Skip to main content
SpringerLink
Log in
Book cover

Glycosyltransferases pp 249–275Cite as

Creation and Characterization of Glycosyltransferase Mutants of Trypanosoma brucei

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1022))

Abstract

The survival strategies of protozoan parasites frequently involve the participation of glycoconjugates. Trypanosoma brucei expresses complex glycoproteins throughout its life cycle and a review of its repertoire of glycosidic linkages suggests a minimum of 38 glycosyltransferase activities. Here we describe a functional characterization workflow in which we create glycosyltransferase null or conditional null mutants in both the bloodstream and procyclic life-cycle forms of the parasite. Subsequently, we characterize the biochemical phenotype of the mutant strains generated and assign precise functions to the genes involved in glycoconjugate biosynthesis and processing in T. brucei. In this way, a comprehensive picture of ­T. brucei glycosylation associated genes, their specificities and their relationship to similar genes in other organisms can be obtained.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Cross GA (1996) Antigenic variation in trypanosomes: secrets surface slowly. Bioessays 18(4):283–291

    Article  PubMed  CAS  Google Scholar 

  2. Pays E, Vanhamme L, Perez-Morga D (2004) Antigenic variation in Trypanosoma brucei: facts, challenges and mysteries. Curr Opin Microbiol 7(4):369–374. doi:10.1016/j.mib.2004.05.001, S1369527404000645 [pii]

    Article  PubMed  CAS  Google Scholar 

  3. Horn D (2004) The molecular control of antigenic variation in Trypanosoma brucei. Curr Mol Med 4(6):563–576

    Article  PubMed  CAS  Google Scholar 

  4. Stockdale C, Swiderski MR, Barry JD, McCulloch R (2008) Antigenic variation in Trypanosoma brucei: joining the DOTs. PLoS Biol 6(7):e185. doi:08-PLBI-P-2322 [pii], 10.1371/journal.pbio.0060185

    Article  PubMed  Google Scholar 

  5. Ferguson MA, Homans SW, Dwek RA, Rademacher TW (1988) Glycosyl-­phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science 239(4841 Pt 1):753–759

    Article  PubMed  CAS  Google Scholar 

  6. Mehlert A, Richardson JM, Ferguson MA (1998) Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei. J Mol Biol 277(2):379–392

    Article  PubMed  CAS  Google Scholar 

  7. Zamze SE, Ashford DA, Wooten EW, Rademacher TW, Dwek RA (1991) Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins. J Biol Chem 266(30):20244–20261

    PubMed  CAS  Google Scholar 

  8. Zamze SE, Wooten EW, Ashford DA, Ferguson MA, Dwek RA, Rademacher TW (1990) Characterisation of the asparagine-­linked oligosaccharides from Trypanosoma brucei type-I variant surface glycoproteins. Eur J Biochem 187(3):657–663

    Article  PubMed  CAS  Google Scholar 

  9. Mehlert A, Zitzmann N, Richardson JM, Treumann A, Ferguson MA (1998) The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei. Mol Biochem Parasitol 91(1):145–152

    Article  PubMed  CAS  Google Scholar 

  10. Jones DC, Mehlert A, Guther ML, Ferguson MA (2005) Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein. J Biol Chem 280(43):35929–35942

    Article  PubMed  CAS  Google Scholar 

  11. Manthri S, Guther ML, Izquierdo L, Acosta-­Serrano A, Ferguson MA (2008) Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei. Glycobiology 18(5):367–383

    Article  PubMed  CAS  Google Scholar 

  12. Guther ML, Lee S, Tetley L, Acosta-Serrano A, Ferguson MA (2006) GPI-anchored proteins and free GPI glycolipids of procyclic form Trypanosoma brucei are nonessential for growth, are required for colonization of the tsetse fly, and are not the only components of the surface coat. Mol Biol Cell 17(12):5265–5274

    Article  PubMed  CAS  Google Scholar 

  13. Roditi I, Schwarz H, Pearson TW, Beecroft RP, Liu MK, Richardson JP, Bühring HJ, Pleiss J, Bülow R, Williams RO, Overath P (1989) Procyclin gene expression and loss of the variant surface glycoprotein during ­differentiation of Trypanosoma brucei. J Cell Biol 108(2):737–746

    Article  PubMed  CAS  Google Scholar 

  14. Treumann A, Zitzmann N, Hulsmeier A, Prescott AR, Almond A, Sheehan J, Ferguson MA (1997) Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J Mol Biol 269(4):529–547

    Article  PubMed  CAS  Google Scholar 

  15. Acosta-Serrano A, Cole RN, Mehlert A, Lee MG, Ferguson MA, Englund PT (1999) The procyclin repertoire of Trypanosoma brucei. Identification and structural characterization of the Glu-Pro-rich polypeptides. J Biol Chem 274(42):29763–29771

    Article  PubMed  CAS  Google Scholar 

  16. Mehlert A, Treumann A, Ferguson MA (1999) Trypanosoma brucei GPEET-PARP is phosphorylated on six out of seven threonine residues. Mol Biochem Parasitol 98(2):291–296

    Article  PubMed  CAS  Google Scholar 

  17. Schlaeppi AC, Malherbe T, Butikofer P (2003) Coordinate expression of GPEET procyclin and its membrane-associated kinase in Trypanosoma brucei procyclic forms. J Biol Chem 278(50):49980–49987

    Article  PubMed  CAS  Google Scholar 

  18. Ferguson MA, Murray P, Rutherford H, McConville MJ (1993) A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-­phosphatidylinositol membrane anchor. Biochem J 291(Pt 1):51–55

    PubMed  CAS  Google Scholar 

  19. Engstler M, Reuter G, Schauer R (1993) The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein. Mol Biochem Parasitol 61(1):1–13

    Article  PubMed  CAS  Google Scholar 

  20. Pontes de Carvalho LC, Tomlinson S, Vandekerckhove F, Bienen EJ, Clarkson AB, Jiang MS, Hart GW, Nussenzweig V (1993) Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor. J Exp Med 177(2):465–474

    Article  PubMed  CAS  Google Scholar 

  21. Montagna G, Cremona ML, Paris G, Amaya MF, Buschiazzo A, Alzari PM, Frasch AC (2002) The trans-sialidase from the african trypanosome Trypanosoma brucei. Eur J Biochem 269(12):2941–2950

    Article  PubMed  CAS  Google Scholar 

  22. Montagna GN, Donelson JE, Frasch AC (2006) Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane. J Biol Chem 281(45):33949–33958

    Article  PubMed  CAS  Google Scholar 

  23. Nagamune K, Acosta-Serrano A, Uemura H, Brun R, Kunz-Renggli C, Maeda Y, Ferguson MA, Kinoshita T (2004) Surface sialic acids taken from the host allow trypanosome survival in tsetse fly vectors. J Exp Med 199(10):1445–1450

    Article  PubMed  CAS  Google Scholar 

  24. Acosta-Serrano A, Vassella E, Liniger M, Kunz Renggli C, Brun R, Roditi I, Englund PT (2001) The surface coat of procyclic Trypanosoma brucei: programmed expression and proteolytic cleavage of procyclin in the tsetse fly. Proc Natl Acad Sci USA 98(4):1513–1518

    Article  PubMed  CAS  Google Scholar 

  25. Guther ML, Beattie K, Lamont DJ, James J, Prescott AR, Ferguson MA (2009) Fate of ­glycosylphosphatidylinositol (GPI)-less procyclin and characterization of sialylated non-GPI-­anchored surface coat molecules of procyclic-­form Trypanosoma brucei. Eukaryot Cell 8(9):1407–1417. doi:EC.00178-09 [pii], 10.1128/EC.00178-09

    Article  PubMed  CAS  Google Scholar 

  26. Urwyler S, Studer E, Renggli CK, Roditi I (2007) A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei. Mol Microbiol 63(1):218–228

    Article  PubMed  CAS  Google Scholar 

  27. Ziegelbauer K, Overath P (1992) Identification of invariant surface glycoproteins in the bloodstream stage of Trypanosoma brucei. J Biol Chem 267(15):10791–10796

    PubMed  CAS  Google Scholar 

  28. Steverding D (2000) The transferrin receptor of Trypanosoma brucei. Parasitol Int 48(3):191–198. doi:S1383-5769(99)00018-5 [pii]

    Article  PubMed  CAS  Google Scholar 

  29. Steverding D, Stierhof YD, Fuchs H, Tauber R, Overath P (1995) Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei. J Cell Biol 131(5):1173–1182

    Article  PubMed  CAS  Google Scholar 

  30. Mehlert A, Ferguson MA (2007) Structure of the glycosylphosphatidylinositol anchor of the Trypanosoma brucei transferrin receptor. Mol Biochem Parasitol 151(2):220–223

    Article  PubMed  CAS  Google Scholar 

  31. Kelley RJ, Brickman MJ, Balber AE (1995) Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes. Mol Biochem Parasitol 74(2):167–178

    Article  PubMed  CAS  Google Scholar 

  32. Izquierdo L, Schulz BL, Rodrigues JA, Guther ML, Procter JB, Barton GJ, Aebi M, Ferguson MA (2009) Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases. EMBO J 28(17):2650–2661. doi:emboj2009203 [pii], 10.1038/emboj.2009.203

    Article  PubMed  CAS  Google Scholar 

  33. Acosta-Serrano A, O’Rear J, Quellhorst G, Lee SH, Hwa KY, Krag SS, Englund PT (2004) Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant. Eukaryot Cell 3(2):255–263

    Article  PubMed  CAS  Google Scholar 

  34. Atrih A, Richardson JM, Prescott AR, Ferguson MA (2005) Trypanosoma brucei glycoproteins contain novel giant ­poly-N-­acetyllactosamine carbohydrate chains. J Biol Chem 280(2):865–871

    Article  PubMed  CAS  Google Scholar 

  35. Roper JR, Guther ML, Milne KG, Ferguson MA (2002) Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei. Proc Natl Acad Sci U S A 99(9):5884–5889

    Article  PubMed  CAS  Google Scholar 

  36. Roper JR, Guther ML, Macrae JI, Prescott AR, Hallyburton I, Acosta-Serrano A, Ferguson MA (2005) The suppression of galactose metabolism in procylic form Trypanosoma brucei causes cessation of cell growth and alters procyclin glycoprotein structure and copy number. J Biol Chem 280(20):19728–19736

    Article  PubMed  CAS  Google Scholar 

  37. Urbaniak MD, Turnock DC, Ferguson MA (2006) Galactose starvation in a bloodstream form Trypanosoma brucei UDP-glucose 4′-epimerase conditional null mutant. Eukaryot Cell 5(11):1906–1913

    Article  PubMed  CAS  Google Scholar 

  38. Stokes MJ, Guther ML, Turnock DC, Prescott AR, Martin KL, Alphey MS, Ferguson MA (2008) The synthesis of UDP-N-­acetylglucosamine is essential for bloodstream form trypanosoma brucei in vitro and in vivo and UDP-N-acetylglucosamine starvation reveals a hierarchy in parasite protein glycosylation. J Biol Chem 283(23):16147–16161

    Article  PubMed  CAS  Google Scholar 

  39. Marino K, Guther ML, Wernimont AK, Qiu W, Hui R, Ferguson MA (2011) Characterization, localization, essentiality, and high-resolution crystal structure of glucosamine 6-phosphate N-acetyltransferase from Trypanosoma brucei. Eukaryot Cell 10(7):985–997. doi:EC.05025-11 [pii], 10.1128/EC.05025-11

    Article  PubMed  CAS  Google Scholar 

  40. Narimatsu H (2006) Human glycogene cloning: focus on beta 3-glycosyltransferase and beta 4-glycosyltransferase families. Curr Opin Struct Biol 16(5):567–575. doi:S0959-­440X(06)00148-5 [pii], 10.1016/j.sbi.2006.09.001

    Article  PubMed  CAS  Google Scholar 

  41. Izquierdo L, Nakanishi M, Mehlert A, Machray G, Barton GJ, Ferguson MA (2009) Identification of a glycosylphosphatidylinositol anchor-modifying beta1-3 N-acetylglucosaminyl transferase in Trypanosoma brucei. Mol Microbiol 71(2):478–491

    Article  PubMed  CAS  Google Scholar 

  42. Wirtz E, Leal S, Ochatt C, Cross GA (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominant-­negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99(1):89–101

    Article  PubMed  CAS  Google Scholar 

  43. Wirtz E, Clayton C (1995) Inducible gene expression in trypanosomes mediated by a prokaryotic repressor. Science 268(5214):1179–1183

    Article  PubMed  CAS  Google Scholar 

  44. Hirumi H, Hirumi K (1989) 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(6):985–989

    Article  PubMed  CAS  Google Scholar 

  45. Brun R, Schonenberger M (1979) Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop 36(3):289–292

    PubMed  CAS  Google Scholar 

  46. Barnes RL, McCulloch R (2007) Trypanosoma brucei homologous recombination is dependent on substrate length and homology, though displays a differential dependence on mismatch repair as substrate length decreases. Nucleic Acids Res 35(10):3478–3493. doi:gkm249 [pii], 10.1093/nar/gkm249

    Article  PubMed  CAS  Google Scholar 

  47. Izquierdo L, Atrih A, Rodrigues JA, Jones DC, Ferguson MA (2009) Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase has unusual substrate specificity and protects the parasite from stress. Eukaryot Cell 8(2):230–240

    Article  PubMed  CAS  Google Scholar 

  48. Ferguson MA, Duszenko M, Lamont GS, Overath P, Cross GA (1986) Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor. J Biol Chem 261(1):356–362

    PubMed  CAS  Google Scholar 

  49. Merkle RK, Cummings RD (1987) Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains. J Biol Chem 262(17):8179–8189

    PubMed  CAS  Google Scholar 

  50. Baenziger JU, Fiete D (1979) Structural determinants of Ricinus communis agglutinin and toxin specificity for oligosaccharides. J Biol Chem 254(19):9795–9799

    PubMed  CAS  Google Scholar 

  51. Nett IR, Mehlert A, Lamont D, Ferguson MA (2010) Application of electrospray mass spectrometry to the structural determination of glycosylphosphatidylinositol membrane anchors. Glycobiology 20(5):576–585. doi:cwq007 [pii], 10.1093/glycob/cwq007

    Article  PubMed  CAS  Google Scholar 

  52. Richardson JP, Beecroft RP, Tolson DL, Liu MK, Pearson TW (1988) Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol Biochem Parasitol 31(3):203–216

    Article  PubMed  CAS  Google Scholar 

  53. Richardson JP, Jenni L, Beecroft RP, Pearson TW (1986) Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface ­antigens identified by monoclonal antibodies. J Immunol 136(6):2259–2264

    PubMed  CAS  Google Scholar 

  54. Ferguson MAJ (1994) Glycobiology: a practical approach. GPI membrane anchors: isolation and analysis, vol Chapter 8. Fukuda, M. and Kobata, A. (eds.) IRL Press at Oxford University Press, Oxford

    Google Scholar 

  55. Baldwin MA (2005) Analysis of glycosylphosphatidylinositol protein anchors: the prion protein. Methods Enzymol 405:172–187

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Angela Mehlert, Isabelle Nett, Sujatha Manthri, Deuan Jones, and Alvaro Acosta-Serrano who all contributed to the development of the analyses described herein. This work was supported by a programme grant (085622) and a strategic award (083481) from the Wellcome Trust.

Author information

Authors and Affiliations

  1. Barcelona Centre for International Health Research, (CRESIB, Hospital Clinic-Universitat de Barcelona), Barcelona, Spain

    Luis Izquierdo

  2. Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, Scotland, UK

    M. Lucia S. Güther & Michael A. J. Ferguson

Authors
  1. Luis Izquierdo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Lucia S. Güther
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael A. J. Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Medicine, Queen's University, Kingston, Ontario, Canada

    Inka Brockhausen

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Izquierdo, L., Güther, M.L.S., Ferguson, M.A.J. (2013). Creation and Characterization of Glycosyltransferase Mutants of Trypanosoma brucei . In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-465-4_19

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-464-7

  • Online ISBN: 978-1-62703-465-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation