Human Alpha Galactosidases Transiently Produced in Nicotiana benthamiana Leaves: New Insights in Substrate Specificities with Relevance for Fabry Disease.

Deficiency of alpha-galactosidase A (alpha-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 alpha-N-acetyl-galactosaminidase (alpha-NAGAL) with increased alpha-galactosidase activity (alpha-NAGALEL) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional alpha-GAL, alpha-NAGAL, and alpha-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 alpha-NAGALEL is improved in activity toward artificial 4MU-alpha-galactopyranoside. Recombinant alpha-NAGALEL and alpha-NAGAL are not neutralized by Ab-positive FD serum tested and are more stable in human plasma than alpha-GAL. Both enzymes hydrolyze the lipid substrates Gb3 and Lyso-Gb3 accumulating in Fabry patients. The addition to FD sera of alpha-NAGALEL, and to a lesser extent that of alpha-NAGAL, results in a reduction of the toxic Lyso-Gb3. In conclusion, our study suggests that modified alpha-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.