Identification of proNeuropeptide FF
A
peptides processed
in neuronal and non-neuronal cells and in nervous tissue
Elisabeth Bonnard, Odile Burlet-Schiltz, Bernard Monsarrat, Jean-Philippe Girard
and Jean-Marie Zajac
Institut de Pharmacologie et de Biologie Structurale, Toulouse, France
Peptides which should be generated from the neuropeptide
FF (NPFF) precursor were identified in a neuronal (human
neuroblastoma SH-SY5Y) cell line and in COS-7 cells after
transient transfection of the human proNPFF
A
cDNA and
were compared with those detected in the mouse spinal cord.
After reverse-phase high performance liquid chromatogra-
phy of soluble material, NPFF-related peptides were im-
munodetected with antisera raised against NPFF and
identified by using on-line capillary liquid chromatography/
nanospray ion trap tandem mass spectrometry. Neuronal
and non-neuronal cells generated different peptides from
the same precursor. In addition to NPFF, SQA-NPFF
(Ser-Gln-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide)
and NPAF were identified in the human neuroblastoma
while only NPFF was clearly identified in COS-7 cells. In
mouse, in addition to previously detected NPFF and NPSF,
SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-
amide), the homologous peptide of SQA-NPFF, were
characterized. These data on intracellular processing of
proNeuropeptide FFA are discussed in regard to the known
enzymatic processing mechanisms.
Keywords: neuropeptide FF; electrospray tandem mass

spectrometry; precursor processing; neuroblastoma.
Neuropeptide FF (NPFF, FLFQPQRFamide) is a mam-
malian amidated neuropeptide, originally isolated from
bovine brain and characterized as a modulator of endo-
genous opioid functions [1,2]. Two precursors, proNPFF
A
and proNPFF
B
encoding peptides possessing the PQRF-
amide sequence, have been cloned in mammals [3,4]. The
proNPFF
A
precursor at basic proteolytic sites should
generate two PQRFamide containing peptides [3] and the
proNPFF
B
, also called RFamide-related peptides precursor
[5], contains a PQRFa sequence and an LPLRFa-contain-
ing peptide.
There is a large body of evidence that NPFF exhibits
antiopioid properties; in rodents, morphine-induced anal-
gesia decreased following administration of NPFF or
NPFF analogues and increased, as stress-induced analgesia,
in response to anti-NPFF antibody administration [6–8]. In
contrast, intrathecal injections of NPFF analogues induced
a long-lasting analgesia [9,10] by increasing opioid peptide
release in the spinal cord through the functional blockade of
presynaptic delta-opioid autoreceptors [11,12]. Recent data
provided evidence that opioid and NPFF endogenous
systems exert a tonic activity, NPFF counteracting tonic

opioid analgesia under resting conditions [13]. NPFF is also
implicated in morphine tolerance, morphine abstinence and
also in several physiological processes, such as body
thermoregulation, food intake and blood pressure regula-
tion [7,14–21].
These pharmacological effects are mediated by two
G-protein-coupled receptors, NPFF
1
and NPFF
2
, cloned
in human and rat [22–25]. Pharmacological characteriza-
tion of these receptors in recombinant cell lines showed a
better selectivity of peptides deduced from proNPFF
A
sequence for NPFF
2
receptors binding, whereas proN-
PFF
B
-derived peptides displayed a greater affinity for
NPFF
1
receptors [26]. Autoradiographic studies per-
formed on rat CNS with highly selective radioligands
revealed the localization of both receptors in central
nervous areas implicated in pain transmission [27]. The
existence of two peptidergic systems of neurotransmission,
mediated through NPFF
1

and NPFF
2
receptor stimula-
tion by peptides generated by proNPFF
B
and proNPFF
A
processing, respectively, could explain the complex
pharmacological effects of NPFF.
The characterization of NPFF-related peptides generated
by NPFF precursors processing is essential to identify
peptides candidate to the role of neurotransmitter. This study
focused on proNPFF
A
processing. In humans, bovines and
rodents, proNPFF
A
contains consensus sequences for a
processing by protein convertases [28,29] (Fig. 1). A sequen-
tial recruitment of carboxypeptidases and peptidylglycine-a
amidating monooxygenase could be implicated in the
production of amidated active NPFF-related peptides.
According to these processing rules, two families of NPFF-
related peptides should be generated by proNPFF
A
process-
ing: (a) N-terminal extended NPFF undecapeptides and (b)
N-terminal extended NPSF (SLAAPQRFamide)-derived
peptides, 11 or 18 amino acids long. In previous studies,
NPFF and NPAF (AGEGLSSPFWSLAAPQRFamide)

were isolated from bovine brain [6]. More recently, NPFF
and NPSF were identified in rodents [30] and a longer
Correspondence to J M. Zajac, Institut de Pharmacologie et de Bio-
logie Structurale, 205 route de Narbonne, 31077 Toulouse, France.
Fax: + 33 5 61175994, Tel.: + 33 5 61175911,
E-mail:
Abbreviations: MS/MS, tandem mass spectrometry; NPFF, neuro-
peptide FF (FLFQPQRFamide); CNS, central nervous system.
(Received 23 June 2003, revised 22 August 2003,
accepted 3 September 2003)
Eur. J. Biochem. 270, 4187–4199 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03816.x
peptide, NPA-NPFF (NPAFLFQPQRFamide), was quan-
tified as the most abundant in rat spinal cord [31]. NPFF and
NPSF were also identified in the mouse spinal cord [31],
NPAF and NPSF in human cerebrospinal fluid [32]. These
reports suggested a processing of proNPFF
A
to both
octapeptides (NPFF and NPSF) despite the absence of
consensus processing sites at their N-terminal end.
The identification of NPFF-related peptides actually
synthesized in neurones should help to understand their role
in neurotransmision. Thus, we investigated the processing of
proNPFF
A
in SH-SY5Y human neuroblastoma cells and
COS-7 (nonneuronal) cells transiently transfected with the
human proNPFF
A
cDNA. The pattern of intracellular

NPFF-related peptides isolated from cell extracts was
compared with that observed in mouse spinal cord.
NPFF-related peptides from cells and tissues were extracted
and purified by RP-HPLC, assessed by radioimmunoassay
and identified by using on-line capillary HPLC/nanospray
ion trap tandem mass spectrometry (nanospray MS/MS).
Materials and methods
Chemicals
NPFF-related peptides (Table 1) were synthesized by the
solid-phase method using Fmoc chemistry with an automatic
synthesizer (430 A Applied Biosystem) and purified by
reverse-phase HPLC as described previously [33]. Fmoc
amino acid derivatives were purchased from Bachem,
France. Iodination of 1DMe ([D.Tyr1(NMe)Phe3]NPFF)
was performed according to Dupouy et al. [34].
hproNPFF
A
cDNA cloning and vector construction
The SMART PCR cDNA synthesis kit (Clontech, Palo
Alto, CA, USA) was used to generate high yields of full-
length cDNA from 1 lg human lymph node total RNA
(Clontech). Amplification of the hproNPFF
A
cDNA from
human LymphNode cDNA was performed by PCR with
an Advantage cDNA PCR kit (Clontech), using
100 ngÆmL
)1
of cDNA and 400 nm of each primer:
5¢Bgl II-hproNPFF

A
:5¢-CGCAGATCTAGCATGGATT
CTAGGCAGGCTGCTGC-3¢ and 3¢Apa-hproNPFF
A
:
5¢-GCGGGGCCCTTCTTCCCAAAGCGTTGAGGGG
CAG-3¢, targeted to the 5¢-and3¢-end of the hproNPFF
A
coding sequence, respectively, in a PTC-150 MiniCycler
(MJ Research Inc.), with 25 cycles consisting of 30 s at
94 °Cand30sat68°C. PCR products were cloned in
pEGFPn3 (Clontech) and sequenced on both strands.
Tissue extraction
Mouse spinal cord tissue. Animals were handled in
accordance with standard ethical guidelines (NIH Guide
for Care and Use of Laboratory Animals,1985).Threemice
were killed by decapitation and the cervical segment of
spinal cord was dissected in ice-cold 0.9% NaCl (106 mg of
tissue), and frozen. All tissues were stored at )80 °C until
used. Cervical segments were chosen for their high NPFF-
like immunoreactivity content [31]. The extraction proce-
dure was performed on frozen tissue: sonication in 0.1
M
HCl, followed by boiling for 10 min. Tissue homogenates
were buffered to pH 7.4 with 2
M
Tris pH 7.4, at a final
concentration of 25 mg tissueÆmL
)1
and centrifuged at

10 000 g for 10 min at 4 °C. The supernatant was stored at
)80 °C until radioimmunoassay.
SH-SY5Y cells. SH-SY5Y cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with Glutamax-1,
glucose (4.5 gÆL
)1
), 10% fetal bovine serum, penicillin
(100 UÆmL
)1
) and streptomycin (100 lgÆmL
)1
). Cells were
seeded into 35 mm Petri dishes, at a density of 7.5 10
5
cells
per dish. Twenty-four hours later, the medium was
removed; cells were washed with cold NaCl/P
i
and scraped
in 0.1
M
HCl. After sonication, lysates were centrifuged at
Fig. 1. Partial amino acid sequence of proNPFF
A
in human and mouse.
NPFF-related peptides predicted by consensus dibasic processing sites
are shown in bold. NPFF and NPSF are eight amino acid peptides
common in mammals (boxed). In mice, an eleven amino acid long
NPSF-derived peptide could be processed: QFW-NPSF.
Table 1. Analytical parameters of synthetic NPFF-related peptides. The IC

50
values were obtained from independent experiments (n ‡ 3). The RIA
detection limit was 10 fmol for NPFF-derived peptides, 55 fmol for hNPAF and 520 fmol for QFW-NPSF. HPLC fractions were collected every
60 s for gradient 1(HPLC-1) and 2 (HPLC-2), every 30 s for gradient 3 (HPLC-3).
Theoretical
monoisotopic
mass (Da)
RIA IC
50
(fmol)
HPLC retention time (min)
Observed
[M + 2H]
2+
(m/z)HPLC-1 HPLC-2 HPLC-3
SQA-NPFF (SQAFLFQPQRFamide) 1366.7 32.8 ± 1.3 – 41.85 54.91 684.5
NPFF (FLFQPQRFamide) 1080.6 28.5 ± 1.3 33.7 42.79 56.42 541.3
hNPAF (AGEGLNSQFWSLAAPQRFamide) 1977.0 264.4 ± 29.5 – 44.42 66.33 990.0
SPA-NPFF (SPAFLFQPQRFamide) 1335.7 29.9 ± 3.2 36.78 – – 668.9
QFW-NPSF (QFWSLAAPQRFamide) 1348.7 1500.2 ± 149.3 36.12 – – 675.4
4188 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
10000 g for 20 min at 4 °C. Surpernatant was stored at
)20 °C until radioimmunoassay.
COS-7 cells. COS-7 cells were grown in 35 mm Petri dishes
in Dulbecco’s modified Eagle’s medium supplemented with
Glutamax-1, glucose (1 gÆL
)1
), 10% fetal calf serum,
penicillin (100 UÆmL
)1

) and streptomycin (100 lgÆmL
)1
).
When cells reached 50–80% confluence, they were transi-
ently transfected with hpro-NPFF
A
cDNA using Lipofect-
AMINE Reagent (Invitrogen). Cells were transfected with
4 lg per dish of pEGFP-hpro-NPFF
A
according to the
manufacturer’s instruction. Forty hours later, the medium
was removed; cells were washed with cold NaCl/P
i
,
extractedin1
M
acetic acid, sonicated and centrifuged at
10 000 g for 20 min at 4 °C. Supernatant was stored at
)20 °C until radioimmunoassay was performed.
Radioimmunoassay
The procedure was carried out as described previously [13].
Dilutions of synthetic peptides, cell or tissue extracts were
incubated overnight at 4 °C with NPFF antiserum
(1 : 150 000 final dilution) and [
125
I][D.Tyr1(NMe)-
Phe3]NPFF (40 pmol). Non-specific binding was deter-
mined with synthetic NPFF (100 pmol per assay). The limit
of detection of NPFF-IR material was estimated to be

between 8 and 12 fmol.
Analysis of the binding characteristics of the NPFF
antiserum indicated that, among all the possible derivatives
of proNPFF
A
and proNPFF
B
, only NPAFLFQPQRF-
amide, SQAFLFQPQRFamide and SPAFLFQPQRF-
amide interfered in the assay (100, 100 and 86%
cross-reactivity as compared with 100% with NPFF,
respectively). SLAAPQRFamide-derived peptide cross-
reacted at 3–10%. Other RFamide peptides, like Met-
EnkRFamide, FMRFamide and the nonamidated NPFF
(NPFF-OH) showed less than 0.1% cross-reactivity.
Degradation of synthetic SQA-NPFF in COS-7 cells
COS-7 cells that had reached 50–80% confluency were
washed with NaCl/P
i
andincubatedwithNaCl/P
i
plus
EDTA, 1 m
M
at 37 °C for 5 min. Cells were then
centrifuged at 500 g, 5 min, resuspended in NaCl/P
i
and
disrupted by nitrogen cavitation. Briefly, cells are intro-
duced in a bomb and equilibrated with nitrogen gas at

30 atm pressure for 10 min. Sudden decompression resul-
ted in a complete disruption of cells with minimum damage
of intracellular organelles [35,36]. Cells lysates were incu-
bated at 37 °C for 10, 30, 60 120, 180 min with or without
protease inhibitors (phenylmethylsulfonyl fluoride, 2 m
M
,
bestatin, 0.1 m
M
) and 50 pmol of synthetic SQA-NPFF.
At the end of the incubation period, cell lysates were
extractedinto1
M
acetic acid and centrifuged at 4 °Cand
8000 g for 20 min. Supernatant was stored at )20 °C until
analytic procedure.
Reverse-phase high pressure liquid chromatography
(RP-HPLC)
Mouse spinal cord. The procedure was carried out as
described previously [13]. Tissue extracts were purified on
C18 Sep-Pak cartridges (Waters). Samples were loaded
on cartridge and washed with 0.088% trifluoroacetic acid
in H
2
O/CH
3
CN (80 : 20 v/v) and eluted with 0.088%
trifluoroacetic acid in H
2
O/CH

3
CN (25 : 75 v/v). The
eluates were lyophilized, diluted in 500 lL of mobile phase
and applied to a C8 Aquapore RP300 Brownlee
(4.6 · 220 mm, Perkin Elmer) equilibrated previously with
70% A and 30% B at a flow rate of 400 lLÆmin
)1
.
Solvent A consisted of 0.088% trifluoroacetic acid in H
2
O
and solvent B was 0.088% trifluoroacetic acid in H
2
O/
CH
3
CN (25 : 75 v/v). Separation was performed by using
isocratic elution at 30% B for 6 min, followed by a linear
gradient of 30–60% B for 50 min (gradient 1). HPLC
fractions (HPLC-1) corresponding to the retention time of
synthetic NPFF-related peptides were collected and
concentrated for radioimmunoassay. NPFF-IR fractions
were subjected to a second lHPLC separation on a C18
column, as previously described [31] before MS/MS
analyses.
Cell extracts
Cell extracts were lyophilized, diluted in 500 lL of mobile
phase and applied to a C8 Spheri-5 RP-8S 5 lmBrownlee
(2.1 · 220 mm) previously equilibrated with 98% of
mobile phase A and 2% of mobile phase B, at a flow

rate of 400 lLÆmin
)1
. Separation of SH-SY5Y cells extract
was achieved using isocratic elution at 2% B for 6 min,
followed by a linear gradient of 2–80% B for 50 min
(gradient 2). Fractions (HPLC-2) co-eluted with SQA-
NPFF, NPFF and hNPAF were subjected to a second
HPLC separation using a linear gradient of 0–44% B for
45 min, followed by an isocratic elution for 15 min. B
reached 54% by 1 min and an isocratic elution was
achieved for 10 min (gradient 3). HPLC fractions
(HPLC-3) corresponding to the retention time of synthetic
NPFF-related peptides were collected and concentrated for
radioimmunoassay. Separation of COS-7 cells extract was
achieved by gradient 1 procedure, followed by gradient 3
procedure. To ensure that tissue and cell extracts were not
contaminated by NPFF-IR material, a blank run on the
RP-HPLC column was performed before each sample
RP-HPLC run and assessed by RIA.
On-line capillary HPLC/nanospray ionization MS/MS
HPLC fractions were concentrated under vacuum and
analyzed by on-line capillary HPLC/nanospray ionization
MS/MS. The sample was injected onto a C18 PepMap
TM
(LC Packings) column (75 lm · 150 mm). The separation
was performed using an isocratic elution at 0% B for
2 min, followed by a linear gradient of 0–40% B in 30 or
40 min, at a flow rate of 150 nLÆmin
)1
. Two different

gradient slopes were used in 40 min. Solvent A consisted of
0.1% formic acid in H
2
O/CH
3
CN (99 : 1 v/v) and B was
0.1% formic acid in H
2
O/CH
3
CN (10 : 90 v/v). The eluent
was injected into an LCQ Deca ion trap mass spectrometer
(ThermoFinnigan, San Jose, CA, USA) through a nano-
flow needle (New Objective, Cambridge, MA, USA) at
2.0 kV. MS/MS data were acquired using a three m/z unit
ion isolation window and a relative collision energy of
35%.
Ó FEBS 2003 Processing of proNeuropeptide FF
A
(Eur. J. Biochem. 270) 4189
Results
RP-HPLC and mass spectrometry analyses
of NPFF-related synthetic peptides
In an attempt to identify NPFF-related peptides in cell
and tissue extracts, analytical characteristics of synthetic
peptides were initially determined on RP-HPLC and mass
spectrometry. Table 1 shows the retention times of human
and mouse synthetic peptides in three different RP-HPLC
procedures. Each peptide was further analyzed by on-line
capillary HPLC/nanospray MS/MS (Fig. 2). The fragmen-

tation of each synthetic NPFF-related peptide (double-
charged precursor ion reported in the Table 1) gave rise to a
Fig. 2. Mass spectrometry analyses of synthetic NPFF-related peptides. One hundred femtomoles of each synthetic NPFF-related peptide were
analyzed by on-line capillary HPLC/nanospray ion trap MS/MS. The MS/MS spectra of NPFF (A), SPA-NPFF (B), SQA-NPFF (C), QFW-
NPSF (D) and hNPAF (E) were acquired from the [M + 2H]
2+
precursor ion at m/z 541.3 (NPFF), m/z 668.9 (SPA-NPFF), m/z 684.5 (SQA-
NPFF), m/z 675.4 (QFW-NPSF) and m/z 990.0 (hNPAF). Fragment ion peaks are labelled according to Biemann’s nomenclature [49]. *Loss of
NH
3
from the y and b ions. The peptide sequence and fragmentation pattern for each peptide is indicated at the top.
4190 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
series of y and b fragment ions, from which the most
abundant was in each case the singly charged y
4
fragment
ion at m/z 546.3, corresponding to the C-terminal tetrapep-
tide PQRFamide. These observations led us to consider the
y
4
fragment ion in the MS/MS spectrum of each precursor
ion, as a criterion for the identification of NPFF-related
peptides in biological samples. Other criteria for the
identification of NPFF-related peptides were the retention
time in each HPLC system, the MS/MS fragmentation
pattern of the double-charged precursor ion which is
characteristic of each peptide and the RIA signal.
RP-HPLC profiles of NPFF-IR in cell extracts
SH-SY5Y cell extracts were applied on gradient 2 and
fractions containing NPFF-immunoreactivity were separ-

ated on gradient 3 (Fig. 3A). Three immunoreactive peaks
corresponding to retention time of 51, 55 and 67 min were
observed. The second peak coeluted with synthetic SQA-
NPFFandthethirdwithhNPAF(Table1).
COS-7 cell extracts were separated on gradient 1 and
NPFF-IR fractions were subjected to a second HPLC
procedure on gradient 3 (Fig. 3B). Three immunoreactive
peaks were obtained with retention times of 55.5, 60 and
67 min. Two corresponded to the retention time of
synthetic peptides: SQA-NPFF or NPFF for peak 1
and hNPAF for peak 3. Quantification of NPFF-IR in
HPLC fractions was assessed by radioimmunoassay
(Table 2). No NPFF-IR material was detected in COS-7
cells either non transfected or transiently transfected with
pEGFPn3 vector (data not shown). The identification of
NPFF-IR molecular forms in SH-SY5Y (peaks 2 and 3)
and COS-7 transfected cells (peaks 1 and 3) was provided
using MS/MS analyses.
Identification of SQA-NPFF and hNPAF by capillary
HPLC/nanospray MS/MS
HPLC fractions corresponding to the NPFF-IR peak 2
from SH-SY5Y cells extract were pooled, concentrated
under vacuum and analyzed by on-line capillary HPLC/
nanospray MS/MS. No peak at m/z 684.5 corresponding to
the expected double-charged ion of SQA-NPFF could be
detected in the MS spectrum. However, the search for the
specific y
4
fragment ion at m/z 546.3 in the MS/MS
spectrum allowed to extract a signal of low intensity from

the background noise at the retention time of synthetic
SQA-NPFF (Figs 4A,B). The corresponding MS/MS spec-
trum (Fig. 4C) displayed, in addition to the y
4
fragment ion,
b and y fragment ions compatible with the fragmentation
Fig. 3. HPLC profile of NPFF-IR in SH-SY5Y and COS-7 cell
extracts. Acid SH-SY5Y extracts were applied on a C8 column and
separated first on gradient 2 at a flow rate of 400 lLÆmin
)1
. Collected
NPFF-immunoreactive fractions were pooled, concentrated and sep-
arated on gradient 3 (A). Acid COS-7 extracts were first separated on a
C8 column on gradient 1. Collected fractions corresponding to the
retention time of synthetic NPFF-related peptides were pooled and
subjected to a second HPLC separation on gradient 3 (B). Elution
positions of synthetic NPFF-related peptides are indicated by arrows.
The gradient is represented by a dotted line.
Table 2. Separation by RP-HPLC of cell and tissue extracts and quantitative analyses of NPFF-IR peaks by RIA. SH-SY5Y cell extract was applied
on gradient 2 and collected fractions corresponding to the retention time of synthetic NPFF-related peptides were pooled and separated on the
gradient 3. COS-7 cells transiently transfected by hpro-NPFF
A
were extracted and separated on gradient 1 and gradient 3. Mouse cervical spinal
cord extracts were purified on a Sep-Pack Cartridge before separated on gradient 1. NPFF-related peptides were estimated in HPLC-3 and HPLC-1
fractions by RIA.
SH-SY5Y neuroblastoma hpro-NPFF
A
transfected COS-7 Mouse cervical spinal cord
HPLC-3 retention
time (min)

Cell extract
(fmolÆmL
)1
)
HPLC-3 retention
time (min)
Cell extract
(fmolÆmL
)1
)
HPLC-1 retention
time (min)
Tissue
(fmolÆmg
)1
)
SQA-NPFF 54.5–55.5 54.6 54.5–55.5 195.7 – –
NPFF 56–57 <10.4 56–57 189.6 34–35 2.36
hNPAF 66–67 34.0 66–67 31.9 – –
SPA-NPFF/QFW-NPSF – – – – 38 1.43
Ó FEBS 2003 Processing of proNeuropeptide FF
A
(Eur. J. Biochem. 270) 4191
pattern of the synthetic SQA-NPFF (Fig. 2C). HPLC
fractions corresponding to the peak 1 from COS-7 cell
extract were subjected to the same procedure. In this case,
neither MS nor MS/MS analyses showed SQA-NPFF.
Further analyses were performed on the HPLC fractions
flanking the peak 1, without identifying SQA-NPFF.
HPLC fractions corresponding to the NPFF-IR peak 3

from SH-SY5Y cell extract were pooled, concentrated
under vacuum and analyzed by on-line capillary
HPLC/nanospray MS/MS. The reconstructed ion chroma-
togram of the specific y
4
fragment ion generated by the
fragmentation of the double-charged ion at m/z 990.0 of the
expected hNPAF (Fig. 4E) shows a peak at the retention
time of synthetic hNPAF (Fig. 4D). The corresponding
MS/MS spectrum (Fig. 4F) shows a fragmentation pattern
superimposable to that obtained with synthetic hNPAF
(Fig. 2E) thus unambiguously identifying this peptide in
SH-SY5Y cell extracts. HPLC fractions corresponding to
Fig. 4. Identification of SQA-NPFF and hNPAF in SH-SY5Y cell extracts. Reconstructed ion chromatograms of the fragment ion at m/z 546.3
generated during the MS/MS analysis of double-charged precursor ions at m/z 684.5 from 100 fmol of synthetic SQA-NPFF (A), at m/z 684.5 from
HPLC fractions of NPFF-IR peak 2 (B), at m/z 990.0 from 100 fmol of synthetic hNPAF (D) and at m/z 990.0 from HPLC fractions of NPFF-IR
peak3(E)andMS/MSspectraofthe[M+2H]
2+
precursor ions at m/z 684.5 (C), and at m/z 990.0 (F) in HPLC fractions of SH-SY5Y extract.
Fragment ion peaks are labelled according to Biemann’s nomenclature. *Loss of NH
3
from the y and b ions. The peptide sequence and
fragmentation pattern for each peptide is indicated at the top.
4192 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the peak 3 from COS-7 cell extract were subjected to the
same procedure. Neither MS nor MS/MS analyses unam-
biguously identified hNPAF.
Identification of NPFF by capillary HPLC/nanospray MS/MS
NPFF was searched in HPLC fractions corresponding to
the NPFF-IR peak 1 from COS-7 cell extract. The capillary

HPLC/nanospray MS/MS analysis of these fractions
allowed to detect the y
4
fragment ion at m/z 546.3 in the
MS/MS spectrum of the double-charged ion at m/z 541.3
corresponding to NPFF (Fig. 5B) at the retention time of
synthetic NPFF (Fig. 5A). The MS/MS spectrum obtained
from the cell extract (Fig. 5C) was identical to the
fragmentation pattern of the synthetic NPFF (Fig. 2A)
identifying NPFF in the samples.
Fig. 5. Identification of NPFF in SH-SY5Y and COS-7 cell extracts. Reconstructed ion chromatograms of the fragment ion at m/z 546.3 generated
during the MS/MS analysis of double-charged precursor ions at m/z 541.3 from 100 fmol of synthetic NPFF (A,D), HPLC fractions of NPFF-IR
peak 1 of COS-7 cell extract (B), HPLC fractions of NPFF-IR peak 2 of SH-SH5Y extracts (E), and MS/MS spectra of the [M + 2H]
2+
precursor
ions at m/z 541.3, in HPLC fractions of COS-7 (C) and SH-SY5Y (F) cell extracts.
Ó FEBS 2003 Processing of proNeuropeptide FF
A
(Eur. J. Biochem. 270) 4193
The same procedure was applied to the HPLC fractions
corresponding to the NPFF-IR peak 2 of SH-SY5Y cell
extract. A weak signal of the m/z 546.3 specific y
4
fragment
ion was detected at the retention time of synthetic NPFF
(Fig. 5D,E). The corresponding MS/MS analysis of this
peak (Fig. 5F) revealed, in addition to the y
4
fragment ion,
a fragmentation pattern compatible with that of the

synthetic NPFF (Fig. 2A). Taking into account the
retention times and the fragmentation pattern observed,
these results indicate that NPFF is present in the SH-SY5Y
cell extracts.
Degradation of SQA-NPFF
Synthetic SQA-NPFF was incubated in COS-7 cell lysates
in order to investigate the putative enzymatic degradation
of SQA-NPFF into NPFF. Time-course experiments per-
formed at 37 °C indicated that synthetic SQA-NPFF
Fig. 6. Degradation of synthetic SQA-NPFF in COS-7 cells. COS-7 cell lysate was incubated with 50 pmol of synthetic SQA-NPFF, 2 m
M
phenylmethanesulfonyl fluoride and 0.1 m
M
bestatin for 10 min at 37 °C. After extraction and separation on gradient 1, 34–35 min HPLC fraction
was analyzed by capillary HPLC/nanospray ion trap MS/MS. Reconstructed ion chromatograms of the fragment ion at m/z 546.3 generated during
the MS/MS analysis of double-charged precursor ions at m/z 541.3 from 100 fmol of synthetic NPFF (A), at m/z 684.5 from 100 fmol of synthetic
SQA-NPFF (D), at m/z 541.3 (B) and m/z 684.5 (E) in HPLC fraction of COS-7 cell extract. MS/MS spectra of the [M + 2H]
2+
precursor ions at
m/z 541.3 (C) and m/z 684.5 (F) in HPLC fractions of COS-7 cell extract.
4194 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(50 pmol) was quickly degraded in the absence of protease
inhibitors, as no NPFF-IR was detected 10 min after
incubation with cell lysates. At this time, the incubation in
the presence of 2 m
M
phenylmethanesulfonyl fluoride and
0.1 m
M
bestatin prevented partially SQA-NPFF degrada-

tion, as 208 fmol of NPFF-IR were detected in HPLC
fraction coeluting with both synthetic peptides SQA-NPFF
and NPFF. After 30-min incubation with SQA-NPFF and
cell lysates in the presence of phenylmethanesulfonyl
fluoride and bestatin, no NPFF-IR was detected.
The capillary HPLC/nanospray MS/MS analysis of cell
lysates incubated for 10 min with peptidase inhibitors was
performed and allowed to detect the specific y
4
fragment ion
at m/z 546.3 in the MS/MS spectrum of the double-charged
ion at m/z 541.3 (Fig. 6B) at the retention time of synthetic
NPFF (Fig. 6A). The corresponding MS/MS fragmenta-
tion pattern (Fig. 6C) was identical to the fragmentation
pattern of synthetic NPFF (Fig. 2A). Similarly, the MS/MS
analysis of the double-charged ion at m/z 684.5, corres-
ponding to the SQA-NPFF (Fig. 6D–F) indicated that
SQA-NPFF was not totally degraded. Even though the
signal intensity of SQA-NPFF was weak, signal intensities
observed for the m/z 541.3 and 684.5 ions also indicated
that NPFF was detected in an approximately 20-fold higher
level than SQA-NPFF.
Identification of NPFF-related peptides in mouse spinal
cord tissue extracts
The proNPFF
A
processing was investigated in mouse spinal
cord extracts. We have reported previously the presence of
NPFF-related octapeptides NPFF and NPSF in mouse
spinal cord [31] but larger peptides should be present in this

tissue as the mouse proNPFF
A
contains cleavage sites
predicted to generate the undecapeptide SPA-NPFF and
the N-terminal extended form of NPSF, QFW-NPSF.
The RP-HPLC profile of NPFF-IR in mouse cervical
spinal cord extract is reported on Fig. 7. Three immuno-
reactive peaks with retention times of 34, 38 and 41 min
were obtained (Table 1). Peaks 1 and 2 coeluted with
synthetic NPFF and SPA-NPFF, respectively. Because
the difference between the retention time of synthetic
SPA-NPFF and QFW-NPSF was very tight, endogenous
QFW-NPSF was also searched in the peak 2.
On line capillary HPLC/nanospray MS/MS analyses of
NPFF-immunoreactive peaks 1 and 2 are reported in
Fig. 8. The data obtained from the NPFF-IR peak 1
HPLC fraction show that NPFF is identified in mouse
spinal cord (Fig. 8A–C). Similarly, data obtained from the
NPFF-IR peak 2 HPLC fraction unambiguously identified
SPA-NPFF (Fig. 8D–F). The identification of QFW-
NPSF in peak 2 was more difficult. However, the detection
of the characteristic y
4
fragment ion at m/z 546.3 in the
MS/MS spectrum of the precursor ion of QFW-NPSF at
m/z 675.4 and the signal retention time corresponding to
the synthetic peptide (Fig. 8G–I) were convincing data for
the identification of QFW-NPSF in the sample. The
coelution of SPA-NPFF and QFW-NPSF did not allow
the quantification of each peptide in the HPLC fractions.

Considering the poor affinity of QFW-NPSF for the
antibody used (Table 1), it seems likely that the immuno-
reactivity detected in the peak 2 corresponds to SPA-
NPFF.
Discussion
It is well documented that pro-neuropeptides are synthe-
sized as inactive precursors that are processed during
intracellular transport [37–40]. At the present time, the
enzymatic pathway responsible for the conversion of NPFF
precursors NPFF
A
and NPFF
B
[41] to smaller biologically
active peptides is completely unknown. The first step in this
knowledge is the description of the peptides actually
generated in neurones before extracellular degradation
processing by a great variety of peptidases.
The key finding of the present study is that the pattern of
NPFF-related peptides processed from the proNPFF
A
is
similar in neuronal cell line and nervous tissue. The SH-
SY5Y human neuroblastoma cell line, used in this study
as an in vitro model for human neurones, expressed and
processed the hproNPFF
A
to generate SQA-NPFF, NPFF
and NPAF. These results showed for the first time that three
different NPFF-related active peptides, NPFF, SQA-NPFF

and NPAF, could be generated by intracellular processing
of hproNPFF
A
in the human neuroblastoma. The presence
of some of these peptides has not been described previously.
Only NPFF was clearly detected in COS-7 cells while in the
mouse spinal cord, SPA-NPFF was detected in addition to
NPFF.
These data were obtained by the combination of sensitive
complementary methods, in particular mass spectrometry,
which has permitted the precise identification of the
different NPFF-related peptides. Nanospray ionization
and MS/MS analyses allowed the identification of femto-
moles quantities of deduced NPFF-related peptides enco-
ded by mouse proNPFF
A
, in particular SPA-NPFF, which
was not previously detected in spinal cord with MS analyses
[31]. This study exemplified that on-line capillary HPLC/
nanospray ion trap tandem mass spectrometry was a
powerful analytical technique, giving rise to the character-
ization of minute amounts of endogenous neuropeptides
[32].
Fig. 7. HPLC profile of NPFF-immunoreactivity in mouse cervical
spinal cord extract. Acid extract prepared from three cervical spinal
cord segments (106 mg) was purified on a Sep-Pack Cartridge, applied
on a C8 column and separated on gradient 1 at a flow rate of
400 lLÆmin
)1
. NPFF-IR was assessed by radioimmunoassay. Elution

positions of synthetic NPFF-related peptides are indicated by arrows.
Ó FEBS 2003 Processing of proNeuropeptide FF
A
(Eur. J. Biochem. 270) 4195
The physiological relevance of these observations is that
the three peptides, NPFF, SQA-NPFF and hNPAF, could
act as neurotransmitters in human as they exhibit a high
affinity and a high activity towards NPFF
2
receptors
[26,42]. We have compared previously the affinities and
antiopioid activities of the different peptides putatively
produced by the rat NPFF precursor and reveal that the
undecapeptides are likely to be the physiologically active
Fig. 8. Identification of SPA-NPFF, NPFF and QFW-NPSF in mouse spinal cord extract. HPLC fractions corresponding to the NPFF-IR peaks 1
and 2 on gradient 1 were separated on a C18 column, as previously described [31]. Collected fractions corresponding to the retention time of NPFF,
SPA-NPFF and QFW-NPQF were analyzed by capillary HPLC/nanospray ion trap MS/MS. Reconstructed ion chromatograms of the fragment
ion at m/z 546.3 generated during the MS/MS analysis of double-charged precursor ions at m/z 541.3 from 100 fmol of synthetic NPFF (A), at m/z
668.9 from 100 fmol of synthetic SPA-NPFF (D), at m/z 675.4 from 100 fmol of synthetic QFW-NPSF (G), at m/z 541.3 from HPLC fractions of
NPFF-IR peak 1 (B), at m/z 668.9 (E) and at m/z 675.4 (H) from HPLC fractions of NPFF-IR peak 2 of tissue extracts. MS/MS spectra of the
[M + 2H]
2+
precursor ions at m/z 541.3 (C), at m/z 668.9 (F) and at m/z 675.4 (I) of tissue extract.
4196 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
neurotransmitters in brain as they exhibit high affinity for
NPFF
2
receptor, functional activity and they could be
generated from proNPFF
A

precursor in neuronal cells.
NPFF and NPA-NPFF exhibited a very high affinity for
NPFF
2
receptor of the rat spinal cord in contrast to shorter
peptides such as NPSF [42]. Similarly NPFF and NPA-
NPFF maximally reduce the inhibitory effect of nociceptin
on the [Ca
2+
]
i
transients triggered by depolarization in the
dorsal raphe
´
while NPSF was inactive in the same test.
Isolation of the octapeptide NPFF in cell extracts was
surprising, insofar that no consensus cleavage site by the
protein convertases was designed at its N-terminal extremity
(Table 1). Thus, different possibilities may be considered to
explain the generation of NPFF: NPFF could be generated
by processing of the hproNPFF
A
by enzymes recognizing
a nonbasic motif. This hypothesis is supported by the
presence of a conserved motif -RXXAFL- that extend the
N-terminal sequence of NPFF in mammals and recent
reports demonstrating the importance of a protein conver-
tase, the subtilisin/kexin isozyme SKI-1, in the processing of
prohormones at specific nonbasic residues [43]. The neces-
sity of an arginine residue at the P4 position and the fact

that an alanine at P1 or phenylalanine at P¢1 position did
not affect the cleavage further support this possibility [44].
NPFF was also isolated from extracts of non-neuronal
COS-7 cells indicating that processing of hproNPFF
A
at a
specific nonbasic residue should be considered to explain the
presence of NPFF in cell extracts. In contrast, degradation
experiments performed with COS-7 lysates showed a
degradation of SQA-NPFF into NPFF. Thus, the cleavage
of SQA-NPFF by membrane-associated or cytosolic
enzymes could explain the presence of NPFF in cells. In a
previous study, we have shown that the degradation of
synthetic SQA-NPFF, observed in mouse brain slices,
produces only a low amount of NPFF [45] and that the
degradation of the synthetic SQA-NPFF was not in
agreement with a sequential enzymatic mechanism, sug-
gesting rather the action of a serine protease, like a
tripeptidyl peptidase (TPP). A similar enzyme could be
involved in the degradation of synthetic SQA-NPFF in
COS-7 cells. In fact, our data demonstrate that the SQA-
NPFF degradation was partially prevented by protease
inhibitors, like phenylmethanesulfonyl fluoride, which effi-
ciently inhibits tripeptidyl peptidase II (TPP II) activity.
Moreover, TPP II, which cleaves at the third peptidic bond
from N-terminal extremity, has a slight preference for a
hydrophobic residue at the P1 position [46]. All these
observations strongly support the involvement of the TPP II
in the production of NPFF. However, tripeptidyl peptidases
could further degrade NPFF-derived undecapeptides dur-

ing exocytosis from brain neurones in mammals. In that
case, the NPFF in vivo activity could be mediated by an
active metabolite of mature NPFF-related peptides [47].
The processing of proneuropeptide and prohormones
occurs in most cases in subcellular compartments within the
intracellular secretory pathways [39,40]. It seems likely that
NPFF, isolated from SH-SY5Y and COS-7 cells, was
produced in specific compartments, where are localized
peptidylglycine-a amidating monooxygenase, carboxy-
peptidases and proteins convertases [29,48] enzymes likely
to be key for the processing of proNPFF
A
to the amidated
NPFF-related peptides. In this respect, two enzymatic
pathways for the production of NPFF should be consid-
ered.
Clearly COS-7 and SH-SY5Y cells generate different
proNPFF
A
fragments indicating that a neuronal cell line
possess a capacity of processing not found in non-neuronal
cells. The undecapeptide generated in a neuronal cell line
such as SH-SY5Y corresponds to the activity of known
maturation enzymes. As these undecapeptides are active
peptides, this processing pathway is likely to be similar to
the one present in neurones in vivo. In contrast, NPFF,
which is observed in mouse and rat tissues as well as in both
cell lines tested, could correspond to a metabolite of the
undecapeptide or to a non-neuronal production by tripep-
tidylpeptidase.

The high concentration of SQA-NPFF observed in
SH-SY5Y cells, relative to NPFF, further supports the
possibility that the long peptides are synthesized before
degradation into NPFF. In contrast in non-neuronal cells,
NPFF is present at a concentration similar to that of SQA-
NPFF, suggesting that another processing pathway is
responsible for NPFF synthesis in non-neuronal cells.
In mice, the long form SPA-NPFF contributes only
weakly to the total NPFF immunoreactivity. This could
be due, as in COS-7 cells, to the fact that a majority of
peptides observed in tissues correspond to a degradation
into shorter forms such as NPFF and represents for a
great part an extracellular pool. An important point is the
discovery in mice of SPA-NPFF that displays a high
affinity and a high activity towards NPFF
2
receptors. The
presence of this longer peptide in the brain suggests that it
could be, with NPFF, an active neurotransmitter pro-
duced by neurones.
Acknowledgements
This work was supported by the Centre National de la Recherche
Scientifique (CNRS) and grants from the Association pour la
Recherche contre le Cancer (ARC 4523) and The Re
´
gion Midi-
Pyre
´
ne
´

es. We thank F. Noble for kindly providing SH-SY5Y cells.
References
1. Roumy, M. & Zajac, J.M. (1998) Neuropeptide FF, pain and
analgesia. Eur. J. Pharmacol. 345, 1–11.
2. Panula, P., Kalso, E., Nieminen, M., Kontinen, V.K., Brandt, A.
& Pertovaara, A. (1999) Neuropeptide FF and modulation of
pain. Brain Res. 848, 191–196.
3. Perry, S.J., Huang, E.Y., Cronk, D., Bagust, J., Sharma, R.,
Walker, R.J., Wilson, S. & Burke, J.F. (1997) A human gene
encoding morphine modulating peptides related to NPFF and
FMRFamide. FEBS Lett. 409, 426–430.
4. Vilim, F.S., Aarnisalo, A.A., Nieminen, M.L., Lintunen, M.,
Karlstedt,K.,Kontinen,V.K.,Kalso,E.,States,B.,Panula,P.&
Ziff, E. (1999) Gene for pain modulatory neuropeptide NPFF:
induction in spinal cord by noxious stimuli. Mol. Pharmacol. 55,
804–811.
5. Hinuma, S., Shintani, Y., Fukusumi, S., Iijima, N., Matsumoto,
Y.,Hosoya,M.,Fujii,R.,Watanabe,T.,Kikuchi,K.,Terao,Y.,
Yano, T., Yamamoto, T., Kawamata, Y., Habata, Y., Asada, M.,
Kitada, C., Kurokawa, T., Onda, H., Nishimura, O., Tanaka, M.,
Ibata, Y. & Fujino, M. (2000) New neuropeptides containing
carboxy-terminal RFamide and their receptor in mammals. Nat.
Cell Biol. 2, 703–708.
Ó FEBS 2003 Processing of proNeuropeptide FF
A
(Eur. J. Biochem. 270) 4197
6. Yang, H.Y., Fratta, W., Majane, E.A. & Costa, E. (1985) Iso-
lation, sequencing, synthesis, and pharmacological characteriza-
tion of two brain neuropeptides that modulate the action of
morphine. Proc. Natl Acad. Sci. USA 82, 7757–7761.

7. Gelot, A., Frances, B., Gicquel, S. & Zajac, J.M. (1998) Antisense
oligonucleotides to human SQA-neuropeptide FF decrease mor-
phine tolerance and dependence in mice. Eur. J. Pharmacol. 358,
203–206.
8. Kavaliers, M. & Yang, H.Y. (1989) IgG from antiserum against
endogenous mammalian FMRF-NH
2
-related peptides augments
morphine- and stress-induced analgesia in mice. Peptides. 10,
741–745.
9. Gouarderes, C., Sutak, M., Zajac, J.M. & Jhamandas, K. (1993)
Antinociceptive effects of intrathecally administered F8Famide
and FMRFamide in the rat. Eur. J. Pharmacol. 237, 73–81.
10. Gouarderes, C., Jhamandas, K., Sutak, M. & Zajac, J.M. (1996)
Role of opioid receptors in the spinal antinociceptive effects of
neuropeptide FF analogues. Br. J. Pharmacol. 117, 493–501.
11. Ballet, S., Mauborgne, A., Gouarderes, C., Bourgoin, A.S., Zajac,
J.M., Hamon, M. & Cesselin, F. (1999) The neuropeptide FF
analogue, 1DME, enhances in vivo met-enkephalin release from
the rat spinal cord. Neuropharmacology 38, 1317–1324.
12. Mauborgne, A., Bourgoin, S., Polienor, H., Roumy, M., Simon-
net, G., Zajac, J.M. & Cesselin, F. (2001) The neuropeptide FF
analogue, 1DMe, acts as a functional opioid autoreceptor anta-
gonist in the rat spinal cord. Eur. J. Pharmacol. 430, 273–276.
13. Bonnard, E., Mazarguil, H. & Zajac, J.M. (2002) Peptide nucleic
acids targeted to the mouse proNPFF(A) reveal an endogenous
opioid tonus. Peptides. 23, 1107–1113.
14. Malin,D.H.,Lake,J.R.,Fowler,D.E.,Hammond,M.V.,Brown,
S.L., Leyva, J.E., Prasco, P.E. & Dougherty, T.M. (1990) FMRF-
NH

2
-like mammalian peptide precipitates opiate-withdrawal
syndrome in the rat. Peptides. 11, 277–280.
15. Malin, D.H., Lake, J.R., Arcangeli, K.A.R., Deshotel, K.D.,
Hausam, D.D., Witherspoon, W.E., Carter, V.A., Yang, H Y.T.,
Pal, B. & Burgess, K. (1993) Subcutaneous injection of an analog
of neuropeptide FF precipitates morphine abstinence syndrome.
Life Sci. 53, 261–266.
16. Desprat, C. & Zajac, J.M. (1997) Hypothermic effects of neuro-
peptide FF analogues in mice. Pharmacol. Biochem. Behav. 58,
559–563.
17. Frances, B., Lahlou, H. & Zajac, J.M. (2001) Cholera and per-
tussis toxins inhibit differently hypothermic and anti-opioid effects
of neuropeptide FF. Reg. Pept. 98, 13–18.
18. Sunter, D., Hewson, A.K., Lynam, S. & Dickson, S.L. (2001)
Intracerebroventricular injection of neuropeptide FF, an opioid
modulating neuropeptide, acutely reduces food intake and sti-
mulates water intake in the rat. Neurosci. Lett. 313, 145–148.
19. Murase,T.,Arima,H.,Kondo,K.&Oiso,Y.(1996)Neuro-
peptide FF reduces food intake in rats. Peptides 17, 353–354.
20. Takeuchi,T.,Fujita,A.,Roumy,M.,Zajac,J.M.&Hata,F.
(2001) Effect of 1DMe, a neuropeptide FF analog, on acetylcho-
line release from myenteric plexus of guinea pig ileum. Jpn
J. Pharmacol. 86, 417–422.
21. Laguzzi, R., Nosjean, A., Mazarguil, H. & Allard, M. (1996)
Cardiovascular effects induced by the stimulation of neuropeptide
FF receptors in the dorsal vagal complex: an autoradiographic
and pharmacological study in the rat. Brain Res. 711, 193–202.
22. Cikos, S., Gregor, P. & Koppel, J. (1999) Sequence and tissue
distribution of a novel G-protein-coupled receptor expressed

prominently in human placenta. Biochem. Biophys. Res. Commun.
256, 352–356.
23. Bonini, J.A., Jones, K.A., Adham, N., Forray, C., Artymyshyn,
R., Durkin, M.M., Smith, K.E., Tamm, J.A., Boteju, L.W.,
Lakhlani, P.P., Raddatz, R., Yao, W.J., Ogozalek, K.L., Boyle, N.,
Kouranova, E.V., Quan, Y., Vaysse, P.J., Wetzel, J.M., Branchek,
T.A., Gerald, C. & Borowsky, B. (2000) Identification and char-
acterization of two G protein-coupled receptors for neuropeptide
FF. J. Biol. Chem. 275, 39324–39331.
24. Elshourbagy, N.A., Ames, R.S., Fitzgerald, L.R., Foley, J.J.,
Chambers, J.K., Szekeres, P.G., Evans, N.A., Schmidt, D.B.,
Buckley, P.T., Dytko, G.M., Murdock, P.R., Milligan, G., Gro-
arke, D.A., Tan, K.B., Shabon, U., Nuthulaganti, P., Wang,
D.Y., Wilson, S., Bergsma, D.J. & Sarau, H.M. (2000) Receptor
for the pain modulatory neuropeptides FF and AF is an orphan
G protein-coupled receptor. J. Biol. Chem. 275, 25965–25971.
25. Kotani, M., Mollereau, C., Detheux, M., Le Poul, E., Brezillon,
S., Vakili, J., Mazarguil, H., Vassart, G., Zajac, J.M. & Parmen-
tier, M. (2001) Functional characterization of a human receptor
for neuropeptide FF and related peptides. Br. J. Pharmacol. 133,
138–144.
26. Mollereau, C., Mazarguil, H., Marcus, D., Quelven, I., Kotani,
M.,Lannoy,V.,Dumont,Y.,Quirion,R.,Detheux,M.,
Parmentier, M. & Zajac, J.M. (2002) Pharmacological character-
izationofhumanNPFF(1)andNPFF(2)receptorsexpressedin
CHO cells by using NPY Y(1) receptor antagonists. Eur. J.
Pharmacol. 451, 245–256.
27. Gouarderes, C., Quelven, I., Mollereau, C., Mazarguil, H., Rice,
S.Q. & Zajac, J.M. (2002) Quantitative autoradiographic dis-
tribution of NPFF1 neuropeptide FF receptor in the rat brain and

comparison with NPFF2 receptor by using [
125
I]YVP and
[
125
I]EYF as selective radioligands. Neuroscience 115, 349–361.
28. Nakayama, K., Watanabe, T., Nakayama, T., Kim, W., Nagah-
ama, M., Hosaka, H., Hatsuzawa, K., Kondoh-Hashiba, K. &
Murakami, K. (1992) Consensus sequence for precursor proces-
sing at mono-arginyl sites: evidence for the involvment of a Kex2-
like endoprotease in precursor cleavages at both dibasic and
mono-arginyl sites. J. Biol. Chem. 267, 16335–16340.
29. Seidah, N.G. & Chretien, M. (1999) Proprotein and prohormone
convertases: a family of subtilases generating diverse bioactive
polypeptides. Brain Res. 848, 45–62.
30. Yang, H Y. & Martin, B. (1995) Isolation and characterization of
a neuropeptide from brain and spinal cord of rat. Soc. Neurosci.
Abstr. 21, 760.
31. Bonnard, E., Burlet-Schiltz, O., Frances, B., Mazarguil, H.,
Monsarrat, B., Zajac, J.M. & Roussin, A. (2001) Identification of
neuropeptide FF-related peptides in rodent spinal cord. Peptides
22, 1085–1092.
32. Burlet-Schiltz, O., Mazarguil, H., Sol, J.C., Chaynes, P.,
Monsarrat, B., Zajac, J.M. & Roussin, A. (2002) Identification of
neuropeptide FF-related peptides in human cerebrospinal fluid by
mass spectrometry. FEBS Lett. 532, 313–318.
33. Gicquel, S., Mazarguil, H., Allard, M., Simonnet, G. & Zajac,
J.M. (1992) Analogues of F8Famide resistant to degradation, with
high affinity and in vivo effects. Eur. J. Pharmacol. 222, 61–67.
34. Dupouy, V. & Zajac, J.M. (1996) Neuropeptide FF receptors in

rat brain: a quantitative light- microscopic autoradiographic study
using [
125
I][D.Tyr1,(NMe)Phe
3
]NPFF. Synapse 24, 282–296.
35. Dowben, R.M., Gaffey, A. & Lynch, P.M. (1968) Isolation of liver
and muscle polyribosomes in high yield after cell disruption by
nitrogen cavitation. FEBS Lett. 2, 1–3.
36. Cezanne, L., Navarro, L. & Tocanne, J.F. (1992) Isolation of the
plasma membrane and organelles from Chinese hamster ovary
cells. Biochim. Biophys. Acta 1112, 205–214.
37. Seidah, N.G. & Chretien, M. (1997) Eukaryotic protein proces-
sing: endoproteolysis of precursor proteins. Curr. Opin. Bio-
technol. 8, 602–607.
38. Seidah, N.G., Benjannet, S., Hamelin, J., Mamarbachi, A.M.,
Basak,A.,Marcinkiewicz,J.,Mbikay,M.,Chretien,M.&Mar-
cinkiewicz, M. (1999) The subtilisin/kexin family of precursor
convertases: emphasis on PC1, PC2/7B2, POMC and the novel
enzyme SKI-1. Ann. NY Acad. Sci. 885, 57–74.
4198 E. Bonnard et al. (Eur. J. Biochem. 270) Ó FEBS 2003
39. Zhou, A., Webb, G., Zhu, X. & Steiner, D.F. (1999) Proteolytic
processing in the secretory pathway. J. Biol. Chem. 274, 20745–
20748.
40. Glombik, M.M. & Gerdes, H.H. (2000) Signal-mediated sorting
of neuropeptides and prohormones: secretory granule biogenesis
revisited. Biochimie 82, 315–326.
41. Zajac, J.M. (2001) Neuropeptide FF: new molecular insights.
Trends Pharmacol. Sci. 22, 63.
42. Roumy, M., Gouarderes, C., Mazarguil, H. & Zajac, J.M. (2000)

Are neuropeptides FF and SF neurotransmitters in the rat?
Biochem. Biophys. Res. Commun. 275, 821–824.
43. Seidah, N.G., Mowla, S.J., Hamelin, J., Mamarbachi, A.M.,
Benjannet, S., Toure, B.B., Basak, A., Munzer, J.S.,
Marcinkiewicz, J., Zhong, M., Barale, J.C., Lazure, C., Murphy,
R.A., Chretien, M. & Marcinkiewicz, M. (1999) Mammalian
subtilisin/kexin isozyme SKI-1: a widely expressed proprotein
convertase with a unique cleavage specificity and cellular locali-
zation. Proc. Natl Acad. Sci. USA 96, 1321–1326.
44. Duncan, E.A., Brown, M.S., Goldstein, J.L. & Sakai, J. (1997)
Cleavage site for sterol-regulated protease localized to a Leu-Ser
bond in the lumenal loop of sterol regulatory element-binding
protein-2. J. Biol. Chem. 272, 12778–12785.
45. Sol, J.C., Roussin, A., Proto, S., Mazarguil, H. & Zajac, J.M.
(1999) Enzymatic degradation of neuropeptide FF and SQA-
neuropeptide FF in the mouse brain. Peptides 20, 1219–1227.
46. Balow, R.M., Tomkinson, B., Ragnarsson, U. & Zetterqvist, O.
(1986) Purification, substrate specificity, and classification of tri-
peptidyl peptidase II. J. Biol. Chem. 261, 2409–2417.
47. Rose, C., Vargas, F., Facchinetti, P., Bourgeat, P., Bambal, R.B.,
Bishop, P.B., Chan, S.M.T., Moore, A.N.J., Ganellin, C.R. &
schwartz, J.C. (1996) Characterization and inhibition of a chole-
cystokinin-inactivating serine peptidase. Nature 380, 403–409.
48. Eipper, B.A., Milgram, S.L., Husten, E.J., Yun, H.Y. & Mains,
R.E. (1993) Peptidylglycine alpha-amidating monooxygenase: a
multifunctional protein with catalytic, processing, and routing
domains. Protein Sci. 2, 489–497.
49. Biemann, K. (1990) Appendix 5. Nomenclature for peptide frag-
ment ions (positive ions). Methods Enzymol. 193, 886–887.
Ó FEBS 2003 Processing of proNeuropeptide FF

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