Research article

Cloning and characterization of human liver cytosolic β-glycosidase

Michelle de GRAAF, Irene C. van VEEN, Ida H. van der MEULEN-MUILEMAN, Winald R. GERRITSEN, Herbert M. PINEDO, Hidde J. HAISMA

Abstract

Cytosolic β-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of β-d-glycosides, including β-d-glucoside and β-d-galactoside. We therefore refer to this enzyme as cytosolic β-glycosidase. We cloned the cDNA encoding the human cytosolic β-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic β-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.

  • β-galactosidase
  • β-glucosidase
  • glycoside
  • mammalian

Footnotes

  • 2 Present address: Department for Therapeutic Gene Modulation, University Center of Pharmacy, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

  • Abbreviations used: EST, expressed sequence tag; LPH, lactase phlorizin hydrolase; 4-MuGal, 4-methylumbelliferyl-β-d-galactopyranoside; 4-MuGlc, 4-methylumbelliferyl-β-d-glucopyranoside.