Human acid beta-glucosidase. N-glycosylation site occupancy and the effect of glycosylation on enzymatic activity.

The five potential N-glycosylation sites (sequons) of human acid beta-glucosidase were individually mutated to determine site occupancy and the effect of site occupancy on selected catalytic and stability properties of this enzyme. Each N-glycosylation consensus sequence [Asn-Xaa-(Ser/Thr)] was obliterated by individually substituting glutamine (Q) for asparagine (N). By expression of the normal and mutated cDNAs in insect (Sf9) and COS-1 cells and subsequent immunoblotting with anti-human acid beta-glucosidase antibodies, the four sequons at Asn-19, Asn-59, Asn-146, and Asn-270 were shown to be glycosylated in either source. The sequon at Asn-462 was never occupied. The mutant enzymes N59Q, N146Q, and N270Q were catalytically active and had normal interactions with active site-directed inhibitors as well as with the activators, phosphatidylserine and saposin C. Of the occupied sequons, N-glycosylation of the first was critical to the synthesis of a catalytically active enzyme. Alteration of this sequon, Asn-19-Ala-20-Thr-21, by the substitutions N19Q, N19D, N19E, or T21G led to a lack of glycosylation at this site. Enzymes containing N19Q, N19E, or T21G had significant decreases (3- to 60-fold) in intrinsic enzyme activity. The N19D enzyme had nearly normal catalytic activity and had enhanced activation by phosphatidylserine. These results show that sequon occupancy as well as steric effects at residue 19 are important for the development of an active conformer of this enzyme. This is the first example of a lysosomal hydrolase that requires sequon occupancy for the synthesis of a catalytically active enzyme.