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Characterization of human glucocerebrosidase from different mutant alleles

1991; Elsevier BV; Volume: 266; Issue: 6 Linguagem: Inglês

10.1016/s0021-9258(19)67845-2

1083-351X

Toya Ohashi, Christine Hong, Solly Weiler, John M. Tomich, Johannes M. F. G. Aerts, J. M. Tager, J. A. Barranger,

Biochemical and Molecular Research

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocerebrosidase.The mutant genes were expressed in NIH 3T3 cells.The abnormal human enzymes were purified by immunoaffinity chromatography and characterized.The Asd7' + Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site.In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme.The Arg4s3 + Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine.The ArgI2' Gln mutant protein was catalytically inactive.The + Pro, the pseudopattern, and the Pro4" + Arg mutants appear to have reduced amounts of enzyme protein in cells.The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.An inherited deficiency of glucocerebrosidase (EC 3.2.1.45)is the basis for the lysosomal storage of glucosylceramide in the heterogeneous group of disorders known collectively as Gaucher disease.Biochemical analyses of the enzyme from tissues and cells have been inconclusive, but have suggested that the clinical phenotype is related to a specific abnormality in either the amount or activity of the mutant enzyme (1-3).In a molecular approach to the study of the mutations responsible for Gaucher disease, the cDNA for glucocerebrosidase (GC)' was cloned ( 5 ) , and its sequence was determined (6, 7).Sequencing of mutant GC genes has been carried out, and six different mutant alleles have been identified codon + Pro (S), codon Am3?* + Ser (9), codon Arg'** + Gln (lo), codon Pro4'* + Arg (11), codon Arg463 "+ Cys (12), and a crossover with the pseudogene (12,41).Only recently have