Glycobiology, 2001, Vol. 11, No. 9 741-750
© 2001 Oxford University Press
Glycosidase active site mutations in human 
-L-iduronidase
3Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, King William Road, North Adelaide, SA 5006, Australia; 4INSERM U458, Hopital Robert Debré, 48 Bd Serurier, 75019 Paris, France; 5Systèmes Moléculaires et Biologie Structurale, Laboratoire de Minéralogie-Cristallographie, CNRS UMR 7590, Universités Paris VI-Paris VII, T16, case 115, 4 place Jussieu, 75252 Paris Cedex 5, France; 6Federal University of Rio Grande do Sul, Porto Alegre, Brazil; and 7Architecture et Fonction des Macromolécules Biologiques, CNRS UMR 6098, Universités d’Aix-Marseille I and II, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Mucopolysaccharidosis type I (MPS I; McKusick 25280) results from a deficiency in 
-L-iduronidase activity. Using a bioinformatics approach, we have previously predicted the putative acid/base catalyst and nucleophile residues in the active site of this human lysosomal glycosidase to be Glu182 and Glu299, respectively. To obtain experimental evidence supporting these predictions, wild-type -L-iduronidase and site-directed mutants E182A and E299A were individually expressed in Chinese hamster ovary–K1 cell lines. We have compared the synthesis, processing, and catalytic properties of the two mutant proteins with wild-type human -L-iduronidase. Both E182A and E299A transfected cells produced catalytically inactive human -L-iduronidase protein at levels comparable to the wild-type control. The E182A protein was synthesized, processed, targeted to the lysosome, and secreted in a similar fashion to wild-type -L-iduronidase. The E299A mutant protein was also synthesized and secreted similarly to the wild-type enzyme, but there were alterations in its rate of traffic and proteolytic processing. These data indicate that the enzymatic inactivity of the E182A and E299A mutants is not due to problems of synthesis/folding, but to the removal of key catalytic residues. In addition, we have identified a MPS I patient with an E182K mutant allele. The E182K mutant protein was expressed in CHO-K1 cells and also found to be enzymatically inactive. Together, these results support the predicted role of E182 and E299 in the catalytic mechanism of -L-iduronidase and we propose that the mutation of either of these residues would contribute to a very severe clinical phenotype in a MPS I patient.
1 To whom correspondence should be addressed
2 Present address: Hybrigenics S.A., 180 avenue Daumesnil, 75012 Paris, France
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