Antonie van Leeuwenhoek

, Volume 101, Issue 2, pp 313–322 | Cite as

Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii

  • Blanca I. Torres-Rodríguez
  • Karina Flores-Berrout
  • Julio C. Villagómez-Castro
  • Everardo López-RomeroEmail author
Original Paper


The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-d-glucopyranoside with high affinity as judged from Km and Vmax values of 0.3 μM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-d-glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.


Sporothrix schenckii N-glycan assembly Processing α-glucosidase 











Endoplasmic reticulum


Mixed membrane fraction






Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Solubilized fraction



This work was supported by grant SEP-CONACyT-2002-CO1-39528/A-1 from Secretaría de Educación Pública and Consejo Nacional de Ciencia y Tecnología, México, and by Dirección de Apoyo a la Investigación y al Posgrado (DINPO), Universidad de Guanajuato, México. ELR and JCVC are members of the Sistema Nacional de Investigadores, México.


  1. Bause E, Erkens R, Schweden J, Jaenicke L (1986) Purification and characterization of trimming glucosidase I from Saccharomyces cerevisiae. Fed Eur Biochem Soc Lett 206:208–212CrossRefGoogle Scholar
  2. Berthelot K, Delmotte FM (1999) Purification and characterization of an α-glucosidase from Rhizobium sp. (Robinia pseudoacacia L.) strain USDA 4280. Appl Environ Microbiol 65:2907–2911PubMedGoogle Scholar
  3. Bonifaz A (2010) Micología médica básica. McGraw-Hill, MéxicoGoogle Scholar
  4. Brada D, Dubach UC (1984) Isolation of a homogenous glucosidase II from pig kidney microsomes. Eur J Biochem 141:149–156PubMedCrossRefGoogle Scholar
  5. Brada D, Kerjaschki D, Roth J (1990) Cell type-specific post-Golgi apparatus localization of a “resident” endoplasmic reticulum glycoprotein, glucosidase II. J Cell Biol 110:309–318PubMedCrossRefGoogle Scholar
  6. Bravo-Torres JC, Calvo-Méndez C, Flores-Carreón A, López-Romero E (2003) Purification and biochemical characterization of a soluble α-glucosidase from the parasite Entamoeba histolytica. Antonie van Leeuwenhoek 84:169–178PubMedCrossRefGoogle Scholar
  7. Bravo-Torres JC, Villagómez-Castro JC, Calvo-Méndez C, Flores-Carreón A, López-Romero E (2004) Purification and biochemical characterization of a membrane-bound α-glucosidase from the parasite Entamoeba histolytica. Int J Parasitol 34:455–462PubMedCrossRefGoogle Scholar
  8. Burns DM, Touster O (1982) Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis. J Biol Chem 257:9991–10000Google Scholar
  9. D’Alessio C, Fernández F, Trombetta ES, Parodi AJ (1999) Genetic evidence for the heterodimeric structure of glucosidase II. The effect of disrupting the subunit-encoding genes on glycoprotein folding. J Biol Chem 274:25899–25905PubMedCrossRefGoogle Scholar
  10. Dhanawansa R, Faridmoayer A, van der Merwe G, Li YX, Scaman CH (2002) Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I. Glycobiology 12:229–234PubMedCrossRefGoogle Scholar
  11. Elbein AD (1991) Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing. Fed Am Soc Exp Biol J 5:3055–3063Google Scholar
  12. Frade-Pérez MD, Hernández-Cervantes A, Flores-Carreón A, Mora-Montes HM (2010) Biochemical characterization of Candida albicans α-glucosidase I heterologously expressed in Escherichia coli. Antonie van Leeuwenhoek 98:291–298PubMedCrossRefGoogle Scholar
  13. Goldstein A, Lampen JO (1985) β-D-Fructofuronoside fructo-hydrolase from yeast. Methods Enzymol 9:504–511Google Scholar
  14. Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049PubMedCrossRefGoogle Scholar
  15. Hentges A, Bause E (1997) Affinity purification and characterization of glucosidase II from pig liver. Biol Chem 378:1031–1038PubMedCrossRefGoogle Scholar
  16. Herscovics A (1999a) Importance of glycosidases in mammalian glycoprotein biosynthesis. Biochim Biophys Acta 1473:96–107PubMedCrossRefGoogle Scholar
  17. Herscovics A (1999b) Processing glycosidases of Saccharomyces cerevisiae. Biochim Biophys Acta 1426:275–285PubMedCrossRefGoogle Scholar
  18. Hettkamp H, Legler G, Bause E (1984) Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagines linked oligosaccharides. Eur J Biochem 142:85–90PubMedCrossRefGoogle Scholar
  19. Kaushal GP, Pastuszak I, Hatanaka K, Elbein AD (1990) Purification to homogeneity and properties of glucosidase II from mung bean seedlings and suspension-cultured soybean cells. J Biol Chem 265:12671–12679Google Scholar
  20. Kilker RD, Saunier B, Tkacz JS, Herscovics A (1981) Partial purification from Saccharomyces cerevisiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3Man9GlcNAc2. J Biol Chem 256:5299–5303PubMedGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  22. Lopes-Bezerra LM, Schubach A, Costa RO (2006) Sporothrix schenckii and sporotrichosis. Ann Braz Acad Sci 78:293–308Google Scholar
  23. López-Romero E, Flores-Carreón A, Arroyo-Flores BL, Torre-Bouscoulet ME, Bravo-Torres JC, Villagómez-Castro JC, Balcázar-Orozco R (2000) Glycosyl transferases and glycosidases of glycoprotein biosynthesis with emphasis on Candida albicans and Entamoeba histolytica. Rec Res Dev Microbiol 4:667–681Google Scholar
  24. López-Romero E, Reyes-Montes MR, Pérez-Torres A, Ruiz-Baca E, Villagómez-Castro JC, Mora-Montes HM, Flores-Carreón A, Toriello C (2011) Sporothrix schenckii complex and sporotrichosis, an emerging health problem. Future Microbiol 6:85–102PubMedCrossRefGoogle Scholar
  25. Merril CR, Goldman D, Van Keuren ML (1984) Gel protein stains: silver stain. Methods Enzymol 104:441–446PubMedCrossRefGoogle Scholar
  26. Mora-Montes HM, Bates S, Netea MG, Díaz-Jiménez DF, López-Romero E, Zinker S, Ponce-Noyola P, Kullberg BJ, Brown AJ, Odds FC, Flores-Carreón A, Gow NAR (2007) Endoplasmic reticulum α-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction. Eukaryot Cell 6:2184–2193PubMedCrossRefGoogle Scholar
  27. Mora-Montes HM, Ponce-Noyola P, Villagómez-Castro JC, Gow NAR, Flores-Carreón A, López-Romero E (2009) Protein glycosylation in Candida. Future Microbiol 4:1167–1183PubMedCrossRefGoogle Scholar
  28. Moremen KW, Trimble RB, Herscovics A (1994) Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology 4:113–125PubMedCrossRefGoogle Scholar
  29. Pelletier MF, Mercil A, Sevigny G, Jakob CA, Tessier DC, Chevet E, Menard R, Bergeron JJ, Thomas DY (2000) The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo. Glycobiology 10:815–827PubMedCrossRefGoogle Scholar
  30. Rippon JW (1990) Tratado de micología médica. Interamericana McGraw-Hill, MéxicoGoogle Scholar
  31. Ruiz-Baca E, Villagómez-Castro JC, Leal-Morales CA, Sabanero-López M, Flores-Carreón A, López-Romero E (2005) Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases. Antonie van Leeuwenhoek 88:221–230PubMedCrossRefGoogle Scholar
  32. Ruiz-Baca E, Toriello C, Pérez-Torres A, Sabanero-López M, Villagómez-Castro JC, López-Romero E (2009) Isolation and some properties of a glycoprotein of 70 kDa (Gp70) from the cell wall of Sporothrix schenckii involved in fungal adherence to dermal extracellular matrix. Med Mycol 47:185–196PubMedCrossRefGoogle Scholar
  33. Ruiz-Baca E, Mora-Montes M, Mojica-Marín V, López-Romero E, Urtíz-Estrada N (2011) 2D-immunoproteomic analysis of the Sporothrix schenckii cell wall. Mem Inst Osvaldo Cruz 106:248–250CrossRefGoogle Scholar
  34. Saunier B, Kilker RD Jr, Tkacz JS, Quaroni A, Herscovics A (1982) Inhibition of N-linked complex oligosaccharide formation by1-deoxynojirimicin, an inhibitor of processing glycosidases. J Biol Chem 257:14155–14161PubMedGoogle Scholar
  35. Saxena S, Shailubhai K, Don-Yu B, Vijay IK (1987) Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland. Biochem J 247:563–570PubMedGoogle Scholar
  36. Shailubhai K, Pratta MA, Vijay IK (1987) Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland. Biochem J 247:555–562PubMedGoogle Scholar
  37. Simons JF, Ebersold M, Helenius A (1998) Cell wall 1, 6-β-glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p. EMBO J 17:396–405PubMedCrossRefGoogle Scholar
  38. Stigliano ID, Caramelo JJ, Labriola CA, Parodi AJ, D’Alessio C (2009) Glucosidase II β subunit modulates N-glycan trimming in fission yeasts and mammals. Mol Biol Cell 20:3974–3984PubMedCrossRefGoogle Scholar
  39. Szumilo T, Kaushal GP, Elbein AD (1986) Purification and properties of α-glucosidase I from mung bean seedlings. Arch Biochem Biophys 247:261–271PubMedCrossRefGoogle Scholar
  40. Takesue Y, Takesue S (1996) Purification and characterization of α-glucosidase complex from the intestine of the frog, Rana japonica. Biochim Biophys Acta 1296:152–158PubMedCrossRefGoogle Scholar
  41. Torre-Bouscoulet ME, López-Romero E, Balcázar-Orozco R, Calvo-Méndez C, Flores-Carreón A (2004) Partial purification and biochemical characterization of a soluble α-glucosidase II-like activity from Candida albicans. FEMS Microbiol Lett 236:123–128PubMedCrossRefGoogle Scholar
  42. Treml K, Meimaroglou D, Hentges A, Bause E (2000) The alpha-and beta-subunits are required for expression of catalytic activity in the heterodimeric glucosidase II complex from human liver. Glycobiology 10:493–502PubMedCrossRefGoogle Scholar
  43. Trombetta ES, Fleming KG, Helenius A (2001) Quaternary and domain structure of glycoprotein processing glucosidase II. Biochemistry 40:10717–10722PubMedCrossRefGoogle Scholar
  44. Yet MG, Shao MC, Wold F (1988) Effects of the protein matrix on glycan processing in glycoproteins. FASEB J 2:22–31PubMedGoogle Scholar
  45. Zamarripa-Morales S, Villagómez-Castro JC, Calvo-Méndez C, Flores-Carreón A, López-Romero E (1999) Entamoeba histolytica: Identification and properties of membrane-bound and soluble α-glucosidases. Exp Parasitol 93:109–115PubMedCrossRefGoogle Scholar
  46. Zeng YC, Elbein AD (1998) Purification to homogeneity and properties of plant glucosidase I. Arch Biochem Biophys 355:26–34PubMedCrossRefGoogle Scholar
  47. Zhang L, Zhou H, Ouyang H, Li Y, Jin C (2008) Afcwh41 is required for cell wall synthesis, conidiation, and polarity in Aspergillus fumigatus. FEMS Microbiol Lett 289:155–165PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Blanca I. Torres-Rodríguez
    • 1
  • Karina Flores-Berrout
    • 1
  • Julio C. Villagómez-Castro
    • 1
  • Everardo López-Romero
    • 1
    Email author
  1. 1.Departamento de Biología, División de Ciencias Naturales y ExactasUniversidad de GuanajuatoGuanajuatoMexico

Personalised recommendations