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Volume 140, Issue 7

Purification and characterization of two forms of -acetylglucosaminidase from showing widely different outer chain glycosylation Free

Abstract

Two forms of -acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of ATCC 10261 culture filtrates. Synthesis and secretion of -acetylglucosaminidase were induced by incubating starved yeast cells at 37 °C in medium containing -acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. -Acetylglucosaminidase A (32% total carbohydrate, 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and -acetylglucosaminidase B (56% carbohydrate, 132000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350000; B form, 600000; A and B forms after endoglycosidase H (endo H) treatment, 180 000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of centred at ~ 100 000 (A) and ~ 150 000 (B) but were reduced to a single 58 000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of ~ 3000 that were endo H resistant. Therefore the difference in the size of -acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. -Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both -nitrophenylGIcNAc (NPGIcNAc) and NPGalNAc at pH 4·0. The kinetic parameters (s) and (mM) of -acetylglucosaminidase A using the following substrates were, respectively: NPGIcNAc, 740, 0·77; NPGalNAc, 910, 1·26; '-diacetylchitobiose 620, 0·20; and -triacetylchitotriose, 170, 0·044. The enzyme showed substrate inhibition with all substrates above 0·5 mM except for NPGalNAc.

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Keyword(s): Candida albicans , glycosylation , N-acetylglucosaminidase and N-acetylhexosaminidase
© Society for General Microbiology 1994
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1994-07-01
2025-05-15
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References

  1. Andrews P. Estimation of molecular size and molecular weights of biological compounds by gel filtration. Methods Biochem Anal 1970; 18:1–53
     [Google Scholar]
  2. Aronson N.N., Kuranda M.J. Lysosomal degradation of Asn-linked glycoproteins. FASEB J 1989; 3:2615–2622
     [Google Scholar]
  3. Bahl O.P., Agrawal K.M.L. Glycosidases of Aspergillus niger I. Purification and characterization of a and yS-galactosidases and β-N-acetylglucosaminidase. J Biol Chem 1969; 244:2970–2978
     [Google Scholar]
  4. Ballou C.E. Yeast cell wall and cell surface. In Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression 1982 Cold Spring Harbor, New York: Cold Spring Harbor Laboratory; pp 335–360
     [Google Scholar]
  5. Ballou L., Cohen R.E., Ballou C.E. Saccharomycescerevisiae mutants that make mannoproteins with a truncated outer chain. J Biol Chem 1980; 255:5986–5991
     [Google Scholar]
  6. Barrett-Bee K., Hayes Y., Wilson R.G., Ryley J.F. A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts. J Gen Microbiol 1985; 131:1217–1221
     [Google Scholar]
  7. Chu F.K., Watorek W., Maley F. Factors affecting the oligomeric structure of yeast external invertase. Arch Biochem Biophys 1983; 223:543–555
     [Google Scholar]
  8. Cohen R.E., Ballou C.E. Mannoproteins: structure. In Encyclopedia of Plant Physiology: New Series 1981 New York: Springer-Verlag; pp 441–483
     [Google Scholar]
  9. Dixon M., Webb E.C. Engymes 1979 London: Longman;
     [Google Scholar]
  10. Douglas L.J. Adhesion of Candida albicans to host surfaces. In Candida and Candidamycosis 1991 Edited by Timbay E., Seeliger H.P.R., Ang O. New York: Plenum Press; pp 43–47
     [Google Scholar]
  11. Dubois M., Gilles R.A., Hamilton J.K., Rebers P.A., Smith F. Colorimetric method for the determination of sugars and related substances. Anal Chem 1956; 28:350–356
     [Google Scholar]
  12. Eggstein M., Kreutz F.H. In Techniques in Protein Chemistry 1967 London: Elsevier;340
     [Google Scholar]
  13. Eisenthal R., Cornish-Bowden A. The direct linear plot. Biochem J 1974; 138:715–720
     [Google Scholar]
  14. Esmon P.C., Esmon B.E., Schauer L.E., Taylor A., Schekman R. Structure, assembly, and secretion of octomeric invertase. J Biol Chem 1987; 262:4387–4394
     [Google Scholar]
  15. Fersht A. Enzyme Structure and Mechanism 1985 New York: W. H. Freeman;
     [Google Scholar]
  16. Fisher K.J., Aronson N.N. Cloning and expression of the cDNA sequence encoding the lysosomal glycosidase β-N-acetylchitobiase. J Biol Chem 1992; 267:19607–19616
     [Google Scholar]
  17. Ghannoum M.A., Abu-Elteen K.H. Pathogenicity determinants of Candida. Mycoses 1990; 33:265–282
     [Google Scholar]
  18. Gopal P., Sullivan P.A., Shepherd M.G. Enzymes of N- acetylglucosamine metabolism during germ-tube formation in Candida albicans. J Gen Microbiol 1982; 128:2319–2326
     [Google Scholar]
  19. Hayashi S., Nakamura S. Multiple forms of glucose oxidase with different carbohydrate compositions. Biochim Biophys Acta 1981; 657:40–51
     [Google Scholar]
  20. Jenkinson H.F., Shepherd M.G. A mutant of Candida albicans deficient in β-N-acetylglucosaminidase (chitobiase). J Gen Microbiol 1987; 133:2097–2106
     [Google Scholar]
  21. Johnson W.C., Lindsey A.J. An improved universal buffer. Analyst 1939; 64:490–492
     [Google Scholar]
  22. Jones C.S., Kosman D.J. Purification, properties, kinetics, and mechanism of β-N-acetylglucosaminidase from Aspergillus niger. J Biol Chem 1980; 255:11861–11869
     [Google Scholar]
  23. Kobayashi H., Shibata N., Nakada M., Chaki S., Mizugami K., Ohkubo Y., Suzuki S. Structural study of cell wall phosphomannan of Candida albicans NIH B-792 (serotype B) strain, with special reference to XH and 13C NMR analyses of acid-labile oligomannosyl residues. Arch Biochem Biophys 1990; 278:195–204
     [Google Scholar]
  24. Kwon-Chung K.J., Lehman D., Good C., Magee P.T. Genetic evidence for role of extracellular proteinase in virulence of Candida albicans. Infect Immun 1985; 49:571–575
     [Google Scholar]
  25. Laemmli U.K. Clevage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
     [Google Scholar]
  26. Lehle L., Cohen R.E., Ballou C.E. Carbohydrate structure of yeast invertase. Demonstration of a form with only core oligosaccharides and a form with completed polysaccharide chains. J Biol Chem 1979; 254:12209–12218
     [Google Scholar]
  27. Mega T., Ikenaka T., Matsushima Y. Studies on N- acetyl- β -D-glucosaminidase of Aspergillus oryzae I Purification and characterization of IV-acetyl-β-D-glucosaminidase obtained from Takadiastase. J Biochem 1970; 68:109–117
     [Google Scholar]
  28. Moore S. On the determination of cysteine as cysteic acid. J Biol Chem 1963; 238:235–237
     [Google Scholar]
  29. Morrissey J.H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 1981; 117:307–310
     [Google Scholar]
  30. Odds F.C. Candida and Candidosis 1988 2nd edn London: Bailliere Tindall;
     [Google Scholar]
  31. Odds F.C., Hierholzer J.C. Purification and properties of a glycoprotein acid phosphatase from Candida albicans. J Bacteriol 1973; 114:257–266
     [Google Scholar]
  32. Oktakara A., Yoshida M., Murakami M., Izumi T. Purification and characterization of β-N-acetylhexosaminidase from Picnoporus cinnabarinus. Agric Biol Chem 1981; 45:239–247
     [Google Scholar]
  33. Pugh D., Cawson R.A. The cytochemical localization of acid hydrolases in four common fungi. Cell Mol Biol 1977; 22:125–132
     [Google Scholar]
  34. Ram S.P., Sullivan P.A., Shepherd M.G. The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation. J Gen Microbiol 1983; 129:2367–2378
     [Google Scholar]
  35. Ram S.P., Romana L.K., Shepherd M.G., Sullivan P.A. Exo-(l → 3)- β-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans. J Gen Microbiol 1984; 130:1227–1236
     [Google Scholar]
  36. Reissig J.L., Strominger J.L., Leloir L.F. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 1955; 217:959–966
     [Google Scholar]
  37. Ross I.K., De Bernardis F., Emerson G.W., Cassone A., Sullivan P.A. The secreted aspartate proteinase of Candida albicans-, physiology of secretion and virulence of a proteinase-deficient strain. J Gen Microbiol 1990; 136:687–694
     [Google Scholar]
  38. Sandhoff K., Conzelmann E., Neufeld E.F., Kaback M.M., Suzuki K. The GM2 gangliosides. In The Molecular Basis of Inherited Disease 1989 New York: McGraw Hill; 3 pp 1807–1839
     [Google Scholar]
  39. Shepherd M.G., Chiew Y.Y., Ram S.P., Sullivan P.A. Germ-tube induction in Candida albicans. Can J Microbiol 1980; 26:21–26
     [Google Scholar]
  40. Shepherd M.G., Poulter R.T.M., Sullivan P.A. Candida albicans-, biology, genetics, and pathogenicity. Annu Rev Microbiol 1985; 39:579–614
     [Google Scholar]
  41. Sullivan P.A., McHugh N.J., Romara L.K., Shepherd M.G. The secretion of IV-acetylglucosaminidase during germ-tube formation in Candida albicans. J Gen Microbiol 1984; 130:2213–2218
     [Google Scholar]
  42. Tammi M., Ballou L., Taylor A., Ballou C.E. Effect of glycosylation on yeast invertase oligomer stability. J Biol Chem 1987; 262:4395–4401
     [Google Scholar]
  43. Trimble R.B., Atkinson P.H. Structure of yeast external invertase Man8_14 GlcNAc processing intermediates by 500-megahertz NMR spectroscopy. J Biol Chem 1986; 261:9815–9824
     [Google Scholar]
  44. Trimble R.B., Maley F. Optimising hydrolysis of N-linked high mannose oligosaccharides by endo- β-N-acetylgluco-saminidase H. Anal Biochem 1984; 141:515–522
     [Google Scholar]
  45. Trimble R.B., Maley F., Chu F.K. Glycoprotein biosynthesis in yeast. Protein conformation affects processing of high mannose oligosaccharides on carboxypeptidase Y and invertase. J Biol Chem 1983; 238:2562–2567
     [Google Scholar]
  46. Weitz G., Proia R.L. Analysis of the glycosylation and phosphorylation of the a-subunit of the lysozomal enzyme β-hexosaminidase A by site-directed mutagenesis. J Biol Chem 1992; 267:10039–10044
     [Google Scholar]
  47. Wright R.J., Carne A., Hieber A.D., Lamont I., L, Emerson G.W., Sullivan P.A. A second gene for a secreted aspartate proteinase in Candida albicans. J Bacteriol 1992; 174:7848–7853
     [Google Scholar]
  48. Yamamoto K., Lee K.M., Kumagai M., Tochikura T. Purification and characterization of β-N-acetylhexosaminidase from Penicillium oxalicum. Agric Biol Chem 1985; 49:611–619
     [Google Scholar]
/content/journal/micro/10.1099/13500872-140-7-1543
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Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation
Microbiology 140, 1543 (1994); https://doi.org/10.1099/13500872-140-7-1543
/content/journal/micro/10.1099/13500872-140-7-1543
/content/journal/micro/10.1099/13500872-140-7-1543
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