Selective detection of Cathepsin E proteolytic activity

Abstract

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.

General significance: This study forms the foundation of Cath E specific inhibitor development in further studies.

Figure 1

Changes in the fluorescence intensity of substrates a–e (200 µM) with 23 picomole of (A) Cath E (B) Cath D in 50 mM NaOAc buffer containing 150 mM NaCl (pH 4.0). Solid filled and unfilled markers denote the enzyme treated and untreated substrates, respectively. Without enzymes, fluorescence intensities of all tested substrates remain at the base line level. Values represent the mean of triplicate measurements.

Figure 2

Profile of net fluorescence signals ratio (Cath E/Cath D) of substrates a–e encountered at 1 minute after starting the enzymatic catalytic cleavage. Net fluorescence signals represent the signals after correction for the substrates quenched background signals.

Figure 3

(A) Structure of intramolecular quenched substrate e, Mca-Ala-Gly-Phe-Ser-Leu**Pro-Ala-Lys(Dnp)-D-Arg-CONH₂; (B) Change in the fluorescence intensity of substrate e (200 µM) during the incubation with 23 picomole of Cath E, Cath D, Cath S and Cath B in 50 mM NaOAc buffer of pH 4.0 for Cath E and D and 100 mM NaOAc buffer of pH 6.5 for Cath S and Cath B. Values represent the mean of at least three independent experiments. Error bars represent the upper and lower values of the Standard Error Mean (SEM). Asterisks represent the statistical significance of the two tailed P-values (***, P ≤ 0.001).

Figure 4

(A) RP-HPLC Profile of peptide fragments obtained after digestion of 100 µM fluorogenic substrate e with Cath E (~119 picomole) in 50 mM sodium acetate buffer, pH 4.0, 150 mM NaCl at 37 °C for 3 h. UV absorbance detected at 280 nm. (B) The identified proteolytic fragments of substrate e and their ESI-MS characteristics.

Figure 5

Effect of inhibition the enzymatic catalytic activity of Cath E and Cath D. Substrate e (200 µM) in 150 mM NaCl, 50 mM NaOAc buffer (pH 4.0) with 1µL of 1 mM pepstatin A/Methanol (panel A) and Selective immunoprecipitation using Cath E specific antibody in 1× PBS (panel B). All fluorescent measurements were collected with λ_ex=340 nm and λ_em=405 nm.

Figure 6

(A) Dose response of proteolysis. Substrate e (10 µM) was incubated with various amount of Cathepsin E (2.27, 4.55, 6.82 nM). (B) Hanes-Woolf kinetic transformation diagrams of Cathepsin E (6.82 nM). Values represent the mean of triplicate measurements.