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

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

  • J Biol Chem. 2018 Jun 29;293(26):10042-10058. doi: 10.1074/jbc.RA118.001774.
Kassiani Kytidou 1 Jules Beekwilder 2 Marta Artola 3 Eline van Meel 1 Ruud H P Wilbers 2 Geri F Moolenaar 4 Nora Goosen 4 Maria J Ferraz 1 Rebecca Katzy 1 Patrick Voskamp 5 Bogdan I Florea 3 Cornelis H Hokke 6 Herman S Overkleeft 3 Arjen Schots 2 Dirk Bosch 2 Navraj Pannu 5 Johannes M F G Aerts 7
Affiliations

Affiliations

  • 1 From the Departments of Medical Biochemistry.
  • 2 the Plant Sciences Group, Wageningen University and Research, Droevendaalsesteeg 1, 6708 PB Wageningen, and.
  • 3 Bio-organic Synthesis, and.
  • 4 Cloning and Protein Purification Facility, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden.
  • 5 Biophysical Structural Chemistry and.
  • 6 the Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
  • 7 From the Departments of Medical Biochemistry, j.m.f.g.aerts@lic.leidenuniv.nl.
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.

Keywords

Fabry disease; Nicotiana benthamiana; enzyme inhibitor; enzyme purification; enzyme structure; enzyme-replacement therapy; fluorescence; glycolipid; glycoside hydrolase; glycosphingolipid; glycosphingolipids; homologue; human; lysosomal storage disorder; plant; protein expression; recombinant enzyme; α-galactosidase.

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