Expression of functional human acid beta-glucosidase in COS-1 and Spodoptera frugiperda cells.
A cDNA encoding human acid beta-glucosidase (N-acylsphingosyl-1-O-beta-D-glucoside: glucohydrolase, EC 3.2.1.45) expressed catalytically active enzyme in transfected COS-1 or infected Spodoptera frugiperda (Sf9) cells. The expression plasmid p91023(B) (p91023B/Glc) and a Baculovirus (AcMNPV/Glc) containing the cDNA were constructed and used with the respective cells. By immunoblotting a glycosylated, 63-kilodalton human acid-beta-glucosidase was detected in the transfected or infected cells. A 56-kilodalton human polypeptide was obtained after complete deglycosylation with N-Glycanase. The expressed human enzymes also had partial endoglycosidase H sensitivity. The human enzyme expressed at high levels in Sf9 cells and had normal immunologic properties. With the partially purified enzyme from Sf9 cells, intact function of active site was indicated by normal kcat and Kmapp or Kiapp values for alternative substrates or potent inhibitors, respectively. The expressed enzyme was also activated normally by the negatively charged lipid, taurocholate. The results of these studies indicate that the Baculovirus expression system could provide a convenient source of normal human enzyme for structure/function investigations. In addition, this expression system should prove useful for the identification and evaluation of putative etiologic point mutations in Gaucher disease variants with kinetically altered residual enzymes.
Grabowski
G A
GA
Department of Pediatrics, Mount Sinai School of Medicine, New York, N.Y.
White
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WR
Grace
M E
ME
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant / Q000378:metabolism; D005959:Glucosidases / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D007915:Lepidoptera; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005959:Glucosidases; D001617:beta-Glucosidase