Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.     

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.     

The inactivation and reactivation of an expression-linked gene copy for a variant surface glycoprotein in Trypanosoma brucei.

We have previously shown that the gene for variant surface glycoprotein 118 of Trypanosoma brucei (strain 427) is activated by a duplicative transposition to a telomeric expression site. In chronically-infected animals, this expression-linked copy is lost when the 118 gene is replaced at the expression site by another variant surface glycoprotein gene. We show here that expression of the 118 gene can also be switched off without loss of the extra expression-linked copy. In two variants, called 1.8b and 1.8c, we find expression of the variant surface glycoprotein 1.8 gene, notwithstanding the continued presence of the 118 expression-linked copy. The 1.8 gene activated has a telomeric location, like the 118 expression-linked copy. In variant 1.8b, activation is accompanied by duplication of the 1.8 gene, resulting in an extra telomeric gene copy; in variant 1.8c it is not. Variants 1.8b and 1.8c both switch back preferentially to expression of the 118 gene. The 5'-flanking regions of the active, inactive and reactivated versions of the 118 expression-linked copy are indistinguishable by restriction mapping up to 28 kb. We conclude that there are at least two separate telomeric expression sites in our T. brucei strain. How these are switched on and off is unclear. The ability to retain expression-linked copies in inactive form may allow the trypanosome to re-programme the order in which variant surface glycoprotein genes are expressed.     

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 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.     

A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site.

A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of bloodstream forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in bloodstream forms is discussed.     

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.