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. 1996 Jul;64(7):2577–2584. doi: 10.1128/iai.64.7.2577-2584.1996

Purification and biochemical characterization of a 65-kilodalton mannoprotein (MP65), a main target of anti-Candida cell-mediated immune responses in humans.

M J Gomez 1, A Torosantucci 1, S Arancia 1, B Maras 1, L Parisi 1, A Cassone 1
PMCID: PMC174113  PMID: 8698482

Abstract

A 65 kDa-constituent (MP65) of a whole-cell mannoprotein (MP) fraction of Candida albicans was purified by immunoaffinity chromatography with monoclonal antibodies directed against periodate-insensitive, protease-sensitive MP epitopes, putatively polypeptide in nature. These antibodies were obtained by immunization of mice with concanavalin A bead-coupled, low-glycosylated MP from hyphal cells of C. albicans grown in the presence of a subinhibitory dose of tunicamycin. The immunoaffinity-purified MP65 molecule had a pI of 4.1 and a protein/polysaccharide ratio of 1.8:1. It was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, or N-glycoffanases but still reactive with concanavalin A. The polysaccharide moiety of MP65 was composed exclusively of mannose and glucose at a ratio of 12.7 to 1. The protein moiety showed numerous potential O-glycosidic linkage sites as suggested by the high proportion of serine and threonine (together accounting for more than 20% of the total amino acid composition) and susceptibility to diluted alkali. This treatment and digestion with alpha-mannosidase caused a reduction in the MP65 molecular mass to around 54 kDa. The N-terminal sequence of MP65 protein moiety was rich in alanine and valine (7 of 13 amino acids) and did not show any significant homology with deposited sequences in data banks. Purified MP65, at doses of a few nanograms, induced extensive T-cell proliferation of human peripheral blood mononuclear cells. This proliferation was specifically inhibited, in a dose-response fashion, by the antigen-binding fragment of the monoclonal antibody used for immunoaffinity purification. Overall, these results highlight biochemical and molecular details of MP65, a main target of human T-cell response to C.albicans.

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Selected References

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

  1. Angiolella L., Facchin M., Stringaro A., Maras B., Simonetti N., Cassone A. Identification of a glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J Infect Dis. 1996 Mar;173(3):684–690. doi: 10.1093/infdis/173.3.684. [DOI] [PubMed] [Google Scholar]
  2. Ausiello C. M., Palma C., Spagnoli G. C., Piazza A., Casciani C. U., Cassone A. Cytotoxic effectors in human peripheral blood mononuclear cells induced by a mannoprotein complex of Candida albicans: a comparison with interleukin 2-activated killer cells. Cell Immunol. 1989 Jul;121(2):349–359. doi: 10.1016/0008-8749(89)90033-6. [DOI] [PubMed] [Google Scholar]
  3. Ausiello C. M., Spagnoli G. C., Boccanera M., Casalinuovo I., Malavasi F., Casciani C. U., Cassone A. Proliferation of human peripheral blood mononuclear cells induced by Candida albicans and its cell wall fractions. J Med Microbiol. 1986 Nov;22(3):195–202. doi: 10.1099/00222615-22-3-195. [DOI] [PubMed] [Google Scholar]
  4. Ausiello C. M., Urbani F., Gessani S., Spagnoli G. C., Gomez M. J., Cassone A. Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans. Infect Immun. 1993 Oct;61(10):4105–4111. doi: 10.1128/iai.61.10.4105-4111.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bendel C. M., St Sauver J., Carlson S., Hostetter M. K. Epithelial adhesion in yeast species: correlation with surface expression of the integrin analog. J Infect Dis. 1995 Jun;171(6):1660–1663. doi: 10.1093/infdis/171.6.1660. [DOI] [PubMed] [Google Scholar]
  6. Bouchara J. P., Tronchin G., Annaix V., Robert R., Senet J. M. Laminin receptors on Candida albicans germ tubes. Infect Immun. 1990 Jan;58(1):48–54. doi: 10.1128/iai.58.1.48-54.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bromuro C., Torosantucci A., Gomez M. J., Urbani F., Cassone A. Differential release of an immunodominant 65 kDa mannoprotein antigen from yeast and mycelial forms of Candida albicans. J Med Vet Mycol. 1994 Dec;32(6):447–459. doi: 10.1080/02681219480000601. [DOI] [PubMed] [Google Scholar]
  8. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  9. Calderone R. A., Linehan L., Wadsworth E., Sandberg A. L. Identification of C3d receptors on Candida albicans. Infect Immun. 1988 Jan;56(1):252–258. doi: 10.1128/iai.56.1.252-258.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cantorna M. T., Balish E. Role of CD4+ lymphocytes in resistance to mucosal candidiasis. Infect Immun. 1991 Jul;59(7):2447–2455. doi: 10.1128/iai.59.7.2447-2455.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Casanova M., Gil M. L., Cardeñoso L., Martinez J. P., Sentandreu R. Identification of wall-specific antigens synthesized during germ tube formation by Candida albicans. Infect Immun. 1989 Jan;57(1):262–271. doi: 10.1128/iai.57.1.262-271.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cassone A. Cell wall of Candida albicans: its functions and its impact on the host. Curr Top Med Mycol. 1989;3:248–314. doi: 10.1007/978-1-4612-3624-5_10. [DOI] [PubMed] [Google Scholar]
  13. Cassone A., Palma C., Djeu J. Y., Aiuti F., Quinti I. Anticandidal activity and interleukin-1 beta and interleukin-6 production by polymorphonuclear leukocytes are preserved in subjects with AIDS. J Clin Microbiol. 1993 May;31(5):1354–1357. doi: 10.1128/jcm.31.5.1354-1357.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cassone A., Torosantucci A., Boccanera M., Pellegrini G., Palma C., Malavasi F. Production and characterisation of a monoclonal antibody to a cell-surface, glucomannoprotein constituent of Candida albicans and other pathogenic Candida species. J Med Microbiol. 1988 Dec;27(4):233–238. doi: 10.1099/00222615-27-4-233. [DOI] [PubMed] [Google Scholar]
  15. Cutler J. E. Putative virulence factors of Candida albicans. Annu Rev Microbiol. 1991;45:187–218. doi: 10.1146/annurev.mi.45.100191.001155. [DOI] [PubMed] [Google Scholar]
  16. Domer J. E. Candida cell wall mannan: a polysaccharide with diverse immunologic properties. Crit Rev Microbiol. 1989;17(1):33–51. doi: 10.3109/10408418909105721. [DOI] [PubMed] [Google Scholar]
  17. Durandy A., Fischer A., Le Deist F., Drouhet E., Griscelli C. Mannan-specific and mannan-induced T-cell suppressive activity in patients with chronic mucocutaneous candidiasis. J Clin Immunol. 1987 Sep;7(5):400–409. doi: 10.1007/BF00917018. [DOI] [PubMed] [Google Scholar]
  18. Fisher-Hoch S. P., Hutwagner L. Opportunistic candidiasis: an epidemic of the 1980s. Clin Infect Dis. 1995 Oct;21(4):897–904. doi: 10.1093/clinids/21.4.897. [DOI] [PubMed] [Google Scholar]
  19. Fukayama M., Wadsworth E., Calderone R. Expression of the C3d-binding protein (CR2) from Candida albicans during experimental candidiasis as measured by lymphoblastogenesis. Infect Immun. 1992 Jan;60(1):8–12. doi: 10.1128/iai.60.1.8-12.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gilmore B. J., Retsinas E. M., Lorenz J. S., Hostetter M. K. An iC3b receptor on Candida albicans: structure, function, and correlates for pathogenicity. J Infect Dis. 1988 Jan;157(1):38–46. doi: 10.1093/infdis/157.1.38. [DOI] [PubMed] [Google Scholar]
  21. Hostetter M. K. Handicaps to host defense. Effects of hyperglycemia on C3 and Candida albicans. Diabetes. 1990 Mar;39(3):271–275. doi: 10.2337/diab.39.3.271. [DOI] [PubMed] [Google Scholar]
  22. Klein R. S., Harris C. A., Small C. B., Moll B., Lesser M., Friedland G. H. Oral candidiasis in high-risk patients as the initial manifestation of the acquired immunodeficiency syndrome. N Engl J Med. 1984 Aug 9;311(6):354–358. doi: 10.1056/NEJM198408093110602. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Lee K. L., Buckley H. R., Campbell C. C. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida Albicans. Sabouraudia. 1975 Jul;13(2):148–153. doi: 10.1080/00362177585190271. [DOI] [PubMed] [Google Scholar]
  25. Malavasi F., Caligaris-Cappio F., Milanese C., Dellabona P., Richiardi P., Carbonara A. O. Characterization of a murine monoclonal antibody specific for human early lymphohemopoietic cells. Hum Immunol. 1984 Jan;9(1):9–20. doi: 10.1016/0198-8859(84)90003-x. [DOI] [PubMed] [Google Scholar]
  26. Matsudaira P. Limited N-terminal sequence analysis. Methods Enzymol. 1990;182:602–613. doi: 10.1016/0076-6879(90)82047-6. [DOI] [PubMed] [Google Scholar]
  27. Mencacci A., Torosantucci A., Spaccapelo R., Romani L., Bistoni F., Cassone A. A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice. Infect Immun. 1994 Dec;62(12):5353–5360. doi: 10.1128/iai.62.12.5353-5360.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Molinari A., Gomez M. J., Crateri P., Torosantucci A., Cassone A., Arancia G. Differential cell surface expression of mannoprotein epitopes in yeast and mycelial forms of Candida albicans. Eur J Cell Biol. 1993 Feb;60(1):146–153. [PubMed] [Google Scholar]
  29. Nelson R. D., Shibata N., Podzorski R. P., Herron M. J. Candida mannan: chemistry, suppression of cell-mediated immunity, and possible mechanisms of action. Clin Microbiol Rev. 1991 Jan;4(1):1–19. doi: 10.1128/cmr.4.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Odds F. C. Candida infections: an overview. Crit Rev Microbiol. 1987;15(1):1–5. doi: 10.3109/10408418709104444. [DOI] [PubMed] [Google Scholar]
  31. Piccolella E., Lombardi G., Morelli R. Generation of suppressor cells in the response of human lymphocytes to a polysaccharide from Candida albicans. J Immunol. 1981 Jun;126(6):2151–2155. [PubMed] [Google Scholar]
  32. Podzorski R. P., Gray G. R., Nelson R. D. Different effects of native Candida albicans mannan and mannan-derived oligosaccharides on antigen-stimulated lymphoproliferation in vitro. J Immunol. 1990 Jan 15;144(2):707–716. [PubMed] [Google Scholar]
  33. Ponton J., Marot-Leblond A., Ezkurra P. A., Barturen B., Robert R., Senet J. M. Characterization of Candida albicans cell wall antigens with monoclonal antibodies. Infect Immun. 1993 Nov;61(11):4842–4847. doi: 10.1128/iai.61.11.4842-4847.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Puccetti P., Mencacci A., Cenci E., Spaccapelo R., Mosci P., Enssle K. H., Romani L., Bistoni F. Cure of murine candidiasis by recombinant soluble interleukin-4 receptor. J Infect Dis. 1994 Jun;169(6):1325–1331. doi: 10.1093/infdis/169.6.1325. [DOI] [PubMed] [Google Scholar]
  35. Romani L., Mencacci A., Cenci E., Spaccapelo R., Mosci P., Puccetti P., Bistoni F. CD4+ subset expression in murine candidiasis. Th responses correlate directly with genetically determined susceptibility or vaccine-induced resistance. J Immunol. 1993 Feb 1;150(3):925–931. [PubMed] [Google Scholar]
  36. Romani L., Mocci S., Bietta C., Lanfaloni L., Puccetti P., Bistoni F. Th1 and Th2 cytokine secretion patterns in murine candidiasis: association of Th1 responses with acquired resistance. Infect Immun. 1991 Dec;59(12):4647–4654. doi: 10.1128/iai.59.12.4647-4654.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saxena A., Calderone R. Purification and characterization of the extracellular C3d-binding protein of Candida albicans. Infect Immun. 1990 Feb;58(2):309–314. doi: 10.1128/iai.58.2.309-314.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shibata N., Arai M., Haga E., Kikuchi T., Najima M., Satoh T., Kobayashi H., Suzuki S. Structural identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B-792 (serotype B) and J-1012 (serotype A) as beta-1,2-linked oligomannosyl residues. Infect Immun. 1992 Oct;60(10):4100–4110. doi: 10.1128/iai.60.10.4100-4110.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sundstrom P., Jensen J., Balish E. Humoral and cellular immune responses to enolase after alimentary tract colonization or intravenous immunization with Candida albicans. J Infect Dis. 1994 Aug;170(2):390–395. doi: 10.1093/infdis/170.2.390. [DOI] [PubMed] [Google Scholar]
  40. Torosantucci A., Bromuro C., Gomez M. J., Ausiello C. M., Urbani F., Cassone A. Identification of a 65-kDa mannoprotein as a main target of human cell-mediated immune response to Candida albicans. J Infect Dis. 1993 Aug;168(2):427–435. doi: 10.1093/infdis/168.2.427. [DOI] [PubMed] [Google Scholar]
  41. Torosantucci A., Gomez M. J., Bromuro C., Casalinuovo I., Cassone A. Biochemical and antigenic characterization of mannoprotein constituents released from yeast and mycelial forms of Candida albicans. J Med Vet Mycol. 1991;29(6):361–372. doi: 10.1080/02681219180000591. [DOI] [PubMed] [Google Scholar]
  42. Torosantucci A., Palma C., Boccanera M., Ausiello C. M., Spagnoli G. C., Cassone A. Lymphoproliferative and cytotoxic responses of human peripheral blood mononuclear cells to mannoprotein constituents of Candida albicans. J Gen Microbiol. 1990 Nov;136(11):2155–2163. doi: 10.1099/00221287-136-11-2155. [DOI] [PubMed] [Google Scholar]
  43. Trinel P. A., Faille C., Jacquinot P. M., Cailliez J. C., Poulain D. Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies. Infect Immun. 1992 Sep;60(9):3845–3851. doi: 10.1128/iai.60.9.3845-3851.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tronchin G., Bouchara J. P., Annaix V., Robert R., Senet J. M. Fungal cell adhesion molecules in Candida albicans. Eur J Epidemiol. 1991 Jan;7(1):23–33. doi: 10.1007/BF00221338. [DOI] [PubMed] [Google Scholar]
  45. Yu L., Lee K. K., Ens K., Doig P. C., Carpenter M. R., Staddon W., Hodges R. S., Paranchych W., Irvin R. T. Partial characterization of a Candida albicans fimbrial adhesin. Infect Immun. 1994 Jul;62(7):2834–2842. doi: 10.1128/iai.62.7.2834-2842.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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