Identification of proNeuropeptide FFA peptides processedin neuronal and non-neuronal cells and in nervous tissue Elisabeth Bonnard, Odile Burlet-Schiltz, Bernard Monsarrat, Jean-Philippe
Trang 1Identification of proNeuropeptide FFA 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 proNPFFAcDNA 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, proNPFFA
and proNPFFB encoding peptides possessing the
PQRF-amide sequence, have been cloned in mammals [3,4] The
proNPFFA precursor at basic proteolytic sites should
generate two PQRFamide containing peptides [3] and the
proNPFFB, 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, NPFF1 and NPFF2, 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 proNPFFA
sequence for NPFF2 receptors binding, whereas proN-PFFB-derived peptides displayed a greater affinity for NPFF1 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 NPFF1 and NPFF2 receptor stimula-tion by peptides generated by proNPFFBand proNPFFA 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 proNPFFAprocessing In humans, bovines and rodents, proNPFFA 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 proNPFFA 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: jean-marie.zajac@ipbs.fr
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)
Trang 2peptide, 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 proNPFFA 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
proNPFFAin SH-SY5Y human neuroblastoma cells and
COS-7 (nonneuronal) cells transiently transfected with the
human proNPFFA 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]
hproNPFFAcDNA 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 hproNPFFAcDNA 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-hproNPFFA: 5¢-CGCAGATCTAGCATGGATT CTAGGCAGGCTGCTGC-3¢ and 3¢Apa-hproNPFFA: 5¢-GCGGGGCCCTTCTTCCCAAAGCGTTGAGGGG CAG-3¢, targeted to the 5¢- and 3¢-end of the hproNPFFA
coding sequence, respectively, in a PTC-150 MiniCycler (MJ Research Inc.), with 25 cycles consisting of 30 s at
94C and 30 s at 68 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) Three mice 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 0.1M
HCl, followed by boiling for 10 min Tissue homogenates were buffered to pH 7.4 with 2MTris pH 7.4, at a final concentration of 25 mg tissueÆmL)1 and centrifuged at
10 000 g for 10 min at 4C 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 105cells per dish Twenty-four hours later, the medium was removed; cells were washed with cold NaCl/Piand scraped
in 0.1MHCl 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)
Trang 310000 g for 20 min at 4C 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-NPFFAcDNA using
Lipofect-AMINE Reagent (Invitrogen) Cells were transfected with
4 lg per dish of pEGFP-hpro-NPFFA according to the
manufacturer’s instruction Forty hours later, the medium
was removed; cells were washed with cold NaCl/Pi,
extracted in 1M acetic acid, sonicated and centrifuged at
10 000 g for 20 min at 4C 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 4C 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 proNPFFA and proNPFFB, 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/Pi and incubated with NaCl/Pi plus
EDTA, 1 mM at 37C for 5 min Cells were then
centrifuged at 500 g, 5 min, resuspended in NaCl/Piand
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 37C for 10, 30, 60 120, 180 min with or without
protease inhibitors (phenylmethylsulfonyl fluoride, 2 mM,
bestatin, 0.1 mM) and 50 pmol of synthetic SQA-NPFF
At the end of the incubation period, cell lysates were
extracted into 1Macetic acid and centrifuged at 4C and
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 H2O/CH3CN (80 : 20 v/v) and eluted with 0.088% trifluoroacetic acid in H2O/CH3CN (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 H2O and solvent B was 0.088% trifluoroacetic acid in H2O/
CH3CN (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 lm Brownlee (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 PepMapTM (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 H2O/CH3CN (99 : 1 v/v) and B was 0.1% formic acid in H2O/CH3CN (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%
Trang 4RP-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 from the y and b ions The peptide sequence and fragmentation pattern for each peptide is indicated at the top.
Trang 5series of y and b fragment ions, from which the most
abundant was in each case the singly charged y4fragment
ion at m/z 546.3, corresponding to the C-terminal
tetrapep-tide PQRFamide These observations led us to consider the
y4fragment 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-NPFF and the third with hNPAF (Table 1)
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 y4 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 y4fragment 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 sepsep-arated 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)
Trang 6pattern 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 y4 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 peak 3 (E) and MS/MS spectra of the [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.
Trang 7the 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 y4 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.
Trang 8The 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 y4fragment
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 y4fragment 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 37C 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.
Trang 9(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 mMphenylmethanesulfonyl fluoride and
0.1 mMbestatin 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 y4fragment 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 fragmentafragmenta-tion
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 proNPFFAprocessing 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 proNPFFA 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 y4 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 NPFFAand NPFFB[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 proNPFFA 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 hproNPFFAto 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 hproNPFFAin 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 proNPFFA, 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.
Trang 10The 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 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.