Selective detection of Cathepsin E proteolytic activity.
BACKGROUND
Aspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide-substrate that is only sensitive to Cath E but not Cath D.


METHODS
Substrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH₂, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence.


RESULTS
Substrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (K(cat)/K(M)) of Cath E for substrate e was 16.7 μM⁻¹S⁻¹. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B.


CONCLUSIONS
This study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement.


CONCLUSIONS
This study forms the foundation of Cath E specific inhibitor development in further studies.
Abd-Elgaliel
Wael R
WR
Department of Radiology, The Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX, USA.
Tung
Ching-Hsuan
CH
D002384:Catalysis; D002402:Cathepsin D / Q000737:chemistry / Q000378:metabolism; D020059:Cathepsin E / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015195:Drug Design; D005453:Fluorescence; D006801:Humans; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D020059:Cathepsin E; C533922:CTSD protein, human; C533925:CTSE protein, human; D002402:Cathepsin D