A new proteinase 3 substrate with improved selectivity over human neutrophil elastase

doi:10.1016/j.ab.2013.07.028

Analytical Biochemistry

Volume 442, Issue 1, 1 November 2013, Pages 75-82

https://doi.org/10.1016/j.ab.2013.07.028 Get rights and content

Abstract

We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X₁′-X₂′-X₃′-Tyr(3-NO₂)-NH₂ using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO₂)-NH₂ with a high specificity constant (k_cat/K_M = 1534 × 10³ M⁻¹ s⁻¹) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.

Section snippets

Peptide synthesis

All peptides were synthesized manually via the solid-phase method using Fmoc chemistry, as described previously [10]. TentaGel S RAM (substitution 0.24 meq/g) (RAPP Polymere, Germany) was used as a solid support. The α-amino groups of amino acids were Fmoc protected. The Fmoc-protected amino acid residues were attached to the resin using the DIPCI/HOBt (diisopropylcarbodiimide/hydroxybenzotriazole) method. Briefly, the mixture of N-protected amino acid derivative, DIPCI and HOBt (molar ratio,

Results

The synthesized library underwent parallel iterative deconvolution against PR3 and HNE. The enzyme concentration was adjusted in order to obtain a fluorescence increase for both of the studied enzymes. This forced us to increase the HNE concentration during the progress of deconvolution. After incubation of each sublibrary with the experimental enzyme, the fluorescence increase was followed at 450 nm, reflecting the emission of liberated ABZ attached to the peptide fragment.

In position X₁′ in

Discussion

The obtained results indicate that PR3 and HNE have different preferences in terms of the substrate prime region. PR3 displays high affinity toward hydrophilic/negatively charged residues (Asn/Thr-Glu) located in substrate P₁′ and P₂′ positions. In contrast to Asn-based libraries that were resistant to HNE, we observed the intense hydrolysis of sublibraries with Thr in position P₁′. Such findings did not correlate with data presented by Hajjar and coworkers [16], [17]. This group postulated the

Acknowledgment

This project was supported by the National Council of Science (NCN) under registration number UMO-2012/07/N/ST5/00178 (J.P.-S.).

References (18)

P.C. Grayson et al.
Antineutrophil cytoplasmic antibodies, autoimmune neutropenia, and vasculitis
Semin. Arthritis Rheum.
(2011)
I. Schechter et al.
On the size of the active site in proteases: I. Papain
Biochem. Biophys. Res. Commun.
(1967)
M. Wysocka et al.
Design of selective substrates of proteinase 3 using combinatorial chemistry methods
Anal. Biochem.
(2008)
T. Chase et al.
P-Nitrophenyl-p′-guanidinobenzoate HCl: a new active site titrant for trypsin
Biochem. Biophys. Res. Commun.
(1967)
C. Epinette et al.
A selective reversible azapeptide inhibitor of human neutrophil proteinase 3 derived from a high affinity FRET substrate
Biochem. Pharmacol.
(2012)
E. Hajjar et al.
Differences in the substrate binding sites of murine and human proteinase 3 and neutrophil elastase
FEBS Lett.
(2007)
M. Wysocka et al.
The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region
Anal. Biochem.
(2010)
B. Korkmaz et al.
Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases
Pharmacol. Rev.
(2010)
N.C. Perera et al.
NSP4, an elastase-related protease in human neutrophils with arginine specificity
Proc. Natl. Acad. Sci. USA
(2012)

There are more references available in the full text version of this article.

Cited by (17)

Constitutive and induced forms of membrane-bound proteinase 3 interact with antineutrophil cytoplasmic antibodies and promote immune activation of neutrophils
2023, Journal of Biological Chemistry
Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA–associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3^mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3^mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3^mb from the cell surface. Incubation of TNFα-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3^mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigen-binding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3^mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3^mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA–associated vasculitis.
Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe
2021, Bioconjugate Chemistry
A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)₅ and polycationic (d-Arg)₅ sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (k_cat/K_m= 440 000 ± 5500 M^–1·s^–1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.
Proteinase 3 phosphonic inhibitors
2019, Biochimie
Citation Excerpt :
For a long time the substrate specificity of PR3 had not been thoroughly established and it was considered as generally broad and overlapping with other closely related NSPs (mainly HNE). Previous studies were focused on detailed analysis of the PR3 binding sites specificity using its natural substrates (e.g. alpha-1 protease inhibitor (α1PI) or monocyte neutrophil elastase inhibitor, MNEI) and on the enzyme binding pockets characteristics [17–22] as well as on the application of the combinatorial substrate libraries (Table 1) [23–25]. They described the PR3 substrate preference for both non-primed (S3-S1) and primed (S1′-S3’; Schechter and Berger notation) [26] binding pockets developing the selective FRET-type substrate ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2.
Neutrophils are one of the most important military services of the armed forces of the immune system, a crucial line of defense against bacterial or fungal onslaughts. One of their mechanisms of action relies on the production of serine proteases. One of these enzymes is proteinase 3 (PR3), which is engaged in the processing of pro-inflammatory cytokines, receptors, heat shock proteins and in the generation of antibacterial peptides. Despite its protective function, uncontrolled activity of PR3 has been associated with the progression of inflammation and tissue injury.
Although PR3 was characterized at the beginning of 90's of the last century for the first time, the research on the development of its inhibitors is barely noticeable. Here we present the recent findings on the design, synthesis and the activity of phosphonic PR3 inhibitors together with the historical perspective.
Design, Synthesis, and Enzymatic Evaluation of Novel ZnO Quantum Dot-Based Assay for Detection of Proteinase 3 Activity
2018, Bioconjugate Chemistry
Herein, the synthesis and application of functionalized quantum dot-based protease probes is described. Such probes are composed of nontoxic ZnO nanocrystals decorated by amino groups followed by linker and labeled peptide attachment. Spherical NH₂-terminated ZnO quantum dots (QDs) with the average size ranging from 4 to 8 nm and strong emission centered at 530 nm were prepared using the sol–gel method. The fluorescence of ZnO QDs was quenched by the BHQ1 moiety present on the N-terminal amino group of the peptide. The enzymatic cleavage of the peptide mediated by the proteinase 3 (PR3) bond resulted in an increase in the QD probe fluorescence. This observation was verified using both model and biological systems; and the picomolar detection limit was found to be more than 30 times lower than that of the previously reported internally quenched peptide (a decrease in detection limit from 43 to 1.3 pmol was observed).
Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples
2016, Archives of Biochemistry and Biophysics
Citation Excerpt :
Firstly, we have approached two different fluorescence donors that could possibly enable us to measure Cat C activity: o-aminobenzoic acid (ABZ) and β-(7-methoxy-coumarin-4-yl)-l-alanine (Ala(Mca)). Several findings have already shown the successful application of FRET pair ABZ/Tyr(3-NO2) for sensitive measurement of different endopeptidases activity [31–33]. Despite the fact that Cat C requires the presence of free N-terminal amino group of the substrate to be bound with high affinity, Cat C is able to recognize substrates containing the N-terminal protection such as Z-Phe-Arg-AMC [34] and Sar-Phe-AMC (where Sar is N-methyl glycine) [6].
Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S′ specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO₂)-NH₂, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.
Design and synthesis of new substrates of HtrA2 protease
2015, Analytical Biochemistry
Citation Excerpt :
Similar to previous reports, position P2′ was found to be successfully occupied by a set of bulky aromatic residues, whereas positions P3′ and P4′ showed the highest preference toward aliphatic residues and aromatic as the secondary specificity. In silico analysis of the HtrA2–substrate 1 binding mode was based on the crystal structure of HtrA2/Omi mutant with a catalytic serine residue substituted by alanine (S306A) [7]. In the proposed model, the hydrolyzed peptide bond (Thr3-Abu4) is located in close proximity to the catalytic residues with an orientation favoring the reaction (Fig. 7).
HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.

View all citing articles on Scopus

View full text

A new proteinase 3 substrate with improved selectivity over human neutrophil elastase

Abstract

Section snippets

Peptide synthesis

Results

Discussion

Acknowledgment

Semin. Arthritis Rheum.

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Biochem. Pharmacol.

FEBS Lett.

Anal. Biochem.

Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases

Pharmacol. Rev.

NSP4, an elastase-related protease in human neutrophils with arginine specificity

Proc. Natl. Acad. Sci. USA