Investigation of the α‐Galactosidase Deficiency in Fabry's Disease Using Antibodies against the Purified Enzyme

Abstract

1. α‐Galactosidase A, the enzyme deficient in Fabry's disease, was purified from normal human urine. The final preparation hydrolysed about 50 μmol p‐nitrophenyl‐α‐galactoside per h per mg protein at 37 °C. An antiserum against this enzyme was raised in rabbits. Preincubation of preparations of normal kidney and urine with the antiserum, followed by centrifugation, led to a marked reduction of the α‐galactosidase activity of the preparations. 2. There was no influence of the antiserum on the residual α‐galactosidase activity in Fabry kidney or urine, or on an α‐galactosidase B preparation from normal urine. 3. Pretreatment of the antiserum with urine or kidney preparations from a Fabry patient did not result in detectable loss of antibodies reacting with α‐galactosidase A, as shown by the unimpaired ability of the antiserum to diminish α‐galactosidase activity in normal kidney and urine. Furthermore, double immunodiffusion of the pretreated antiserum with a partially purified α‐galactosidase preparation, showed that antibodies not absorbable by the Fabry material were still present in the antiserum. 4. Incubation of normal urinary α‐galactosidase with antiserum, followed by centrifugation and enzyme assay in the supernatant, led to the same reduction of α‐galactosidase activity, regardless of whether a Fabry urine or kidney preparation was simultaneously present or not. 5. It is concluded that no detectable cross‐reacting material was present in this case of Fabry's disease.

Glycolipid and Glycoprotein Degradation

“…Only a-galactosidase A, the major isozyme in most tissues, is deficient in Fabry's disease (33). Immunochemical analysis showed no cross-reactivity between the A and B forms (29,34,35). Independently, Dean et al (36) and Schram et al (37) identified a-galactosidase B as an a-N-acetyl-D--galactosaminidase.…”

“…The enzyme has been purified from human liver (37,40,41), spleen (42), placenta (42,43), blood plasma (42), and urine (34) employing conventional techniques as well as affinity chromatography with an a-galactosylamine ligand (42).…”

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

“…After cleavage of the signal peptide and carbohydrate modifications in the Golgi and lysosomes, mature enzyme subunits of 46 kD form the active, homodimeric enzyme (20,21). In Fabry disease, early studies revealed the presence of nonfunctional, immunologically cross-reactive enzyme protein in some classically affected hemizygotes (with essentially no detectable enzymatic activity), while others had no detectable enzyme protein (22,23). Rare, atypical variants with residual a-galactosidase activity and milder phenotypes had enzyme protein (24)(25)(26)(27)(28), however, the amounts were not quantitated.…”

Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region.

“…In affected males, the progressive deposition of these substrates in lysosomes of vascular endothelial and smooth muscle cells leads to early demise due to occlusive disease of the heart, kidney, and brain. Classically affected males have no detectable a-Gal A activity and immunologic studies have revealed both the presence and absence of enzyme protein (1,(7)(8)(9). Atypical variants with residual a-Gal A activity and immunologically detectable enzyme protein have milder or no clinical manifestations (1,7,(10)(11)(12)(13)(14).…”