Toolbox of Fluorescent Probes for Parallel Imaging Reveals Uneven Location of Serine Proteases in Neutrophils

Article abstract of DOI:10.1021/jacs.7b04394

Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that play roles in the control of cell-signaling pathways and defense against pathogens and whose imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end-stage cells with a short half-life and minimal ongoing protein synthesis. To gain insight into the regulation of NSP activity we have generated a small-molecule chemical toolbox consisting of activity-based probes with different fluorophore-detecting groups with minimal wavelength overlap and highly selective natural and unnatural amino acid recognition sequences. The key feature of these activity-based probes is the ability to use them for simultaneous observation and detection of all four individual NSPs by fluorescence microscopy, a feature never achieved in previous studies. Using these probes we demonstrate uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually excluded from each other, suggesting the existence of unknown granule-targeting mechanisms.

Full text of DOI:10.1021/jacs.7b04394

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Article
A toolbox of fluorescent probes for parallel imaging
reveals uneven location of serine proteases in neutrophils
Paulina Kasperkiewicz, Yoav Altman, Maximiliano D'Angelo, Guy S. Salvesen, and Marcin Drag
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b04394 • Publication Date (Web): 03 Jul 2017
Downloaded from http://pubs.acs.org on July 3, 2017
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A toolbox of fluorescent probes for parallel imaging reveals uneven location of
serine proteases in neutrophils
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,*
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Paulina Kasperkiewicz†,# , Yoav Altman#, Maximiliano D’Angelo#, Guy S. Salvesen# , Marcin
,*
Drag†
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†Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and
Technology, Wyb. Wyspianskiego 27, 50ꢀ370 Wroclaw, Poland
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#NCIꢀdesignated Cancer Center, SanfordꢀBurnham Prebys Medical Discovery Institute, La Jolla,
CA 92037, USA
*Corresponding authors
Author for manuscript correspondence – Marcin Drąg (marcin.drag@pwr.edu.pl)
Abstract
Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that
play roles in the control of cell signaling pathways, defense against pathogens, and whose
imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is
problematic because neutrophils are end stage cells with a short half life and minimal ongoing
protein synthesis. To gain insight into the regulation of NSPs activity we have generated a small
molecule chemical toolbox consisting of activityꢀbased probes with different fluorophore
detecting groups with minimal wavelength overlap, and highly selective natural and unnatural
amino acid recognition sequences. The key feature of these activityꢀbased probes is the ability to
use them for simultaneous observation and detection of all four individual NSPs by fluorescence
microscopy, a feature never achieved in previous studies. Using these probes we demonstrate
uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually
excluded from each other, suggesting the existence of unknown granule targeting mechanisms.
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1. INTRODUCTION
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Neutrophils are terminal effector cells that constitute the first line innate immune defense against
infection. They contain specialized sets of vesicles, or granules, that incorporate a number of
proteins important for pathogen killing and other immune cell functions 1. The most abundant of
these are the unique azurophil (primary) granules that accommodate four serine proteases found
mainly in neutrophils (NSPs), namely neutrophil elastase (NE), cathepsin G (CATG), proteinase
3 (PR3), and neutrophil proteinase 4 (NSP4) – also known as PRSS57 1ꢀ2. These proteases have
been implicated in the control of cellular signaling by chemokine activation, activation of cells
receptors and nonꢀoxidative pathway of intracellular pathogen destruction, and the generation of
Neutrophil Extracellular Traps (NETs) 3ꢀ4. Mice knockouts of NE and CATG present with a
phenotype consistent with a role for these enzymes in clearance of Gramꢀnegative bacteria and
fungal pathogens 5ꢀ7. A mutation of one of these proteases, NE, is associated with episodes of
cyclic neutropenia 8ꢀ9 and severe congenital neutropenia 10.
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NSPs constitute a subset of the hematopoietic serine proteases (HeSPs) 11 related by around 40%
amino acid sequence identity, that also encompasses the granzymes and chymase 12. Catalytic
(proteolytic) and nonꢀcatalytic roles have been proposed, and several endogenous substrates have
been identified, although it is not entirely clear how important proteolysis of individual
substrates is for cell physiological functions. However, it is evident that the activity of these
proteases presents a major proteolytic burden that must be closely regulated in vivo, primarily by
endogenous potent protease inhibitors.
Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end
stage cells with a short half life and minimal ongoing protein synthesis – precluding genetic
manipulations. Antibody related techniques yield information on total protein (protease) amount,
however they lack the ability to differentiate between active and nonꢀactive enzyme forms and
cannot be used in live cell imaging. To overcome these shortcomings we developed a set of
highly selective NSP small molecule chemical tools (substrates, inhibitors and fluorescent
activityꢀbased probes) that allow us to monitor each NSP acting individually or in parallel in
neutrophils. We sought to generate probes that would enable simultaneous imaging of all four
NSPs, with the criteria that these probes should be highly selective and penetrate to the cellular
location where the active proteases are stored.
NSPs are members of the trypsin/chymotrypsin protease family and thus their primary substrate
specificity is dominated by their S1 pockets (Schechter and Berger nomenclature 13), an enzyme
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feature that dictates which amino acids are preferred adjacent to the peptide bond that will be cut.
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NE and PR3 have a common preference for Val or Ala 14ꢀ18, CATG prefers Tyr, Phe, or Lys 17, 19
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and NSP4 prefers Arg 20. Accordingly, it should be readily possible to distinguish CATG and
NSP4 from each other and the other NSPs, but the overlap in specificity between NE and PR3
presents a problem. To accomplish the goals of selectivity visualizing NSPs we deployed a
Hybrid Combinatorial Substrate Library (HyCoSuL) positional scanning approach that has
recently proven useful for interrogating specificity of several endopeptidase families21ꢀ24. Here
we describe a unique toolbox that allows us to investigate the location of individual NSPs in
neutrophils.
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2. EXPERIMENTAL SECTION
2.1. HyCoSuL P1 Phe library synthesis
The P1ꢀPhe HyCoSuL library was synthesized as previously described 21ꢀ22, with the exception
of the P1 position, where Phe was incorporated. The library contains three sublibraries (P4ꢀP2),
with the general structures AcꢀP4ꢀmixꢀmixꢀPheꢀACC, AcꢀmixꢀP3ꢀmixꢀ PheꢀACC and Acꢀmixꢀ
mixꢀP2ꢀPheꢀACC, where mix is an equimolar mixture of natural amino acids (without cysteine,
methionine was replaced with norleucine), P4ꢀP2 are natural or unnatural amino acid, ACC is the
fluorogenic reporter 7ꢀaminoꢀ4ꢀcarbamoylmethylcoumarin. Libraries were dissolved in dry,
biochemical grade DMSO to a final concentration of 10mM and stored at ꢀ20°C until use.
2.2. CATG specificity determination with HyCoSuL screening
Specificity screening was carried out in the Cathepsin G buffer (0.1 mM Tris/HCl, 0.5 M NaCl,
pH 7.5) in the same manner as previously described for NE, PR3 and NSP4 21ꢀ22. The final
library concentration was 100ꢁM, the final CATG concentration was 100nM, and the reaction
volume was 100ꢁL. Library screening was carried out using fMax spectrofluorimeter (Molecular
Devices) and 96ꢀwell Opaque Corning plates (Corning®). The excitation wavelength was 355 nm
and the emission wavelength was 460 nm (cutoff at 455 nm). 1ꢁL of each library component was
loaded into each of 96 wells, CATG was preincubated in assay buffer for 15 minutes at 37°C
prior to addition to each well. Substrate hydrolysis was recorded in kinetic mode at 37°C for at
least 20 minutes and the linear portion of each progress curve was used to calculate Relative
Fluorescence Units per second (Rfu/s). All experiments were repeated at least three times and the
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results are presented as mean values. Specificity was calculated by normalizing Rfu/s to the
highest value (set at 100%). Calculations were made in Microsoft Excel.
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2.3. General method of individual fluorogenic substrates synthesis (1-4)
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Individual substrates were synthesized as previously described
using ACC as a reporter
group 25. After every coupling and deprotection reaction, efficiency was analyzed by a ninhydrin
test.
Synthesis: 100mg of the FmocꢀRA resin was activated with dry DCM, followed by Nꢀterminal
amine group deprotection with 25% PIP/DMF. Next, 2.5 eq of FmocꢀACCꢀOH (made in house)
was dissolved in DMF and activated with 2.5 eq of HOBt and 2.5eq of DICI for 3 minutes in a
microfuge tube and added to the resin. After 24 hours of reaction uncoupled components were
removed, the resin was washed 3 times with DMF and the coupling reaction was repeated using
1.5 eq of reagents. After Fmoc deprotection 2.5 eq of FmocꢀP1ꢀOH was dissolved in DMF and
activated with 2.5 eq of HATU and 2.5 eq of 2,4,6ꢀtrimethylpiridine and added to the resin. After
24 hours of agitation, the resin was washed and FmocꢀP1ꢀOH coupling was repeated using 1.5 eq
of the above chemicals. Following Fmocꢀdeprotection the peptide chain was elongated with 2.5
eq of FmocꢀP2ꢀOH, 2.5 eq of HOBT and 2.5 eq of DICI. Elongation was continued until H2Nꢀ
P4ꢀP3ꢀP2ꢀP1ꢀACC. Afterwards, a free amine at the Nꢀterminal end of the peptide was acetylated
with 5 eq of AcOH mixed with 5 eq of HBTU and 5eq of DIPEA, and, added to the resin after 3
minutes of activation, 45 minutes at room temperature. The resin was washed six times with
DMF, three times with DCM and three times with MeOH and dried over P2O5 for at least 3 hours.
A cleavage mixture (TFA:H2O:TIPS (95%:2.5%:2.5%, v:v:v)) was cooled to ꢀ20°C for one hour
and added to the dry resin and shaken vigorously once per 10 minutes in 60 minutes period
(room temperature). The first filtrate was collected, the resin was washed with cleavage mixture,
filtrates were combined, and peptide substrates were precipitated in cold Et2O, centrifuged for 5
minutes and the precipitate was washed with an additional portion of Et2O, spun down for 5
minutes and dried overnight at room temperature, followed by HPLC purification (Waters,
column: Spherisorb, 5 µm particle size, L × I.D. 25 cm × 4.6 mm) using an H2O:MeOH gradient,
and lyophilized. Each product was dissolved in dry DMSO to a concentration of 10mM and
stored at ꢀ20°C until use. Compounds were analyzed by analytical HPLC and HRMS.
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2.4. Fluorogenic substrates kinetic analysis
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Kinetic parameters of selected NE, PR3 and NSP4 substrates and activityꢀbased probes were
determined in 50 mM Hepes, 0.1 M NaCl, 0.1%TRITON Xꢀ100, pH 7.45 assay buffer, while for
CATG assays were carried out in Tris/HCl (0.1 mM, pH 7.5), NaCl (0.5 M). Buffers were
prepared at room temperature prior to analysis.
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Assays were carried out as previously described 21ꢀ22. Briefly, stock substrates in DMSO were
serially diluted in the assay buffer and 20ꢁL was added to the wells of a 96 well plate prior to
addition of 80ꢁL of enzyme (preincubated for 20 minutes at 37°C in buffer). Measurements were
carried out using a plate reader (Molecular Device) operating in the kinetic mode for a minimum
of 20 minutes with the optimal for ACC excitation/emission wavelengths 355 nm/460 nm, and
cutoff of at 455 nm. Km, kcat and kcat/Km parameters were calculated using GraphPad Prism and
Excel software. Each experiment was repeated three times, and the results are presented as an
average ± S.D.
2.5. Fluorescent activity-based probe synthesis
General method for the hydrobromide salt of H2N-PheP(OPh)2, H2N-AbuP(OPh)2 and H2N-
NvaP(OPh)2 synthesis (9)
Electrophilic covalent diphenyl phosphonate warheads were synthesized using previously
described methodology 26. In a round bottom flask, 1 eq of triphenyl phosphate, 1.1 eq of
adequate aldehyde (propionaldehyde for CbzꢀAbuP(OPh)2, butyraldehyde for CbzꢀNvaP(OPh)2
and phenylacetaldehyde for CbzꢀPheP(OPh)2) and 1.1 eq of benzylcarbamate (in house) were
dissolved in MeOH. The reaction mixture was cooled in an ice bath, mixed using a magnetic
stirrer and HBF4 (10% in Et2O, (v/v)) was slowly added. After half an hour, the ice bath was
removed and the reaction was stirred at room temperature for 4 h, followed by the evaporation of
volatile components. Crude product as an oil was left for precipitation in cold MeOH at ꢀ20°C
for 2ꢀ7 days. The white precipitate was filtered under reduced pressure, washed twice with
MeOH, dried at room temperature, and used in the next step without additional purification.
The Cbz protecting group was removed with 30% HBr/AcOH (v:v) (1 h, room temperature).
The solvent was evaporated using a rotary evaporator and the crude product was precipitated in
Et2O for 1ꢀ3 days at 4°C. the white precipitate was filtered, washed twice with a small portion of
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Et2O, dried at room temperature, and used in the next step without additional purification. A
general method for the H2NꢀArgP(OPh)2 synthesis has been described previously 21.
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General method for Boc-linker-P4-P3-P2-OH peptide synthesis (8)
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400 mg of 2ꢀchlorotrityl chloride resin (loading 1.6 mmol/g) was placed in a glass peptide
synthesis vessel and activated for 1 hour with dry DCM by shaking for 10 minutes, followed by
washing (three times with dry DCM), and addition of 3 eq of FmocꢀP2ꢀOH (1.92 mmol) (5)
activated with 6 eq of DIPEA (3.84 mmol) in dry DCM. The resin was agitated for 6 hours under
argon atmosphere, followed by filtration and washing with DMF (3 times) (6). After the Fmocꢀ
group removal with 20% PIP/DMF for 5 minutes, 5 minutes and 25 minutes, the resin was
washed 6 times with dry DMF. Next, 2.5 eq of FmocꢀP3ꢀOH (1.6 mmol) was preactivated for
three minutes with 2.5 eq of HOBt (1.6 mmol) and 2.5 eq of DICI (1.6 mmol) in DMF and
poured onto the resin. After Fmoc group removal (as above), the peptide chain was elongated in
the same manner using 2.5 eq of FmocꢀP4ꢀOH (1.6 mmol), 2.5 eq of HOBt (1.6 mmol) and 2.5
eq of DICI (1.6 mmol) (for P4 coupling), followed by Nꢀterminal amine group deprotection and
coupling of 2.5eq of BocꢀlinkerꢀOH (1.6 mmol) with 2.5 eq of HOBt (1.6 mmol) and 2.5 eq of
DICI (1.6 mmol) (7).
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The resin was washed 6 times with DMF, 3 times with DCM and 3 times with MeOH, dried over
P2O5 for at least 3 hours and peptide derivative (BocꢀlinkerꢀP4ꢀP3ꢀP2ꢀOH) was cleaved from the
resin using solution of TFE:AcOH:DCM (1:1:4; v/v/v). Reaction was carried out for 1.5 hour at
room temperature. Filtrate was collected and the resin was washed with the cleaving mixture and
filtrates were combined and the volatiles were removed on a rotary evaporator under reduced
pressure. Peptide product was dissolved in MeOH:H2O (1:1, v/v), lyophilized and used for the
next experiment without further purification.
General method for H2N-linker-P4-P3-P2-P1P(OPh)2 (10) synthesis
Crude peptide product (8) was dissolved in DMF and activated with 1 eq of HATU and 5 eq of
2,4,6ꢀtrimethylpiridine for three minutes and added to a pearꢀshaped flask with 1.2 eq of H2Nꢀ
P1P(OPh)2 (9) under an argon atmosphere, constantly stirring with magnetic stirrer and
monitored by analytical HPLC for 2 hours. After the reaction was complete, crude Bocꢀlinkerꢀ
P4ꢀP3ꢀP2ꢀNH2ꢀP1P(OPh)2 was extracted to ethyl acetate, washed twice with 5% NaHCO3, twice
with 5% citric acid and twice with brine. The organic layer was dried over MgSO4 for half an
hour. Solvent was removed on a rotary evaporator under reduced pressure and all Boc protecting
groups (from Ahx and Phe(guan)) were removed with TFA:DCM (1:1, v/v) for 30 minutes, while
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the reaction progress was monitored by analytical HPLC. The volatiles were removed on a rotary
evaporator under reduced pressure. H2NꢀlinkerꢀP4ꢀP3ꢀP2ꢀP1P(OPh)2 (10) was purified by HPLC
(Discovery BIO Wide Pore C8ꢀ10, semiꢀpreparative column), lyophilized and its molecular
weight was confirmed by HRMS.
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Coupling of fluorogenic dyes to the peptide sequences
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1 eq of ester of fluorogenic dye (BODIPY FLꢀNHS, Cy3ꢀNHS, Cy5ꢀNHS or Cy7ꢀNHS) was
dissolved in DMSO and 2 eq of DIPEA was added. After 3 minutes of preincubation, the mixture
was added to a pearꢀshaped flask with 1.2 eq of (10) H2NꢀP4ꢀP3ꢀP2ꢀP1P(OPh)2 (dissolved in
DMSO). The reaction was carried out for one hour and monitored by analytical HPLC
(Discovery BIO Wide Pore C8, analytical column). Crude product was then purified by HPLC
(Discovery BIO Wide Pore C8ꢀ10, semiꢀpreparative column), lyophilized, dissolved to a final
concentration of 10mM and stored at ꢀ80°C until use. Purity of the compounds (12) was
confirmed by analytical HPLC and HRMS (for HPLC and MS analysis spectra see Supporting
Information, Compounds Analysis).
2.6. Determination of inhibition rate constant (kobs/I) of activity-based probes
Neutrophils contain four structurallyꢀrelated serine proteases NE, PR3, CatG and NSP4 that are
stored in azurophilic granules 2, 27. To estimate the degree of eventual cross reactivity of the
optimal probe designed for targeted enzyme, we tested each champion activityꢀbased probe
against each NSPs member. Serial probe dilutions in assay buffer were added in 96 well format
in an opaque plate (Corning®) and incubated with enzyme for 5 minutes. Substrate was added
and the increase of fluorescence was monitored for 15 minutes. Probes exhibiting inhibitory
activity were further characterized to determine second order inhibition rates (kobs/I).
NSPs were incubated with increasing concentrations of probes in at least a twoꢀfold excess over
enzyme in appropriate assay buffers (for NE, PR3 and NSP4; 50 mM Hepes, 0.1 M NaCl, 0.1%
TRITON Xꢀ100, pH 7.45, for CATG 0.1mM Tris/HCl, 0.5M NaCl, pH 7.5) in the presence of
champion substrates. Fluorophore release (Rfu) was monitored over time using a BMG
ClarioStar®. The pseudoꢀfirst order rate constants kobs were determined using GraphPad Prism
and the second order rate constants (kobs/I) were calculated by adjusting for the substrate
competition factor (1 + [S]/Km), where S is the concentration of substrate in the assay and Km is
the Michaelis constant for that substrate/enzyme reaction.
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2.7. Selectivity factor determination for individual fluorescent NSP probes
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Analysis using purified enzymes.
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NE, CATG and PR3 and NSP4 were diluted to a final concentration of 100nM. Probes from
PKX05 series were diluted to final concentration of 100nM, with the first dilution in DMSO and
subsequent ones in appropriate buffer (see kinetic assay buffers). To test specificity of the PK105
probe NE (100nM) was diluted in assay buffer with 100nM PK105 in a microfuge tube. In
parallel, the remaining NSPs (100nM) were incubated for 10 minutes at 37°C with 100nM of
PK105. Reactions were terminated for 5 minutes in 95°C in SDS buffer, and run in SDS PAGE
(Gradient gels 4ꢀ12%, Bolt buffer system, Invitrogen), and visualized using LiꢀCor Odyssey
Infrared Imaging System Model 9120.
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Analysis using neutrophil exocytosis extracts.
Preparation of exocytosis extracts. Exocytosis of azurophil granule proteins (extracts) was
induced by treating 2.5 × 107neutrophils/mL with diphenyleneiodonium chloride (10µM) and
cytochalasin B for 10 minutes at 37°C, followed by fMLP addition (100nM) for additional 20
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minutes at 37°C and centrifuged for 5 minutes, 200 × g . The supernatant was harvested and
stored at ꢀ80°C until use. In parallel, Protein A/GꢀPlus agarose resin, (Santa Cruz Biotechnology)
and NSP antibodies were mixed and rocked for 5 hours at 4°C.
Activity-based probe assisted immunocapture of NSPs. To each of 4 microfuge tubes 600ꢁL
portions of neutrophil extracts (2.5 × 107cells/mL) was added. The first portion of extracts was
treated with Cy5 NE probe (PK105, final concentration 100nM), the second portion was treated
with Cy5 CatG probe (PK205, final concentration 100nM), the third portion was treated with
Cy5 PR3 probe (PK305, final concentration 100nM), and the fourth portion was treated with Cy5
NSP4 probe (PK405, final concentration 100nM). The treated extracts were mixed gently and
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incubated for 20 minutes at room temperature. A protease inhibitor cocktail
(1,10ꢀ
phenanthroline ꢀ 1mM final concentration, 3,4ꢀdichloroisocoumarin ꢀ 50ꢁM final concentration,
and Eꢀ64 ꢀ 10ꢁM final concentration) was added and mixed gently to prevent adventitious
proteolysis. Each portion was divided into four microfuge tubes and antiꢀNSP coated protein
A/G beads were added as appropriate, followed by incubation at 4°C overnight.
Mixtures were centrifuged at 4°C, the supernatant was gently removed with a needle and beads
were washed four times in lysis buffer and three times with TE buffer, followed by addition of
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SDS buffer, captured proteins were released at 95°C for 5 minutes and run in SDS PAGE
(Gradient gels 4ꢀ12%, Bolt buffer system, Invitrogen), and visualized using LiꢀCor Odyssey
Infrared Imaging System Model 9120.
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50ꢁL of PK307 (50nM final) probe was incubated with 50ꢁL of neutrophil extracts for 10
minutes at room temperature, followed by addition of 50ꢁL PK105 (50nM final) probe and
additional 10 minutes incubation. Parallel, 50ꢁL of PK305 (50nM final) probe was incubated
with 50ꢁL of extracts for 10 minutes at room temperature, followed by addition of 50ꢁL PK107
(50nM final) probe and additional 10 minutes of incubation. 50ꢁL of PK107 (50nM final) probe
was incubated with 50ꢁL of extracts for 10 minutes at room temperature, followed by addition of
50ꢁL of Hank’s Buffered Salt Solution (HBSS) buffer. 50ꢁL of PK307 probe (50nM final) was
incubated with 50ꢁL of extracts for 10 minutes at room temperature, followed by addition of
50ꢁL of HBSS buffer. In the same manner, NSP4 and CATG probes were investigated. Reaction
mixtures were boiled in SDSꢀpage buffer containing DTT for 5 minutes, before being resolved
by 4ꢀ12% SDSꢀpage (Bolt buffer system, Invitrogen). Inꢀgel detection of fluorescently labeled
enzymes was carried out directly in the wet gel using LiꢀCor Odyssey Infrared Imaging System
Model 9120 (700nm and 800nm).
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2.8. Titration of neutrophil lysates with activity-based probes
Freshly isolated neutrophils were washed with Dulbecco’s Phosphate Buffered Saline (DPBS)
buffer. 5 × 106 neutrophils/mL (for NE, CATG and PR3) or 2 × 107 neutrophils/mL for NSP4
were spun down, the pellet was resuspended in cold lysis buffer (50mM Tris, 1M NaCl and 0.5%
Triton Xꢀ100), and cells were lysed for three freeze/thaw cycles. 12ꢁL lysate portions were
added and treated with 12ꢁL of appropriate inhibitor (PK101, PK201, PK301, PK401) in the
rage 46ꢀ3000nM and incubated for 30 minutes. Next 12ꢁL of appropriate probe (PK105, PK205,
PK305 and PK405 respectively, 200nM final) was added to the samples and incubated for
additional 30 minutes. Reaction mixtures were boiled in SDSꢀpage buffer containing DTT for 5
minutes, before being resolved by 4ꢀ12% SDSꢀpage (Bolt buffer system, Invitrogen), followed
by immunobloting, using rabbit antiꢀNE antibody (ab21595, abcam, 1:300), rabbit antiꢀCATG
antibody (ab131407, abcam, 1:300), rabbit antiꢀPR3 antibody (ab21592, abcam, 1:300).,
followed by secondary antibody donkey antiꢀrabbit IRDye®8—CW (926ꢀ32213, LIꢀCOR,
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1:10000). Inꢀgel detection of fluorescently labeled enzymes was carried out directly in the wet
gel using LiꢀCor Odyssey Infrared Imaging System Model 9120 (700nm).
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2.9. NSP imaging in live neutrophils with fluorescent activity-based probes
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Parallel imaging of individual NSPs in neutrophils: Freshly isolated neutrophils (5 × 105/mL)
were incubated on cover slips 2.5%FBS/RPMI at 37°C, 5% CO2 with or without 50nM PMA for
2 hours. PK203 was added and incubated for an additional 20 minutes followed by PK302 for an
additional 10 minutes, followed by addition of PK407 and PK105. All probes were added to a
final concentration of 50nM. Cells were incubated for additional 30 minutes. Thus, PK203 was
in contact with the neutrophils for 1 hour, PK302 was in contact with the neutrophils for 40
minutes, and PK407 and PK105 probes were in contact with the neutrophils for 30 minutes. The
solution was aspirated and cells were fixed with 4% paraformaldehyde (Sigma) for 40 minutes,
mounted with Fluoromount G and slides were imaged using a Leica confocal microscope with
488nm laser (for BODIPY FL), 552nm laser (for Cy3), 648nm laser (for Cy5, Cy7).
Selective labeling with activity-based probes with different fluorescent dyes coupled to the same
specific sequence
Slides were prepared in a similar manner as described above, however two probes (100nM of
PK105 and 100nM of PK102, 100nM of PK205 and 100nM of PK202, 100nM of PK305 and
100nM of PK302, 100nM of PK405 and 100nM of PK402) were mixed prior to addition to the
neutrophils. Probes were incubated with neutrophils for 30 minutes, and the procedure described
above was followed.
2.10. Immunolabeling of NSPs
Freshly isolated neutrophils (5 × 105 cells/mL) were allowed to settle on polyꢀDꢀlysine treated
cover slips 30 minutes, while PMA (50nM) stimulated neutrophils were allowed to settle for 3
hours at 37°C, 5% CO2. Neutrophils were fixed with 4% PFA for 40 minutes and blocked with
10% BSA in HBSS for one hour at room temperature, followed by washing with DPBS. 1:200
dilution of antibodies (sheep antiꢀNE (ab80043), chicken antiꢀCATG (ab47787) and mouse antiꢀ
PR3 (ab91181), Abcam) in 3% BSA/0.5% TRITON Xꢀ100 in DPBS were added to the cover
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slips and incubated overnight at 4°C. Coverslips were washed three times with 0.5%TRITON Xꢀ
100 in DPBS and secondary antibodies were added for 1 hour (AF488 goat antiꢀchicken, AF568
goat antiꢀmouse and AF680 donkey antiꢀsheep). Cover slips were washed twice with 3%
BSA/0.5%TRITON Xꢀ100 in DPBS and mounted with Fluoromount G (for mounting details see
Supporting Information Methods). Labeled neutrophils were imaged using a Leica confocal
microscope
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2.11. Quantitation of NSPs activity-based probes colocalization
Freshly isolated neutrophils (1 × 106/mL) were treated with mixture of probes (100nM of each:
PK107, PK205, PK303 and PK40BD) or with only one probe as a control (100nM) and
incubated for 30 minutes at 37°C with constant gentle agitation. Cells were centrifuged for 5
minutes at 200 × g and the supernatant was aspirated. 250ꢁL of 4% parafolmaldehyde was added
to the pellet and incubated for 40 minutes at room temperature. Cells were centrifuged, the
supernatant was removed, and cells were washed in 0.5%TRITON Xꢀ100/DPBS buffer. The
concentration of the cells was equilibrated to 2 × 107/mL and sorted with ImageStream, mk II,
AMNIS, filters 405nm (violet), 488nm (blue) and 642nm (red).
3. RESULTS AND DISCUSSION
3.1. NSPs Substrate Specificity Matrix Using the HyCoSuL Approach
HyCoSuL is a platform for selection of optimally active and selective protease peptideꢀbased
substrates (Figure 1), consisting of a large group (over 100) of unnatural amino acids in addition
to natural amino acids, and yields a substrate preference matrix of virtually any protease whose
specificity determinants reside on the Nꢀterminal side (the nonꢀprime side) of the scissile bond.
This technology explores a large number of possible structural variations including large and
small aromatic, large and small aliphatic, branched, basic, acidic, hydrophilic and hydrophobic
groups, Dꢀamino acids, and amino acids with posttranslational modifications. Previous studies
revealed that HyCoSuL screening resulted in highly active substrates and probes for NE, PR3,
and NSP4 21ꢀ22. To profile the fourth member of the NSP family (CATG) we synthesized a
library with Phe fixed at P1 using the same scaffold as previous HyCoSuL screens (Supporting
Information Figure S1). We deployed this approach to select optimal substrates that would
distinguish the NSPs from each other and carried out detailed kinetic analyses of each champion
substrate (Figure 1). The champion sequence obtained from the CATG screen was synthesized
as a substrate with a kcat/Km value of 8.8 (±0.8) × 103 Mꢀ1sꢀ1.
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The comprehensive HyCoSuL procedure for all four NSPs followed by synthesis of optimal
sequences allowed us to generate a substrate matrix (Figure 1) for all four NSPs in terms of
optimal activity and, most importantly, selectivity: Nle(OꢀBzl)ꢀMet(O)2ꢀOicꢀAbu for NE, hChaꢀ
Phe(guan)ꢀOicꢀArg for NSP4, His(Bzl)ꢀValꢀProꢀPhe for CATG and Glu(OꢀBzl)ꢀLys(Ac)ꢀ
hPro(Bzl)ꢀNva for PR3 (Figure 1). We were able to develop favorably selective sequences for
CATG and NSP4, while the sequence for NE was slightly crossꢀreacting with PR3 (ratio around
1:900) and the champion sequence for PR3 also recognized NE (ratio around 1:14) (Figure 1).
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Figure 1. Screening methodology and structures of selected champion sequences for
individual NSPs. The first step in the procedure is to screen HyCoSuL libraries where the P1
residue (X) is fixed according to the known primary specificity of each NSP – Phe, Arg or Ala,
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and the P2, P3, P4 are randomized with natural and unnatural amino acids. In the second step a
selection of optimal sequences are synthesized as individual substrates and scanned against all
NSPs to discover the most selective sequence. These optimal and selective substrates are called
champions, characterized for kcat/Km values and selectivity ratios, and constitute the sequence
basis for further analysis. kcat/Km values were calculated using GraphPad Prism software.
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3.2. Development and Validation of Highly Selective Enzyme Activity-Based Probes for
NSPs
We sought to develop imaging probes that would interact with only the active form of NSPs,
allowing us to evaluate their individual activity status during neutrophil functions and disease.
We converted the champion substrate sequences to activityꢀbased probes that would allow
selective labeling of each NSP in a complex mixture. All activityꢀbased probes were synthesized
using the mixed solid phase and solution phase strategy outlined in Figure 2a. We equipped each
probe with Nꢀterminal biotin, or with one of four fluorophores (BODIPYFL, Cy3, Cy5 or Cy7)
29ꢀ31
that have minimal wavelength overlap
to allow for simultaneous imaging by fluorescence
microscopy. As an electrophilic covalent warhead selective for serine proteases we employed
diphenyl phosphonate derivatives related to the P1 preference of each NSPs (Figure 2b) 32ꢀ33. To
avoid interaction of the detection tag with the recognition sequence we incorporated either PEG
or Ahx linkers (For structures, see Supporting Information Figure S2). We determined second
order inhibition rates for each probe, and to simplify the descriptions throughout the paper we
gave code names to each probe (Table 1).
Based on the large selectivity factors for champion probes with Cy5 equipped probes (PKX05
series) as examples (Table 1, Figure 2c,d) we predicted that we would be able to specifically
identify PR3, CATG and NSP4 in complex mixtures. However, crossꢀreactivity of the optimal
NE probe with PR3 requires an alternative approach to allow distinction between these two
closely related proteases. To validate this we incubated each Cy5 probe with its targeted isolated
enzyme or a mixture of the remaining three NSPs (Figure 3a). CATG, PR3 and NSP4 were all
uniquely labeled by their respective Cy5 probes, but the NE probe PK105 also labeled a
component in the NEꢀdeficient mixture, likely PR3 based on our kinetic results. As a further step
of validation we induced exocytosis of azurophil granule proteins to obtain a mixture of NSPs,
incubated these extracts with individual probes and captured each enzyme with specific
antibodies (Figure 3b). Using these extracts, obtained from fresh peripheral neutrophils isolated
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from healthy donors, we confirmed that PK205 and PK305 probes bind exclusively to the
targeted enzymes, while PR3 binds PK305 and PK105 probes. On the basis of these specificity
checks we conclude that series CATG, PR3 and NSP4 probes are highly selective, and that the
NE probes are optimal, but require additional considerations to be selective.
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Table 1. Activity-based probes equipped with different N-terminal fluorescent tags.
kobs/I (M-1 s-1)
fluorophore
PKcode
NE
CATG
NI
PR3
NSP4
NI
106
106
4.2 ± 0.48
4.6 ± 0.51
1.9 ± 0.10
3.6 ± 0.80
×
×
×
×
105
105
105
105
BODIPYFL
Cy3
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103
105
107
202
203
205
207
302
303
305
307
402
403
405
407
2.7 ± 0.20
2.3 ± 0.21
8.1 ± 0.68
7.6 ± 0.89
×
×
×
×
NI
NI
Cy5
106 NI
NI
106
NI
Cy7
NI
103
103
103
BODIPYFL
Cy3
NI
NI
NI
NI
NI
NI
4.3 ± 0.3
×
NI
NI
3.5 ± 20
1.6 ± 15
×
Cy5
NI
NI
×
×
103
Cy7
NI
NI
1.4 ± 0.23
×
106
BODIPYFL
Cy3
300 ± 50
1200 ± 30
80 ± 20
600 ± 200
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
1.4 ± 11
4.7 ± 0.45
7.2 ± 0.12
7.8 ± 0.56
NI
×
×
×
105
105
105
NI
Cy5
NI
Cy7
NI
3.8 ± 0.5
4.0 ± 0.2
4.7 ± 0.3
5.2 ± 0.2
×
×
×
×
106
104
104
104
BODIPYFL
Cy3
NI
NI
Cy5
NI
NI
Cy7
NI
NI
Inhibition rate constants (kobs/I) were calculated under pseudo first order conditions 34. All
calculations were repeated in triplicate represent (± SD). Code numbers are given to simplify
discussion of the probes throughout the paper. NI – no inhibition observed.
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Figure 2. (a) Scheme of activity-based probe synthesis using PK305 as an example. The
respective P1 amino acid was loaded on the Chlorotrityl resin using DIPEA in dry DCM,
followed by coupling of the P2ꢀP4 residues by standard solid phase elongation. Following
deprotection each tetrapeptide was cleaved from the resin with and attached to the hydrobromide
salt of NvaP(OPh)2. The Boc protecting group was removed, followed by lyophilization and
attachment of Cyanine5ꢀNHS. The crude product was purified by HPLC, lyophilized and stored
at ꢀ80°C until use. (b) Structures of the components of each activityꢀbased probe used in this
study. We utilized 16 fluorescent probes, and they are coded throughout the manuscript
according to the Nꢀterminal fluorescent tag (X) and specificity sequence as indicated in the panel.
Thus, NE series probes are always PK10X, where X is one of the four fluorophores in the panel,
CATG are PK20X, etc. for the other enzyme probes. (c) Second order rate constants for each
Cy5 coded champion probe (PKX05 series) with each NSP. The log scale compresses the S.D.
error bars from biological replicates, which were all within 10% of the mean. (d) The overall
principle of activityꢀbased probes.
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There are essentially two ways that the activityꢀbased probes can be used to address neutrophil
protease function. One would require complete inhibition of each enzyme by saturating with
probe, and the other would be to add trace amounts of probe to follow the location and
disposition of each enzyme without affecting their overall function. Because the NE probe is so
efficient (kobs/I > 107 Mꢀ1sꢀ1) it also reacts with the closely related PR3, and so we needed to
develop a reliable protocol that would diminish labeling of PR3 by NE probes (10X series). We
found that the most efficient way to do this was by incubating the PR3 probe with neutrophils
before adding the NE probe, and providing the NE probe at traceꢀlabeling concentrations. With
this caveat, all probes are now stringently selective for their individual target enzymes (Figure 3c,
Supporting Information Figure S3). To date the total amount of active NSPs has not been
reported, due to lack of available reagents. We titrated neutrophil lysates with different
concentrations of biotinylated probes (PK101, PK201, PK301 and PK401) and after 45 minutes
incubation we added Cy5 probes (PK105, PK205, PK305 and PK405 respectively). The total
amount of targeted enzyme is thus defined by the concentration of covalent PKX01 inhibitors
that prevent PKX05 binding. NSP antibodies were used to ensure that all lanes contain
equivalent amount of protein (Figure 3d, for full length gels, please see Supporting Information
Figure S4). The fluorescent probes signals were quantitated and plotted against inhibitor
concentration to derive IC50 values from at least two biological replicates (individual blood
donors). Interestingly, we observed quiet of broad range and we attribute this to the donor effect,
since each of the biological replicates was from separate donors. Active NSP concentrations
(measured in 107 cells/mL) ranged from 230ꢀ340 nM for NE, 150ꢀ530 nM for PR3, 75ꢀ188 nM
for CATG and 17ꢀ34 nM for NSP4 (Figure 3d), in agreement with literature data that concluded
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NSP4 is present at about 5ꢀ10 % the amount of NE . The large amounts of active NSPs in
neutrophils mitigated against saturating with probe, so we conducted experiments where trace
amounts of NE and PR3 selective probes were added sequentially (50ꢀ100nM). As demonstrated
in Figure 3c, the trace labeling experiments provided good selectivity of PK10X series probes for
NE, even in the presence of PR3.
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Figure 3. Selectivity of champion NSP probes. (a) Champion probes (100nM) were incubated
with their target NSP or with a mixture of the other NSPs for 10 minutes followed by SDS
PAGE and fluorescence imaging of the gel. (b) Neutrophil extracts were treated with probes
(100nM), followed by a protease inhibitor cocktail (1mM 1,10ꢀphenanthroline, 50µM DCI,
10µM Eꢀ6) and the indicated specific antibodies, captured by Protein A/GꢀPlus agarose beads,
followed by SDS PAGE of the captured proteins, and fluorescence imaging of the gels. No bands
were detected with the NSP4 antibody capture, likely because the antibody is poor at recognizing
the native protein. (c) PK10X series probes preferentially label NE in the presence of PR3. PR3
probes were added to neutrophil extracts at time = 0, followed in lanes 3ꢀ4 by a second NE probe
after 10 min as indicated above the gel. Each sample was incubated for a total of 20 min before
SDS page, and gels were analyzed for Cy5 and Cy7 fluorescence. All probes were used at 50nM
final concentration. PK107 labels NE glycoforms and a small amount of PR3, whereas PK305
labels PR3 exclusively. The small amount of cross reactivity of PK10X series probes (see the
faint overlaid band in lane 3) represents off target labeling of PR3 by PK107. Images describe
the individual channels that form the basis of the overlay. (d) Lysates were treated with PKX01
(biotinꢀtagged) activityꢀbased probes to act as titration inhibitors (I) in the indicated
concentration range for 45 minutes at 37°C, followed by 200nM Cy5 probes (PK X05 series) and
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an additional 30 minutes incubation. For NE, CATG and PR3, western blots were performed and
membranes were stained with appropriate antibodies. The membranes were imaged for the Cy5
probes (red) and IRDye800W® (green).
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3.3. Localization of active NSPs in neutrophils
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NSPs are processed to their active forms in the transꢀGolgi prior to targeting to, and storage in
azurophil granules: first removal of a short Nꢀterminal dipeptide and second by removal of a Cꢀ
terminal peptide 35ꢀ36. To test whether NSPs are sorted and localize to the same azurophil granule
we coded each specific probe with a different fluorophore and incubated them with freshly
prepared unstimulated and PMAꢀstimulated neutrophils. After fixing, washing and mounting, we
imaged neutrophils with confocal microscopy, reducing the emission wavelengths to diminish
potential fluorophore signal overlap.
The probes revealed that individual NSPs appeared to have a nonꢀoverlapping punctate
distribution (Figure 4a). Since this observation was unexpected, we hypothesized that some
property of the probes was leading toward an uneven distribution. To test this possibility we
chose three strategies. In the first we incubated two different fluorophoreꢀtagged probes (Cy5
and BODIPY FL) for each individual NSP asking whether the different tags may alter
localization (Figure 4c). In the second we used antibodies specific for the individual enzymes
combined with three secondary antibodies equipped with different fluorescent tags (Figure 4d).
In the third we employed selective NSP probes followed by fixation and coꢀstaining with specific
antibodies (Figure 4e). In each case these experiments demonstrated that probes with the same
selectivity sequence, regardless of visualization tag, are targeted to the same localization within
the neutrophil confirming, by complementary methods, the dislocated distribution of NSPs
within single neutrophils (See Supporting Information Figure S4).
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Figure 4. Localization of active NSPs in neutrophil granules. (a) Freshly isolated neutrophils
were allowed to settle on cover slips, treated or untreated with PMA for 3 hours, incubated with
the indicated probes (50nM) for 30 min and fixed (b) scheme of experimental flow (c)
Neutrophils labeled with two probes (100nM each) with the same sequence and two different
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fluorescent dyes and fixed. Each signal overlays well, demonstrating specific probe labeling of
the targeted enzyme. For separate channels see Supporting Information Figure S6. (d) Neutrophil
staining with antibodies for three individual NSPs. Neutrophils were prepared as in (a) without
probe labeling, stained with antiꢀNE, antiꢀPR3 and antiꢀCATG followed by secondary antibodies
with the fluorophores indicated in the figure. (e) Coꢀstaining with activityꢀbased probes and
antibodies. Neutrophils were allowed to settle on cover slips, treated with 50nM of the indicated
probes, fixed and stained with antiꢀNE, antiꢀPR3, antiꢀCATG and antiꢀNSP4 respectively,
followed by a secondary antibodies with the indicated fluorophores. (For separate channels see
Supporting Information Figure S7). All slides were mounted and imaged by confocal microscopy
with 488nm, 552nm and 648nm lasers. Images are representative of 3 separate donors (For more
examples and zꢀstack movies see Supporting Information Figure S5ꢀS7 and Supporting
Information movies S1ꢀS4)
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These observations imply that NSPs are localized separately in distinctive azurophilic neutrophil
granules, and by way of quantifying this finding we sorted cells according to their
contents/loading using imaging flow cytometry (Figure 5a). To quantitate at a population level
the degree of colocalization of NSPs revealed by the probes we utilized flow cytometry (Amnis
ImageStream), which provides qualitative and quantitative microscopic image data of every
event acquired in flow in bright field and all fluorophore channels utilized by our probes (Figure
5). The level of magnification does not allow for quantitation of individual puncta (azurophil
granules), however we were able to assess the degree of colocalization between probes on a per
cell basis using IDEAS image cytometry analysis software. The Bright Detail Similarity R3
feature (BDS) calculates the log transformed Pearson’s correlation coefficient of the localized
bright spots in two input images, which removes diffuse signals and compares the spatial
distribution of the bright puncta from two NSP probes 37. BDS was calculated in a pairwise
fashion for every combination of the four NSP probes (Figure 5b).
With this experiment we calculated only 0.2% of the total amount of neutrophils have granules
containing all four enzymes, while the majority of the total amount of neutrophils demonstrate
individual loading of the granules (Figure 5b, c) – confirming at a population level that
neutrophil enzymes are loaded separately. Indeed the extent of colocalization was so low that it
seemed to be that the NSPs are antiꢀlocalized (dislocated or positively segregated) by a mutual
exclusion mechanism.
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Azurophil granules were once considered to be neutrophil lysosomes, but later shown to have
functional characteristics of a regulated secretory granule 38. Normally considered to constitute a
homogeneous population, there has been tentative evidence that there are two distinct
subpopulations of azurophil granules based on centrifugation analysis 39, later expanded to
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between 10 and 13 suggestive of both functional and maturational differences . Thus there is
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precedence for distinct neutrophil granule heterogeneity, but the degree of nonꢀlocalization of
contents of the azurophil granules was unexpected. Indeed, we were not able to find any
evidence in the literature for specialized protein targeting in any post Golgi vesicle system
similar to that we have demonstrated for the spatial compartmentalization of the four NSPs.
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Mechanisms for protein targeting to neutrophil granules from the transꢀGolgi network remain
unidentified, primarily because these cells are terminally differentiated and do not synthesize
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granule proteins . NE and CATG undergo two processing steps during packaging. The first is
the removal of an Nꢀterminal dipeptide that generates proteolytic activity. The second is the
removal of a Cꢀterminal peptide, and it is this latter event that may pertain to packaging
mechanisms 35ꢀ36. Thus, the Cꢀterminus of NE contains an adaptor protein 3 (APꢀ3) signal that
has been suggested to provide sorting signals 42. Little if anything is known about potential
sorting signals in the Cꢀterminal peptides of the other NSPs. Significantly, these Cꢀterminal
extensions seem to be unique to NSPs among the broader family of HeSPs and are distinct for
each NSP (Figure 5d). They exist, but are not well conserved, in NSPs throughout mammals, and
so we would predict that they represent a specialized neutrophil sorting function. As yet there is
no understanding of why they are removed following packaging, although it has been suggested
that the removal in NE reveals a cryptic sorting signal 43. Our demonstration of dislocated
packaging of NSPs is concrete visual and quantifiable evidence related to the previous finding
that there may be as many as 13 varieties of azurophil granule 40.
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Figure 5. Quantitation of NSP probe colocalization and potential sorting signals encoded
within NSPs.
Freshly isolated neutrophils from a healthy donor were treated with PK107, PK205, PK303 and
PK402, followed by washing, fixation, and analysis by imaging flow cytometry with 60X
objective and EDF. Single inꢀfocus neutrophils were selected in IDEAS software with the
Brightfield Gradient RMS, Brightfield Area, Brightfield Aspect Ratio, and Side Scatter Intensity
features prior to NSP colocalization analysis. (a) Bright detail similarity (BDS) was calculated
for each pair of probes according to fluorescence intensity from specifically labeled NSPs in
neutrophils. Cells were considered colocalized for a probe pair if BDS ≥ 1.5. (b) Compilation of
colocalized BDS events – final colocalization of all four BDSs is 0.2%. The analysis is a
representative of experiments from 2 healthy donors. (c) An example of 5 of the 9,394 events
used for quantitation. Each row represents images from one neutrophil. (d) Alignment of the Cꢀ
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terminal tails of HeSPs with the archetype of the family chymotrypsin. Red text indicates Cꢀ
terminal extensions that are known (PR3, CATG, NE) or suspected (NSP4) to be cleaved during
protease maturation.
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In recent years, development of activityꢀbased probes has led to a significant improvement in the
imaging of active proteases in situ 44ꢀ45. A daunting challenge has been to image in parallel
several enzymes in their active forms at the same time and location 46, a problem hindered by the
absense of sequences that can distinguish between closely related enzymes47. In addressing this
challenge we set out to design a toolbox to reliably image the four known neutrophil serine
proteases. Initial experiments demonstrated the feasibility of selecting highly active substrates
and biotinꢀlabeled probes for NE and NSP4 through HyCoSuL screening 21ꢀ22, 48. We have now
utilized this technique to develop selective probes for the other two human NSPs, CATG and
PR3. NE and PR3 probes are very efficient (kobs/I>107 and 5 × 105 respectively, while NSP4 and
CATG probes are less efficient (kobs/I> 4 × 104 and 1.4 × 103 respectively) yet show high
discrimination between NSPs. The stringency of the probes was successfully demonstrated by
labeling of active NSPs in neutrophil exocytosis extracts and lysates.
Armed with these selective fluorescent probes we were now able to address the question of the
localization of active NSPs in neutrophils. The degree of colocalization is so low that it appears
that a discriminatory packaging system operates at the stage of myelopoiesis when azurophil
granules are populated. A number of mechanisms seem plausible, and need to be investigated in
future studies. For example, neutrophils may be released from the bone marrow with an even
NSP distribution that deviates as they age. Formation of individual azurophil granules may
proceed in kinetic or spatial waves that tend to translate messages at different times or locations
within neutrophil progenitors. Equally likely is that each NSP is sorted into preꢀazurophil
vesicles based on information in their distinct Cꢀterminal peptides (Figure 5d). Importantly, all
prior work has considered that NSPs are coꢀsorted, and the discriminatory localization based on
NSP content, revealed in our work thus has no precedent.
In summary, we have developed a first in class toolbox for reliable parallel imaging of active
neutrophil serine proteases and demonstrated that these enzymes are unevenly located in the
granules of human neutrophils to an extent that implies a hitherto unknown discriminatory
packaging system. The utility of this approach can be readily extended to other groups of
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proteases using HyCoSuL as a tool for selection of champion recognition sequences in
combination with different fluorophores with non overlapping emission spectra.
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SUPPORTING INFORMATION
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Supporting figures: CATG substrate specificity, structure of PKX0X activityꢀbased probes,
experimental methods and characterisation of all compounds
ACKNOWLEDGEMENTS
This work was supported by grants NIH R01GM099040 to GS, and 2014/14/M/ST5/00619 by
National Science Centre in Poland to MD. PK is the beneficiary of a START scholarship from
the Foundation for Polish Science. We would like to thank Daniel Kirchhofer, Ph.D. for kind gift
of the NSP4 protease. We would like to thank to Scott Snipas for his technical assistance.
COMPETING INTEREST STATEMENT
The authors declare no competing financial interests
DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding authors
upon request.
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