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Author(s): J. N. Reeves (corresponding author) [1]; D. Watson [1]; J. P. Osborne [1]; K. A. Pounds [1]; P. T. O'Brien [1]; A. D. T. Short [1]; M. J. L. [...]

The variable domain of the trypanosome variant surface glycoprotein (VSG) ILTat 1.24 has been shown by X-ray crystallography to resemble closely the structures of VSG MITat 1.2, despite their low sequence similarity. Specific structural features of these VSGs, including substitution of carbohydrate for an alpha-helix, can be found in other VSG sequences. Thus antigenic variation in trypanosomes is accomplished by sequence variation, not gross structural alteration; the extensive sequence differences among VSGs may be required for another reason, such as the avoidance of recognition by helper T cells. Additionally, VSG sequences are found to define families, within a VSG superfamily, which have evolved in the trypanosome genome.

African trypanosomes are parasitic protozoa which are enveloped by a surface coat consisting of a matrix of identical glycoprotein molecules. Variations in the composition of these variant surface glycoproteins (VSGs) allow the parasite to escape the host's immune system and render effective immunoprophylaxis improbable. However, underlying the surface coat, all variant antigen types contain common membrane components, some of which can activate complement by the alternative pathway, leading to lysis of uncoated trypanosomes. Hence, stimulation of VSG release in vivo should be a potential form of chemotherapy, and we have therefore investigated the mode of attachment of VSG to the plasma membrane. Biochemical characterization of VSGs from several species has been performed on material purified after release from the cell surface following rupture of the trypanosome. We demonstrate here that VSGs of Trypanosoma brucei when bound to the membrane exist in a form which differs both biochemically and immunochemically from VSGs purified in the conventional manner. After rupture of the cell, membrane-form VSG (mfVSG) is enzymatically transformed into the commonly isolated water-soluble released form (sVSG). In conditions in which this modification does not take place, purified VSGs have amphiphilic properties and behave as integral membrane proteins by the criterion of charge-shift electrophoresis. The difference between the two forms lies in the C-terminal domain, which is phosphorylated in both forms. This domain in sVSGs contains an immunogenic oligosaccharide known as the cross-reacting determinant (CRD), attached to the C-terminal amino acid. Recognition of this determinant by anti-CRD antibodies is impaired in the membrane form.

Antigenic variation in the African trypanosomes involves the sequential expression of genes coding for different variant surface glycoproteins (VSGs) (reviewed in refs 1-3). When expression of some VSG genes is switched on, a newly duplicated copy of the expressed gene has been observed within the trypanosome genome, which is not found after the gene's expression is switched off again. The duplicated copy has therefore been called an expression-linked copy (ELC). The expression of the gene appears to be strictly coupled to the presence of the ELC. This has led to the hypothesis that the duplicative transposition generating the ELC may itself be responsible for the control of VSG expression. With other VSG genes, expression-linked duplication has not been observed, and expression is clearly not controlled in this way. Data are presented here which demonstrate that either of these observations may be obtained with a single VSG gene, depending on the chance selection of particular clones from antigenically switched populations. Thus, the different observations do not imply the existence of two distinct classes of VSG gene controlled by different mechanisms, but different aspects of processes common to all VSG genes.