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Glycobiology, 2000, Vol. 10, No. 11 1217-1224
© 2000 Oxford University Press

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

Sylvie Fabrega, Patrick Durand2, Patrice Codogno3, Chantal Bauvy3, Claudine Delomenie, Bernard Henrissat4, Brian M. Martin5, Cindy McKinney5, Edward I. Ginns5, Jean-Paul Mornon2 and Pierre Lehn1

INSERM U 458, Hôpital Robert Debré, 48 Bd Sérurier, 75019 Paris, France, 2Systèmes Moléculaires et Biologie Structurale, Laboratoire de Minéralogie-Cristallographie, CNRS UMR 7590, Universités Paris VI-Paris VII, T16, case 115, 4 place Jussieu, F-75252 Paris Cedex 5, France, 3INSERM U 504, 16 Av Paul Vaillant-Couturier, 94807 Villejuif Cedex, France, 4Architecture et Fonctions des Macromolécules Biologiques, CNRS UPR 9039, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, and 5Clinical Neuroscience Branch, National Institute of Mental Health, Building 49, 49 Convent Drive, MSC 4405, Bethesda, MD 20892, USA

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

1 To whom correspondence should be addressed


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