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. 1994 Dec 15;304(Pt 3):793–802. doi: 10.1042/bj3040793

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.

I C Almeida 1, M A Ferguson 1, S Schenkman 1, L R Travassos 1
PMCID: PMC1137404  PMID: 7818483

Abstract

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.

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Selected References

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

  1. Almeida I. C., Krautz G. M., Krettli A. U., Travassos L. R. Glycoconjugates of Trypanosoma cruzi: a 74 kD antigen of trypomastigotes specifically reacts with lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas disease. J Clin Lab Anal. 1993;7(6):307–316. doi: 10.1002/jcla.1860070603. [DOI] [PubMed] [Google Scholar]
  2. Almeida I. C., Milani S. R., Gorin P. A., Travassos L. R. Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies. J Immunol. 1991 Apr 1;146(7):2394–2400. [PubMed] [Google Scholar]
  3. Andrews N. W., Hong K. S., Robbins E. S., Nussenzweig V. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Exp Parasitol. 1987 Dec;64(3):474–484. doi: 10.1016/0014-4894(87)90062-2. [DOI] [PubMed] [Google Scholar]
  4. Avila J. L., Rojas M., Acosta A. Glycoinositol phospholipids from American Leishmania and Trypanosoma spp: partial characterization of the glycan cores and the human humoral immune response to them. J Clin Microbiol. 1991 Oct;29(10):2305–2312. doi: 10.1128/jcm.29.10.2305-2312.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Avila J. L., Rojas M., Galili U. Immunogenic Gal alpha 1----3Gal carbohydrate epitopes are present on pathogenic American Trypanosoma and Leishmania. J Immunol. 1989 Apr 15;142(8):2828–2834. [PubMed] [Google Scholar]
  6. Avila J. L., Rojas M., Velazquez-Avila G. Characterization of a natural human antibody with anti-galactosyl(alpha 1-2)galactose specificity that is present at high titers in chronic Trypanosoma cruzi infection. Am J Trop Med Hyg. 1992 Oct;47(4):413–421. doi: 10.4269/ajtmh.1992.47.413. [DOI] [PubMed] [Google Scholar]
  7. CAMARGO E. P. GROWTH AND DIFFERENTIATION IN TRYPANOSOMA CRUZI. I. ORIGIN OF METACYCLIC TRYPANOSOMES IN LIQUID MEDIA. Rev Inst Med Trop Sao Paulo. 1964 May-Jun;6:93–100. [PubMed] [Google Scholar]
  8. Cardoso de Almeida M. L., Turner M. J. The membrane form of variant surface glycoproteins of Trypanosoma brucei. Nature. 1983 Mar 24;302(5906):349–352. doi: 10.1038/302349a0. [DOI] [PubMed] [Google Scholar]
  9. Dieckmann-Schuppert A., Bause E., Schwarz R. T. Studies on O-glycans of Plasmodium-falciparum-infected human erythrocytes. Evidence for O-GlcNAc and O-GlcNAc-transferase in malaria parasites. Eur J Biochem. 1993 Sep 15;216(3):779–788. doi: 10.1111/j.1432-1033.1993.tb18198.x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Galili U. Evolution and pathophysiology of the human natural anti-alpha-galactosyl IgG (anti-Gal) antibody. Springer Semin Immunopathol. 1993;15(2-3):155–171. doi: 10.1007/BF00201098. [DOI] [PubMed] [Google Scholar]
  12. Galili U., Shohet S. B., Kobrin E., Stults C. L., Macher B. A. Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J Biol Chem. 1988 Nov 25;263(33):17755–17762. [PubMed] [Google Scholar]
  13. Galvao L. M., Nunes R. M., Cançado J. R., Brener Z., Krettli A. U. Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study. Trans R Soc Trop Med Hyg. 1993 Mar-Apr;87(2):220–223. doi: 10.1016/0035-9203(93)90501-g. [DOI] [PubMed] [Google Scholar]
  14. Greis K. D., Turco S. J., Thomas J. R., McConville M. J., Homans S. W., Ferguson M. A. Purification and characterization of an extracellular phosphoglycan from Leishmania donovani. J Biol Chem. 1992 Mar 25;267(9):5876–5881. [PubMed] [Google Scholar]
  15. Haltiwanger R. S., Blomberg M. A., Hart G. W. Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase. J Biol Chem. 1992 May 5;267(13):9005–9013. [PubMed] [Google Scholar]
  16. 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]
  17. Hanover J. A., Cohen C. K., Willingham M. C., Park M. K. O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J Biol Chem. 1987 Jul 15;262(20):9887–9894. [PubMed] [Google Scholar]
  18. Holt G. D., Hart G. W. The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc. J Biol Chem. 1986 Jun 15;261(17):8049–8057. [PubMed] [Google Scholar]
  19. Krettli A. U., Brener Z. Protective effects of specific antibodies in Trypanosoma cruzi infections. J Immunol. 1976 Mar;116(3):755–760. [PubMed] [Google Scholar]
  20. Krettli A. U., Brener Z. Resistance against Trypanosoma cruzi associated to anti-living trypomastigote antibodies. J Immunol. 1982 May;128(5):2009–2012. [PubMed] [Google Scholar]
  21. McConville M. J., Blackwell J. M. Developmental changes in the glycosylated phosphatidylinositols of Leishmania donovani. Characterization of the promastigote and amastigote glycolipids. J Biol Chem. 1991 Aug 15;266(23):15170–15179. [PubMed] [Google Scholar]
  22. 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]
  23. Patel T., Bruce J., Merry A., Bigge C., Wormald M., Jaques A., Parekh R. Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry. 1993 Jan 19;32(2):679–693. doi: 10.1021/bi00053a037. [DOI] [PubMed] [Google Scholar]
  24. Previato J. O., Andrade A. F., Pessolani M. C., Mendonça-Previato L. Incorporation of sialic acid into Trypanosoma cruzi macromolecules. A proposal for a new metabolic route. Mol Biochem Parasitol. 1985 Jun;16(1):85–96. doi: 10.1016/0166-6851(85)90051-9. [DOI] [PubMed] [Google Scholar]
  25. Previato J. O., Jones C., Gonçalves L. P., Wait R., Travassos L. R., Mendonça-Previato L. O-glycosidically linked N-acetylglucosamine-bound oligosaccharides from glycoproteins of Trypanosoma cruzi. Biochem J. 1994 Jul 1;301(Pt 1):151–159. doi: 10.1042/bj3010151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schenkman S., Ferguson M. A., Heise N., de Almeida M. L., Mortara R. A., Yoshida N. Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalyzed by trans-sialidase in metacyclic forms of Trypanosoma cruzi. Mol Biochem Parasitol. 1993 Jun;59(2):293–303. doi: 10.1016/0166-6851(93)90227-o. [DOI] [PubMed] [Google Scholar]
  27. Schenkman S., Jiang M. S., Hart G. W., Nussenzweig V. A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells. Cell. 1991 Jun 28;65(7):1117–1125. doi: 10.1016/0092-8674(91)90008-m. [DOI] [PubMed] [Google Scholar]
  28. Schneider P., Ferguson M. A., McConville M. J., Mehlert A., Homans S. W., Bordier C. Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease. J Biol Chem. 1990 Oct 5;265(28):16955–16964. [PubMed] [Google Scholar]
  29. 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]
  30. Smith R., Braun P. E., Ferguson M. A., Low M. G., Sherman W. R. Direct measurement of inositol in bovine myelin basic protein. Biochem J. 1987 Nov 15;248(1):285–288. doi: 10.1042/bj2480285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Souto-Padron T., Almeida I. C., de Souza W., Travassos L. R. Distribution of alpha-galactosyl-containing epitopes on Trypanosoma cruzi trypomastigote and amastigote forms from infected Vero cells detected by Chagasic antibodies. J Eukaryot Microbiol. 1994 Jan-Feb;41(1):47–54. doi: 10.1111/j.1550-7408.1994.tb05933.x. [DOI] [PubMed] [Google Scholar]
  32. Travassos L. R., Almeida I. C. Carbohydrate immunity in American trypanosomiasis. Springer Semin Immunopathol. 1993;15(2-3):183–204. doi: 10.1007/BF00201100. [DOI] [PubMed] [Google Scholar]
  33. Yamashita K., Mizuochi T., Kobata A. Analysis of oligosaccharides by gel filtration. Methods Enzymol. 1982;83:105–126. doi: 10.1016/0076-6879(82)83008-5. [DOI] [PubMed] [Google Scholar]
  34. Zamze S. E., Ferguson M. A., Collins R., Dwek R. A., Rademacher T. W. Characterization of the cross-reacting determinant (CRD) of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoprotein. Eur J Biochem. 1988 Oct 1;176(3):527–534. doi: 10.1111/j.1432-1033.1988.tb14310.x. [DOI] [PubMed] [Google Scholar]

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