Furin, a specialized endoproteinase, transforms proproteins into biologically active
proteins.
Furin function is important for normal cells and also in multiple pathologies including
malignancy and
anthrax.
Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of
anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface
furin. To monitor
furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent
Proteins were linked by the
peptide sequence SNSRKKR / STSAGP derived from
anthrax PA83. Both because of the sensitivity and selectivity of the
anthrax sequence to
furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of
furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface
furin are present in the intact cells and in the cells with the enforced
furin overexpression. This observation was in a sharp contrast with the existing concepts about the
furin presentation on cell surfaces and
anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by
furin in the extracellular milieu and that only then the resulting PA63 bound the
anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional
peptide substrates, allowed continuous monitoring of
furin activity in
cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface
furin and, accordingly, that the mechanisms of
anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of
furin activity in
cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other
proteinase-activated biosensors.