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

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.

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease

“…The purified enzyme is an active monomer, based on gel filtration behaviour. This feature is common to rice α-galactosidase, but the human enzyme occurs as a homodimer (49). As predicted by the absence of appropriate Asn-X-Ser/Thr/Cys motives, A1.1 is not N-glycosylated: endoglycanase digestion points to no N-linked glycans.…”

“…We earlier used N. benthamiana for expression of wild type and mutagenized human α-galactosidase and α-galactosaminidase (49). The N. benthamiana plant is a convenient production platform for recombinant proteins given the ease of cultivation and transfection with Agrobacterium tumefaciens (49).…”

“…The N. benthamiana plant is a convenient production platform for recombinant proteins given the ease of cultivation and transfection with Agrobacterium tumefaciens (49). In the previous study we employed a cyclophellitoltype activity-based probe (TB474) to visualize the recombinant human α-galactosidases.…”

“…The α-galactopyranose configured cyclophellitol aziridine grafted with Cy5 as fluorophore ABP (TB474) was synthesized as previously described (49). The biotinylated α-galactopyranoside cyclophellitol aziridine (ME741) was synthesized by copper-catalyzed click chemistry of azido cyclophellitol aziridine intermediate with biotin alkyne (Scheme S1).…”

“…Confirmation of sequences was done by sequencing (Macrogen, the Netherlands) and the complete open reading frame of the gene was inserted into pHYG, plant expression vector as previously described (58). The construct was under the control of cauliflower mosaic virus 35S constitutive promoter, with duplicate enhancer and the nopaline synthase terminator (49). The pHYG vectors harboring the genes were used for transformation of Agrobacterium tumefaciens strain MOG101, following N. benthamiana plant leaf infiltrations.…”