1887

Abstract

Aspartyl proteinase (AP) is an extracellular enzyme of implicated as a pathogenic factor. Previous reports on the purification and characterization of AP suggested that a single DEAE-Sephadex chromatographic step was sufficient for the removal of extraneous proteins and that the final product was glycosylated. We purified AP using a chromatographic series consisting of DEAE-Sephadex A25, Sephadex G75 and rechromatography on DEAE-Sephadex A25. Use of DEAE-Sephadex alone did not remove extraneous proteins and removed little contaminating mannoprotein (MP). The addition of a Sephadex G75 column to the purification scheme removed the majority of contaminating MP and proteins. The final DEAE-Sephadex A25 chromatographic step resulted in () removal of detectable extraneous proteins, () removal of immunologically detectable MP by dot blot and Western blot enzyme immunoassay,() loss of periodic acid-silver stain positivity, and () a high AP yield (1295 U I) and specific activity (1749 U mg). We conclude that a single DEAE-Sephadex A25 purification step is insufficient to remove extraneous proteins and MP, which could interfere with the production of AP-specific antibodies and the dissection of moieties responsible for immune reactivity. Reports of periodic acid-Schiff or anthrone positivity of AP preparations may reflect the presence of extraneous MP, which can be removed by the chromatographic series we describe.

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1993-06-01
2024-12-08
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References

  1. Anderson M. L., Odds F. C. 1985; Adherence of Candida albicans to vaginal epithelia: significance of morphological form and effect of ketoconazole. Mycosen 28:531–540
    [Google Scholar]
  2. Barnes J. L., Osgood R. W., Lee J. C., King R. D., Stein J. H. 1983; Host-parasite interactions in the pathogenesis of experimental renal candidosis. Laboratory Investigation 49:460–467
    [Google Scholar]
  3. Bradford M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  4. Crandall M., Edwards J. E. JR 1987; Segregation of proteinasenegative mutants from heterozygous Candida albicans . Journal of General Microbiology 133:2817–2824
    [Google Scholar]
  5. Domer J. E., Garner R. E. 1989; Immunomodulation in response to Candida . Immunology Series 47:293–317
    [Google Scholar]
  6. Dubray G., Bezard G. 1982; A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Analytical Biochemistry 119:325–329
    [Google Scholar]
  7. Duksin D., Mahoney W. C. 1982; Relationship of the structure and biological activity of the natural homologues of tunicamycin. Journal of Biological Chemistry 257:3105–3109
    [Google Scholar]
  8. Ganesan K., Banerjee A., Datta A. 1991; Molecular cloning of the secretory acid proteinase gene from Candida albicans and its use as a species-specific probe. Infection and Immunity 59:2972–2977
    [Google Scholar]
  9. Garner R. E., Childress A. M., Human L. G., Domer J. E. 1990; Characterization of Candida albicans mannan-induced, mannan-specific delayed hypersensitivity suppressor cells. Infection and Immunity 58:2613–2620
    [Google Scholar]
  10. Ghannoun M. A., Abu Elteen K. 1986; Correlative relationship between proteinase production, adherence, and pathogenicity of various strains of Candida albicans . Journal of Medical and Veterinary Mycology 24:407–413
    [Google Scholar]
  11. Hellwege W. H., Fischer K., Blaker F. 1972; Diagnostic value of Candida precipitins. Lancet 2:386
    [Google Scholar]
  12. Hube B., Turver C. J., Odds F. C., Eiffert H., Boulnois G. J., Kochel H., Rüchel R. 1991; Sequence of the Candida albicans gene encoding the secretory aspartate kinase. Journal of Medical and Veterinary Mycology 29:129–132
    [Google Scholar]
  13. Kaufman L., Reiss E. 1992; Serodiagnosis of fungal diseases. In Manual of Clinical Immunology, 4th. pp. 506–528 Edited by Rose N. R., De Macario E. C., Fahey J. L., Friedman H., Penn G. M. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  14. Kregar I., Urh I., Umezawa H., Turk V. 1977; Purification of cathepsin D by affinity chromatography on pepstatin sepharose column. Croatica Chemica Acta 49:587–592
    [Google Scholar]
  15. Kwon-Chung K. J., Lehman D., Good C., Magee P. T. 1985; Genetic evidence for role of extracellular proteinase in virulence of Candida albicans . Infection and Immunity 49:571–575
    [Google Scholar]
  16. Lam M., Peterkin V., Reiss E., Morrison C. J. 1991; Effect of growth conditions on the extracellular production of the aspartic proteinase by Candida albicans . Advances in Experimental Medicine and Biology 306:265–267
    [Google Scholar]
  17. Lerner C. G., Goldman R. C. 1992; Environmental stimuli that induce production of Candida albicans extracellular aspartyl proteinase. Abstracts of the 92nd Annual Meeting of the American Society for Microbiology 1992 abstract F-51 p. 507
    [Google Scholar]
  18. Mccourtie J., Douglas L. J. 1984; Relationship between cell surface composition, adherence, and virulence of Candida albicans . Infection and Immunity 45:6–12
    [Google Scholar]
  19. Macdonald F., Odds F. C. 1980; Inducible proteinase of Candida albicans in diagnostic serology and the pathogenesis of systemic candidosis. Journal of Medical Microbiology 13:423–435
    [Google Scholar]
  20. Miyasaki S. H., White T. C., Agabian N. 1992; Genomic organization and transcriptional regulation of the Candida albicans secreted acid protease gene. Conference on Fungal Dimorphism, Cambridge, UK, September 2nd, 1992 abstract P18 pp. 218–219
    [Google Scholar]
  21. Morrison A. J. JR. Freer C. V., Searcy M. A., Landry S. M., Wenzel R. P. 1986; Nosocomial bloodstream infections: secular trends in a statewide surveillance program in Virginia. Infection Control 7:550–553
    [Google Scholar]
  22. Morrow B., Srikantha T., Soll D. R. 1992; Transcription of the gene for a pepsinogen, PEP1, is regulated by white-opaque switching in Candida albicans . Molecular and Cellular Biology 12:2997–3005
    [Google Scholar]
  23. Negi M., Tsuboi R., Matsui T., Ogawa H. 1984; Isolation and characterization of proteinase from Candida albicans: substrate specificity. Journal of Investigative Dermatology 83:32–36
    [Google Scholar]
  24. Odds F. C. 1988; Factors that predispose the host to candidosis. In Candida and Candidosis, 2nd. pp. 93–114 London: Bailliere Tindall;
    [Google Scholar]
  25. Peat S., Whalen W. J., Edwards T. E. 1961; Polysaccharides of baker’s yeast. IV. Mannan. Journal of the Chemical Society (London)29–34
    [Google Scholar]
  26. Ray T., Payne C. D. 1990; Comparative production and rapid purification of Candida acid proteinase from protein-supplemented cultures. Infection and Immunity 58:508–514
    [Google Scholar]
  27. Ray T., Payne C. D. 1991; Candida acid proteinases: characterization and role in candidiasis. Advances in Experimental Medicine and Biology 306:173–183
    [Google Scholar]
  28. Reingold A. L., Lu X. D., Plikaytis B. D., Ajello L. 1986; Systemic mycoses in the United States 1980–1982. Journal of Medical and Veterinary Mycology 24:433–436
    [Google Scholar]
  29. Reiss E., Stone S. H., Hasenclever H. F. 1974; Serological and cellular immune activity of peptidoglucomannan fractions of Candida albicans cell walls. Infection and Immunity 9:881–890
    [Google Scholar]
  30. Reiss E., Hutchinson H., Pine L., Ziegler D. W., Kaufman L. 1977; Solid-phase competitive binding radioimmunoassay for detecting antibody to the M antigen of histoplasmin. Journal of Clinical Microbiology 6:598–604
    [Google Scholar]
  31. Reiss E., De Repentigny L., Kuykendall R. J., Carter A. W., Galindo R., Auger P., Bragg S. L., Kaufman L. 1986; Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence. Journal of Clinical Microbiology 24:796–802
    [Google Scholar]
  32. Remold H., Fasold H., Staib F. 1968; Purification and characterization of a proteolytic enzyme from Candida albicans . Biochimica et Biophysica Acta 167:399–406
    [Google Scholar]
  33. Richardson M. D. 1991; Opportunistic and pathogenic fungi. Journal of Antimicrobial Chemotherapy 28: suppl. A 1–11
    [Google Scholar]
  34. Ross I. K., De Bernardis F., Emerson G. W., Cassone A., Sullivan P. A. 1990; The secreted aspartate proteinase of Candida albicans: physiology of secretion and virulence of a proteinase-deficient mutant. Journal of General Microbiology 136:687–694
    [Google Scholar]
  35. Rüchel R. 1981; Properties of a purified proteinase from the yeast Candida albicans . Biochimica et Biophysica Acta 659:99–113
    [Google Scholar]
  36. Rüchel R., Böning B. 1983; Detection of Candida proteinase by enzyme immunoassay and interaction of the enzyme with alpha-2-macroglobulin. Journal of Immunological Methods 61:107–116
    [Google Scholar]
  37. Rüchel R., Tegeler R., Trost M. 1982; A comparison of secretory proteinases from different strains of Candida albicans . Sabouraudia 20:233–244
    [Google Scholar]
  38. Rüchel R., Böning B., Borg M. 1986; Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of enzyme during infection in vitro . Infection and Immunity 53:411–419
    [Google Scholar]
  39. Rüchel R., Böning-Stutzer B., Mari A. 1988; A synoptical approach to the diagnosis of candidosis, relying on serological antigen and antibody tests, on culture, and on evaluation of clinical data. Mycoses 31:87–106
    [Google Scholar]
  40. Rüchel R., De Bernardis F., Ray T. L., Sullivan P. A., Cole G. T. 1992; Candida acid proteinases. Journal of Medical and Veterinary Mycology 30: Suppl 1 123–132
    [Google Scholar]
  41. Sartorelli A. C., Fischer D. S., Downs W. G. 1966; Use of sarcoma 180/TG to prepare hyperimmune ascitic fluid in the mouse. Journal of Immunology 96:676–682
    [Google Scholar]
  42. Shimizu K., Kondoh Y., Tanaka K. 1987; Proteinase production and pathogenicity of Candida albicans. I. Invasion into chorioallantoic membrane by C. albicans strains of different proteinase activity. Microbiology and Immunology 31:1045–1060
    [Google Scholar]
  43. Staib F. 1969; Proteolysis and pathogenicity of Candida albicans strains. Mycopathologia et Mycologia Applicata 37:345–348
    [Google Scholar]
  44. Taschdjian C. L., Seelig M. S., Kozinn P. J. 1973; Serological diagnosis of candidiasis. Critical Reviews in Clinical Laboratory Sciences 4:19–60
    [Google Scholar]
  45. Todaro-Luck F., Reiss E., Cherniak R., Kaufman L. 1989; Characterization of Cryptococcus neoformans capsular glucuronoxylomannan polysaccharide with monoclonal antibodies. Infection and Immunity 57:3882–3887
    [Google Scholar]
  46. Tsai C.-M, Frasch C. E. 1982; A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Analytical Biochemistry 119:115–119
    [Google Scholar]
  47. Tsang V. C. W. 1987; The enzyme-linked immuno-electrotransfer blot (‘Western blot’): technical considerations. In Integration and Control of Metabolic Processes: Pure and Applied Aspects pp. 489–500 Kon O. L., Chung M. C.-M., Hwang P. L. H., Leong S.-F., Loke K. H., Thiyagarajah P., Wong P. T.-H. Edited by Epidemiological and Molecular Aspects on Cholera Cambridge: ICSU Press(Cambridge University Press);
    [Google Scholar]
  48. Tsang V. C. W., Peralta J. M., Simons A. R. 1983; Enzyme-linked immuno-electro-transfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods in Enzymology 92:377–391
    [Google Scholar]
  49. Tsang V. C. W., Hancock K., Wilson M., Palmer D. F., Whaley S. D., Mcdougal J. S., Kennedy S. 1986; Enzyme-linked immunoelectrotransfer blot technique (Western blot) for human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) antibodies. Immunology Series No. 15. Procedural Guide. Atlanta, GA, USA: Centers for Disease Control;
    [Google Scholar]
  50. Wright R. J., Carne A., Hieber A. D., Lamont I. L., Emerson G. W., Sullivan P. A. 1992; A second gene for a secreted aspartate proteinase in Candida albicans . Journal of Bacteriology 174:7848–7853
    [Google Scholar]
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