Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy.
Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.
Chen
C S
CS
Molecular Probes, Inc., Eugene, OR 97402, USA. cchen@probes.com
Chen
W N
WN
Zhou
M
M
Arttamangkul
S
S
Haugland
R P
RP
D000818:Animals; D001896:Boron Compounds / Q000138:chemical synthesis / Q000494:pharmacology*; D002402:Cathepsin D / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008264:Macrophages; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D010436:Pepstatins / Q000138:chemical synthesis / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology*
C408343:BODIPY FL-pepstatin A; D001896:Boron Compounds; D005456:Fluorescent Dyes; D010436:Pepstatins; D011480:Protease Inhibitors; D002402:Cathepsin D