Abstract
Deficiency of α-galactosidase A (α-GAL) causes Fabry disease (FD), an X-linked storage disease of the glycosphingolipid globtriaosylcerammide (Gb3) in lysosomes of various cells and elevated plasma globotriaosylsphingosine (Lyso-Gb3) toxic for podocytes and nociceptive neurons. Enzyme replacement therapy is used to treat the disease, but clinical efficacy is limited in many male FD patients due to development of neutralizing antibodies (Ab). Therapeutic use of modified lysosomal α-N-acetyl-galactosaminidase (α-NAGAL) with increased α-galactosidase activity (α-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional α-GAL, α-NAGAL, and α-NAGALEL enzymes for research purposes. All enzymes could be visualized with activity-based probes covalently binding in their catalytic pocket. Characterization of purified proteins indicated that α-NAGALEL is improved in activity toward artificial 4MU-α-galactopyranoside. Recombinant α-NAGALEL and α-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than α-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of α-NAGALEL, and to a lesser extent that of α-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified α-NAGALEL might reduce excessive Lyso-Gb3 in FD serum. This neo-enzyme can be produced in Nicotiana benthamiana and might be further developed for the treatment of FD aiming at reduction of circulating Lyso-Gb3.
Highlights
Deficiencies in lysosomal enzymes are the cause of various inherited lysosomal storage disorders in humans (Futerman and Van Meer, 2004)
We first established the optimal conditions for production of α-galactosidases α-galactosidase A (α-GAL), α-NAGAL, and α-NAGALEL in a transient expression system in N. benthamiana
The amount of enzyme activity was proportional to the amount of active enzyme molecules detected with the fluorescent activity-based probes (ABPs) (Figure 1B) and that detected with western blotting using anti-α-GAL and α-NAGAL antibodies (Figure 1C)
Summary
Deficiencies in lysosomal enzymes are the cause of various inherited lysosomal storage disorders in humans (Futerman and Van Meer, 2004). Examples are Gaucher disease (GD), Pompe disease and Fabry disease (FD) with deficiencies of acid betaglucosidase (glucocerebrosidase, GBA), acid alpha-glucosidase and acid alpha-galactosidase (α-GAL) respectively For each of these diseases, enzyme replacement therapy (ERT) approaches have been designed and are applied with variable success. Two similar approaches were developed for FD using human α-GAL produced in Chinese hamster ovary cells (agalsidase beta; FabrazymeTM; SanofiGenzyme) or human fibroblasts (agalsidase alfa; ReplagalTM; Shire) (Eng et al, 2001; Schiffmann et al, 2001) Both enzyme preparations were registered in August 2001 as orphan drug in Europe, but only Fabrazyme was approved by the FDA in the United States (Desnick, 2004). Fabrazyme and Replagal are infused bi-weekly at a dose of 1.0 and 0.2 mg/kg body weight, respectively (Eng et al, 2001; Schiffmann et al, 2001)
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