African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat


The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and ‘switching’ to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by zero to three hexose residues and is also present in other VSGs. We demonstrate that this O-glycosylation increases parasite virulence by impairing the generation of protective immunity. These data alter the paradigm of antigenic variation by the African trypanosome, expanding VSG variability beyond amino-acid sequence to include surface post-translational modifications with immunomodulatory impact.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Substantial structural divergence between VSGs.
Fig. 2: Structural identification of O-linked carbohydrate on the VSG3 surface.
Fig. 3: Identification of heterogeneous O-linked glycans in surface loops of multiple VSGs.
Fig. 4: The presence of O-linked glycan impairs immune function.


  1. 1.

    Matthews, K. R., McCulloch, R. & Morrison, L. J. The within-host dynamics of African trypanosome infections. Phil. Trans. R. Soc. B (2015).

  2. 2.

    Hsia, R., Beals, T. & Boothroyd, J. C. Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins. Mol. Microbiol. 19, 53–63 (1996).

    CAS  Article  Google Scholar 

  3. 3.

    Schwede, A., Macleod, O. J., MacGregor, P. & Carrington, M. How does the VSG coat of bloodstream form African trypanosomes interact with external proteins? PLoS Pathog. 11, e1005259 (2015).

    Article  Google Scholar 

  4. 4.

    Metcalf, P., Blum, M., Freymann, D., Turner, M. & Wiley, D. C. Two variant surface glycoproteins of Trypanosoma brucei of different sequence classes have similar 6 Å resolution X-ray structures. Nature 325, 84–86 (1987).

    CAS  Article  Google Scholar 

  5. 5.

    Blum, M. L. et al. A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature 362, 603–609 (1993).

    CAS  Article  Google Scholar 

  6. 6.

    Carrington, M. et al. Variant specific glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. J. Mol. Biol. 221, 823–835 (1991).

    CAS  Article  Google Scholar 

  7. 7.

    Bartossek, T. et al. Structural basis for the shielding function of the dynamic trypanosome variant surface glycoprotein coat. Nat. Microbiol. 2, 1523–1532 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Mendonca-Previato, L., Todeschini, A. R., Heise, N. & Previato, J. O. Protozoan parasite-specific carbohydrate structures. Curr. Opin. Struct. Biol. 15, 499–505 (2005).

    CAS  Article  Google Scholar 

  10. 10.

    Takeuchi, H., Kantharia, J., Sethi, M. K., Bakker, H. & Haltiwanger, R. S. Site-specific O-glucosylation of the epidermal growth factor-like (EGF) repeats of notch: efficiency of glycosylation is affected by proper folding and amino acid sequence of individual EGF repeats. J. Biol. Chem. 287, 33934–33944 (2012).

    CAS  Article  Google Scholar 

  11. 11.

    Cross, G. A., Kim, H. S. & Wickstead, B. Capturing the variant surface glycoprotein repertoire (the VSGnome) of Trypanosoma brucei Lister 427. Mol. Biochem. Parasitol. 195, 59–73 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Black, S. J. et al. Regulation of parasitaemia in mice infected with Trypanosoma brucei. Curr. Top. Microbiol. Immunol. 117, 93–118 (1985).

    CAS  Google Scholar 

  13. 13.

    Pinger, J., Chowdhury, S. & Papavasiliou, F. N. Variant surface glycoprotein density defines an immune evasion threshold for African trypanosomes undergoing antigenic variation. Nat. Commun. 8, 828 (2017).

    Article  Google Scholar 

  14. 14.

    Lisowska, E. The role of glycosylation in protein antigenic properties. Cell Mol. Life Sci. 59, 445–455 (2002).

    CAS  Article  Google Scholar 

  15. 15.

    Rangappa, S. et al. Effects of the multiple O-glycosylation states on antibody recognition of the immunodominant motif in MUC1 extracellular tandem repeats. Med. Chem. Commun. 7, 1102–1122 (2016).

    CAS  Article  Google Scholar 

  16. 16.

    Hirumi, H. & Hirumi, K. Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol. 75, 985–989 (1989).

    CAS  Article  Google Scholar 

  17. 17.

    Cross, G. A. Release and purification of Trypanosoma brucei variant surface glycoprotein. J. Cell. Biochem. 24, 79–90 (1984).

    CAS  Article  Google Scholar 

  18. 18.

    Kabsch, W. XDS. Acta Crystallogr D 66, 125–132 (2010).

  19. 19.

    Evans, P. Scaling and assessment of data quality. Acta Crystallogr D 62, 72–82 (2006).

    Article  Google Scholar 

  20. 20.

    Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D 67, 282–292 (2011).

    CAS  Article  Google Scholar 

  21. 21.

    Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr D 69, 1204–1214 (2013).

    CAS  Article  Google Scholar 

  22. 22.

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50, 760–763 (1994).

  23. 23.

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr A 64, 112–122 (2008).

    CAS  Article  Google Scholar 

  24. 24.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171–1179 (2008).

    CAS  Article  Google Scholar 

  26. 26.

    Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D 67, 235–242 (2011).

    CAS  Article  Google Scholar 

  27. 27.

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53, 240–255 (1997).

    CAS  Article  Google Scholar 

  28. 28.

    Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D 67, 355–367 (2011).

    CAS  Article  Google Scholar 

  29. 29.

    Ali, L. et al. The O-glycomap of lubricin, a novel mucin responsible for joint lubrication, identified by site-specific glycopeptide analysis. Mol. Cell Proteom. 13, 3396–3409 (2014).

    CAS  Article  Google Scholar 

  30. 30.

    Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999).

    CAS  Article  Google Scholar 

  31. 31.

    Böhme, U. & Cross, G. A. M. Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei. J. Cell Sci. 115, 805–816 (2002).

    Google Scholar 

  32. 32.

    Burkard, G., Fragoso, C. M. & Roditi, I. Highly efficient stable transformation of bloodstream forms of Trypanosoma brucei. Mol. Biochem. Parasitol. 153, 220–223 (2007).

    CAS  Article  Google Scholar 

  33. 33.

    Wirtz, E., Leal, S., Ochatt, C. & Cross, G. A. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89–101 (1999).

    CAS  Article  Google Scholar 

  34. 34.

    Leal, S. et al. Virulence of Trypanosoma brucei strain 427 is not affected by the absence of glycosylphosphatidylinositol phospholipase C. Mol. Biochem. Parasitol. 114, 245–247 (2001).

    CAS  Google Scholar 

Download references


We thank G. Cross (Rockefeller University) and H. Wardemann (DKFZ) for critical reading of the manuscript and for general advice, M. Sanches-Vaz and L. Figueiredo (IMM, Lisbon) for help with mouse experiments and M. Chandra (DKFZ) for providing us with purified VSG615. We also thank the staff at Argonne National Laboratories (NE-CAT) for beamline support, and D. Oren at the Structural Biology and the High-Throughput Sequencing and Spectroscopy Resource Centers at Rockefeller University. NE-CAT is funded by an NIH/NIGMS grant (P41 GM103403) and the Pilatus 6M detector on 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). The Advanced Photon Source, within which NE-CAT is located, is a US Department of Energy (DOE) User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. This work was also supported by funds to C.E.S. and F.N.P. from the German Cancer Research Center (DKFZ, Heidelberg) and Rockefeller University, by NIH/NIAID (AI085973) to F.N.P. and by a Wellcome Trust Senior Investigator Award (101842) to M.A.J.F. The University of Dundee MS facility is supported by Wellcome Trust grant 097045.

Author information




J.P., D.N., C.E.S., L.A., M.A.J.F. and F.N.P. conceived and designed the experiments. J.P., D.N., M.L., F.N.P. and F.A.B. carried out the protein purification. C.E.S. and F.A.B. performed the structural prediction analyses. D.N. and C.E.S carried out the crystallography analyses. L.A. and M.A.J.F. performed the MS analyses. J.P., S.C., F.A.B., F.N.P., J.V. and J.R. carried out the trypanosome genetics and growth analyses, antibody assays and mouse infection studies. D.N., L.A., J.P., M.L., S.C., F.A.B., H.-S.K., F.N.P. and C.E.S. contributed reagents, materials and analysis tools. J.P., C.E.S., F.N.P., D.N., L.A. and M.A.J.F. wrote the paper.

Corresponding authors

Correspondence to Michael A. J. Ferguson or F. Nina Papavasiliou or C. Erec Stebbins.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1 and 2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pinger, J., Nešić, D., Ali, L. et al. African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat. Nat Microbiol 3, 932–938 (2018).

Download citation

Further reading


Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing