Publications about genetic testing for metabolic disorders
  1. Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A

Fluorescent probes for selective protein labeling in lysosomes: a case of α-galactosidase A

Adam Pomorski, PhD 1 Cornelius Bohl 2 Susanne Seemann 2 Anne-Marie Knospe 2 Chaonan Zheng 2 Artur Krężel, PhD 1 Prof. Arndt Rolfs, MD 2 Jan Lukas, PhD 2
1 University of Wroclaw 2 University of Rostock
December 03, 2017

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.