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*
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
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.
2011, 187: 1475–1485.
erosclerosis (1), arthritis (2), and cancer (3). Therefore, inflam-
matory processes need to be well coordinated and regulated. An
optimal immune response depends in particular on specific leu-
kocyte subsets recruited to the sites of inflammation. Neutrophils,
macrophages, and dendritic cells (DCs) are important cellular
mediators of innate immune defenses (4). Numerous molecules
are able to elicit chemotactic responses, and they can be classified
into different chemical and structural families, among which are
chemokines (5), complement factors C3a and C5a (6), leuko-
trienes, and formylated peptides. These latest factors, such as the
prototypic fMLF, are among the first identified and most potent
chemoattractants for phagocytic leukocytes (7–9).
The Journal of Immunology,
nflammation is a critical response of the organism to
pathogens, damaged cells, or inflammatory stimuli. However,
uncontrolled inflammation can lead to diseases such as ath-
These various families of chemotactic factors activate G protein-
coupledseven transmembrane domains receptors expressed not only
by leukocytes but also by a variety of other cell types. A receptor for
formylated peptides was first described in 1976 (10), and the cDNA
encoding the human formyl peptide receptor (FPR, now called
FPR1) was cloned in 1990 (11, 12). Two closely related human re-
ceptors, FPR-like 1 (FPRL1/FPR2) and FPR-like 2 (FPRL2/FPR3),
were subsequently cloned by low-stringency hybridization, using
the FPR1 cDNA as a probe (13–15). The three genes are clustered
on human chromosome 19q13.3 and encode receptors sharing a
high level of sequence identity (13, 14). N-formylated peptides are
derived either from bacterial proteins or from endogenous mito-
chondrial proteins. FPR1, and presumably the two other members
of the family, are therefore believed to be involved in host defense
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-
finity for FPR2 (15). In addition to formylated peptides, a number
of structurally diverse agonists of FPR1 and/or FPR2 have been de-
scribed during recent years (16). In contrast, FPR3 does not respond
to fMLF (17) and presents a distinctive expression pattern among
leukocyte populations. FPR3 is expressed in monocytes, myeloid
and plasmacytoid DCs, some tissue-specific macrophage subpop-
ulations (particularly in lung, skin, and colon) and eosinophils, but
not in neutrophils (18).
In contrast to FPR1 and FPR2, a very few ligands have been
identified for FPR3. Humanin, a neuroprotective peptide in models
of Alzheimer’s disease, was shown to bind FPR3 and FPR2 with
high affinity (19, 20). The most specific ligand described for FPR3
is, however, the endogenous peptide F2L (21), an acetylated 21-aa
peptide derived from heme-binding protein 1 (HEBP1). F2L was
isolated from porcine spleen extracts on the basis of its bioactivity.
This highly conserved peptide activates FPR3 in the low nano-
molar range, is poorly active on FPR2, and is inactive on FPR1.
On FPR3-expressing cells, F2L triggers intracellular calcium re-
lease, inhibition of cAMP accumulation, and phosphorylation of
ERK1/2 MAPKs through the Giclass of G proteins. When tested
*Institut de Recherche Interdisciplinaire en Biologie Humaine et Mole ´culaire, Uni-
versite ´ Libre de Bruxelles, Campus Erasme, B-1070 Brussels, Belgium;†Institut de
Recherche en Microbiologie Jean-Marie Wiame, B-1070 Brussels, Belgium; and
‡Service d’Anatomie Pathologique, Universite ´ Libre de Bruxelles, Campus Erasme,
B-1070 Brussels, Belgium
Received for publication October 25, 2010. Accepted for publication May 24, 2011.
This work was supported by the Actions de Recherche Concerte ´es of the Commu-
naute ´ Franc ¸aise de Belgique, the Interuniversity Attraction Poles Programme (P6-14)
Belgian State Belgian Science Policy, the Walloon Region (Programme d’excellence
“CIBLES”), the European Union (Grant LSHB-CT-2005-518167/INNOCHEM), the
Fonds de la Recherche Scientifique Me ´dicale of Belgium, and the Fondation Me ´d-
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
Van Buuren Foundation.
Address correspondence and reprint requests to Prof. Marc Parmentier, Institut de
Recherche Interdisciplinaire en Biologie Humaine et Mole ´culaire, Universite ´ Libre
de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.
E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: CTS D, cathepsin D; DC, dendritic cell; FPR,
formyl peptide receptor; FPRL1/FPR2, formyl peptide receptor-like 1; FPRL2/FPR3,
formyl peptide receptor-like 2; HEBP1, heme-binding protein 1; siRNA, small in-
on FPR3-expressing leukocytes, F2L promotes calcium mobili-
zation and chemotaxis (18, 21).
How and in which circumstances F2L is generated in the or-
ganism is, however, still unknown. HEBP1 is an intracellular
tetrapyrrole-binding protein of 22 kDa. Initially purified from
mouse liver, HEBP1 is expressed in many tissues. Knockdown of
the cell heme content, suggesting that HEBP1 may be involved in
heme regulation, biosynthesis, or transport (22, 23). However, no
additional data have reinforced this hypothesis, and the biological
function of HEBP1 remains therefore poorly defined. The three-
dimensional structure of murine p22HBP was determined by nu-
clear magnetic resonance and consists of a 9-stranded distorted
b-barrel flanked by two long a-helices (24, 25). Located outside
the globular structure of HEBP1, residues 1–17 are disordered,
whereas residues 18–23 form a b-strand. This part of the protein is
not found in bacterial homologues of the SOUL/HEBP family. It
is therefore conceivable that cleavage of HEBP1 after the leucine
21 would release the F2L ligand while keeping the heme-binding
domain functional (24).
In the current study, we investigated the potential pathways
leading to the generation of F2L in the organism. According to the
role of macrophages in cleaning up cellular debris and the reso-
lution ofinflammatory processes, we searched for the generation of
F2L when recombinant HEBP1 was submitted to the action of
macrophage proteases or conditioned media. The use of specific
protease inhibitors and purified proteases demonstrated that the
lysosomal aspartyl endopeptidase cathepsin D (CTS D) is able
to process HEBP1 and generate F2L. This observation suggests
that F2L might be generated after tissue damage and macrophage
recruitment, favoring the recruitment of additional monocyte/
macrophages and DCs, which would contribute to tissue repair
and the control of the inflammatory process.
Materials and Methods
Plasmid construction and yeast transformation
The cDNA corresponding to human HEBP1 was amplified by PCR from
pCDNA3–HEBP1 using the primer pair oecj301 and oecj302 and was
introduced in the pCSC2 Saccharomyces cerevisiae expression vector (26)
by homologous recombination. The resulting plasmid, pCSC2–HEBP1
(pCSC294), allows the production of recombinant HEBP1 displaying a
C-terminal tag of six histidines. The S. cerevisiae BY4709 strain (MATa
ura3D0) was transformed with the vector using the lithium acetate pro-
cedure (27). Transformants were selected on YNB plates containing 20
HEBP1 production and purification
Strain BY4709 producing HEBP1 was culturedon YNB containing 20 mg/ml
glucose in a 13 l-batch bioreactor (Biolafitte) to an OD660nmof 1.69. Cells
were harvested by centrifugation and the pellet washed twice in water. Cells
were resuspended in buffer A (300 mM NaCl, 50 mM NaH2PO4) with
EDTA-free Complete Protease Inhibitor Cocktail (Roche) and lysed with
a French press. The lysate was centrifuged at 12,000 rpm (Sorvall RC5B, SS-
34 rotor) for 30 min at 4˚C. The supernatant was purified on an Ni-NTA
Sepharose column (Qiagen) eluted by a step gradient of 0, 90, and 150 mM
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
was loaded on a 12% polyacrylamide gel, and HEBP1 was identified by
Western blotting using a rabbit polyclonal Ab (Phoenix). Fractions of interest
were finally pooled, concentrated to 1 ml, and purified on a Superdex 75
column (GE Healthcare) run with buffer A at 1 ml/min. Fractions were an-
alyzed on a 12% polyacrylamide gel, followed by HEBP1 immunodetection
on Western blots, and purity was checked by Coomassie blue staining.
Truncated synthetic peptides and alanine scanning
Acetylated F2L (Ac-MLGMIKNSLFGSVETWPWQVL) and alanine
variants were synthesized locally by using the solid-phase Fmoc strategy.
Monoisotopic masses and sequences of all peptides were verified by mass
spectrometry. Because of their hydrophobicity, peptides were dissolved in
DMSO at 1 mM, and 25-fold intermediate dilutions were made in 50%
CH3CN, followed by further dilution in assay buffer to working concen-
trations. All peptides were assayed from 0.1 to 3000 nM (2 points per log)
in the aequorin-based assay on FPR3-expressing cells, and the EC50was
determined. The results are presented as the ratio between the EC50of the
peptide and the EC50of native F2L.
Aequorin-based luminescence assay of intracellular calcium
Calcium release was measured by an aequorin-based bioluminescence
assay, as previously described (28, 29). In brief, CHO-K1 cells coex-
pressing apoaequorin, Ga16, and FPR3 or control GPCRs were collected
from culture dishes, pelleted by centrifugation, and resuspended at 5 3 106
cells/ml in DMEM/Ham’s F12 containing 0.1% BSA (aequorin buffer).
The cell suspension was supplemented with 5 mM coelenterazine h
(Promega, Madison, WI) and incubated under shaking for 3 h 30 min at
room temperature in the dark, then diluted 5-fold in aequorin buffer. Fifty
microliters of cell suspension was injected onto 50 ml of agonist-
containing medium in 96-well plates, and light emission was recorded
for 40 s in a Centro LB 960 luminometer (Berthold Technologies). ATP
(20 mM; Sigma) was used as standard to normalize the data.
For the analysis of truncated F2L peptides, a F2L variant containing an
additional C-terminal tyrosine was used as tracer after labeling with125I
using the Iodogen method (the sp. act. was 900 Ci/mmol), as described
previously (16). Afterwards, we obtained a custom-made fluorescent F2L
derivative, containing 5(6)-carboxyfluorescein linked to an additional
C-terminal lysine (F2L–FAM), from JPT Peptide Technologies (Berlin,
Germany). Two hundred thousand FPR3-expressing CHO-K1 cells or par-
ental CHO-K1 cells in 100 ml binding buffer (DMEM-F12, containing
0.5% BSA and 0.1% NaN3; Life Technologies) in duplicate samples were
incubated in siliconized 1.5-ml microcentrifuge tubes (Sigma) with in-
creasing concentrations of F2L–FAM for 1 h at room temperature in the
dark. The cells were then washed with 2 volumes of binding buffer, pel-
leted, resuspended in 250 ml binding buffer, and analyzed by FACS
(FACScan; Becton Dickinson). FACS data were analyzed with the WinMDI
software. Nonspecific binding was determined in the presence of 10 mM
unlabeled F2L. For competition binding assays, cells were incubated with
10 nM F2L–FAM and increasing concentrations of HEBP1, unlabeled F2L,
or variants thereof. The binding data were analyzed with the GraphPad
Proteolytic processing of HEBP1 in conditioned medium from
Monocytes were isolated from venous blood of healthy donors by im-
munomagnetic bead cell sorting (MACS) according to the manufac-
turer’s specifications. These procedures received authorization from the
Ethics Committee of the Free University of Brussels Medical Faculty.
After Ficoll density gradient, monocytes were purified by positive selec-
tion using CD14 microbeads (Miltenyi Biotec). Macrophages were dif-
ferentiated from monocytes in the presence of 50 ng/ml recombinant
human M-CSF (R&D Systems) for 6 to 8 d. The purity of the cell prepa-
rations was evaluated to 95% or more by flow cytometry (CD206+CD14+,
or CD68+for permeabilized cells). Macrophage monolayers were in-
cubated in a proteolysis buffer (25 mM sodium acetate pH 3.6, 100 mM
NaCl) at 37˚C in a humidified atmosphere of 5% CO2during 24 h. The
medium was collected and incubated with HEBP1 with or without 10 mg/
ml pepstatin A (Sigma) for different periods of time at 37˚C. The medium
was then adjusted to pH 7 by 10 mM sodium bicarbonate pH 8, and
samples were engaged in the aequorin-based assay.
Inhibition of CTS D expression
CTS D expression was inhibited in human monocyte-derived macro-
phages by transfection of specific small interfering RNAs (siRNAs; A,
HSS102578; B, HSS175648; C, HSS175649; Invitrogen), using their re-
spective scrambled oligonucleotides as controls. Oligonucleotides (0.1, 1,
and 10 nM) were transfected using the INTERFERin reagent (Polyplus-
transfection) at days 6 and 7 of macrophage differentiation from mono-
cytes, and the purity of fully differentiated macrophages at day 7
was evaluated by flow cytometry (CD68+CD206+cells). Transfection
efficiency was monitored with 100 nM FITC-labeled oligonucleotide
(BLOCK-iT Fluorescent Oligo; Invitrogen). CTS D levels were evaluated
48 h after the second transfection by Western blotting using a mouse anti-
1476 HEBP1 PROCESSING
cells, tissue-specific macrophage subpopulations, and eosinophils. J. Immunol.
19. Harada, M., Y. Habata, M. Hosoya, K. Nishi, R. Fujii, M. Kobayashi, and
S. Hinuma. 2004. N-Formylated humanin activates both formyl peptide receptor-
like 1 and 2. Biochem. Biophys. Res. Commun. 324: 255–261.
20. Ying, G., P. Iribarren, Y. Zhou, W. Gong, N. Zhang, Z. X. Yu, Y. Le, Y. Cui, and
J. M. Wang. 2004. Humanin, a newly identified neuroprotective factor, uses the
G protein-coupled formylpeptide receptor-like-1 as a functional receptor. J.
Immunol. 172: 7078–7085.
21. Migeotte, I., E. Riboldi, J. D. Franssen, F. Gre ´goire, C. Loison, V. Wittamer,
M. Detheux, P. Robberecht, S. Costagliola, G. Vassart, et al. 2005. Identification
and characterization of an endogenous chemotactic ligand specific for FPRL2. J.
Exp. Med. 201: 83–93.
22. Taketani, S., Y. Adachi, H. Kohno, S. Ikehara, R. Tokunaga, and T. Ishii. 1998.
differentiation of urine erythroleukemia cells. J. Biol. Chem. 273: 31388–31394.
23. Zylka, M. J., and S. M. Reppert. 1999. Discovery of a putative heme-binding
protein family (SOUL/HBP) by two-tissue suppression subtractive hybridization
and database searches. Brain Res. Mol. Brain Res. 74: 175–181.
24. Dias, J. S., A. L. Macedo, G. C. Ferreira, F. C. Peterson, B. F. Volkman, and
B. J. Goodfellow. 2006. The first structure from the SOUL/HBP family of heme-
binding proteins, murine P22HBP. J. Biol. Chem. 281: 31553–31561.
25. Gell, D. A., B. J. Westman, D. Gorman, C. K. Liew, J. J. Welch, M. J. Weiss, and
J. P. Mackay. 2006. A novel haem-binding interface in the 22 kDa haem-binding
protein p22HBP. J. Mol. Biol. 362: 287–297.
26. Dutoit, R., E. Dubois, and E. Jacobs. 2010. Selection systems based on
dominant-negative transcription factors for precise genetic engineering. Nucleic
Acids Res. 38: e183.
27. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact
yeast cells treated with alkali cations. J. Bacteriol. 153: 163–168.
28. Button, D., and M. Brownstein. 1993. Aequorin-expressing mammalian cell
lines used to report Ca2+ mobilization. Cell Calcium 14: 663–671.
29. Le Poul, E., S. Hisada, Y. Mizuguchi, V. J. Dupriez, E. Burgeon, and
M. Detheux. 2002. Adaptation of aequorin functional assay to high throughput
screening. J. Biomol. Screen. 7: 57–65.
30. Mortz, E., T. N. Krogh, H. Vorum, and A. Go ¨rg. 2001. Improved silver staining
protocols for high sensitivity protein identification using matrix-assisted laser
desorption/ionization-time of flight analysis. Proteomics 1: 1359–1363.
31. Boyden, S. 1962. The chemotactic effect of mixtures of antibody and antigen on
polymorphonuclear leucocytes. J. Exp. Med. 115: 453–466.
32. Polevoda, B., and F. Sherman. 2000. Nalpha -terminal acetylation of eukaryotic
proteins. J. Biol. Chem. 275: 36479–36482.
33. Polevoda, B., and F. Sherman. 2003. N-terminal acetyltransferases and sequence
requirements for N-terminal acetylation of eukaryotic proteins. J. Mol. Biol. 325:
34. Polevoda, B., and F. Sherman. 2002. The diversity of acetylated proteins. Ge-
nome Biol. 3: reviews0006.
35. Gao, J. L., A. Guillabert, J. Hu, Y. Le, E. Urizar, E. Seligman, K. J. Fang,
X. Yuan, V. Imbault, D. Communi, et al. 2007. F2L, a peptide derived from
heme-binding protein, chemoattracts mouse neutrophils by specifically activating
Fpr2, the low-affinity N-formylpeptide receptor. J. Immunol. 178: 1450–1456.
36. Dias, J. S., A. L. Macedo, G. C. Ferreira, N. Jeanty, S. Taketani,
B. J. Goodfellow, F. C. Peterson, and B. F. Volkman. 2005. 1H, 15N and 13C
resonance assignments of the heme-binding protein murine p22HBP. J. Biomol.
NMR 32: 338.
37. Tapper, H., and R. Sundler. 1990. Role of lysosomal and cytosolic pH in the
regulation of macrophage lysosomal enzyme secretion. Biochem. J. 272: 407–
38. Benes, P., V. Vetvicka, and M. Fusek. 2008. Cathepsin D—many functions of
one aspartic protease. Crit. Rev. Oncol. Hematol. 68: 12–28.
39. Conus, S., R. Perozzo, T. Reinheckel, C. Peters, L. Scapozza, S. Yousefi, and
H. U. Simon. 2008. Caspase-8 is activated by cathepsin D initiating neutrophil
apoptosis during the resolution of inflammation. J. Exp. Med. 205: 685–698.
40. Minarowska, A., M. Gacko, A. Karwowska, and L. Minarowski. 2008. Human
cathepsin D. Folia Histochem. Cytobiol. 46: 23–38.
41. Zavasnik-Bergant, T., and B. Turk. 2007. Cysteine proteases: destruction ability
versus immunomodulation capacity in immune cells. Biol. Chem. 388: 1141–
42. Fruitier, I., I. Garreau, and J. M. Piot. 1998. Cathepsin D is a good candidate for
the specific release of a stable hemorphin from hemoglobin in vivo: VV-hem-
orphin-7. Biochem. Biophys. Res. Commun. 246: 719–724.
43. Cohen, M., I. Fruitier-Arnaudin, and J. M. Piot. 2004. Hemorphins: substrates
and/or inhibitors of dipeptidyl peptidase IV. Hemorphins N-terminus sequence
influence on the interaction between hemorphins and DPPIV. Biochimie 86: 31–
44. Lantz, I., E. L. Gla ¨msta, L. Talba ¨ck, and F. Nyberg. 1991. Hemorphins derived
from hemoglobin have an inhibitory action on angiotensin converting enzyme
activity. FEBS Lett. 287: 39–41.
45. Dagouassat, N., I. Garreau, F. Sannier, Q. Zhao, and J. M. Piot. 1996. Generation
of VV-hemorphin-7 from globin by peritoneal macrophages. FEBS Lett. 382:
46. Deiss, L. P., H. Galinka, H. Berissi, O. Cohen, and A. Kimchi. 1996. Cathepsin D
protease mediates programmed cell death induced by interferon-gamma, Fas/
APO-1 and TNF-alpha. EMBO J. 15: 3861–3870.
47. Conus, S., and H. U. Simon. 2008. Cathepsins: key modulators of cell death and
inflammatory responses. Biochem. Pharmacol. 76: 1374–1382.
48. Nickel, W., and C. Rabouille. 2009. Mechanisms of regulated unconventional
protein secretion. Nat. Rev. Mol. Cell Biol. 10: 148–155.
49. Lkhider, M., R. Castino, E. Bouguyon, C. Isidoro, and M. Ollivier-Bousquet.
2004. Cathepsin D released by lactating rat mammary epithelial cells is involved
in prolactin cleavage under physiological conditions. J. Cell Sci. 117: 5155–
50. Baechle, D., T. Flad, A. Cansier, H. Steffen, B. Schittek, J. Tolson, T. Herrmann,
H. Dihazi, A. Beck, G. A. Mueller, et al. 2006. Cathepsin D is present in human
eccrine sweat and involved in the postsecretory processing of the antimicrobial
peptide DCD-1L. J. Biol. Chem. 281: 5406–5415.
51. Fusek, M., and V. Vetvicka. 2005. Dual role of cathepsin D: ligand and protease.
Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 149: 43–50.
52. Liaudet-Coopman, E., M. Beaujouin, D. Derocq, M. Garcia, M. Glondu-Lassis,
V. Laurent-Matha, C. Pre ´bois, H. Rochefort, and F. Vignon. 2006. Cathepsin D:
newly discovered functions of a long-standing aspartic protease in cancer and
apoptosis. Cancer Lett. 237: 167–179.
53. Hakala, J. K., R. Oksjoki, P. Laine, H. Du, G. A. Grabowski, P. T. Kovanen, and
M. O. Pentika ¨inen. 2003. Lysosomal enzymes are released from cultured human
macrophages, hydrolyze LDL in vitro, and are present extracellularly in human
atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 23: 1430–1436.
The Journal of Immunology1485