cell-fixed and soluble VSG from both parasites elicited similar agglutinating-antibody titers, indicating that the two trypanosomes were equally antigenic. Results from neutralization tests, however, revealed that, per unit of immune mouse serum, 400 times more T. b. gambiense became noninfective than T. b. rhodesiense. Apparently, virulence is related to relative sensitivity of the trypanosomes to immunological assault.     

The role of duplication in the expression of a variable surface glycoprotein gene of Trypanosoma brucei

The variable surface glycoprotein (VSG) genes of Trypanosoma brucei have been classified into two groups depending upon whether or not duplication of the genes is observed when they are expressed. We report here the observation of duplication apparently linked to expression of the ILTaT 1.3 gene in the ETaR 1 trypanosome stock. In the ILTaR 1 stock, expression of the ILTaT 1.3 VSG did not involve a new duplication, but instead activation of a preexisting gene copy that had been apparently generated earlier by a duplication event analogous to that directly observed in the ETaR 1 trypanosomes. The results suggest that the well-characterised gene duplications found with other VSG genes are common to all VSG genes but are not directly responsible for controlling expression. All currently available data can be accommodated by a model that assumes that gene duplication and replacement occurs independently of antigenic switching.     

Selective cleavage of variant surface glycoproteins from Trypanosoma brucei.

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.     

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.     

Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei.

Soluble glycoproteins have been purified from a series of clones of Trypanosoma brucei 427. Each clone yielded a characteristic predominant glycoprotein which induced clone-specific immunity to trypanosome infection in mice. These glycoproteins were shown by specific labelling and enzyme digestion of cells to be the major components of the trypanosome surface coat. Each glycoprotein consisted of a single polypeptide chain having an apparent molecular weight of 65 000 (as measured by SDS-polyacrylamide gel electrophoresis) and containing around 600 amino acid and 20 monosaccharide residues. Preliminary structural studies indicated large changes in amino acid sequence dispersed over a considerable length of the polypeptide chain. Proteolytic activity was demonstrated in semi-purified trypanosome extracts, providing one reason for the heterogeneity sometimes observed in surface glycoprotein antigen preparations.     

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.     

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.     

Structure of the C-terminal domain from Trypanosoma brucei variant surface glycoprotein MITat1.2

The variant surface glycoprotein (VSG) of African trypanosomes has a structural role in protecting other cell surface proteins from effector molecules of the mammalian immune system and also undergoes antigenic variation necessary for a persistent infection in a host. Here we have reported the solution structure of a VSG type 2 C-terminal domain from MITat1.2, completing the first structure of both domains of a VSG. The isolated C-terminal domain is a monomer in solution and forms a novel fold, which commences with a short alpha-helix followed by a single turn of 3(10)-helix and connected by a short loop to a small anti-parallel beta-sheet and then a longer alpha-helix at the C terminus. This compact domain is flanked by two unstructured regions. The structured part of the domain contains 42 residues, and the core comprises 2 disulfide bonds and 2 hydrophobic residues. These cysteines and hydrophobic residues are conserved in other VSGs, and we have modeled the structures of two further VSG C-terminal domains using the structure of MITat1.2. The models suggest that the overall structure of the core is conserved in the different VSGs but that the C-terminal alpha-helix is of variable length and depends on the presence of charged residues. The results provided evidence for a conserved tertiary structure for all the type 2 VSG C-terminal domains, indicated that VSG dimers form through interactions between N-terminal domains, and showed that the selection pressure for sequence variation within a conserved tertiary structure acts on the whole 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.     

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.     

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.     

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.