Abstract
α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.
Highlights
␣-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal ␣-linked galactose residues from glycoconjugate substrates. ␣-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals
Dysfunction or absence of ␣-Gal A leads to Fabry disease (FD), an X-linked lysosomal disorder characterized by accumulation of glycosphingolipids with terminal galactosyl moieties in tissues and body fluids of FD patients [9, 20]
We here report on the outcome of the investigation, including a comparison of the plant ␣-galactosidase with the human enzyme, which is deficient in FD patients
Summary
␣-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal ␣-linked galactose residues from glycoconjugate substrates. ␣-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human ␣-galactosidase A (␣-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Dysfunction or absence of ␣-Gal A leads to Fabry disease (FD), an X-linked lysosomal disorder characterized by accumulation of glycosphingolipids with terminal galactosyl moieties in tissues and body fluids of FD patients [9, 20]. Most male FD patients lack the ␣-Gal A protein and develop neutralizing antibodies against the therapeutic recombinant enzymes that might contribute to the noted poor responses to current treatments [24]. Novel chemical tools have been developed to study different retaining glycosidases, including ␣-galactosidases [33, 34] These activity-based probes (ABPs) are mechanism-based irreversible inhibitors functionalized with a bio-orthogonal tag such as a fluorophore or biotin. We here report on the outcome of the investigation, including a comparison of the plant ␣-galactosidase with the human enzyme, which is deficient in FD patients
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
