Probing the cathepsin D using a BODIPY EL-pepstatin A: applications in fluorescence polarization and microscopy

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, ha s 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. Tn vitro, BODIPY FL-pepstatin A inh ibits 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 t o cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lyso somes, where it co-localized with cathepsin D. This staining was depleted w hen cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysos omes. The staining in the lysosomes can be competed with unlabeled pepstati n 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 an d trafficking of cathepsin D. (C) 2000 Published by Elsevier Science BN. Al l rights reserved.