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J. Biol. Chem., Vol. 281, Issue 7, 4242-4253, February 17, 2006

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Analyses of Variant Acid -Glucosidases

EFFECTS OF GAUCHER DISEASE MUTATIONS^*

Benjamin Liou¹, Andrzej Kazimierczuk¹, Min Zhang^¶, C. Ronald Scott^¶, Rashmi S. Hegde^||, and Gregory A. Grabowski²

From the Division and Program in Human Genetics and the ^||Division of Developmental Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229, the Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, and the ^¶Department of Pediatrics, School of Medicine, University of Washington, Seattle, Washington 98195

Acid -glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo--glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k_cat and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k_cat were present in variant GCases involving residues 48-122, whereas 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys⁴-Cys¹⁶ and Cys¹⁸-Cys²³, and a free Cys³⁴² were essential for activity; the free Cys¹²⁶ and Cys²⁴⁸ were not. Relative k_cat was highly sensitive to a His substitution at Arg⁴⁹⁶ but not at Arg⁴⁹⁵. These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.

Received for publication, October 12, 2005 , and in revised form, November 16, 2005.

The atomic coordinates and structure factors (code 2F61) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This study was supported by National Institutes of Health Grants DK36729 (to G. G.) and EY014648 (to R. S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Division and Program in Human Genetics, Children's Hospital Research Foundation, 3333 Burnet Ave., MLC 4006, Cincinnati, OH 45229-3039. Tel.: 513-636-7290; Fax: 513-636-2261; E-mail: greg.grabowski{at}cchmc.org.

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