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

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

The Elastase-PK101 Structure: Mechanism of an Ultrasensitive Activity-based Probe Revealed

Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle­(<i>O</i>-Bzl) function in the P4 position of PK101 reveals and leverages an “exo-pocket” on HNE as a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly–Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling

Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

<div><p>Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.</p></div

Structures of the optimized NSP4 substrates based on natural (PK417 and PK418) and natural/unnatural amino acids (PK421 and PK431).

<p>The activity-based probe (PK401), a diphenyl phosphonate derived from the optimal substrate sequence—PK421—is shown as the last structure.</p

Visualization of PK401 with purified NSP4 and all NSP’s.

<p>(A) NSP4 was treated with PK401 in a range from 1 to 2000nM. (B) 100nM of NE, PR3, CatG and NSP4 with or without 100nM of PK401. (A, B) Samples were denatured in SDS sample buffer, run in SDS/PAGE followed by membrane transfer. The blot was developed with fluorescently-tagged streptavidin and imaged by fluorescence scanning (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132818#pone.0132818.s001" target="_blank">S1 Text</a>).</p

Scheme of the HyCoSuL P1 Arg library.

<p>The general library structure contains tetrapeptide derivatives with the sequence Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents one of 120 fixed natural or unnatural amino acids and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met) with ACC (7-amino-4-carbamoylmethylcoumarin) as a reporter group.</p

Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.

<p>NA–no activity detected.</p><p>Kinetic parameters/constants for the hydrolysis of Ac-hCha-Phe(guan)-Oic-Arg-ACC substrate by neutrophil serine proteases to three significant digits.</p

Determination of NSP4 substrate specificity.

<p>Preferences in the P4-P2 positions were determined by screening HyCoSuL, which contains tetramer peptides with the general structures Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents fixed natural or unnatural amino acid and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met). Screening was performed on a SpectraMax Gemini plate reader. Substrate hydrolysis rates were normalized to the most active component (100%) y axis. Natural amino acids are colored grey, unnatural black. Results are shown as an average of 3 experiments with S.D.</p

Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).

<p>NI–no inhibition observed; K_m values relate to the substrate used for analysis,</p><p>* K_m for this substrate was above 100μM, the concentration used in the assay. AMC – 7-amino-4-methylcoumarin.</p><p>Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).</p

Kinetic analysis of tetrapeptide substrates for NSP4.

<p>Results are shown as an average of a minimum of 2 separate experiments with S.D.</p