| Autor: |
Kytidou K; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., Beenakker TJM; Department of Bio-organic Synthesis, Leiden Institute of ChemistryLeiden, Netherlands., Westerhof LB; Wageningen University and Research, Plant Sciences GroupWageningen, Netherlands., Hokke CH; Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical CenterLeiden, Netherlands., Moolenaar GF; Cloning and Protein Purification Facility of Leiden Institute of ChemistryLeiden, Netherlands., Goosen N; Cloning and Protein Purification Facility of Leiden Institute of ChemistryLeiden, Netherlands., Mirzaian M; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., Ferraz MJ; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., de Geus M; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., Kallemeijn WW; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., Overkleeft HS; Department of Bio-organic Synthesis, Leiden Institute of ChemistryLeiden, Netherlands., Boot RG; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands., Schots A; Wageningen University and Research, Plant Sciences GroupWageningen, Netherlands., Bosch D; Wageningen University and Research, Plant Sciences GroupWageningen, Netherlands., Aerts JMFG; Department of Medical Biochemistry, Leiden Institute of ChemistryLeiden, Netherlands. |
| Abstrakt: |
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-NAGAL EL ) has therefore been suggested. We transiently produced in Nicotiana benthamiana leaves functional α-GAL, α-NAGAL, and α-NAGAL EL 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-NAGAL EL is improved in activity toward artificial 4MU-α-galactopyranoside. Recombinant α-NAGAL EL 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-NAGAL EL , 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-NAGAL EL 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. |
