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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.

Human lysosomal alpha-galactosidase predominantly hydrolyzes ceramide trihexoside. A transgenic mouse line, C57BL/6CrSIc-TgN(GLA) 1951 Rin, highly expressing human alpha-galactosidase, has been established and investigated biochemically and immunohistochemically in order to clarify the distribution of the expressed enzyme proteins and to evaluate it as a donor model of organ transplantation therapy for Fabry disease caused by a genetic defect of alpha-galactosidase. In these transgenic mice, about five copies of the transgene were integrated, and alpha-galactosidase activity was expressed in liver, kidney, heart, spleen, small intestine, submaxillary gland, skeletal muscle, cerebrum, cerebellum, bone marrow cells and serum. The enzyme activity was about 22 to 11,080-fold higher than that in non-transgenic mice. In liver, heart and kidney tissues, which are important organs for transplantation studies, sufficient amounts of alpha-galactosidase mRNAs were transcribed, and the expressed enzymes, with molecular weights of 54-60 kDa, are abundant in the liver (enzyme activity: 53,965 nmol h-1 mg-1 protein) and heart (39,906 nmol h-1 mg-1 protein), followed by in the kidney tissue (9177 nmol h-1 mg-1 protein), respectively. An immunohistochemical microscopic study clearly demonstrated the distribution of the expressed enzyme proteins in kidney and liver tissues. Highly expressed alpha-galactosidase was detected in glomerular cells, tubular cells and hepatocytes. These transgenic mice will be useful as a donor model for experimental organ transplantation, and also it will enable recurrent biopsies and long-term observation. The organ transplantation data on mice will provide us with important information., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Our previous study on chimeric mutants of alpha-galactosidase suggested that two peptide regions encoded by exons 1-2 and 6 of the enzyme gene contribute to substrate recognition (Ishii, S. et al. (1994) Biochim. Biophys. Acta 1204, 265-270). In this study, we constructed five single amino acid substitutions for functional analysis of the amino acid residues around glutamine-279, the mutation site detected in an atypical Fabry disease patient. Two mutants, Q280S (Gln280-->Ser; CAA-->TCA) and T282A (Thr282-->Ala; ACT-->GCT), showed increased Km and decreased thermostability as compared with normal enzyme. Circular dichroism spectrum was not modified. An additional chimeric mutation in the exon 1-2 region by substitution with the homologous sequence of alpha-N-acetylgalactosaminidase cDNA restored catalytic activity and thermostability in both mutants. These data indicated the functional significance of glutamine-280 and threonine-282 for expressing the activity and stability of alpha-galactosidase molecule, and also the presence of an intramolecular interaction between the two peptide regions encoded by exons 1-2 and 6.

Two proteins with alpha-galactosidase activity, alpha-galactosidase A (alpha-GalA) and alpha-galactosidase B (alpha-GalB, or alpha-N-acetylgalactosaminidase; alpha-NAGA) have a high homology of amino-acid sequence. Point mutations of the alpha-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1-2 and/or 6 sequences of alpha-GalA cDNA were partly substituted by the corresponding regions of alpha-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1-2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved alpha-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl alpha-galactopyranoside were compared with those of alpha-GalA. The catalytic activity was slightly low in CMB12, decreased to 1/10 in CMB6, and restored to a significant degree in CMB126. Km was more than 4-fold higher for CMB6 and CMB126 than for alpha-GalA. The pH optimum was 4.0 for both CMB12 and alpha-GalA, 4.8 for CMB6, and 4.6 for CMB126 and alpha-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in alpha-GalB, CMB126, alpha-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Gal alpha 1-6Glc) and methyl alpha-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for alpha-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal alpha-linked galactose. We conclude that (1) the two regions encoded by exons 1-2 and 6 contribute to the alpha-galactosidic cleavage, and (2) an increase in Km of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal alpha-linked galactose.