title: Functional analysis of variant lysosomal acid glycosidases of Anderson-Fabry and Pompe disease in a human embryonic kidney epithelial cell line (HEK 293 T).
abstract: The functional significance of missense mutations in genes encoding acid glycosidases of lysosomal storage disorders (LSDs) is not always clear. Here we describe a method of investigating functional properties of variant enzymes in vitro using a human embryonic kidney epithelial cell line. Site-directed mutagenesis was performed on the parental plasmids containing cDNA encoding for alpha-galactosidase A (α-Gal A) and acid maltase (α-Glu) to prepare plasmids encoding relevant point mutations. Mutant plasmids were transfected into HEK 293 T cells, and transient over-expression of variant enzymes was measured after 3 days. We have illustrated the method by examining enzymatic activities of four unknown α-Gal A and one α-Glu variants identified in our patients with Anderson-Fabry disease and Pompe diseases respectively. Comparison with control variants known to be either pathogenic or non-pathogenic together with over-expression of wild-type enzyme allowed determination of the pathogenicity of the mutation. One leader sequence novel variant of α-Gal A (p.A15T) was shown not to significantly reduce enzyme activity, whereas three other novel α-Gal A variants (p.D93Y, p.L372P and p.T410I) were shown to be pathogenic as they resulted in significant reduction of enzyme activity. A novel α-Glu variant (p.L72R) was shown to be pathogenic as this significantly reduced enzyme activity. Certain acid glycosidase variants that have been described in association with late-onset LSDs and which are known to have variable residual plasma and leukocyte enzyme activity in patients appear to show intermediate to low enzyme activity (p.N215S and p.Q279E α-Gal A respectively) in the over-expression system.
authors.affiliation: Department of Haematology, Royal Free Campus, University College London Medical School, Rowland Hill Street, London, NW3 2PF, UK.
authors.forename: Hatim Y
authors.initials: HY
authors.lastname: Ebrahim
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Baker
authors.forename: Atul B
authors.initials: AB
authors.lastname: Mehta
authors.forename: Derralynn A
authors.initials: DA
authors.lastname: Hughes
mesh_terms: D016022:Case-Control Studies; D002460:Cell Line; D000795:Fabry Disease / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005260:Female; D006009:Glycogen Storage Disease Type II / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed / Q000379:methods; D020125:Mutation, Missense; D014162:Transfection / Q000379:methods; D000519:alpha-Galactosidase / Q000235:genetics* / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism
chemical_list: D000520:alpha-Glucosidases; D000519:alpha-Galactosidase