1887

Abstract

Initial contact between the opportunistic fungal pathogen and host tissue occurs at the cell surface. Biotin derivatives have been used to label the cell-surface proteins of yeasts, with labelled proteins subsequently detected by avidin–reporter conjugates. Previous work has indicated that avidin can bind to proteins in the absence of biotin, suggesting a possible host-cell-recognition mechanism by fungal cell-surface proteins. To investigate this mechanism, Western blots of proteins extracted from biotinylated and mock-treated cells were probed with avidin or modified-avidin reagents. Each avidin reagent bound to cell-wall proteins extracted from non-biotinylated cells. Binding did not appear to be due to the lectin-like activity of the cell-wall proteins of or to the presence of biotin in the sample itself. Binding was inhibited by added biotin, by the chaotrope KSCN and by NaCl in a concentration-dependent manner, although inhibition varied among the avidin conjugates tested. Thus, the non-specific binding of avidin to the cell-wall proteins of appears to involve hydrophobic and electrostatic interactions, depending on the particular avidin species. These observations demonstrate potential pitfalls in the use of avidin–biotin complexes to identify cell-surface molecules and could provide insights into protein–protein interactions at the cell wall.

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2002-04-01
2020-09-29
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References

  1. Alexandre H., Blanchet S., Charpentier C. 2000; Identification of a 49-kDa hydrophobic cell wall mannoprotein present in velum yeast which may be implicated in velum formation. FEMS Microbiol Lett185:147–150[CrossRef]
    [Google Scholar]
  2. Alon R., Bayer E. A., Wilchek M. 1990; Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin. Biochem Biophys Res Comm170:1236–1241[CrossRef]
    [Google Scholar]
  3. Bayer E. A., Wilchek M. 1980; The use of the avidin–biotin complex as a tool in molecular biology. Methods Biochem Anal26:1–45
    [Google Scholar]
  4. Bayer E. A., Ben-Hur H., Wilchek M. 1987; Enzyme-based detection of glycoproteins on blot transfers using avidin–biotin technology. Anal Biochem161:123–131[CrossRef]
    [Google Scholar]
  5. Beck-Sagué C. M., Jarvis W. R. the National Nosocomial Infections Surveillance System 1993; Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980–1990. J Infect Dis167:1247–1251[CrossRef]
    [Google Scholar]
  6. Casanova M., Lopez-Ribot J. L., Martinez J. P., Sentandreu R. 1992; Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques. Infect Immun60:4898–4906
    [Google Scholar]
  7. Cassone A. 1989; Cell wall of Candida albicans : its function and its impact on the host. Curr Top Med Mycol3:248–314
    [Google Scholar]
  8. Cole S. R., Ashman L. K., Ey P. L. 1987; Biotinylation: an alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol Immunol24:699–705[CrossRef]
    [Google Scholar]
  9. Duhamel R. C., Whitehead J. S. 1990; Prevention of nonspecific binding of avidin. Methods Enzymol184:201–207
    [Google Scholar]
  10. Gale C., Finkel D., Tao N., Meinke M., McClellan M., Olson J., Kendrick K., Hostetter M. 1996; Cloning and expression of a gene encoding an integrin-like protein in Candida albicans . Proc Natl Acad Sci USA93:357–361[CrossRef]
    [Google Scholar]
  11. Hatefi Y., Hanstein W. G. 1969; Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc Natl Acad Sci USA62:1129–1136[CrossRef]
    [Google Scholar]
  12. Hazen K. C., Cutler J. E. 1982; Optimal conditions for breaking medically important yeasts by an inexpensive and simple method. Mycopathologia80:113–116[CrossRef]
    [Google Scholar]
  13. Hazen K. C., Hazen B. W. 1987; Temperature-modulated physiological characteristics of Candida albicans . Microbiol Immunol31:497–508
    [Google Scholar]
  14. Hazen K. C., Hazen B. W. 1992; Hydrophobic surface protein masking by the opportunistic fungal pathogen Candida albicans . Infect Immun60:1499–1508
    [Google Scholar]
  15. Hazen K. C., Hazen B. W. 1993; Surface hydrophobic and hydrophilic protein alterations in Candida albicans . FEMS Microbiol Lett107:83–88[CrossRef]
    [Google Scholar]
  16. Hazen K. C., Lay J.-G., Hazen B. W., Fu R. C., Murthy S. 1990; Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans . Infect Immun58:3469–3476
    [Google Scholar]
  17. Hurley W. L., Finkelstein E., Holst B. D. 1985; Identification of surface proteins on bovine leukocytes by a biotin–avidin protein blotting technique. J Immunol Methods85:195–202[CrossRef]
    [Google Scholar]
  18. Jensen R. G. 1995; Vitamins in milk. B. Water-soluble vitamins in bovine milk. In Handbook of Milk Composition pp688–692 Edited by Jensen R. G.. San Diego, CA: Academic Press;
    [Google Scholar]
  19. Johnson D. A., Elder J. H. 1983; Antibody directed to determinants of a Moloney virus derived MCF GP70 recognizes a thymic differentiation antigen. J Exp Med159:1751–1756
    [Google Scholar]
  20. Kandasamy R., Vediyappan G., Chaffin W. L. 2000; Evidence for the presence of Pir-like proteins in Candida albicans . FEMS Microbiol Lett186:239–243[CrossRef]
    [Google Scholar]
  21. Klotz S. A., Smith R. L. 1991; A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix. J Infect Dis163:604–610[CrossRef]
    [Google Scholar]
  22. Laemmli U. K. 1970; Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  23. Leong M. M. L., Milstein C., Pannell R. 1986; Luminescent detection method for immunodot, Western and Southern blots. J Histochem Cytochem34:1645–1650[CrossRef]
    [Google Scholar]
  24. Marot-Leblond A., Robert R., Loiseau O., Apaire-Marchais V., Senet J. M. 2000; Hydrophobic and hydrophilic cell surface (glyco)proteinic components of Candida albicans . J Mycol Med10:115–122
    [Google Scholar]
  25. Mrsǎ V., Seidl T., Gentzsch M., Tanner W. 1997; Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O -mannosylated proteins of Saccharomyces cerevisiae . Yeast13:1145–1154[CrossRef]
    [Google Scholar]
  26. Nègre E., Vogel T., Levanon A., Guy R., Walsh T. J., Roberts D. D. 1994; The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans . J Biol Chem269:22039–22045
    [Google Scholar]
  27. Odds F. C. 1988; Candida and Candidosis. A Review and Bibliography, 2nd edn. London: Baillière Tindall;
    [Google Scholar]
  28. Pastor F. I. J., Valentı́n E., Herrero E., Sentandreu R. 1984; Structure of the Saccharomyces cerevisiae cell wall mannoproteins released by zymolyase and their contribution to wall architecture. Biochim Biophys Acta802:292–300[CrossRef]
    [Google Scholar]
  29. Reiss E., Hearn V. M., Poulain D., Shepherd M. G. 1992; Structure and function of the fungal cell wall. J Med Vet Mycol30:143–156[CrossRef]
    [Google Scholar]
  30. Singleton D. R., Masuoka J., Hazen K. C. 2001; Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity. J Bacteriol183:3582–3588[CrossRef]
    [Google Scholar]
  31. Sternberg S. 1994; The emerging fungal threat. Science266:1632–1634[CrossRef]
    [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA76:4350–4354[CrossRef]
    [Google Scholar]
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