Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.
Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects. Graphical Abstract ᅟ.
Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, 65428, Rüsselsheim am Main, Germany.|Laboratory of Analytical Chemistry, University of Konstanz, 78457, Konstanz, Germany.
Adrian
A
Moise
Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, 65428, Rüsselsheim am Main, Germany.
Stefan
S
Maeser
ThermoFisher Scientific, Franfurter Strasse 129, 64269, Darmstadt, Germany.
Stephan
S
Rawer
Laboratory of Analytical Chemistry, University of Konstanz, 78457, Konstanz, Germany.
Frederike
F
Eggers
Ametek-Reichert Technologies, 3362 Walden Avenue, 14043, Buffalo, NY, USA.
Mary
M
Murphy
Ametek-Reichert Technologies, 3362 Walden Avenue, 14043, Buffalo, NY, USA.
Jeff
J
Bornheim
Steinbeis Center for Biopolymer Analysis and Biomedical Mass Spectrometry, 65428, Rüsselsheim am Main, Germany. Michael.przybylski@stw.de.|Laboratory of Analytical Chemistry, University of Konstanz, 78457, Konstanz, Germany. Michael.przybylski@stw.de.
Michael
M
Przybylski
D001665:Binding Sites; D000795:Fabry Disease / Q000201:enzymology; D006801:Humans; D013058:Mass Spectrometry; D018832:Molecular Chaperones; D000519:alpha-Galactosidase / Q000737:chemistry*
D018832:Molecular Chaperones; D000519:alpha-Galactosidase
Affinity-mass spectrometry; Binding sites; Chaperones; Fabry disease; Human α-galactosidase; Proteolytic extraction