Abstract
The surface of Trypanosoma brucei brucei insect forms is covered by an invariant protein coat consisting of procyclins. There are six or seven procyclin genes that encode unusual proteins with extensive tandem repeat units of glutamic acid (E) and proline (P) (referred to as EP repeats), and two genes that encode proteins with internal pentapeptide (GPEET) repeats. Although the EP forms of procyclins have been isolated and characterized by several laboratories, evidence for GPEET procyclin has largely been confined to the expression of its mRNA. To characterize GPEET procyclin further, we isolated the protein from T. b. brucei strain 427. We found that label from [3H]myristic acid and [3H]ethanolamine was incorporated into GPEET procyclin and we demonstrated the protein's covalent modification with a glycosylphosphatidylinositol anchor. The major form of GPEET procyclin showed an apparent molecular mass of 22-32 kDa, was susceptible to proteolytic treatment and was found to be phosphorylated. Surprisingly, our results show that GPEET procyclin represents the major form of procyclin in T. b. brucei 427 culture forms and that the ratio of EP to GPEET procyclin can vary considerably between different cell lines.
Full Text
The Full Text of this article is available as a PDF (388.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
- Bütikofer P., Boschung M. Glycosyl inositolphospholipid-anchored structures in Herpetomonas davidi. Mol Biochem Parasitol. 1995 Oct;74(1):65–75. doi: 10.1016/0166-6851(95)02484-0. [DOI] [PubMed] [Google Scholar]
- Bütikofer P., Boschung M., Menon A. K. Production of a nested set of glycosylphosphatidylinositol structures from a glycosylphosphatidylinositol-anchored protein. Anal Biochem. 1995 Jul 20;229(1):125–132. doi: 10.1006/abio.1995.1388. [DOI] [PubMed] [Google Scholar]
- Clayton C. E., Mowatt M. R. The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification. J Biol Chem. 1989 Sep 5;264(25):15088–15093. [PubMed] [Google Scholar]
- Cross G. A., Manning J. C. Cultivation of Trypanosoma brucei sspp. in semi-defined and defined media. Parasitology. 1973 Dec;67(3):315–331. doi: 10.1017/s0031182000046540. [DOI] [PubMed] [Google Scholar]
- Engstler M., Reuter G., Schauer R. The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein. Mol Biochem Parasitol. 1993 Sep;61(1):1–13. doi: 10.1016/0166-6851(93)90153-o. [DOI] [PubMed] [Google Scholar]
- Ferguson M. A., Homans S. W., Dwek R. A., Rademacher T. W. Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science. 1988 Feb 12;239(4841 Pt 1):753–759. doi: 10.1126/science.3340856. [DOI] [PubMed] [Google Scholar]
- Ferguson M. A., Murray P., Rutherford H., McConville M. J. A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor. Biochem J. 1993 Apr 1;291(Pt 1):51–55. doi: 10.1042/bj2910051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Field M. C., Menon A. K., Cross G. A. A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: lipid structure and biosynthesis. EMBO J. 1991 Oct;10(10):2731–2739. doi: 10.1002/j.1460-2075.1991.tb07821.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geigy R., Kauffmann M. Sleeping sickness survey in the Serengeti area (Tanzania) 1971. I. Examination of large mammals for trypanosomes. Acta Trop. 1973;30(1):12–23. [PubMed] [Google Scholar]
- Gibson W. C., Gashumba J. K. Isoenzyme characterization of some Trypanozoon stocks from a recent trypanosomiasis epidemic in Uganda. Trans R Soc Trop Med Hyg. 1983;77(1):114–118. doi: 10.1016/0035-9203(83)90033-0. [DOI] [PubMed] [Google Scholar]
- Hehl A., Pearson T. W., Barry J. D., Braun R., Roditi I. Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker. Mol Biochem Parasitol. 1995 Mar;70(1-2):45–58. doi: 10.1016/0166-6851(95)00003-j. [DOI] [PubMed] [Google Scholar]
- Hoener M. C., Brodbeck U. Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins. Eur J Biochem. 1992 Jun 15;206(3):747–757. doi: 10.1111/j.1432-1033.1992.tb16981.x. [DOI] [PubMed] [Google Scholar]
- König E., Delius H., Carrington M., Williams R. O., Roditi I. Duplication and transcription of procyclin genes in Trypanosoma brucei. Nucleic Acids Res. 1989 Nov 11;17(21):8727–8739. doi: 10.1093/nar/17.21.8727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Matthews K. R., Gull K. Evidence for an interplay between cell cycle progression and the initiation of differentiation between life cycle forms of African trypanosomes. J Cell Biol. 1994 Jun;125(5):1147–1156. doi: 10.1083/jcb.125.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mayor S., Menon A. K., Cross G. A., Ferguson M. A., Dwek R. A., Rademacher T. W. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6164–6173. [PubMed] [Google Scholar]
- McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mowatt M. R., Clayton C. E. Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol Cell Biol. 1987 Aug;7(8):2838–2844. doi: 10.1128/mcb.7.8.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mowatt M. R., Clayton C. E. Polymorphism in the procyclic acidic repetitive protein gene family of Trypanosoma brucei. Mol Cell Biol. 1988 Oct;8(10):4055–4062. doi: 10.1128/mcb.8.10.4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mowatt M. R., Wisdom G. S., Clayton C. E. Variation of tandem repeats in the developmentally regulated procyclic acidic repetitive proteins of Trypanosoma brucei. Mol Cell Biol. 1989 Mar;9(3):1332–1335. doi: 10.1128/mcb.9.3.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Overath P., Czichos J., Stock U., Nonnengaesser C. Repression of glycoprotein synthesis and release of surface coat during transformation of Trypanosoma brucei. EMBO J. 1983;2(10):1721–1728. doi: 10.1002/j.1460-2075.1983.tb01648.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pinna L. A. Casein kinase 2: an 'eminence grise' in cellular regulation? Biochim Biophys Acta. 1990 Sep 24;1054(3):267–284. doi: 10.1016/0167-4889(90)90098-x. [DOI] [PubMed] [Google Scholar]
- Pontes de Carvalho L. C., Tomlinson S., Vandekerckhove F., Bienen E. J., Clarkson A. B., Jiang M. S., Hart G. W., Nussenzweig V. Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor. J Exp Med. 1993 Feb 1;177(2):465–474. doi: 10.1084/jem.177.2.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richardson J. P., Beecroft R. P., Tolson D. L., Liu M. K., Pearson T. W. Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol Biochem Parasitol. 1988 Dec;31(3):203–216. doi: 10.1016/0166-6851(88)90150-8. [DOI] [PubMed] [Google Scholar]
- Richardson J. P., Jenni L., Beecroft R. P., Pearson T. W. Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies. J Immunol. 1986 Mar 15;136(6):2259–2264. [PubMed] [Google Scholar]
- Roberts W. L., Myher J. J., Kuksis A., Low M. G., Rosenberry T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1988 Dec 15;263(35):18766–18775. [PubMed] [Google Scholar]
- Roditi I., Carrington M., Turner M. Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature. 1987 Jan 15;325(6101):272–274. doi: 10.1038/325272a0. [DOI] [PubMed] [Google Scholar]
- Roditi I., Schwarz H., Pearson T. W., Beecroft R. P., Liu M. K., Richardson J. P., Bühring H. J., Pleiss J., Bülow R., Williams R. O. Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei. J Cell Biol. 1989 Feb;108(2):737–746. doi: 10.1083/jcb.108.2.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenberry T. L., Krall J. A., Dever T. E., Haas R., Louvard D., Merrick W. C. Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification. J Biol Chem. 1989 May 5;264(13):7096–7099. [PubMed] [Google Scholar]
- Ruepp S., Furger A., Kurath U., Renggli C. K., Hemphill A., Brun R., Roditi I. Survival of Trypanosoma brucei in the tsetse fly is enhanced by the expression of specific forms of procyclin. J Cell Biol. 1997 Jun 16;137(6):1369–1379. doi: 10.1083/jcb.137.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Serrano A. A., Schenkman S., Yoshida N., Mehlert A., Richardson J. M., Ferguson M. A. The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J Biol Chem. 1995 Nov 10;270(45):27244–27253. doi: 10.1074/jbc.270.45.27244. [DOI] [PubMed] [Google Scholar]
- Treumann A., Zitzmann N., Hülsmeier A., Prescott A. R., Almond A., Sheehan J., Ferguson M. A. Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J Mol Biol. 1997 Jun 20;269(4):529–547. doi: 10.1006/jmbi.1997.1066. [DOI] [PubMed] [Google Scholar]
- Turco S. J., Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol. 1992;46:65–94. doi: 10.1146/annurev.mi.46.100192.000433. [DOI] [PubMed] [Google Scholar]
- Ziegelbauer K., Quinten M., Schwarz H., Pearson T. W., Overath P. Synchronous differentiation of Trypanosoma brucei from bloodstream to procyclic forms in vitro. Eur J Biochem. 1990 Sep 11;192(2):373–378. doi: 10.1111/j.1432-1033.1990.tb19237.x. [DOI] [PubMed] [Google Scholar]
