Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Jul 5;91(14):6443–6447. doi: 10.1073/pnas.91.14.6443

Expression of a glycosylphosphatidylinositol-anchored Trypanosoma brucei transferrin-binding protein complex in insect cells.

M Chaudhri 1, D Steverding 1, D Kittelberger 1, S Tjia 1, P Overath 1
PMCID: PMC44218  PMID: 8022802

Abstract

The expression site-associated gene ESAG 6 was previously implicated in transferrin binding in the protozoan parasite Trypanosoma brucei. ESAG 6 and the closely related ESAG 7 of T. brucei strain AnTat1.3 have now been expressed in insect cells using the baculovirus expression system. Expression of ESAG 6 alone in insect cells gives rise to a glycosylated protein of approximately 52 kDa, which is cell surface-associated through a glycosylphosphatidylinositol anchor at its C terminus. The ESAG 7 product of about 42 kDa is also glycosylated, but lacks the glycosylphosphatidylinositol modification, and is located intracellularly. No transferrin-binding activity is observed when either ESAG is expressed independently. However, their expression results in a cell surface complex of ESAG 6 and 7 products that specifically binds transferrin. This shows that both ESAG 6 and 7 products are necessary and sufficient for binding to transferrin.

Full text

PDF
6444

Images in this article

6444Image
on p.6444
6445Image
on p.6445
6445Image
on p.6445
6446Image
on p.6446
6446Image
on p.6446
6446Image
on p.6446

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bahr V., Stierhof Y. D., Ilg T., Demar M., Quinten M., Overath P. Expression of lipophosphoglycan, high-molecular weight phosphoglycan and glycoprotein 63 in promastigotes and amastigotes of Leishmania mexicana. Mol Biochem Parasitol. 1993 Mar;58(1):107–121. doi: 10.1016/0166-6851(93)90095-f. [DOI] [PubMed] [Google Scholar]
  2. Bülow R., Overath P. Purification and characterization of the membrane-form variant surface glycoprotein hydrolase of Trypanosoma brucei. J Biol Chem. 1986 Sep 5;261(25):11918–11923. [PubMed] [Google Scholar]
  3. Cross G. A. Cellular and genetic aspects of antigenic variation in trypanosomes. Annu Rev Immunol. 1990;8:83–110. doi: 10.1146/annurev.iy.08.040190.000503. [DOI] [PubMed] [Google Scholar]
  4. Cross G. A. Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell Biol. 1990;6:1–39. doi: 10.1146/annurev.cb.06.110190.000245. [DOI] [PubMed] [Google Scholar]
  5. Davidson D. J., Bretthauer R. K., Castellino F. J. alpha-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells. Biochemistry. 1991 Oct 15;30(41):9811–9815. doi: 10.1021/bi00105a001. [DOI] [PubMed] [Google Scholar]
  6. Davidson D. J., Castellino F. J. Asparagine-linked oligosaccharide processing in lepidopteran insect cells. Temporal dependence of the nature of the oligosaccharides assembled on asparagine-289 of recombinant human plasminogen produced in baculovirus vector infected Spodoptera frugiperda (IPLB-SF-21AE) cells. Biochemistry. 1991 Jun 25;30(25):6165–6174. doi: 10.1021/bi00239a013. [DOI] [PubMed] [Google Scholar]
  7. Davies A., Morgan B. P. Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector. Biochem J. 1993 Nov 1;295(Pt 3):889–896. doi: 10.1042/bj2950889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeTomaso A. W., Xie Z. J., Liu G., Mercer R. W. Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells. J Biol Chem. 1993 Jan 15;268(2):1470–1478. [PubMed] [Google Scholar]
  9. Domingo D. L., Trowbridge I. S. Characterization of the human transferrin receptor produced in a baculovirus expression system. J Biol Chem. 1988 Sep 15;263(26):13386–13392. [PubMed] [Google Scholar]
  10. Elliston J. F., Beekman J. M., Tsai S. Y., O'Malley B. W., Tsai M. J. Hormone activation of baculovirus expressed progesterone receptors. J Biol Chem. 1992 Mar 15;267(8):5193–5198. [PubMed] [Google Scholar]
  11. Fox J. A., Soliz N. M., Saltiel A. R. Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action. Proc Natl Acad Sci U S A. 1987 May;84(9):2663–2667. doi: 10.1073/pnas.84.9.2663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goochee C. F., Monica T. Environmental effects on protein glycosylation. Biotechnology (N Y) 1990 May;8(5):421–427. doi: 10.1038/nbt0590-421. [DOI] [PubMed] [Google Scholar]
  13. Hopkins C. R., Trowbridge I. S. Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells. J Cell Biol. 1983 Aug;97(2):508–521. doi: 10.1083/jcb.97.2.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  15. Keller G. A., Siegel M. W., Caras I. W. Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways. EMBO J. 1992 Mar;11(3):863–874. doi: 10.1002/j.1460-2075.1992.tb05124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Korth K. L., Levings C. S., 3rd Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3388–3392. doi: 10.1073/pnas.90.8.3388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pays E., Tebabi P., Pays A., Coquelet H., Revelard P., Salmon D., Steinert M. The genes and transcripts of an antigen gene expression site from T. brucei. Cell. 1989 Jun 2;57(5):835–845. doi: 10.1016/0092-8674(89)90798-8. [DOI] [PubMed] [Google Scholar]
  19. Schell D., Borowy N. K., Overath P. Transferrin is a growth factor for the bloodstream form of Trypanosoma brucei. Parasitol Res. 1991;77(7):558–560. doi: 10.1007/BF00931012. [DOI] [PubMed] [Google Scholar]
  20. Schell D., Evers R., Preis D., Ziegelbauer K., Kiefer H., Lottspeich F., Cornelissen A. W., Overath P. A transferrin-binding protein of Trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site. EMBO J. 1991 May;10(5):1061–1066. doi: 10.1002/j.1460-2075.1991.tb08045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vuori K., Pihlajaniemi T., Marttila M., Kivirikko K. I. Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7467–7470. doi: 10.1073/pnas.89.16.7467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Windhofer V., Varsànyi M., Heilmeyer L. M., Jr Rabbit fast skeletal muscle phospholipase C. Molecular weight determination by renaturation after polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. FEBS Lett. 1992 Nov 16;313(1):51–55. doi: 10.1016/0014-5793(92)81182-l. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES