Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

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2016
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Maeser, Stefan
Rawer, Stephan
Murphy, Mary
Bornheim, Jeff
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Journal of The American Society for Mass Spectrometry. 2016, 27(6), pp. 1071-1078. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1007/s13361-016-1386-0
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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.

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ISO 690MOISE, Adrian, Stefan MAESER, Stephan RAWER, Frederike EGGERS, Mary MURPHY, Jeff BORNHEIM, Michael PRZYBYLSKI, 2016. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry. In: Journal of The American Society for Mass Spectrometry. 2016, 27(6), pp. 1071-1078. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1007/s13361-016-1386-0
BibTex
@article{Moise2016Subst-34899,
  year={2016},
  doi={10.1007/s13361-016-1386-0},
  title={Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry},
  number={6},
  volume={27},
  issn={1044-0305},
  journal={Journal of The American Society for Mass Spectrometry},
  pages={1071--1078},
  author={Moise, Adrian and Maeser, Stefan and Rawer, Stephan and Eggers, Frederike and Murphy, Mary and Bornheim, Jeff and Przybylski, Michael}
}
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