Analyses of Variant Acid β-Glucosidases

Abstract

Acid β-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-β-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 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 enhancement of kcat 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 kcat 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. Acid β-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-β-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 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 enhancement of kcat 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 kcat 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. Acid β-glucosidase (GCase, 3The abbreviations used are: GCase, glucocerebrosidase (acid β-glucosidase, EC 3.2.1.45); 4MUG, 4-methylumbelliferyl-β-d-glucopyranoside; CBE, conduritol B epoxide; MES, 4-morpholineethanesulfonic acid; CRIM, cross-reacting immunological material; CRIM SA, CRIM specific activity. glucocerebrosidase, EC 3.2.1.45) is a membrane-associated lysosomal hydrolase whose defective function leads to Gaucher disease (1.Brady R.O. Kanfer J.N. Shapiro D. Biochem. Biophys. Res. Commun. 1965; 18: 221-225Crossref PubMed Scopus (586) Google Scholar, 2.Beutler E. Grabowski G.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Diseases. 8th Ed. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar). The mature glycoprotein has 497 amino acids that are derived from 517- or 536-amino acid precursors containing leader sequences that are removed during transit through the endoplasmic reticulum membrane (3.Bergmann J.E. Grabowski G.A. Am. J. Hum. Genet. 1989; 44: 741-750PubMed Google Scholar, 4.Erickson A.H. Ginns E.I. Barranger J.A. J. Biol. Chem. 1985; 260: 14319-14324Abstract PubMed Google Scholar). at of Grabowski G.A. J. Biol. Chem. PubMed Google Scholar). is essential for the of a active with of the Grabowski G.A. J. Biol. Chem. PubMed Google Scholar, Grabowski G.A. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). The not provide to the membrane of GCase, are not present in the mature Barranger J.A. Ginns E.I. J. Biol. Chem. PubMed Google Scholar). The is to the and Ginns E.I. Barranger J.A. J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Google Scholar, Grabowski G.A. Genet. PubMed Scopus Google the for to be in the the has a of in and is to Ginns E.I. Barranger J.A. J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Google Scholar, Grabowski G.A. Genet. PubMed Scopus Google Scholar). GCase a of and or for in vitro a 2.Beutler E. Grabowski G.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Diseases. 8th Ed. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar). The is the with the a but GCase to of these in a E. Grabowski G.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Diseases. 8th Ed. McGraw-Hill Inc., New York2001: 3635-3668Google Scholar). GCase is a β-glucosidase, whose catalytic through a of the active site with a of D. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Biochem. J. PubMed Scopus Google Scholar). The in active site function is and the is J. D. Grabowski G.A. J. Biol. Chem. PubMed Google Scholar, of and Acid of Scholar, Ginns E.I. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Scopus Google Scholar). The of is the and is the and the is is of the with of the the and of The membrane with for negatively charged to the in vitro activity from in a The of a acid activator saposin has effects on the activity of the negatively charged GCase J. Biol. Chem. PubMed Scopus Google Scholar, Grabowski G.A. J. Biol. Chem. PubMed Scopus Google Scholar, Grabowski G.A. PubMed Scopus Google Scholar, Grabowski G.A. J. Biol. Chem. PubMed Scopus Google Scholar, E. J. Biol. PubMed Scopus Google Scholar). The activation of the negatively charged and/or saposin C to be but the to the into a active that is to the function of saposin C in Grabowski G.A. J. Biol. Chem. PubMed Scopus Google Scholar, Grabowski G.A. PubMed Scopus Google Scholar, D. Grabowski G.A. PubMed Scopus Google Scholar). The of saposin C leads to a of and a disease 1989; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). About mutations amino acid substitutions in patients with Gaucher but to functional E. Genet. PubMed Scopus Google Scholar). of these mutations the amino acid substitutions are predicted to be highly (e.g. and/or whereas not (e.g. substitutions of amino the of to in Gaucher disease be on functional derived from highly mutations effects on catalytic function and/or to or disease of the of the mutations and functional effects on catalytic or properties of GCase for the and/or of therapeutic for or therapeutic The majority of mutations with expressed in the the of the specific GCase properties. of these in for in vitro of these properties. of mutations in Gaucher disease mutations of cysteine residues that are in disulfide or are and mutations of were to into effects on and function of GCase. 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The and were to the effects of the substitution in the and the in Gaucher disease with the wild-type amino acid of the wild-type from Barranger J.A. Ginns E.I. J. Biol. Chem. PubMed Google of GCase from Gaucher disease patients and Grabowski G.A. Genet. PubMed Scopus Google Scholar, E. Am. J. Hum. Genet. PubMed Scopus Google Grabowski G.A. Am. J. Genet. Scopus Google Grabowski G.A. PubMed Scopus Google PubMed Scopus Google not from from from from E. E. Hum. PubMed Scopus Google from E. Am. J. Hum. Genet. PubMed Scopus Google Grabowski G.A. Hum. PubMed Scopus Google from not Hum. Genet. PubMed Scopus Google J. PubMed Scopus Google from from from C.R. Am. J. Genet. Scopus Google Grabowski G.A. Res. 1989; PubMed Scopus Google J. Ginns E.I. E. Biochem. PubMed Scopus Google Grabowski G.A. Res. 1989; PubMed Scopus Google from J.A. Barranger J.A. Ginns E.I. J. PubMed Scopus Google from not from Grabowski G.A. J. Biol. Chem. 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