Receptor 3 Rise to F2L, the Agonist of Formyl Peptide Processing of HEBP1 by Cathepsin D Gives

Processing of HEBP1 by Cathepsin D Gives Rise to F2L, the Agonist of Formyl Peptide Receptor 3

This information is current as Thalie Devosse, Raphaël Dutoit, Isabelle Migeotte, Patricia of September 27, 2021. De Nadai, Virginie Imbault, David Communi, Isabelle Salmon and Marc Parmentier J Immunol published online 27 June 2011 http://www.jimmunol.org/content/early/2011/06/27/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 27, 2011, doi:10.4049/jimmunol.1003545 The Journal of Immunology

Processing of HEBP1 by Cathepsin D Gives Rise to F2L, the Agonist of Formyl Peptide Receptor 3

Thalie Devosse,* Raphae¨l Dutoit,† Isabelle Migeotte,* Patricia De Nadai,* Virginie Imbault,* David Communi,* Isabelle Salmon,‡ and Marc Parmentier*

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure– function analysis of F2L identified three amino acids, G3,N7, and S8, as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium

from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by Downloaded from pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo. The Journal of Immunology, 2011, 187: 000–000. http://www.jimmunol.org/ nflammation is a critical response of the organism to These various families of chemotactic factors activate G protein- pathogens, damaged cells, or inflammatory stimuli. However, coupled seven transmembrane domains receptors expressed not only I uncontrolled inflammation can lead to diseases such as ath- by leukocytes but also by a variety of other cell types. A receptor for erosclerosis (1), arthritis (2), and cancer (3). Therefore, inflam- formylated peptides was first described in 1976 (10), and the cDNA matory processes need to be well coordinated and regulated. An encoding the human formyl peptide receptor (FPR, now called optimal immune response depends in particular on specific leu- FPR1) was cloned in 1990 (11, 12). Two closely related human re- kocyte subsets recruited to the sites of inflammation. Neutrophils, ceptors, FPR-like 1 (FPRL1/FPR2) and FPR-like 2 (FPRL2/FPR3), macrophages, and dendritic cells (DCs) are important cellular were subsequently cloned by low-stringency hybridization, using mediators of innate immune defenses (4). Numerous molecules the FPR1 cDNA as a probe (13–15). The three genes are clustered by guest on September 27, 2021 are able to elicit chemotactic responses, and they can be classified on human chromosome 19q13.3 and encode receptors sharing a into different chemical and structural families, among which are high level of sequence identity (13, 14). N-formylated peptides are chemokines (5), complement factors C3a and C5a (6), leuko- derived either from bacterial proteins or from endogenous mito- trienes, and formylated peptides. These latest factors, such as the chondrial proteins. FPR1, and presumably the two other members prototypic fMLF, are among the first identified and most potent of the family, are therefore believed to be involved in host defense chemoattractants for phagocytic leukocytes (7–9). mechanisms against invading pathogens and also in the sensing of internal danger signals resulting from cellular dysfunction. The prototypic fMLF displays high affinity for FPR1 (10) and low af- *Institut de Recherche Interdisciplinaire en Biologie Humaine et Molećulaire, Uni- finity for FPR2 (15). In addition to formylated peptides, a number versite´ Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium; †Institut de Recherche en Microbiologie Jean-Marie Wiame, B-1070 Brussels, Belgium; and of structurally diverse agonists of FPR1 and/or FPR2 have been de- ‡Service d’Anatomie Pathologique, Universite´ Libre de Bruxelles, Campus Erasme, scribed during recent years (16). In contrast, FPR3 does not respond B-1070 Brussels, Belgium to fMLF (17) and presents a distinctive expression pattern among Received for publication October 25, 2010. Accepted for publication May 24, 2011. leukocyte populations. FPR3 is expressed in monocytes, myeloid This work was supported by the Actions de Recherche Concerteés of the Commu- and plasmacytoid DCs, some tissue-specific macrophage subpop- naute´ Française de Belgique, the Interuniversity Attraction Poles Programme (P6-14) Belgian State Belgian Science Policy, the Walloon Region (Programme d’excellence ulations (particularly in lung, skin, and colon) and eosinophils, but “CIBLES”), the European Union (Grant LSHB-CT-2005-518167/INNOCHEM), the not in neutrophils (18). Fonds de la Recherche Scientifique Me´dicale of Belgium, and the Fondation Me´d- In contrast to FPR1 and FPR2, a very few ligands have been icale Reine Elisabeth. T.D. was an aspirant of the Belgian Fonds National de la Recherche Scientifique and was also supported by Te´le´vie and the Alice and David identified for FPR3. Humanin, a neuroprotective peptide in models Van Buuren Foundation. of Alzheimer’s disease, was shown to bind FPR3 and FPR2 with Address correspondence and reprint requests to Prof. Marc Parmentier, Institut de high affinity (19, 20). The most specific ligand described for FPR3 Recherche Interdisciplinaire en Biologie Humaine et Molećulaire, Universite´ Libre is, however, the endogenous peptide F2L (21), an acetylated 21-aa de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium. E-mail address: [email protected] peptide derived from heme-binding protein 1 (HEBP1). F2L was The online version of this article contains supplemental material. isolated from porcine spleen extracts on the basis of its bioactivity. Abbreviations used in this article: CTS D, cathepsin D; DC, dendritic cell; FPR, This highly conserved peptide activates FPR3 in the low nano- formyl peptide receptor; FPRL1/FPR2, formyl peptide receptor-like 1; FPRL2/FPR3, molar range, is poorly active on FPR2, and is inactive on FPR1. formyl peptide receptor-like 2; HEBP1, heme-binding protein 1; siRNA, small in- On FPR3-expressing cells, F2L triggers intracellular calcium re- terfering RNA. lease, inhibition of cAMP accumulation, and phosphorylation of Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 ERK1/2 MAPKs through the Gi class of G proteins. When tested

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003545 2 HEBP1 PROCESSING on FPR3-expressing leukocytes, F2L promotes calcium mobili- spectrometry. Because of their hydrophobicity, peptides were dissolved in zation and chemotaxis (18, 21). DMSO at 1 mM, and 25-fold intermediate dilutions were made in 50% How and in which circumstances F2L is generated in the or- CH3CN, followed by further dilution in assay buffer to working concentrations. All peptides were assayed from 0.1 to 3000 nM (2 points per log) ganism is, however, still unknown. HEBP1 is an intracellular in the aequorin-based assay on FPR3-expressing cells, and the EC50 was tetrapyrrole-binding protein of 22 kDa. Initially purified from determined. The results are presented as the ratio between the EC50 of the mouse liver, HEBP1 is expressed in many tissues. Knockdown of peptide and the EC50 of native F2L. mouse HEBP1 by antisense oligonucleotides induces a reduction of Aequorin-based luminescence assay of intracellular calcium the cell heme content, suggesting that HEBP1 may be involved in release heme regulation, biosynthesis, or transport (22, 23). However, no additional data have reinforced this hypothesis, and the biological Calcium release was measured by an aequorin-based bioluminescence function of HEBP1 remains therefore poorly defined. The three- assay, as previously described (28, 29). In brief, CHO-K1 cells coex- pressing apoaequorin, Ga16, and FPR3 or control GPCRs were collected dimensional structure of murine p22HBP was determined by nu- from culture dishes, pelleted by centrifugation, and resuspended at 5 3 106 clear magnetic resonance and consists of a 9-stranded distorted cells/ml in DMEM/Ham’s F12 containing 0.1% BSA (aequorin buffer). b-barrel flanked by two long a-helices (24, 25). Located outside The cell suspension was supplemented with 5 mM coelenterazine h the globular structure of HEBP1, residues 1–17 are disordered, (Promega, Madison, WI) and incubated under shaking for 3 h 30 min at whereas residues 18–23 form a b-strand. This part of the protein is room temperature in the dark, then diluted 5-fold in aequorin buffer. Fifty microliters of cell suspension was injected onto 50 ml of agonist- not found in bacterial homologues of the SOUL/HEBP family. It containing medium in 96-well plates, and light emission was recorded is therefore conceivable that cleavage of HEBP1 after the leucine for 40 s in a Centro LB 960 luminometer (Berthold Technologies). ATP

21 would release the F2L ligand while keeping the heme-binding (20 mM; Sigma) was used as standard to normalize the data. Downloaded from domain functional (24). Binding assays In the current study, we investigated the potential pathways leading to the generation of F2L in the organism. According to the For the analysis of truncated F2L peptides, a F2L variant containing an additional C-terminal tyrosine was used as tracer after labeling with 125I role of macrophages in cleaning up cellular debris and the reso- using the Iodogen method (the sp. act. was 900 Ci/mmol), as described lution of inﬂammatory processes, we searched for the generation of previously (16). Afterwards, we obtained a custom-made ﬂuorescent F2L

F2L when recombinant HEBP1 was submitted to the action of derivative, containing 5(6)-carboxyfluorescein linked to an additional http://www.jimmunol.org/ macrophage proteases or conditioned media. The use of specific C-terminal lysine (F2L–FAM), from JPT Peptide Technologies (Berlin, protease inhibitors and purified proteases demonstrated that the Germany). Two hundred thousand FPR3-expressing CHO-K1 cells or parental CHO-K1 cells in 100 ml binding buffer (DMEM-F12, containing lysosomal aspartyl endopeptidase cathepsin D (CTS D) is able 0.5% BSA and 0.1% NaN3; Life Technologies) in duplicate samples were to process HEBP1 and generate F2L. This observation suggests incubated in siliconized 1.5-ml microcentrifuge tubes (Sigma) with in- that F2L might be generated after tissue damage and macrophage creasing concentrations of F2L–FAM for 1 h at room temperature in the recruitment, favoring the recruitment of additional monocyte/ dark. The cells were then washed with 2 volumes of binding buffer, pelleted, resuspended in 250 ml binding buffer, and analyzed by FACS macrophages and DCs, which would contribute to tissue repair (FACScan; Becton Dickinson). FACS data were analyzed with the WinMDI and the control of the inflammatory process. software. Nonspecific binding was determined in the presence of 10 mM unlabeled F2L. For competition binding assays, cells were incubated with by guest on September 27, 2021 10 nM F2L–FAM and increasing concentrations of HEBP1, unlabeled F2L, Materials and Methods or variants thereof. The binding data were analyzed with the GraphPad Plasmid construction and yeast transformation Prism software.

The cDNA corresponding to human HEBP1 was amplified by PCR from Proteolytic processing of HEBP1 in conditioned medium from pCDNA3–HEBP1 using the primer pair oecj301 and oecj302 and was introduced in the pCSC2 Saccharomyces cerevisiae expression vector (26) monocyte-derived macrophages by homologous recombination. The resulting plasmid, pCSC2–HEBP1 Monocytes were isolated from venous blood of healthy donors by im- (pCSC294), allows the production of recombinant HEBP1 displaying a munomagnetic bead cell sorting (MACS) according to the manufac- C-terminal tag of six histidines. The S. cerevisiae BY4709 strain (MATa D turer’s specifications. These procedures received authorization from the ura3 0) was transformed with the vector using the lithium acetate pro- Ethics Committee of the Free University of Brussels Medical Faculty. cedure (27). Transformants were selected on YNB plates containing 20 After Ficoll density gradient, monocytes were purified by positive selec- mg/ml glucose. tion using CD14 microbeads (Miltenyi Biotec). Macrophages were differentiated from monocytes in the presence of 50 ng/ml recombinant HEBP1 production and purification human M-CSF (R&D Systems) for 6 to 8 d. The purity of the cell prepa- + + Strain BY4709 producing HEBP1 was cultured on YNB containing 20 mg/ml rations was evaluated to 95% or more by flow cytometry (CD206 CD14 , or CD68+ for permeabilized cells). Macrophage monolayers were in- glucose in a 13 l-batch bioreactor (Biolafitte) to an OD660nm of 1.69. Cells were harvested by centrifugation and the pellet washed twice in water. Cells cubated in a proteolysis buffer (25 mM sodium acetate pH 3.6, 100 mM were resuspended in buffer A (300 mM NaCl, 50 mM NaH PO ) with NaCl) at 37˚C in a humidified atmosphere of 5% CO2 during 24 h. The 2 4 m EDTA-free Complete Protease Inhibitor Cocktail (Roche) and lysed with medium was collected and incubated with HEBP1 with or without 10 g/ a French press. The lysate was centrifuged at 12,000 rpm (Sorvall RC5B, SS- ml pepstatin A (Sigma) for different periods of time at 37˚C. The medium 34 rotor) for 30 min at 4˚C. The supernatant was purified on an Ni-NTA was then adjusted to pH 7 by 10 mM sodium bicarbonate pH 8, and Sepharose column (Qiagen) eluted by a step gradient of 0, 90, and 150 mM samples were engaged in the aequorin-based assay. imidazole in buffer A. The serine protease inhibitor PMSF (1 mM; Sigma) was added to the collected fractions (4 ml/fraction). A sample of the fractions Inhibition of CTS D expression was loaded on a 12% polyacrylamide gel, and HEBP1 was identified by Western blotting using a rabbit polyclonal Ab (Phoenix). Fractions of interest CTS D expression was inhibited in human monocyte-derived macro- were finally pooled, concentrated to 1 ml, and purified on a Superdex 75 phages by transfection of specific small interfering RNAs (siRNAs; A, column (GE Healthcare) run with buffer A at 1 ml/min. Fractions were an- HSS102578; B, HSS175648; C, HSS175649; Invitrogen), using their re- alyzed on a 12% polyacrylamide gel, followed by HEBP1 immunodetection spective scrambled oligonucleotides as controls. Oligonucleotides (0.1, 1, on Western blots, and purity was checked by Coomassie blue staining. and 10 nM) were transfected using the INTERFERin reagent (Polyplus- transfection) at days 6 and 7 of macrophage differentiation from mono- Truncated synthetic peptides and alanine scanning cytes, and the purity of fully differentiated macrophages at day 7 was evaluated by flow cytometry (CD68+ CD206+ cells). Transfection Acetylated F2L (Ac-MLGMIKNSLFGSVETWPWQVL) and alanine efficiency was monitored with 100 nM FITC-labeled oligonucleotide variants were synthesized locally by using the solid-phase Fmoc strategy. (BLOCK-iT Fluorescent Oligo; Invitrogen). CTS D levels were evaluated Monoisotopic masses and sequences of all peptides were verified by mass 48 h after the second transfection by Western blotting using a mouse anti- The Journal of Immunology 3

CTS D mAb (clone BC011, IgG2a, 0.1 mg/ml; Calbiochem), a rabbit (Vector Laboratories). HEBP1 and CTS D expression were evaluated by polyclonal Ab against GAPDH as control (1/2000; Cell Signaling), and an two independent observers and assessed by scoring each spot: 0 for no Odyssey infrared imaging system analysis. expression; 1 for low expression; and 2 for high expression. Results are presented as the score means for different donors. Proteolysis of HEBP1 by CTS D Statistics Native CTS D from human spleen was purchased from Calbiochem. HEBP1 was incubated in proteolysis buffer with CTS D at 37˚C with or without For the results of aequorin-based and chemotaxis assays, statistical sig- pepstatin A. The medium was adjusted to pH 7 by sodium bicarbonate pH 8, nificance was determined using the one-way ANOVA test, and p values and samples were tested for activity in the aequorin-based assay. For mass ,0.05 were considered significant. spectrometry analysis, the samples were vacuum dried and resuspended in 1 m l5%CH3CN/0.1% trifluoroacetic acid mixed with matrix mix (2 mg/ml Results 2,5-dihydroxybenzoic acid, 10 mg/ml a-cyano-4-hydroxycinnamic acid, 2 mM fucose). Mass spectrometry analysis was performed on a Q-TOF Ultima HEBP1 production and purification Global mass spectrometer equipped with a MALDI source. In parallel, F2L is to date the only specific ligand of FPR3. However, no in- 3 samples were vacuum-dried, boiled for 10 min in 1 Novex loading buffer formation is available yet regarding the situations and mechanisms (Invitrogen), and loaded on 10–20% polyacrylamide gradient gels in Tricine buffer (Invitrogen). Gels were blotted onto 0.2-mmpore-sizepolyvinyl leading to the generation of F2L in the organism, including the difluoride membranes (Millipore), and HEBP1 fragments were immuno- proteolytic enzymes involved in the cleavage of HEBP1. To gain detected with a rabbit polyclonal Ab directed at the N terminus of the protein insight into this process, we first expressed and purified full-length (Phoenix Pharmaceutical) or stained using an improved Blum’s silver recombinant human HEBP1. staining protocol modified for high-sensitivity protein identification (30). The HEBP1 sequence starts with an acetylated methionine and Preparation of mouse neutrophils contains no apparent signal peptide. N-terminal acetylation occurs Downloaded from Fresh blood samples were collected from posterior vena cava of 8-wk-old in cytosolic mammalian proteins and yeast proteins but rarely in C57BL/6 mice. Neutrophils were recovered on a Ficoll density gradient prokaryotic or archeal proteins (32, 33). In a subset of these pro- (Lymphoprep; Axis-Schield), and erythrocytes were lysed by ammonium teins, acetylation is required for some of their biological properties, chloride. The cell population was consistently composed of .90% neu- + + such as enzymatic activity, stability, DNA binding, protein–protein trophils, as determined by flow cytometry analysis (CD11b Gr1 pop- interaction, or peptide–receptor recognition. Other proteins are ulation). acetylated without known functional consequences. In the case of http://www.jimmunol.org/ Chemotaxis assays F2L, acetylation is not required for binding to FPR3 or activation of Leukocytes were isolated as described earlier and incubated for 30 min in the receptor (21). It might, however, be necessary to stabilize F2L RPMI 1640 containing 5% decomplemented FBS before running the che- and/or HEBP1 in vivo, for the (essentially unknown) functions of motaxis assays. HEBP1 (100 nM) was incubated for 30 min at 37˚C with 60 HEBP1, or for the recognition of the protein by the putative pro- nM CTS D in saline buffer (25 mM acetate buffer, 150 mM NaCl, pH 3.6). tease(s) generating F2L (34). For all these reasons, we produced The medium was neutralized with 1 M sodium bicarbonate pH 8 before testing. Chemotaxis was assayed by a modification of the Boyden micropore recombinant human HEBP1 in the eukaryotic cell S. cerevisiae,so filter technique, as previously described (31). Briefly, the assay was con- that it would be correctly folded and acetylated. As F2L is the N- ducted in 48-well microchemotaxis chambers (Neuroprobe), using 5-mm terminal peptide of HEBP1, we placed a poly-histidine tag for (human monocytes and monocyte-derived macrophages) or 3-mm(mouse purification purposes at the C-terminal end of the protein. by guest on September 27, 2021 neutrophils) pore-size Nuclepore track-etched polycarbonate membranes After selection of a yeast clone, the amount of HEBP1 evaluated (Whatman). The cell suspension (10,000 macrophages/well and 50,000 monocytes or neutrophils/well) was loaded in the upper chamber and the by Western blotting represented close to 1% of the total protein chemoattractant solution or vehicle in the lower chamber. Positive control extract (data not shown). The recombinant protein was purified by of cell migration was 10 nM synthetic F2L. Migration was conducted for an immobilized metal ion affinity chromatography and gel filtration adequate period of time (1 h for monocytes, 1 h 30 min for macrophages, (Fig. 1A–C). Size-exclusion chromatography showed that HEBP1 and 30 min for neutrophils) at 37˚C in humidified air containing 5% CO2. After membrane removal, nonmigrated cells were scraped off its upper side. is monomeric. The purity of the purified protein was evaluated on Then, the filters were fixed in methanol and stained with Hoechst for 2 min. 12% polyacrylamide gels after Coomassie blue staining (Fig. 1C). Micrographs of the lower surface of the filters were taken, and the number Using this procedure, we produced and purified around 300 mg of cells was counted with ImageJ software (version 1.36b). Results are HEBP1 per liter of yeast culture. The integrity of purified HEBP1 expressed as a chemotactic index (ratio of migrated cells in the presence was evaluated by electrospray mass spectrometry. The major peak versus the absence of chemoattractant). Chemotaxis was distinguished from chemokinesis by a checkerboard test, in which the chemoattractant was (21,943.9 Da) corresponded with acetylated HEBP1 and a smaller added to the upper chamber with the cells or to both chambers. peak (21,901.9 Da) with nonacetylated HEBP1 (Fig. 1D, inset), suggesting that most of the purified protein is indeed acetylated. Immunohistochemistry and tissue microarrays Characterization of the binding and functional activities of Normal tissues obtained from 30 different human organs in the Service HEBP1 on FPR3 d’Anatomie Pathologique of the Erasme Hospital were selected using H&E-stained slides. Corresponding paraffin-embedded blocks were then The functional activity of purified full-length HEBP1 was evalu- precisely aligned with the marked slides. Three tissue cores (0.6 mm in ated on FPR3-expressing CHO-K1 cells by using an aequorin- diameter) for each sample were punched using a precision instrument (Beecher) and arrayed into a recipient block. Each organ was represented based calcium mobilization assay. The activity was expressed as by samples from three to five different patients. These procedures received a percentage of the response induced by 10 mM ATP, acting on authorization from the Ethics Committee of the Free University of Brussels endogenous P2Y receptors. No significant activity was detected at Medical Faculty. Sections (5 mm) of formalin-fixed and paraffin-embedded low nanomolar concentrations of HEBP1 compared with the ac- tissue samples or tissue microarrays were subjected to standard immuno- tivity of F2L used as a positive control. A weak activity was, histochemical procedures. Primary Abs were a mouse monoclonal anti- m HEBP1 [aa 1–21] (clone 729, IgG1b; Euroscreen) and a mouse monoclonal however, identified for 1 M HEBP1 (Fig. 2A). anti-CTS D (clone C5, IgG2b; AbD Serotec). Microwave Ag retrieval was We then evaluated the capacity of full-length HEBP1 to bind performed twice in citrate buffer, pH 6, for 5 min. Detection was made FPR3-expressing CHO-K1 cells in competition binding assays by the Vectastain Elite ABC kit (Vector Laboratories) using 3,39-dia- using a fluorescent F2L analogue as tracer. F2L labeled by car- minobenzidine tetrahydrochloride (Dako) as the peroxidase substrate. The slides were counterstained with hematoxylin, dehydrated, and mounted in boxyfluorescein at its C terminus (F2L–FAM) was obtained and DPX mounting medium (Sigma-Aldrich). Negative controls were con- characterized. A saturation binding curve was obtained on FPR3- ducted by replacing primary Abs with corresponding isotype controls expressing cells, with a KD of 20 nM (n = 3), whereas no specific 4 HEBP1 PROCESSING

FIGURE 1. Production and purification of full-length HEBP1. A, A lysate of yeast S. cerevisiae expressing HEBP1 C-terminally tagged with six histidines was loaded on a nickel column and eluted by a step gradient of imidazole (dashed line). The black box indicates the fractions containing HEBP1. B,The three fractions were concentrated and separated on a Superdex 75 column (1 ml/min). The third peak corresponds with HEBP1 (black box). C, Samples from different purification Downloaded from steps were loaded on a 12% polyacrylamide gel stained with Coo- massie blue. D, Electrospray mass spectrometry profile of purified HEBP1. http://www.jimmunol.org/

binding was detected on parental CHO-K1 cells (Fig. 2B). In We further investigated the importance of specific residues of competition binding experiments, unlabeled F2L competed for F2L by testing N- and C-terminally truncated F2L variants in by guest on September 27, 2021 F2L–FAM binding with an IC50 of 17 nM, whereas HEBP1 did binding and functional assays. At the N terminus, removal of the not inhibit F2L–FAM binding (Fig. 2C). first three amino acids had little impact on the activation parameters We also investigated the potential antagonist activity of HEBP1 (Fig. 2J). In contrast, a clear loss of potency was observed after in a calcium mobilization assay. FPR3-expressing CHO-K1 cells deletion of M4, very poor activation was obtained when deleting I5, were stimulated with 50 nM F2L in the presence of various and the peptide starting at K7 was totally inactive. In the binding concentrations of HEBP1. Full-length HEBP1 was unable to an- assay, competition for radioiodinated F2L was significantly im- tagonize the activity of F2L (Fig. 2D), and the weak agonist effect paired by deletion of the first three amino acids, and no compe- of the protein was observed at high concentration (1 mM). In tition was detected for shorter peptides (Fig. 2K).These data are agreement with this agonist activity, preincubation of cells with consistent with the important role of the structure surrounding G3 high concentrations of HEBP1 for 20 min desensitized the cells, in the N terminus of the peptide. which displayed decreased calcium release in response to 90 nM At the C terminus, no differences in activation and binding F2L (Fig. 2E). parameters were observed after deletion of the last three residues. Identification of amino acids involved in F2L binding to FPR3 Peptides 1–17 and 1–16 displayed an EC50 twice as high as that of F2L. EC50 values could still be determined for peptides 1–15 and We investigated the structure–function relationship of F2L using 1–12, but their efficacy was decreased. The activity of peptides modified peptides derived from the F2L sequence. First, an alanine- 1–10 and 1–9 was still detectable at high concentrations, although scanning experiment was devised, in which each amino acid of F2L EC50 values were not measurable. Peptide 1–6 was totally inactive wasreplacedbyanalanine(Fig.2F). In a functional assay, we (Fig. 2H,2I). As for N-terminal truncations, the binding param- 3 7 8 identified three residues (G ,N,andS) as important for FPR3 eters were affected more rapidly with successive truncations. activation. Indeed, replacement of these residues by alanine in- None of the peptides tested behaved as an antagonist, as binding creased significantly the EC50 of the peptides by 2- to 9-fold. and activation parameters decreased consistently in parallel. These 6 7 8 Concurrent replacement of K ,N,andS by alanines increased the results, summarized in Supplemental Table I, fit with the presence EC50 by almost two logs, confirming the important role of these of a core of residues, necessary for binding and activation of amino acids (Fig. 2F,2G). Some individual substitutions (such as FPR3, in the N-terminal part of the F2L peptide. L2,I5, and F10) appeared to improve slightly the potency of the peptide, but the combination of these substitutions did not confirm this tendency (Fig. 2G). The apparent importance of glycine at Proteolysis of HEBP1 in conditioned medium from position 3 suggests that flexibility is required in the N-terminal monocyte-derived macrophages part of the peptide for its interaction with FPR3. Finally, none of To identify conditions allowing proteolysis of HEBP1 and F2L the residues in the C-terminal half of F2L, including some large release, we incubated purified HEBP1 in different media poten- hydrophobic amino acids, appeared to play a critical role. tially containing required proteases. Conditioned media were The Journal of Immunology 5

FIGURE 2. Structure–function analysis of F2L. A, Concentration– action curves for the synthetic F2L peptide and purified HEBP1 on FPR3-expressing CHO-K1 cells using the aequorin-based calcium mobilization assay. B, Saturation binding assay using fluorescent F2L–FAM as tracer on FPR3-expressing CHO-K1 or parental wild-type cells. Bound tracer was determined on individual cells by FACS and expressed as mean fluorescence. Nonspecific binding was determined in the presence of 10 mM F2L. C, Competition binding assay on FPR3-expressing CHO-K1 cells using F2L–FAM as tracer and Downloaded from unlabeled F2L or HEBP1 as competitors. D, Intracellular calcium release was evaluated by the aequorin- based assay on FPR3-expressing cells in response to a constant concentration of F2L (50 nM) and in- http://www.jimmunol.org/ creasing concentrations of HEBP1. E, FPR3-expressing CHO-K1 cells were preincubated for 20 min with increasing concentrations of HEBP1 before testing their response to 90 nM F2L in the aequorin-based assay. F, Each peptide of an alanine scan of F2L was tested on FPR3-expressing cells in the aequorin-based assay.

The results are presented as the ratio by guest on September 27, 2021 between the EC50 of the mutant peptide and that of F2L, as derived from the dose-response curves. G, Functional response of variant F2L peptides with three alanine substitutions (as in F). H and J, Con- centration–action curves on FPR3- expressing CHO-K1 cells using the aequorin-based assay. I and K, Competition binding assay on FPR3- expressing CHO-K1 cells using [125I]F2L as tracer and C-terminally (I) and N-terminally (K) truncated variants of F2L as competitors. Mean 6 SEM, n = 3–5. *p , 0.05, **p , 0.01.

prepared from human monocyte-derived macrophages, human To identify the class of proteolytic enzymes responsible for neutrophils purified from peripheral blood, and purified mouse HEBP1 processing, we investigated the effect of specific protease spleen cells. Incubation of HEBP1 for 30 min at 37˚C in macro- inhibitors on the generation of FPR3 agonists in macrophage phage conditioned medium resulted in the generation of a bi- conditioned medium. The activity was inhibited very efficiently ological activity on FPR3-expressing CHO-K1 cells using the by pepstatin A (1 mg/ml), a potent inhibitor of aspartyl proteases. aequorin-based assay (Fig. 3A). Neither the conditioned medium The vehicle alone (10% ethanol, 1% acetic acid) and other without HEBP1 nor HEBP1 in the unconditioned medium induced protease inhibitors (pMSF and leupeptin) had no effect (Fig. 3C calcium release. Weaker activities were recorded after incubation and data not shown). These data suggest that macrophages re- of HEBP1 in conditioned media from human neutrophils or mouse lease an aspartyl protease able to cleave HEBP1 under acidic spleen cells (Fig. 3B). conditions. 6 HEBP1 PROCESSING

FIGURE 3. Proteolytic processing of HEBP1 in conditioned medium. A, HEBP1 (1 mM) was incubated for 30 min with conditioned medium from macrophages. After incubation, the activity was diluted four times and measured in the aequorin-based assay using FPR3-expressing CHO-K1 cells. No signiﬁcant activity was observed for the medium alone (acetate buffer pH 3.6), HEBP1 in medium, or macrophage conditioned medium in the absence of HEBP1. B, HEBP1 (1 mM) was incubated for 30 min in conditioned media from macrophages, neutrophils, and mouse spleen cells, and the activity was evaluated in the aequorin-based assay on FPR3-expressing CHO-K1 cells. The results are presented as the ratio between the activity observed in the presence of HEBP1 and the activity observed in the absence of HEBP1. C, HEBP1 (1 mM) was incubated for 30 min in conditioned medium from macrophages, with or without pepstatin A (10 mg/ml). The vehicle of pepstatin A (0.01% acetic acid, 0.09% ethanol) did not modify the response. Mean 6 SEM, n = 3. ***p , 0.001. Downloaded from

Proteolysis of HEBP1 by puriﬁed CTS D CTS D and showed, in the aequorin-based assay, a bell-shaped CTS D, a lysosomal aspartyl protease, was considered as a good curve with a maximum corresponding with 60 nM CTS D (Fig. candidate for the processing of HEBP1. Indeed, macrophages 4C). In other experiments, a 15-min incubation of 60 nM CTS D # m contain high amounts of CTS D, and the activity of this enzyme is with increasing concentrations of HEBP1 ( 2 M) resulted in a saturation curve with typical Michaelis–Menten kinetics (Km:

strongly inhibited by pepstatin A. The concentration of CTS D in http://www.jimmunol.org/ the medium of macrophages cultured for 24 h in acetate buffer was 148 nM) (Fig. 4D). Finally, we performed a 24-h time course of evaluated to 63.9 ng/ml (n = 3). Incubation of human HEBP1 (250 the bioactivity generated from HEBP1 (250 nM) by CTS D (60 nM) with puriﬁed human CTS D (10 ng/ml) for 30 min at 37˚C nM). Activity was observed at early time points (5 min) of hy- generated a strong biological activity for FPR3-expressing CHO- drolysis with a peak at 30 min, after which it slowly decreased K1 cells (Fig. 4A). The speciﬁcity of this activity was demon- with time. The same samples were loaded on 12% polyacrylamide strated by the absence of response of parental CHO-K1 cells and gels, showing that most of the substrate was cleaved after 5 min, cell lines expressing FPR2, ChemR23, or GPR88 as control whereas slower degradation was observed afterward (Fig. 4E,4F). receptors (Fig. 4B). Altogether, these results demonstrate a fast cleavage of HEBP1 by

We then evaluated the biological activity resulting from the CTS D, generating an agonist of FPR3, which can be further by guest on September 27, 2021 incubation of HEBP1 (250 nM) with increasing concentrations of degraded by the enzyme, although with slower kinetics.

FIGURE 4. HEBP1 processing by CTS D. A, HEBP1 (250 nM) was incubated with CTS D (60 nM) and tested on FPR3-expressing CHO-K1 cells using the aequorin-based assay. Acetate buffer, CTS D, or HEBP1 alone did not activate FPR3. B, CTS D-treated HEBP1 was also tested on parental CHO-K1 cells or CHO-K1 cell lines expressing other receptors, ChemR23 and GPR88. C, HEBP1 (250 nM) was treated with increasing concentrations of CTS D for 15 min and tested on FPR3-expressing cells. The functional response peaked at 60 nM CTS D. D, Increasing concentrations of HEBP1 were incubated with 60 nM CTS D for 15 min and tested in the aequorin-based assay. The activity follows a typical Michaelis–Menten saturation curve. E and F, HEBP1 (250 nM) was incubated with 60 nM CTS D for a time course of 24 h. The FPR3-stimulatory activity was evaluated in the aequorin based-assay (E) and HEBP1 immunoreactivity by Western blotting (F). All incubations were performed at 37˚C in a heating bath. The data displayed (mean 6 SEM) are repre- sentative of at least three independent experiments. ***p , 0.001. The Journal of Immunology 7

To test whether CTS D is the only (or main) protease contributing and peptides were identified by mass spectrometry. The 2.5- and to HEBP1 processing in macrophage conditioned medium, we 6-kDa peptides contained the N terminus of HEBP1, whereas all inhibited CTS D expression by transfection of three specific other fragments did not. Analysis of the peptides identified two siRNAs during the monocyte-to-macrophage differentiation. The main cleavage sites in the protein: one after Leu21 and the other procedure did not affect differentiation, as macrophages appeared somewhere between residues 60 and 90 (Fig. 6C). We did not fully differentiated 7 d after monocyte selection (Fig. 5A), and recover enough material to perform sequencing and identify more transfection efficiency was shown to be near 100% using a fluo- precisely this second cleavage site. A band around 12 kDa was rescent oligonucleotide as control (Fig. 5B, fluorescence micros- found for some preparations of HEBP1 in the absence of CTS D. copy, 5C, flow cytometry). The three different siRNAs (A, B, and This band did not correspond with a fragment of HEBP1, and we C) significantly reduced the levels of immunoreactive CTS D in could not identify this yeast contaminant by mass spectrometry. monocyte-derived macrophages, as determined by Western blot- The same samples were loaded on a 10–20% polyacrylamide gel ting (Fig. 5D). In contrast, the corresponding control oligonu- and blotted onto 0.2-mm pore-size polyvinyl difluoride mem- cleotides (scrambled sequences) did not affect CTS D immuno- branes. Immunodetection with an anti-F2L Ab (Euroscreen) iden- reactivity. Pretreatment of monocyte-derived macrophages with tified, among others, a diffuse band at 2.5 kDa (Fig. 6B). Cleavage the three siRNAs prevented the hydrolysis of HEBP1 and the leading to F2L generation seems to be among the first events generation of FPR3 agonist in conditioned media prepared from during HEBP1 hydrolysis, as short incubations lead to F2L gen- these cells, as evaluated with the aequorin-based assay on FPR3- eration (Fig. 6B, left panel) whereas longer incubations generate expressing CHO-K1 cells (Fig. 5E). also larger peptides (Fig. 6B, right panel). Characterization of the peptides resulting from the processing Chemotaxis of monocytes toward HEBP1-derived peptides Downloaded from of HEBP1 by CTS D F2L is a chemoattractant agent for FPR3-expressing cells, such as We analyzed by mass spectrometry the nature of the peptides monocytes, DCs, and macrophages (18, 21, 29). We evaluated resulting from the processing of HEBP1 by CTS D to determine therefore the ability of CTS D-treated HEBP1 to recruit FPR3- which proteolytic product(s) activate FPR3. In the absence of expressing leukocytes. In microchemotaxis Boyden’s chambers, tryptic cleavage, we identified in the HEBP1 hydrolysate a pep- processed HEBP1 promoted recruitment of human monocyte- http://www.jimmunol.org/ tide corresponding with acetylated F2L. Silver staining of poly- derived macrophages and human monocytes (Fig. 7B,7C). The acrylamide gels showed that several fragments were generated chemotaxis index was comparable with that obtained for synthetic from HEBP1 after 30 min of digestion by CTS D. The molecular F2L (10 nM) used as a positive control. No chemotaxis was ob- weights of these fragments were evaluated to 2.5, 5, 6, 10, and 22 served toward CTS D alone or unprocessed HEBP1. Checkerboard kDa (Fig. 6A). Each band was excised from the gel, trypsinized, analysis on monocyte-derived macrophages (Fig. 7A) excluded by guest on September 27, 2021

FIGURE 5. Knockdown of CTS D by siRNAs prevents HEBP1 hydrolysis. A, Flow cytometry analysis of human monocyte-derived macrophages showing complete differentiation (CD68+ CD206+) at day 7 after purification of monocytes. B and C, Human monocyte-derived macrophages were transfected with a fluorescent control oligonucleotide, and transfection efficacy was evaluated by fluorescence microscopy (B) and flow cytometry (C). D, Three siRNAs (A, B, and C) and the respective scrambled oligonucleotides (S, 10 nM) as controls were transfected at the indicated concentrations at days 6 and 7 of human monocyte-to-macrophage differentiation. Detection of CTS D and GAPDH (as control) was made on Western blots, showing a decrease in mature CTS D and its precursor after siRNAs, but not scrambled oligonucleotides, transfection. Untransfected cells were used as positive controls of CTS D immunoreactivity (2). E, Conditioned media (CM) from siRNA-treated monocyte-derived macrophages were tested for their ability to generate bioactivity from purified human HEBP1 (1 mM, 30-min incubation). After incubation, the media were diluted four times and the activity measured in the aequorin- based assay using FPR3-expressing CHO-K1 cells. A complete loss of HEBP1 processing was observed for conditioned media resulting from siRNA- treated macrophages. Scrambled oligonucleotides had no effect on HEBP1 hydrolysis. 8 HEBP1 PROCESSING

and lymph nodes, but also in pancreatic islets and proximal kidney tubules. Lower expression was seen in seminal vesicles and Leydig cells of the testis. No expression of HEBP1 was observed in breast, prostate, intestinal segments, ovary, uterus, skin, esoph- agus, and thymus. Using the same approach, we evaluated the distribution of CTS D. In agreement with published data, CTS D was highly expressed in most tissues, particularly in specialized cells such as macrophages. We observed coexpression of high levels of CTS D and HEBP1 in liver, kidney, and spleen, consistent with a potential role of CTS D in HEBP1 processing in vivo.

Discussion Proteolytic regulation of the biological activity of peptides and proteins acting as ligands of GPCRs is not an uncommon process. Many peptidic GPCR ligands are processed by proteases, resulting in their partial or full inactivation. Almost as frequently, proteolysis is required for the generation of GPCR ligands or results in

a significant increase of their affinity and potency for the receptor. Downloaded from We previously identified F2L, a 21-aa peptide, as a high-affinity and specific endogenous ligand for FPR3. F2L corresponds with the N terminus of the heme-binding protein HEBP1, but the mechanisms involved in its production from the precursor protein are unknown. In this work, we investigated how F2L is generated from HEBP1.

Human full-length HEBP1 displays low agonist activity toward http://www.jimmunol.org/ FPR3 and does not act as an antagonist. The recent characterization of HEBP1 tridimensional structure has shown that the F2L peptide is located outside the structured domain of the protein. Its proteolytic release might therefore keep intact the function of HEBP1 (24, 25, 36). We determined the structure–function relations of F2L using point mutants and truncated forms of the peptide. It appears that the important part of the peptide lies near its N ter- FIGURE 6. HEBP1 processing by CTS D generates F2L. A and B, 3 7 8 HEBP1 (6 mg) was processed by 200 ng CTS D for 5 or 30 min at 37˚C minus, the most crucial positions being G ,N, and S . However, by guest on September 27, 2021 and loaded on a 10–20% polyacrylamide gradient gel before silver staining other low-affinity interaction sites are likely spread over the whole (A) or immunoblot detection with an anti-F2L Ab (B). A diffuse band was length of F2L, as it was not possible to design much shorter detected at 2.5 kDa, the size of F2L. This band was more intense after peptides while keeping a reasonable affinity for FPR3. We ob- a short incubation time. C, The silver-stained bands were analyzed by mass served indeed a shift of one log in the EC50 between peptide 1–18 spectrometry after trypsinization. The mass of the tryptic fragments (almost fully active) and peptide 1–15, suggesting a contribution identified are presented in the middle column and the corresponding region of the WPW motif. F2L is highly conserved among mammalian of HEBP1 protein sequence in the left column. D, Schematic representa- species, and the human peptide differs from mouse F2L by only tion of the cleavage sites in HEBP1 by CTS D (triangles) and of the one aa in position 6, where lysine is replaced by arginine. Steric resulting fragments detected. hindrance probably prevents the access of full-length HEBP1 to the binding pocket of F2L, limiting greatly its ability to activate chemokinesis in favor of chemotaxis, as previously demonstrated FPR3. Additional work will be required to identify peptides with for the F2L peptide (21). In mouse, the F2L peptide can also enhanced biological activities or antagonist properties. promote neutrophil chemotaxis, although with a lower efficacy To determine which proteases or protease families are able to than for human leukocytes (18, 35). We tested therefore high cleave HEBP1 and release active FPR3 ligands, human HEBP1 was concentrations of CTS D-treated HEBP1 on mouse neutrophils incubated with a panel of conditioned media. Indeed, no consensus and demonstrated chemotaxis of these cells. This chemotactic sequence for candidate proteases could be identified in HEBP1 activity demonstrated further the production of functional F2L using bioinformatics programs. We selected as a starting point cells peptide and suggests a physiological relevance of the HEBP1 known to contain high amounts of proteases, including macro- processing by CTS D. phages. Macrophages are phagocytic cells, which differentiate from circulating monocytes, and are located at strategic sites in the Distribution of HEBP1 and CTS D in human organs organism. They are involved in innate immune defense and the Expression of the mouse ortholog of HEBP1, p22HBP, was pre- initiation of adaptive immunity. Upon activation, macrophages viously described in various tissues by Northern blot analysis. High release a large set of proteases, including plasminogen activator, expression was found in liver, kidney, and spleen. To evaluate more collagenases, elastase-like enzymes, and other hydrolases, such as precisely the expression of human HEBP1, we characterized a new lysozyme and b-glucuronidase. They also secrete high amounts of Ab directed against the N terminus of HEBP1 and used this Ab on lysosomal enzymes independently of external stimuli. The activity tissue microarrays. For each tissue spot, we assessed an immu- of these hydrolases is generally low at the neutral pH of extra- nohistological score from 0 (no detectable expression) to 2 (high cellular fluids, but macrophages are able to acidify the pericellular expression) and computed the mean of all scores for each organ space by the activity of their plasma membrane proton pumps (Fig. 8). Results are presented in Supplemental Table II. High (V-ATPase) and secretion of lactic acid. This acidification is pos- levels of HEBP1 immunoreactivity were observed in liver, spleen, sibly a prerequisite for degradative processes, and the concentration The Journal of Immunology 9

FIGURE 7. Leukocyte chemotaxis toward HEBP1 processed by CTS D. A, A checkerboard chemotaxis assay was conducted on human monocyte-derived macrophages. The product of HEBP1 hydrolysis by CTS D was added to the upper and/or lower chambers. Cells were added to the upper chamber and allowed to migrate for 90 min at 37˚C. The number of cells having migrated completely through the ﬁlter is indicated. B–D, Chemotaxis of human monocytes (B), monocyte-derived macrophages (C), and mouse neutrophils (D) in response to 1 mM (human cells) or 10 mM (mouse cells) HEBP1 processed by 60 nM CTS D for 30 min at 37˚C. Synthetic F2L at 10 nM (human cells) or 400 nM (mouse cells) was used as positive control of migration. The displayed responses are representa- tive of three experiments, and results are expressed as the ratio of migrated cells in the presence versus the absence of chemoattractant. **p , 0.01,

***p , 0.001. Downloaded from of released enzymes is often sufﬁcient for inducing lytic damage itors) is a factor contributing to various diseases characterized by to other cells (37). Therefore, in the microenvironment of mac- a chronic inﬂammatory state, such as cancer, asthma, atheroscle- rophages, the pH may be lower than in serum or extracellular rosis, Alzheimer’s disease, periodontitis, rheumatoid arthritis, in-

fluids, resulting in the activation of released lysosomal enzymes. flammatory bowel disease, osteoarthritis, and pulmonary fibrosis http://www.jimmunol.org/ The testing of conditioned media allowed us to identify CTS D (reviewed in Ref. 41). as a candidate for HEBP1 processing and F2L generation, as the In our hands, human purified CTS D cleaved, in less than 5 min, activity on FPR3 was totally inhibited by pepstatin A, an aspartyl HEBP1 at two main sites, leading to the generation of N-terminal protease inhibitor, as well as by transfection of siRNAs targeting peptides. One of these peptides was identified by mass spec- CTS D transcripts. Moreover, CTS D is described as an endo- trometry as F2L, the endogenous ligand of FPR3. At this point, we peptidase cleaving mainly after hydrophobic amino acids, partic- cannot exclude the possibility that other proteases might generate ularly leucine and phenylalanine. This is consistent with the VL/SK F2L or other HEBP1-derived peptides active on FPR3. A peptide motif as the cleavage site for F2L generation. CTS D is a lysosomal corresponding with the first 50 aa of HEBP1 was indeed identified aspartic endopeptidase that plays an essential role in cell ho- in extracts from porcine spleen (21), although displaying lower by guest on September 27, 2021 meostasis by degrading aging proteins in the lysosomal com- potency than F2L on FPR3. F2L and other related peptides might, partment and recycling their components. This enzyme is also however, act together in leukocyte recruitment. involved in the activation or inhibition of various hormones, growth CTS D is produced by almost all cells but is expressed at factors, and enzymes, participates in Ag processing, and is involved particularly high levels by macrophages in some tissues, such as in the regulation of programmed cell death (reviewed in Refs. 38– liver, spleen, and kidney. In contrast, our immunohistochemical 40). Deregulated CTS D activity (altered expression, increased screening of tissues has shown that HEBP1 is not expressed secretion, or unbalance between protease and endogenous inhib- ubiquitously but rather specifically in liver, spleen, lymph nodes, as

FIGURE 8. HEBP1 immunodetection on tissue microarrays. A, Overview of a tissue microarray section. A rectangle of 15 cores corresponds with one organ; each column corresponds with one donor, and the three lines correspond with core replicates. B, Immunohis- tochemical staining was scored 0 for no expression (left panel), 1 for low expression or dispersed cells (middle panel, arrows), and 2 for high expression of HEBP1 (right panel). Original magnification 3200. Arrows indicate stained cells. 10 HEBP1 PROCESSING well as in kidney and pancreas, where CTS D is also highly in tumors (reviewed in Refs. 51, 52) and atherosclerotic lesions expressed. The correlated expression of HEBP1 and CTS D in (53). In these pathologies, F2L could participate in the inflam- several tissues supports the physiological relevance of HEBP1 matory response by recruiting leukocytes expressing FPR3, such processing by CTS D. as macrophages, DCs, and eosinophils. HEBP1 appears as a good substrate for CTS D, but we cannot In conclusion, in this work we identified a protease able to exclude a more complex pathway in vivo leading to the production of process HEBP1 and to generate biologically active F2L peptide. FPR3 ligands, with the contribution of other proteases acting in F2L is the only endogenous ligand specific for FPR3 and che- parallel or in cascade. A similar situation prevails for other bioactive motactic for FPR3-expressing leukocyte populations. Further in- peptides such as hemorphins (42), a family of small oligopeptides vestigations will be required to determine in which physiologi- (4-mers to 10-mers) displaying high affinity for atypical m opioid cal and pathological conditions F2L is produced by CTS D. One receptors. The mechanism of their generation is still not well un- such condition is possibly apoptosis, in which F2L could con- derstood, but the first step is likely the hydrolysis of hemoglobin tribute to the resolution of inflammation. Alternatively, F2L might b-chain by CTS D. Then, other hemorphin variants are produced be generated extracellularly by CTS D in pathological conditions by other proteases, including DPP-IV (43) and ACE (44). In this such as cancer or atherosclerosis. In this context, understanding system, macrophages cultured in an acidic environment are able to the mechanisms leading to HEBP1/F2L secretion or release from proteolyze hemoglobin and generate hemorphins (45). the cells will be important. In this study, we showed that the F2L peptide, derived from the processing of nanomolar concentrations of HEBP1, is active on Acknowledgments FPR3 and able to induce the recruitment of FPR3-expressing We thank Dr. Guy Vandenbussche for mass spectrometry analyses of puri- Downloaded from monocytes, monocyte-derived macrophages, as well as mouse fied recombinant HEBP1 and Françoise Gre´goire for the synthesis of F2L neutrophils. It is, however, still unclear how cytosolic HEBP1 can peptide variants. give rise to an extracellular chemoattractant factor. HEBP1 is indeed clearly devoid of signal peptide, and all available data point Disclosures toward an intracellular localization. This raises the question of how The authors have no financial conflicts of interest.

HEBP1 or F2L are secreted or otherwise released from cells. http://www.jimmunol.org/ First, HEBP1 might be processed inside the cell and generate F2L, in a process such as apoptosis. Apoptosis is traditionally References characterized by the maintenance of organelle integrity and con- 1. Lundberg, A. M., and G. K. Hansson. 2010. Innate immune signals in atherosclerosis. Clin. Immunol. 134: 5–24. densation and fragmentation of DNA, ending in the breaking up of 2. Libby, P. 2008. Role of inflammation in atherosclerosis associated with rheu- the cell into apoptotic bodies. It can occur through numerous matoid arthritis. Am. J. Med. 121(10, Suppl 1): S21–S31. 3. Mantovani, A., P. Allavena, A. Sica, and F. Balkwill. 2008. Cancer-related in- pathways, all converging to the activation of the caspase family of flammation. Nature 454: 436–444. proteases. Recent studies have described a significant role for CTS 4. Serbina, N. V., T. Jia, T. M. Hohl, and E. G. Pamer. 2008. 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Schiffmann, S. Aswanikumar, conventional secretory pathway or as a result of necrosis. Sev- B. Corcoran, and E. L. Becker. 1976. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neu- eral distinct nonconventional secretory mechanisms have been trophils. J. Exp. Med. 143: 1154–1169. described (reviewed in Ref. 48). IL-1a is myristoylated, then 11. Boulay, F., M. Tardif, L. Brouchon, and P. Vignais. 1990. The human N- formylpeptide receptor. Characterization of two cDNA isolates and evidence for translocates to the cell membrane and is released by calpain- a new subfamily of G-protein-coupled receptors. Biochemistry 29: 11123–11133. induced proteolysis. IL-1b is generated from its precursor by 12. Boulay, F., M. Tardif, L. Brouchon, and P. Vignais. 1990. Synthesis and use of the IL-converting enzyme, a member of the caspase family. 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