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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

Abstract

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.

Keywords

Sporothrix schenckii N-glycan assembly Processing α-glucosidase 

Abbreviations

AUST

Australine

CNP

Castanospermine

DNJ

1-Deoxynojirimycin

D/P

Detergent/protein

ER

Endoplasmic reticulum

MMF

Mixed membrane fraction

MU

4-Methylumbelliferone

MUαGlc

4-Methylumbelliferyl-α-D-glucopyranoside

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SF

Solubilized fraction

Notes

Acknowledgements

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.

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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

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