Characterization of two α-galactosidase mutants (Q279E and R301Q) found in an atypical variant of Fabry disease

Abstract

The mutant products Q279E ((279)Gln to Glu) and R301Q ((301)Arg to Gln) of the X-chromosomal inherited alpha-galactosidase (EC 3.2.1. 22) gene, found in unrelated male patients with variant Fabry disease (late-onset cardiac form) were characterized. In contrast to patients with classic Fabry disease, who have no detectable alpha-galactosidase activity, atypical variants have residual enzyme activity. First, the properties of insect cell-derived recombinant enzymes were studied. The K(m) and V(max) values of Q279E, R301Q, and wild-type alpha-galactosidase toward an artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, were almost the same. In order to mimic intralysosomal conditions, the degradation of the natural substrate, globotriaosylceramide, by the alpha-galactosidases was analyzed in a detergent-free-liposomal system, in the presence of sphingolipid activator protein B (SAP-B, saposin B). Kinetic analysis revealed that there was no difference in the degradative activity between the mutants and wild-type alpha-galactosidase activity toward the natural substrate. Then, immunotitration studies were carried out to determine the amounts of the mutant gene products naturally occurring in cells. Cultured lymphoblasts, L-57 (Q279E) and L-148 (R301Q), from patients with variant Fabry disease, and L-20 (wild-type) from a normal subject were used. The 50% precipitation doses were 7% (L-57) and 10% (L-148) of that for normal lymphoblast L-20, respectively. The residual alpha-galactosidase activity was 3 and 5% of the normal level in L-57 and L-148, respectively. The quantities of immuno cross-reacting materials roughly correlated with the residual alpha-galactosidase activities in lymphoblast cells from the patients. Compared to normal control cells, fibroblast cells from a patient with variant Fabry disease, Q279E mutation, secreted only small amounts of alpha-galactosidase activity even in the presence of 10 mM NH(4)Cl. It is concluded that Q279E and R301Q substitutions do not significantly affect the enzymatic activity, but the mutant protein levels are decreased presumably in the ER of the cells.

Structural characterization of mutant α-galactosidases causing Fabry disease

“…These results suggest that binding of a substrate analogue to a mutant enzyme protein in which a small structural change has occurred on the surface of the molecule reduces its folding defect and increases its stability in cells. Previously, we expressed mutant GLAs including M72V, L156V, L166V, Q279E, and R301Q in COS-1 cells and Sf9 cells and examined their biochemical characteristics (Ishii et al 1993;Okumiya et al 1995aOkumiya et al , 1998Kase et al 2000). The expressed products had GLA activity, but they were unstable and easily lost their activity in vitro, suggesting that their structural changes are located far from the active site and that the degree of the changes is not so large.…”

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

“…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.…”

Clinical and genetic analysis of Fabry disease: Report of six cases including three heterozygous females

“…Lastly,the p. R301Q mutation detected in Case 1 (a 43-year-old female) was previously reported in hemizygous males with cardiomyopathy and renal dysfunction but no skin abnormalities [7,10]. Interestingly heterozygous females in this family did not have any clinical abnormalities but slightly reduced leukocyte GLA activity [7].…”

“…1). Among them, c.758delT, p.Q250P and p.G171C were novel mutations, whereas p.R301Q, has been previously reported [7,10].…”