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

The mutant products Q279E ((279)Gln to Glu) and R301Q ((301)Arg to Gin) of the X-chromosomal inherited alpha-galactosidase (EC 3.2.1.22) gene, found i n unrelated male patients with variant Fabry disease (late-onset cardiac fo rm) were characterized. In contrast to patients with classic Fabry disease, who have no detectable alpha-galactosidase activity, atypical variants hav e residual enzyme activity. First, the properties of insect cell-derived re combinant enzymes were studied. The K-m and y(max) values of Q279E, R301Q, and wild-type alpha-galactosidase toward an artificial substrate, 4-methylu mbelliferyl-alpha-D-galactopyranoside, were almost the same. In order to mi mic intralysosomal conditions, the degradation of the natural substrate, gl obotriaosylceramide, by the alpha-galactosidases was analyzed in a detergen t-free-liposomal system, in the presence of sphingolipid activator protein B (SAP-B, saposin B). Kinetic analysis revealed that there was no differenc e in the degradative activity between the mutants and wild-type alpha-galac tosidase activity toward the natural substrate. Then, immunotitration studi es were carried out to determine the amounts of the mutant gene products na turally 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 residua l alpha-galactosidase activity was 3 and 5% of the normal level in L-57 and L-148, respectively. The quantities of immune cross-reacting materials rou ghly correlated with the residual alpha-galactosidase activities in lymphob last cells from the patients. Compared to normal control cells, fibroblast cells from a patient with variant Fabry disease, Q279E mutation, secreted o nly small amounts of alpha-galactosidase activity even in the presence of 1 0 mM NH4Cl. It is concluded that Q279E and R301Q substitutions do not signi ficantly affect the enzymatic activity, but the mutant protein levels are d ecreased presumably in the ER of the cells. (C) 2000 Elsevier Science B.V. All rights reserved.