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. 1997 Feb 15;322(Pt 1):49–56. doi: 10.1042/bj3220049

The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein.

P Papanastasiou 1, M J McConville 1, J Ralton 1, P Köhler 1
PMCID: PMC1218157  PMID: 9078242

Abstract

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis (syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CR1SP-90) from a cloned Giardia isolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is posttranslationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by beta-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of[3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

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Selected References

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  1. Adam R. D., Aggarwal A., Lal A. A., de La Cruz V. F., McCutchan T., Nash T. E. Antigenic variation of a cysteine-rich protein in Giardia lamblia. J Exp Med. 1988 Jan 1;167(1):109–118. doi: 10.1084/jem.167.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aley S. B., Gillin F. D. Specialized surface adaptations of Giardia lamblia. Infect Agents Dis. 1995 Sep;4(3):161–166. [PubMed] [Google Scholar]
  3. Almeida I. C., Ferguson M. A., Schenkman S., Travassos L. R. Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. Biochem J. 1994 Dec 15;304(Pt 3):793–802. doi: 10.1042/bj3040793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bizzozero O. A., Fridal K., Pastuszyn A. Identification of the palmitoylation site in rat myelin P0 glycoprotein. J Neurochem. 1994 Mar;62(3):1163–1171. doi: 10.1046/j.1471-4159.1994.62031163.x. [DOI] [PubMed] [Google Scholar]
  5. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  6. Bouvier M., Chidiac P., Hebert T. E., Loisel T. P., Moffett S., Mouillac B. Dynamic palmitoylation of G-protein-coupled receptors in eukaryotic cells. Methods Enzymol. 1995;250:300–314. doi: 10.1016/0076-6879(95)50080-4. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Bruderer T., Papanastasiou P., Castro R., Köhler P. Variant cysteine-rich surface proteins of Giardia isolates from human and animal sources. Infect Immun. 1993 Jul;61(7):2937–2944. doi: 10.1128/iai.61.7.2937-2944.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Das S., Traynor-Kaplan A., Reiner D. S., Meng T. C., Gillin F. D. A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor. J Biol Chem. 1991 Nov 5;266(31):21318–21325. [PubMed] [Google Scholar]
  10. Dieckmann-Schuppert A., Bender S., Odenthal-Schnittler M., Bause E., Schwarz R. T. Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum. Eur J Biochem. 1992 Apr 15;205(2):815–825. doi: 10.1111/j.1432-1033.1992.tb16846.x. [DOI] [PubMed] [Google Scholar]
  11. Ey P. L., Khanna K. K., Manning P. A., Mayrhofer G. A gene encoding a 69-kilodalton major surface protein of Giardia intestinalis trophozoites. Mol Biochem Parasitol. 1993 Apr;58(2):247–257. doi: 10.1016/0166-6851(93)90046-z. [DOI] [PubMed] [Google Scholar]
  12. Gillin F. D., Hagblom P., Harwood J., Aley S. B., Reiner D. S., McCaffery M., So M., Guiney D. G. Isolation and expression of the gene for a major surface protein of Giardia lamblia. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4463–4467. doi: 10.1073/pnas.87.12.4463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gustafson G. L., Milner L. A. Occurrence of N-acetylglucosamine-1-phosphate in proteinase I from Dictyostelium discoideum. J Biol Chem. 1980 Aug 10;255(15):7208–7210. [PubMed] [Google Scholar]
  14. Handman E., Barnett L. D., Osborn A. H., Goding J. W., Murray P. J. Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein. Mol Biochem Parasitol. 1993 Nov;62(1):61–72. doi: 10.1016/0166-6851(93)90178-z. [DOI] [PubMed] [Google Scholar]
  15. Harris R. J., Spellman M. W. O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology. 1993 Jun;3(3):219–224. doi: 10.1093/glycob/3.3.219. [DOI] [PubMed] [Google Scholar]
  16. Ilg T., Overath P., Ferguson M. A., Rutherford T., Campbell D. G., McConville M. J. O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans. J Biol Chem. 1994 Sep 30;269(39):24073–24081. [PubMed] [Google Scholar]
  17. Luján H. D., Mowatt M. R., Wu J. J., Lu Y., Lees A., Chance M. R., Nash T. E. Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties. J Biol Chem. 1995 Jun 9;270(23):13807–13813. doi: 10.1074/jbc.270.23.13807. [DOI] [PubMed] [Google Scholar]
  18. McConville M. J., Thomas-Oates J. E., Ferguson M. A., Homans S. W. Structure of the lipophosphoglycan from Leishmania major. J Biol Chem. 1990 Nov 15;265(32):19611–19623. [PubMed] [Google Scholar]
  19. Mowatt M. R., Aggarwal A., Nash T. E. Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia. Mol Biochem Parasitol. 1991 Dec;49(2):215–227. doi: 10.1016/0166-6851(91)90065-e. [DOI] [PubMed] [Google Scholar]
  20. Nash T. E., Gillin F. D., Smith P. D. Excretory-secretory products of Giardia lamblia. J Immunol. 1983 Oct;131(4):2004–2010. [PubMed] [Google Scholar]
  21. Nash T. E., Mowatt M. R. Characterization of a Giardia lamblia variant-specific surface protein (VSP) gene from isolate GS/M and estimation of the VSP gene repertoire size. Mol Biochem Parasitol. 1992 Apr;51(2):219–227. doi: 10.1016/0166-6851(92)90072-r. [DOI] [PubMed] [Google Scholar]
  22. Nash T. Surface antigen variability and variation in Giardia lamblia. Parasitol Today. 1992 Jul;8(7):229–234. doi: 10.1016/0169-4758(92)90119-m. [DOI] [PubMed] [Google Scholar]
  23. Papanastasiou P., Hiltpold A., Bommeli C., Köhler P. The release of the variant surface protein of Giardia to its soluble isoform is mediated by the selective cleavage of the conserved carboxy-terminal domain. Biochemistry. 1996 Aug 6;35(31):10143–10148. doi: 10.1021/bi960473b. [DOI] [PubMed] [Google Scholar]
  24. Pimenta P. F., da Silva P. P., Nash T. Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and label fracture immunocytochemistry survey. Infect Immun. 1991 Nov;59(11):3989–3996. doi: 10.1128/iai.59.11.3989-3996.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Previato J. O., Jones C., Xavier M. T., Wait R., Travassos L. R., Parodi A. J., Mendonça-Previato L. Structural characterization of the major glycosylphosphatidylinositol membrane-anchored glycoprotein from epimastigote forms of Trypanosoma cruzi Y-strain. J Biol Chem. 1995 Mar 31;270(13):7241–7250. doi: 10.1074/jbc.270.13.7241. [DOI] [PubMed] [Google Scholar]
  26. Schneider P., Ralton J. E., McConville M. J., Ferguson M. A. Analysis of the neutral glycan fractions of glycosyl-phosphatidylinositols by thin-layer chromatography. Anal Biochem. 1993 Apr;210(1):106–112. doi: 10.1006/abio.1993.1158. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Stanley S. L., Jr, Tian K., Koester J. P., Li E. The serine-rich Entamoeba histolytica protein is a phosphorylated membrane protein containing O-linked terminal N-acetylglucosamine residues. J Biol Chem. 1995 Feb 24;270(8):4121–4126. doi: 10.1074/jbc.270.8.4121. [DOI] [PubMed] [Google Scholar]
  29. Strandén A. M., Eckert J., Köhler P. Electrophoretic characterization of Giardia isolated from humans, cattle, sheep, and a dog in Switzerland. J Parasitol. 1990 Oct;76(5):660–668. [PubMed] [Google Scholar]
  30. Torres C. R., Hart G. W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem. 1984 Mar 10;259(5):3308–3317. [PubMed] [Google Scholar]
  31. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]

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