Fluorescent Probes for Selective Protein Labeling in Lysosomes: A Case of α-Galactosidase A

FASEB J. 2017 Dec;31(12):5258-5267. doi: 10.1096/fj.201700058RRRR. Epub 2017 Aug 15.

Abstract:

Fluorescence-based live-cell imaging (LCI) of lysosomal glycosidases is often hampered by unfavorable pH and redox conditions that reduce fluorescence output. Moreover, most lysosomal glycosidases are low-mass soluble proteins that do not allow for bulky fluorescent protein fusions. We selected α-galactosidase A (GALA) as a model lysosomal glycosidase involved in Anderson-Fabry disease (AFD) for the current LCI approach. Examination of the subcellular localization of AFD-causing mutants can reveal the mechanism underlying cellular trafficking deficits. To minimize genetic GALA modification, we employed a biarsenical labeling protocol with tetracysteine (TC-tag) detection. We tested the efficiency of halogen-substituted biarsenical probes to interact with C-terminally TC-tagged GALA peptide at pH 4.5 in vitro and identified F2FlAsH-EDT2 as a superior detection reagent for GALA. This probe provides improved signal/noise ratio in labeled COS-7 cells transiently expressing TC-tagged GALA. The investigated fluorescence-based LCI technology of TC-tagged lysosomal protein using an improved biarsenical probe can be used to identify novel compounds that promote proper trafficking of mutant GALA to lysosomal compartments and rescue the mutant phenotype.

Authors

  • Adam Pomorski , PhD
  • Cornelius Bohl
  • Susanne Seemann
  • Anne-Marie Knospe
  • Chaonan Zheng
  • Artur Krężel , PhD
  • Prof. Arndt Rolfs , MD
  • Jan Lukas , PhD

Topics

Tagged as
  • α-galactosidase A
  • Fabry disease
  • Lysosomal Storage Disorders (LSD)

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