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Papers In Press, published online ahead of print November 17, 2005
Human Genetics, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039
Corresponding Author: greg.grabowski{at}cchmc.org
Acid
J. Biol. Chem, 10.1074/jbc.M511110200
Submitted on October 12, 2005
Revised on November 16, 2005
Accepted on November 17, 2005
Analyses of variant acid
-glucosidases: effects of gaucher disease mutations
-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, 42 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 kcat, 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, the natural GCase protein activator, enhancement of kcat were present in variant GCases involving residues 48-122, and ~2-fold increases were obtained with an L264I GCase. About 50% of variant GCases each had wild-type or decreased 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, C4-C16 and C18-C23, and a free C342 were essential for activity; the free C126 and C248 were not. Relative kcat was highly sensitive to a His substitution at R496, but not at R495. 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 non-disruptive (e.g., VL). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.
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