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

Abstract

The mutant products Q279E (279Gln to Glu) and R301Q (301Arg to Gln) of the X-chromosomal inherited α-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 α-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 α-galactosidase toward an artificial substrate, 4-methylumbelliferyl-α-D-galactopyranoside, were almost the same. In order to mimic intralysosomal conditions, the degradation of the natural substrate, globotriaosylceramide, by the α-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 α-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 α-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 α-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 α-galactosidase activity even in the presence of 10 mM NH4Cl. 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. Copyright (C) 2000 Elsevier Science B.V.