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

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

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

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

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

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

Recognition of SdgB-dependent epitope by human antibodies.

<p>(<b>A</b>) Four different human IgG preparations were reacted with plate-bound CWP from WT or Δ<i>sdgB</i> USA300 by ELISA. To calculate the specific anti-staphylococcal IgG content, data were normalized using a calibration curve with known IgG concentrations of a mAb against peptidoglycan, which has the same reactivity with both USA300 strains by ELISA. Data are expressed as µg/mL of anti-staphylococcal IgG in the serum. The reduction in reactivity observed for CWP from Δ<i>sdgB</i> (red bars) as compared to wild-type CWP (black bars) reflects IgG specific for SdgB-dependent epitopes. Asterisks indicate significant differences (p < 0.05) from WT CWP. (<b>B</b>) CWP from WT, Δ<i>sdgA</i>, or Δ<i>sdgB</i>, Δ<i>sdgAΔsdgB</i> USA300 were immunoblotted with rF1 and three additional human mAbs (SD2, SD3, and SD4) from different patients. All four mAbs showed similar epitope specificity.</p

SdgB is the key rF1 epitope-modifying enzyme.

<p>(<b>A</b>) SdgB is necessary for rF1 reactivity. Cell wall lysates from WT and various putative glycosyltransferase mutants were immunoblotted with mAbs rF1, anti-ClfA (9E10), anti-SdrD (17H4) or anti-panSDR (9G4 α-SDR; recognizes the unmodified SDR-domain. (<b>B</b>) Complementation of Δ<i>sdgB</i> with exogenous SdgB confers rF1 reactivity. Cell wall lysates from WT, glycosyltransferase mutants, and the SdgB-complemented strain were immunoblotted with rF1, anti-ClfA, and anti-SDR mAbs as in (A). (<b>C</b>) Binding of rF1 to whole USA300 bacteria requires SdgB. Binding of mAbs to Δ<i>sdgB</i> USA300 was assessed by flow cytometry as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1A</a>. (<b>D</b>) rF1-mediated killing of USA300 activity requires SdgB. Wild-type USA300 bacteria preopsonized with rF1 (closed square) or anti-gD (closed circle), and Δ<i>sdgB</i> preopsonized with rF1 (closed triangle) or anti-gD (open circle), were incubated with PMN, and bacterial killing was determined as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003653#ppat-1003653-g001" target="_blank">Figure 1C</a>. (<b>E</b>) MBP-SDR-His construct was expressed in WT, Δ<i>sdgA</i>, Δ<i>sdgB</i>, or Δ<i>sgdAΔsdgB S. aureus</i>, and whole cell lysates were immunoblotted with rF1, anti-His and anti-SDR. (<b>F</b>) Preliminary model for step-wise glycosylation of SDR-proteins by SdgB and SdgA. SDR-domains are first glycosylated by SdgB, which appends sugar modifications creating the epitope of mAb rF1. SdgA further modifies these epitopes with additional sugar moieties (left panel). The Δ<i>sdgA S. aureus</i> mutant shows that SdgA-mediated modifications do not influence rF1-binding activity (middle panel). In Δ<i>sdgB or</i> Δ<i>sgdAΔsdgB S. aureus</i>, the unmodified SDR-region is now recognized by the anti-pan-SDR mAb (9G4).</p