Matrix metalloproteinases inactivate the proinflammatory functions of secreted moonlighting tryptophanyl-tRNA synthetase

Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical “moonlighting” functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFα-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated Δ1–47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.

Macrophages adopt a spectrum of cellular phenotypes depending on their mode of activation (30,31), ranging from the most polarized proinflammatory (M1) macrophages to anti-inflammatory (M2) macrophages that contribute to the resolution of inflammation and promote healing and extracellular matrix reformation. Differentiation to M1 macrophages is induced by IFN␥ as well as tumor necrosis factor ␣ (TNF␣) (32)(33)(34). IFN␥-activated macrophages express several matrix metalloproteinases (MMPs), including MMP7 that has long been associated with macrophage function and is often stated to be destructive in inflammation (35,36). M2 macrophage polarization is induced by interleukin 4 (IL4) (32,33).
MMPs not only degrade extracellular matrix proteins but also process virtually all chemokines and a multitude of other signaling factors (1,34,(37)(38)(39)(40)(41). This has led to a shift in interest to their signaling roles, especially in dampening inflammation that defines MMPs as antitargets in many pathologies (42). We have previously identified WRS as a candidate MMP substrate using degradomics (40,43,44), i.e. proteomics techniques for the analysis of proteolysis, with quantification enabled by isotope-coded affinity tags (ICAT) (43), isobaric tags for relative and absolute quantification (iTRAQ) (44), or terminal amine isotopic labeling of substrates (TAILS) that identifies the cleavage sites themselves (38,40). Here, we report the effects of IFN␥ on WRS expression and secretion from macrophages as well as proteolytic processing of WRS by MMPs. We describe how cleavage of the N terminus of WRS to the ⌬1-47 WRS proteoform by MMP7 and MMP8 abrogates its proinflammatory functions, aligning with other well-documented anti-inflammatory activities of MMPs.

Secretion of WRS is induced by IFN␥
To investigate potential inflammatory roles of WRS, we profiled the secretion of WRS in response to cytokine stimulation of macrophages. THP1 monocytes were differentiated to a macrophage-like phenotype (THP1 M0) using phorbol 12-myristate 13-acetate (PMA). After treatment with IFN␥ (20 ng/ml), TNF␣ (40 ng/ml), or IL4 (40 ng/ml), we collected cytosolic, membrane, and conditioned medium fractions. Immunoblot analysis using antibodies recognizing the N or C terminus of WRS (␣N-WRS and ␣C-WRS, respectively) showed that only the proinflammatory IFN␥ increased cytosolic WRS levels (N ϭ 3; Fig. 1A). IFN␥ also increased WRS in the cell membrane and conditioned medium fractions, indicating that WRS translocated to the plasma membrane and was secreted to the medium upon IFN␥ stimulation and M1 macrophage polarization (Fig. 1A). Several lower-molecular-weight proteoforms of WRS were identified in the cytosol and conditioned medium following IFN␥ stimulation. Predominant detection by the ␣C-WRS antibody revealed that these were N-terminally truncated. The effect on WRS was IFN␥-selective as neither IL4, TNF␣ (Fig. 1A), IFN␣, nor IFN␤ (N ϭ 3; Fig. S1A) had any effect on WRS protein levels or secretion. The absence of tubulin in the conditioned media supported secretion of WRS rather than cell lysis, and the absence of the plasma membrane protein Na ϩ /K ϩ -ATPase in the cytosolic fractions confirmed the fidelity of cell fractionation.
We isolated human monocytes from the peripheral blood mononuclear cells (PBMCs) of three healthy human subjects. Monocytes were differentiated into primary M0 macrophages using monocyte colony-stimulating factor and then cultured with IFN␥ (20 ng/ml) or IL4 (40 ng/ml) to induce polarization to proinflammatory M1-or M2-type macrophages, respectively. Polarization was confirmed using the M1 marker indoleamine 2,3-dioxygenase and M2 marker transglutaminase 2 (Fig. 1B). Immunoblot analysis of cell lysates showed that, as with the THP1-derived macrophages, full-length WRS and additional smaller WRS proteoforms were markedly increased only in response to IFN␥. This included a form migrating like ⌬1-47 WRS that was only detected by the ␣C-WRS antibody. Again, the absence of tubulin in the conditioned culture medium confirmed that there was little, if any, nonspecific release of cytoplasmic proteins by cell lysis.
A time-dependent increase in WRS expression and secretion up to 48 h occurred in response to 20 ng/ml IFN␥ in THP1 M0 cells, endothelial cells (N ϭ 3; Fig. 1, C and D), and BJ human skin fibroblasts (N ϭ 2; Fig. 1E), again with no change in tubulin protein. WRS secretion depended on IFN␥ concentration, with 10 ng/ml IFN␥ inducing maximal WRS secretion (N ϭ 3; Fig.  S1B). The secreted proteoforms detected by each antibody were consistent in size among cell types (␣N-WRS, ϳ41, 39, and 32 kDa; ␣C-WRS, ϳ48 and 36 kDa), and these matched the electrophoretic migration of intracellular forms.

WRS stimulates proinflammatory monocyte and macrophage activities
Phosphorylation of the p65 subunit of NF-B leads to upregulated transcription of multiple cytokines and chemokines, including TNF␣, in macrophages and a variety of other cells (45). WRS (100 nM) treatment of THP1 M0 cells induced phosphorylation of p65 coincident with a decrease in the level of NF-B pathway inhibitor IB-␣ protein (N ϭ 3; Fig. 2, A-C). Both responses were maximal at the later time point of 60 min, indicating that NF-B was activated by an indirect pathway.
By ELISA, we showed that WRS stimulated TNF␣ release from THP1 M0 cells, with maximal release at 100 nM WRS (N ϭ 2; Fig. 2D). Neither ⌬1-47 WRS nor heat-denatured WRS promoted TNF␣ release at this concentration (N ϭ 3; Fig. 2E), indicating that both the N terminus and tertiary structure of WRS are important for signaling. We used an antibody array to EDITORS' PICK: MMPs inactivate proinflammatory WRS screen for the secretion of 36 different cytokines and chemokines from human PBMC-derived primary macrophages in response to 100 nM WRS (Fig. S2). The array confirmed that WRS increased TNF␣ secretion in addition to MIP-1␣/␤, CXCL8 (IL8), and CXCL1 (N ϭ 2; Fig. 2F). Recombinant ⌬1-47 WRS did not stimulate cytokine and chemokine release from PBMCs (Fig. 2F).
Dose-response analyses showed that WRS displayed maximal chemoattractant activity for monocytes in transwell migration assays at 100 nM (N ϭ 2; Fig. 2G) (37). As with chemokine secretion, WRS chemoattractant activity was lost upon removal of the N-terminal 47 residues of WRS and heat denaturation (N ϭ 3; Fig. 2H). Thus, the N-terminal WHEP domain of WRS is essential for its signaling and proinflammatory responses.

EDITORS' PICK: MMPs inactivate proinflammatory WRS
⌬1-47 WRS on TNF␣ release from THP1-derived M0 macrophages (Fig. 2E). Likewise, boiling of WRS led to loss of activity. Thus, the N-terminal sequence and folding of WRS are critical for TLR2 and TLR4 receptor engagement and signaling.
The efficacy of the signaling inhibitors in blocking TLR2 and TLR4 signaling at the concentrations used for the TLR reporter cells was investigated in macrophages in response to 100 nM WRS. By ELISA, we measured TNF␣ release from THP1-derived M0 macrophages incubated in the absence and presence of TLR signaling inhibitors. Each of the inhibitors blocked TNF␣ release from WRS-treated THP1 M0 cells (N ϭ 2; Fig.  4A), as did the TLR2-and TLR4-blocking antibodies. TNF␣ release in THP1 M0 cells was reduced 40% by ␣TLR2 antibody, 25% by ␣TLR4, and 80% by a combination of these antibodies compared with antibody isotype controls (each at 5 g/ml) (N ϭ 2; Fig. 4B). Thus, these results and those from the reporter cells demonstrated that WRS triggers TLR2 especially, with a minor contribution by TLR4, to stimulate NF-B activation and TNF␣ release in macrophages.
We had removed lipopolysaccharide (LPS), a potent heatstable proinflammatory TLR stimulator, from our Escherichia coli-expressed recombinant WRS using Triton X-114 during bacterial cell lysis and using polymyxin B-agarose columns after purification. Furthermore, in all experiments, 10 g/ml polymyxin B was added to all cultures where WRS was used. To be fully confident that WRS, and not any contaminating LPS, drove the proinflammatory effects seen, the recombinant WRS and ⌬1-47 WRS used for all presented experiments was expressed in E. coli ClearColi BL21 (DE3) cells, which produce a modified LPS lacking both the oligosaccharide chain and two of the six acyl chains required for endotoxin signaling in human cells (52,53). As the Limulus amoebocyte lysate test for endotoxin is ineffective for proteins prepared from ClearColi (52), we further confirmed that the recombinant WRS proteins were free of LPS by demonstrating that the removal of 10 g/ml polymyxin B from cell culture experiments did not increase TNF␣ secretion in response to 100 nM WRS, whereas addition of 100 ng/ml LPS did (N ϭ 3; Fig. S3A). Heat denaturation of WRS also eliminated TNF␣ release even in the absence of polymyxin B, confirming the absence of heat-stable LPS (N ϭ 3; Fig. S3B).

WRS is cleaved by MMPs
MMPs regulate inflammation by processing bioactive proteins to alter their function (34,36,54). MMP cleavage of WRS

EDITORS' PICK: MMPs inactivate proinflammatory WRS
was explored using nine recombinant MMPs (Fig. 5A), which generated stable cleavage products rather than degrading WRS (Fig. 5, A and B): MMP7, MMP8 and MMP13 processed WRS most efficiently with little intact WRS remaining. MMP2, MMP3, MMP9, and MMP14 also cleaved WRS, whereas MMP1 and MMP10 cleaved WRS poorly. Activity for all MMPs was confirmed using quenched fluorescent peptide cleavage assays (not shown), demonstrating that the lack of activity on WRS by MMP1 and MMP10 was due to specificity differences. The spectrum of activity toward WRS was reflected by kinetic analyses (Fig. 5C), which showed that MMP8 and MMP7 cleaved WRS most efficiently with k cat /K m rate constants of 9.28 ϫ 10 3 and 7.01 ϫ 10 3 M Ϫ1 s Ϫ1 , respectively, 10-fold higher than MMP14. Plasmin and neutrophil elastase were included as positive controls.
Edman N-terminal sequencing of the major MMP7 and MMP8 cleavage products (Fig. S4) revealed that WRS was cleaved at Met 48 by both MMPs to generate ⌬1-47 WRS (ϳ48 kDa), homologous to the ⌬1-47 WRS alternatively spliced proteoform lacking the WHEP domain, and by MMP8 at Val 90 to generate ⌬1-90 WRS (ϳ39 kDa). Both MMPs produced a smaller C-terminal WRS proteoform (⌬1-334 WRS) commencing at Leu 335 at the junction of the catalytic and anticodon recognition domains (Fig. S4). Peptide mapping using LC-tandem MS (LC-MS/MS) analysis of SDS-PAGE-resolved MMPcleaved WRS fragments confirmed both N-and C-terminal truncations of WRS (Fig. S5). Other members of the MMP family generated similar proteoforms of WRS: MMP2, MMP3, MMP7, MMP8, MMP9, MMP13, and MMP14 also produced a ϳ48-kDa fragment matching the ⌬1-47 WRS alternatively spliced WRS. MMP2, MMP3, MMP8, MMP9, MMP13, and MMP14 generated a ϳ43-kDa proteoform, and MMP3, MMP8, MMP9, MMP13, and MMP14 generated a ϳ39-kDa band comparable with the ⌬1-93 WRS product of neutrophil elastase cleavage (Fig. 5B). Because peptide mapping by shotgun proteomics does not necessarily identify the actual neo N-terminal P1Ј residue of the cleavage site, amino-terminal oriented mass spectrometry of substrates (ATOMS), a sensitive targeted MS sequencing method for identifying proteolytically generated N termini (55,56), was employed. Like Edman degradation, ATOMS identifies N-terminal residues without consideration of their relative abundances in a proteolytically  EDITORS' PICK: MMPs inactivate proinflammatory WRS digested mixture, but ATOMS is much more sensitive. ATOMS identified Ͼ30 cleavage sites, including those characterized by Edman sequencing, many of which were common to several MMPs and resulted in N-and C-terminal deletions (Fig. 6).

MMP cleavage attenuated the WRS-induced proinflammatory response
We tested how cleavage of WRS by MMP7 and MMP8 affected the WRS-induced proinflammatory responses of monocytes and macrophages (Fig. 7, A and B). Cleavage of WRS by MMP7 or MMP8 decreased chemotaxis of THP1 monocytes by 87 and 76%, respectively, in transwell migration assays (N ϭ 3; Fig. 7, A and C). Similarly, TNF␣ release from THP1-derived M0 macrophages was reduced by 99 and 84% upon MMP7 or MMP8 cleavage, respectively (100 nM WRS) (N ϭ 3; Fig. 7D). Thus, the removal of the N terminus of WRS by MMP7 or MMP8 abrogated both chemotaxis and TNF␣ stimulatory responses in THP1 monocytes/macrophages. In other words, proinflammatory TLR signaling by extracellular WRS was inactivated by MMP-mediated removal of the WHEP domain.
TNF␣ is known to induce the expression of MMPs (57). Because we showed that WRS induced TNF␣ secretion from macrophages, we tested whether WRS promoted MMP expres- EDITORS' PICK: MMPs inactivate proinflammatory WRS sion via TNF␣. Infliximab (100 ng/ml), a mAb inhibitor of TNF␣, was added to THP1-derived M0 macrophages treated with 100 nM WRS or ⌬1-47 WRS or 40 ng/ml TNF␣ as a positive control. Using a quenched fluorescent peptide cleavage assay, we assayed for MMP activity in the conditioned medium. WRS treatment increased MMP activity with similar potency to TNF␣ (N ϭ 3; Fig. 8), but ⌬1-47 WRS had no effect, indicating that the increase in MMPs depended on the N-terminal WHEP domain of WRS. Furthermore, the inclusion of infliximab with WRS abrogated the increase in MMP activity. Therefore, WRS mediates increased MMP protein secretion and activity through the downstream mediator TNF␣.

Discussion
Our study reveals that WRS, one of 37 nuclearly and mitochondrially encoded tRNA synthetases (58), is specifically secreted from human macrophages and other cell types in response to IFN␥, a cytokine critical to both innate and adaptive immunity that is produced during the initiation of immune responses (59,60). The mechanism of WRS secretion remains uncertain. In endothelial cells stimulated with IFN␥, cytosolic WRS interacts with two known exocytosis-regulating proteins, cytoplasmic annexin A2 and S100A10 (9), to control WRS secretion. In proteomics studies of macrophage secretomes, WRS was identified in extracellular vesicles and exosomes from human primary macrophages (61,62), PMA-differentiated THP1 cells (63), and exosomes from the murine macrophage cell line Ana-1 (64). It is likely that local concentrations of WRS, like MMPs, could be high because the influx of neutrophils and macrophages can be considerable. 100 nM WRS is ϳ5 g/ml, a concentration that is likely attainable in inflammation. Indeed, concentrations of other innate immune regulatory proteins, such as pentraxin 3 (itself an MMP substrate (65)) and C-reactive protein, increase in concentration 100 -1,000-fold during inflammation, up to 5,000 g/ml for C-reactive protein in plasma (66, 67).
We found several extracellular proinflammatory functions for WRS, including induction of monocyte chemotaxis and stimulation of the release of proinflammatory TNF␣ and several other chemokines from macrophages, most notably the potent neutrophil chemokine IL8. We also determined that WRS activated TLR2 and, to a lesser extent, TLR4 followed by phosphorylation of NF-B subunit p65 and loss of the NF-B inhibitor IB-␣. This is consistent with two recent reports showing that WRS engages the TLR4 -myeloid differentiation factor 2 complex to stimulate an innate immune response in murine bone marrow-derived macrophages (13,14). These authors found that the first 154 residues of WRS were necessary to form the TLR4 complex (13). Our data refined this observation where we showed that recombinant ⌬1-47 WRS, corresponding to the WRS splice form that was up-regulated in the cytosol by IFN␥ and the major cleavage product of WRS generated by both MMP7 and MMP8, did not activate TLR2 or TLR4 and was inactive in all signaling and inflammatory pathways that we investigated.
In our study, the maximal NF-B response occurred at 1 h, which is considerably later than is typical for an immediate direct response (68), suggesting a signaling relay, which we showed included TNF␣. Indeed, TLR2-and TLR4-dependent release of TNF␣ into the medium led to increased MMP expression and activity. We showed that MMPs, particularly MMP7 and MMP8 that are secreted from macrophages and neutrophils, respectively, cleaved in the N-terminal domain of WRS to generate ⌬1-47 WRS as one of their products. This major cleaved form of WRS did not activate TLR2 or TLR4. In line with these observations, MMP cleavage of WRS reduced TNF␣ release and monocyte chemotaxis, revealing a negative feedback loop mediated by MMPs that suppresses the proinflammatory actions of WRS (Fig. 9). Thus, MMPs in this context dampen inflammatory responses.
The distinct activities of full-length WRS and truncated proteoforms suggested that MMPs modulate IFN␥-induced inflammatory processes through proteolysis of WRS. It is interesting that both MMPs and serine proteases (neutrophil elastase (20) and plasmin (9)) from different cellular and tissue sources generate similar lower-molecular-weight forms through extracellular proteolysis as alternative splicing does within cells, namely the N-terminal truncation of WRS to ⌬1-47 WRS. Redundancy of proteases cleaving WRS would afford different cell types and tissues the ability to temporally control the resolution of innate immune responses, further indicating the importance of WRS in host defense. As initially proposed by Jin (17) and because both alternatively spliced ⌬1-47 WRS and neutrophil elastasegenerated WRS proteoforms exhibit antiangiogenic activity (20), we hypothesize that increased secretion of WRS and accumulation of MMP-generated N-terminally truncated antiangiogenic WRS proteoforms could pause angiogenesis at a site of injury to allow proper resolution of inflammation prior to angiogenesis. Furthermore, neutrophils, which quickly infiltrate a site of tissue injury along IL8 chemoattractant gradients and play an important role in wound healing, secrete neutrophil elastase and MMP8, which potently truncate the N terminus of WRS, terminating its proinflam- EDITORS' PICK: MMPs inactivate proinflammatory WRS matory function while promoting angiogenesis (20). Therefore, cleavage of WRS by immune cell MMPs to generate ⌬1-47 WRS expands the potential functions of MMPs in regulating angiogenesis and wound healing.
In conclusion, our study shows that, in human cells, MMPs exert post-translational control over moonlighting WRS to down-regulate inflammation. Our degradomics analyses previously indicated that MMPs cleave WRS in secretomes of human MDA-MB-231 cells expressing MMP14 (43) and murine MMP2 Ϫ/Ϫ fibroblasts (44), and we have now elucidated a new MMP-WRS-TNF␣ axis for regulating inflammation. MMPs process a variety of substrates to regulate inflammation (36, 54), and our data here show that WRS is another such substrate. It would be interesting to examine WRS processing in inflammatory disease, for example both WRS and MMPs are increased in the blood of sepsis patients (13,69), so it is possible that processing of WRS by MMPs during sepsis has a role in regulating systemic inflammation. To establish the significance of WRS proteolysis by MMPs in the immune response, noncleavable mutant WRS animal models would be valuable because knocking out MMPs would likely be ineffective due to proteolytic redundancy. Overall, our data reveal that WRS is an MMP substrate that is entwined in inflammation due to its moonlighting functions, eclipsing mere matrix-modeling roles for MMPs.
HEK-Blue TM (human embryonic kidney 293) cells coexpressing the NF-B reporter system and TLR2, TLR4, TLR9, or receptor-null counterparts (Null1 and Null2) were cultured in DMEM with selective antibiotics. Cells and reagents were from InvivoGen, and assays were carried out according to their instructions. For TLR reporter assays, 96-well plates were seeded with TLR2 and Null1, TLR4 and Null2, or TLR9 and Null1 HEK-Blue cells and incubated with 100 nM WRS, ⌬1-47 WRS, heat-denatured WRS, or PBS with 10 g/ml polymyxin B for 18 h. Conditioned media were assayed for NF-B activation in QUANTI-Blue TM detection medium. For TLR inhibitor experiments, cells were incubated with antibodies and inhibitors for 1 h prior to treatment with 100 nM WRS for 18 h as for THP1 M0 cells above.

WRS secretion assays
Serum-free media containing various concentrations of human IFN␣ (Cedarlane), IFN␤, IFN␥, IL4 (all Peprotech), TNF␣ (Millipore-Sigma), or combinations thereof were added to PBMC-derived M0 cells, PMA-differentiated THP1 M0 cells, HUVECs, and fibroblasts at 80% confluence. Clarified EDITORS' PICK: MMPs inactivate proinflammatory WRS conditioned media were harvested at times shown, and protease inhibitor mixture (Biotool) and 1 mM EDTA were added. PBS-washed cells were lysed with Zwittergent buffer containing protease inhibitor mixture as above. Membrane and cytosolic fractions were isolated from THP1 M0 cells using a Mem-PER TM Plus membrane protein extraction kit (Thermo Fisher Scientific) according to the manufacturer's instructions.
To precipitate proteins, 15% (v/v) TCA was added to conditioned media samples and centrifuged, and the protein pellet was washed with 100% acetone. Pellets were air-dried, resolubilized by boiling for 5 min in 4ϫ SDS-PAGE loading buffer (0.5 M Tris, 8 M urea, 8% (w/v) SDS, pH 6.8), and diluted 4-fold. Protein concentrations were determined by A 280 nm .
C-terminally FLAG-tagged murine MMP10 in pGW1GH was expressed and purified from CHO-K1 cells. Conditioned medium was loaded onto a green Sepharose column and eluted as described for MMP7. Eluate, dialyzed against 50 mM Tris, 150 mM NaCl, pH 7.4, was loaded onto an ␣FLAG-agarose column (Millipore-Sigma) and washed with dialysis buffer, and MMP10 was eluted with 100 mM glycine, pH 3.5, into tubes containing 50 l of 1 M Tris, pH 8. Fractions containing MMP10 were exchanged into 50 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , pH 7.2, using Ultra-4 centrifugal filter units (Amicon). Activity was assayed as described below, and aliquots were stored at Ϫ80°C.
To assess MMP activity in conditioned medium, THP1 M0 cells were cultured in phenol red-free, serum-free growth medium for 24 h with 10 g/ml polymyxin B plus 100 nM WRS, TNF␣ 40 g/ml, or PBS Ϯ 100 g/ml infliximab (Novus Biologicals, catalog number NBP2-52655). Clarified conditioned medium was concentrated 20ϫ using Ultra-4 centrifugal filter units, and 40 g of protein was assayed for MMP activity using the quenched fluorescence peptide cleavage activity as described above.

WRS cleavage assays
Pro-MMPs were activated for 20 min at 25°C with 1 mM p-aminophenylmercuric acetate (APMA) in HEPES buffer (50 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , pH 7.2). APMA was removed by dialysis against HEPES buffer at 4°C for 1 h. WRS cleavage assays were performed at protease:WRS molar ratios described in the figures. Human serine proteases neutrophil elastase (Millipore-Sigma) and plasmin (Biovision) were reconstituted in 50 mM HEPES, 150 mM NaCl, 5 mM CaCl 2 , pH 7.2. For kinetic analyses, electrophoretic bands of WRS cleavage fragments were quantified by densitometry as described previously (74) using NIH ImageJ software, and the results were fitted to the following equation.

LC-MS/MS analysis of MMP cleavage of WRS
WRS (1 g) was digested at 37°C ϮMMP for 18 h at molar ratios selected from prior digests. Reactions were diluted into sample buffer (0.5 M Tris, 8% (w/v) SDS, pH 6.8, 20% (v/v) ␤-mercaptoethanol), boiled for 5 min, and resolved by 10% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue G-250. Cleaved WRS bands were excised, destained, lyophilized, and rehydrated in 15 l of MS-grade trypsin (12 ng/l in 50 mM ammonium bicarbonate) for 45 min at 4°C. Excess buffer was removed, and the gel plugs were resuspended in 15 l of ammonium bicarbonate for 18 h at 37°C. Centrifuged EDITORS' PICK: MMPs inactivate proinflammatory WRS plugs were discarded, and supernatants were desalted using StageTips (75). Samples were run on an Easy nLC-1000 (Thermo Fisher Scientific) online-coupled to a UHR Q-TOF Impact II mass spectrometer (Bruker Daltonics) with a Cap-tiveSpray nanoBooster ionization interface. Peptides (1 g) were loaded on to a 75-m ϫ 400-mm analytical column containing ReproSil-Pur C 18 1.8-m stationary phase resin (packed in house; Dr. Maisch GmbH), and the column temperature was maintained at 50°C. Samples were automatically loaded onto the analytical column at 800 bars using buffer A (0.1% formic acid) and 8-l injection flush volume. Peptides were eluted using a 125-min gradient established with the nLC at 200 nl/min from 2 to 24% buffer B (99.9% acetonitrile, 0.1% formic acid) over 90 min, then increased to 30% over a 10-min period, further increased to 95% buffer B over 5 min, and finally held at 95% for 15 min. Alternatively, a 60-min gradient with 20-min separation was utilized with similar washing parameters. Peptides were ionized by electrospray ionization (2.2 kV), and MS analysis was performed in positive ion polarity with precursor ions detected from 150 to 1750 m/z. Spectra were acquired using a Top17 data-dependent method with precursor intensity-adjusted MS/MS summation time (Compass oTOF control 1.9, Bruker Daltonics).
For database searching, Bruker .d files were converted to .mgf using DataAnalysis v4.3 (Bruker Daltonics) and searched against the reverse concatenated Homo sapiens UniProt database (downloaded January 10, 2014; 69,085 sequences) with the Mascot search algorithm (Matrix Science) using the following parameters: 25-ppm MS1 tolerance; 0.08-Da MS2 tolerance; semitryptic enzyme specificity; and variable modifications oxidation (Met), propionamide (Cys), N-terminal ammonia loss, and N-terminal cyclization at glutamine and glutamic acid. Result files were imported into Scaffold v4.8.7 (Proteome Software) and searched with X!Tandem (76). Search results were filtered for 1% false discovery rate at the peptide and protein level. The N-terminal residue of the most N-terminal semitryptic peptide detected and C-terminal residue of the most C-terminal semitryptic peptide detected were defined as the N and C termini of the excised band, respectively. The MS proteomics data have been deposited with the ProteomeXchange Consortium (77) via the PRIDE partner repository (78) with the data set identifier PXD013217.

Determination of WRS cleavage sites
WRS cleavage sites were determined by Edman degradation (performed by Tufts University Core Facility) as described previously (55,56) and by positional MS using ATOMS (55,56). Briefly, WRS (100 g) was incubated ϮMMP (at molar ratios selected from prior digests) for 18 h at 37°C. Digests were denatured with 4 M guanidine HCl. Cysteines were reduced with 5 mM DTT for 1 h at 37°C and alkylated with 15 mM iodoacetamide for 15 min at room temperature in the dark. Excess iodoacetamide was quenched with 15 mM DTT for 30 min at 37°C. Lysine and N termini were labeled with 40 mM heavy ( 13 CD 2 O) (ϩMMP) or light (CH 2 O) (control) formaldehyde with 20 mM sodium cyanoborohydride for 18 h at 37°C. Excess formaldehyde was quenched with 50 mM ammonium bicarbonate for 2 h at 37°C. Samples were mixed, split in half, and digested for 16 h at 37°C with either MS-grade trypsin (1 g/ ml; Thermo Fisher Scientific) or Glu-C (Staphylococcus aureus protease V8; 1 g/ml; Worthington). Samples were desalted and analyzed by LC-MS/MS as described above.
Data were analyzed using MaxQuant software v1.6.0.1 (79) and searched against a custom protein database including WRS, all proteases used, and 247 protein contaminants frequently observed in MS experiments. Enzyme specificity was set as semispecific free N terminus. Quantitation of peptides was performed using the MS1 signal from heavy ( 13 C 2 D 4 ; 34.063 Da) and light (C 2 H 4 ; 28.031 Da) dimethylated peptides. Carbamidomethylation on cysteine was set as a fixed modification, and methionine oxidation and asparagine deamidation were set as variable modifications. Delta score-based false discovery rates of 1 and 5% were set for peptide and protein identification, respectively. Peptide spectrum matches corresponding to N-terminal dimethylated peptides from WRS were manually validated. P1Ј residues of the cleavage site were identified from peptides dimethylated at the N terminus that can only be labeled after a cleavage event, with a heavy to light ratio Ͼ2, and identified (i) in both trypsin and Glu-C analyses, (ii) in either trypsin and Glu-C analyses based on three or more peptide spectrum matches, or (iii) by Edman degradation. Cleavage sites beginning at the P1Ј position with a charged or a hydrophilic residue with the exception of glutamine and cysteine were excluded due to known substrate preferences for MMPs (72). The data have been deposited as above under the data set identifier PXD013367.

Statistics
All statistical tests were performed using GraphPad Prism version 5.0b software.