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

Abstract

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.

Fig 1. Structure of VSG221 (MITat1.2).

(A) Illustrated model of VSG221 dimer showing the structures of the N-terminal domain, one monomer in blue and one in grey, and the two C-terminal domains in purple (PDB: 1VSG and 1XU6) [1,9]. The N-linked oligosaccharide in the N-terminal domain is shown in red. Three residues are shown that form the core; there are between one and three further residues not shown. The relative positions of the N- and C-terminal domains are not known, and this illustration is a model [1]. (B) Space-filling model of VSG221 viewed from the x-, y-, and z-axes. The maximum width dimensions of the VSG are shown below, and the fit of an ellipse of approximately 28 Ų is shown around the structure of a VSG viewed from outside the cell.

Fig 2. Comparison of VSG structure with modeled structures for ESAG4 and ISG65.

The line diagram at the top shows the location in the extracellular part of the proteins of the real and putative structured domains. The structures below are coloured from blue at the N-terminus to red at the C-terminus. VSG structures are from PDB: 1VSG and 1XU6. The ESAG4 models were made using Phyre2 [22] and default parameters; the programme gave a 100% confidence model for both domains. The ISG65 model was made using an initial structural alignment using Fugue Profile Library Search [32], and small adjustments were made to align cysteines for disulphide bridge formation. Subsequently, Modeller was used to generate 100 models using standard Modeller scripts [31], and the model with the lowest Discrete Optimized Protein Energy (DOPE) assessment score [33] was selected to be shown here.

Fig 3. VSG models.

(A) A model of VSG121 showing the location of the cyanogen bromide fragment p19 (blue) that contains the epitopes for MoAbs that bound live trypanosomes. From the left, one monomer orientated so the dimerization interface runs vertically up the page; second, rotated approximately 90° so that the dimerization interface has turned away from the observer; third, same view with the surface added. There are potential surface-exposed epitopes along the entire length of the domain. (B) A model of VSG117 showing in blue the location that contained the epitope recognised by a MoAb that bound live cells. (C) Model of VSG WATat1.1 showing the location of differences with the related VSG WATat1.12. A monoclonal antibody that recognises an epitope in WATat1.1 does not bind WATat1.12, so the epitope probably contains one of these differences. An envelope for one possible position of the C-terminal domain is shown in purple.

Fig 4. Packing of the VSG on the plasma membrane and comparison with an immunoglobulin G.

The VSG spacing is based on experimentally determined copy number and surface area estimates. The widest part of the VSG N-terminal domain is shown in red and the C-terminal domain in purple. Together, these two features probably form the barrier that restricts access of immunoglobulins to the plasma membrane. One heavy and one light chain in the IgG is shown in green, the other pair in grey. The VSG structure is derived from PDB 1VSG and 1XU6, the IgG from PDB 1IGY.