Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells

Using bioinformatics methods, we have previously identified Glu235 and Glu3 40 as the putative acid/base catalyst and nucleophile? respectively, in the active site of human glucocerebrosidase. Thus, rye undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predi ctions. Recombinant retroviruses were used to express wild-type and E235A a nd E340A mutant proteins in glucocerebrosidase-deficient murine cells. In c ontrast to wild-type enzyme, the mutants were found to be catalytically ina ctive. We also report the results of various studies (Western blotting, gly cosylation analysis, subcellular fractionation, and confocal microscopy) in dicating that the wild-type and mutant enzymes are identically processed an d sorted to the lysosomes, Thus, enzymatic inactivity of the mutant protein s is not the result of incorrect folding/processing. These findings indicat e that Glu235 plays a keg role in the catalytic machinery of human glucocer ebrosidase and may indeed be the acidi base catalyst. As concerns Glu340, t he results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by u se of active site labeling techniques. Finally, our findings may help to be tter understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.