Mutant α-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

Abstract

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

Mutations of the GLA gene in Korean patients with Fabry disease and frequency of the E66Q allele as a functional variant in Korean newborns

“…9,11,17,20 However, the clinical manifestations of patients with p.E66Q in the cited reports were not sufficiently convincing to confirm p.E66Q as a disease-causing mutation. For instance, Nakao et al 11 suggested that renal variants have a phenotype intermediate between cardiac variants and classical phenotypes.…”

“…8,17,19,20 Indeed, we have reported p.E66Q activities as high as 40% of mean normal activity in the transiently overexpressed COS-7 cells. 19 X-ray crystallography shows that GLA is a homodimeric glycoprotein in which each monomer is composed of two domains (Figure 2).…”

“…The protein is predicted to show more residual enzyme activity when a mutation occurs in a more surface-accessible residue. 17,31 The location of p.E66 is distant from both the active site and the dimer interface, and has a more accessible surface area of 3.1 Å 2 than have other mutations of atypical Fabry disease (Figure 2). 31,32 Although predicted to be unstable at neutral pH, 17 which is similar to the pH of the ER, p.Glu66 The pocket for active site Domain 1…”

“…Most mutations in classical Fabry disease affect the catalytic activity of the enzyme, whereas mutations in atypical Fabry disease do not affect enzyme activity per se but rather lead to improper protein folding and, subsequently, instability of the tertiary structure. 7,[16][17][18] These mutant proteins retain some residual enzyme activity, which is related to the milder phenotypes of atypical Fabry disease.…”

“…19 In this report, we found remarkably high residual enzyme activity of a protein with the p.E66Q mutation, previously known as a pathogenic mutation in patients with atypical Fabry disease. 9,11,17,20 p.E66Q synthesizes 40% of wild-type enzyme activity when overexpressed in COS-7 cells, indicating that p.E66Q might be a mild mutation or a functional single nucleotide polymorphism (SNP). 19 Herein, we report 25 distinct mutations in GLA, including six novel mutations, identified from 28 unrelated Korean families with Fabry disease.…”