Novel fish hypothalamic neuropeptide
Cloning of a cDNA encoding the precursor polypeptide and identification
and localization of the mature peptide
Kaori Sawada
1,2
, Kazuyoshi Ukena
1,2
, Honoo Satake
3
, Eiko Iwakoshi
3
, Hiroyuki Minakata
3
and Kazuyoshi Tsutsui
1,2
1
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, Japan,
2
Core Research of Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo, Japan and
3
Suntory Institute for Bioorganic Research, Wakayamadai 1-1-1, Shimamoto-cho, Mishima-gun, Osaka, Japan
Recently, we identified novel avian and amphibian
hypothalamic neuropeptides that inhibited gonadotropin
release and stimulated growth hormone release. They were
characterized by a similar structure including the C-terminal
LPLRF-NH
2
motif. To clarify that the expression of these
novel hypothalamic neuropeptides is a conserved property in
vertebrates, we characterized a cDNA encoding a similar
novel peptide, having LPLRF-NH

2
(FMRFamide) was found in the ganglia of the venus clam
[1], immunohistochemical studies using the antiserum
against FMRFamide suggested that the vertebrate hypo-
thalamus possesses some unknown neuropeptide similar to
FMRFamide. In fact, neuropeptides having the RFamide
motif at their C-termini (RFamide peptides) have been
identified in the brains of several vertebrates. For the first
time Leu-Pro-Leu-Arg-Phe-NH
2
(LPLRFamide), a chicken
pentapeptide, has been purified from the vertebrate brain
[2]. Two pain modulatory neuropeptides, NPFF and NPAF
[3], prolactin-releasing peptide (PrRP) [4] and its fish
counterpart, Carassius RFamide, [5] are also RFamide
peptides. To date, these RFamide peptides have been shown
to have important physiological roles in neuroendocrine,
behavioral, sensory and autonomic functions [6–8].
We have also identified a novel hypothalamic RFamide
peptide (SIKPSAYLPLRF-NH
2
) inhibiting gonadotropin
release in the quail brain and termed this dodecapeptide
gonadotropin-inhibitory hormone (GnIH) [9]. Subse-
quently, we have cloned a cDNA encoding GnIH from
the quail brain [10]. Interestingly, the GnIH cDNA encoded
GnIH and its related peptides (GnIH-RP-1 and GnIH-
RP-2), which contained a C-terminal -LPXRF-NH
2
(X is L

ventral telencephalon; OTec, optic tectum.
Note: The nucleotide sequence data are available in the DDBJ, EMBL
and GenBank Nucleotide Sequence Databases under the accession
number AB078976.
(Received 13 May 2002, revised 16 July 2002, accepted 24 July 2002)
Eur. J. Biochem. 269, 6000–6008 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03351.x
novel RFamide-related peptides (RFRPs) similar to GnIH
and fGRP in mammalian brains and the deduced RFRPs
have been suggested to participate in neuroendocrine [12]
and pain mechanisms [13] in the rat. Two peptides have
been predicted to be encoded in the cDNA of rodent
RFRPs. More recently, we have identified an octadecapep-
tide (ANMEAGTMSHFPSLPQRF-NH
2
) as one of rodent
RFRPs [14]. In addition, a pentatriacontapeptide (SLTFEE
VKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH
2
)
has been isolated as one of the bovine RFRPs [15].
Collectively, these peptides identified from the brain of
several vertebrates are characterized by the LPXRF-NH
2
motif at their C-termini.
The presence of novel neuropeptides featuring the
C-terminal -LPXRF-NH
2
sequence (LPXRFamide pep-
tides) may be a conserved property of vertebrate brains, in
particular the hypothalamus. In view of the immunohisto-

thermal cycler (Program Temp Control System PC-700,
Astec, Fukuoka, Japan). First-strand cDNA was synthes-
ized with the oligo(dT)-anchor primer supplied in the 5¢/3¢
rapid amplification of cDNA ends (RACE) kit (Roche
Diagnostics, Basal, Switzerland) and amplified with the
anchor primer (Roche Diagnostics) and the first degenerate
primers 5¢-(T/C)TIAA(A/G)CCIGCIGCIAA(T/C)(T/C)
TICC-3¢ (I represents inosine), corresponding to the fGRP
sequence sequence, Leu2-Lys3-Pro4-Ala5-Ala6-Asn7-Leu8-
Pro9 [11]. First-round PCR products were reamplified with
the anchor primer and the second degenerate primers
5¢-GCIAA(T/C)(T/C)TICCI(T/C)TI(A/C)GITT(T/C)GG-3¢,
corresponding to the fGRP Ala6-Asn7-Leu8-Pro9-Leu10-
Arg11-Phe12-Gly13 [11]. Both first- and second-round
PCRs consisted of five cycles for 30 s at 94 °C, 30 s at
45 °C and 2.5 min at 72 °C, and of 30 cycles for 30 s at
94 °C, 30 s at 50 °C and 2.5 min at 72 °C(5.5minforlast
cycle). The second-round PCR products were subcloned
into a TA-cloning vector in accordance with the manufac-
turer’s instructions (Invitrogen, San Diego, CA, USA). The
DNA inserts of the positive clones were amplified by PCR
with universal M13 primers.
Determination of the cDNA 5¢-end sequence
Template cDNA was synthesized with an oligonucleotide
primer complementary to nucleotides 444–463 (5¢-GGTCT
AAAGGAAATATGTTC-3¢), followed by dA-tailing of
the cDNA with dATP and terminal transferase (Roche
Diagnostics). The tailed cDNA was amplified with the
oligo(dT)-anchor primer (Roche Diagnostics) and gene-
specific primer 1 (5¢-TATGTTCCTCCTCCCAAACC-3¢,

+
membrane (Amersham Pharmacia Life Science, Uppsala,
Sweden) by UV irradiation. Hybridization and detection
were performed in accordance with the manufacturer’s
standard procedure (Roche Diagnostics). RNA size was
estimated with the use of DIG-labelled RNA molecular
markers (Roche Diagnostics).
Immunoaffinity purification and mass spectrometry (MS)
To identify endogenous mature peptides in the brain, we
carried out affinity purification and immunoassay with the
antiserum raised against fGRP, which cross-reacted with
three putative peptides [goldfish LPXRFamide peptide-1,
-2 and -3 (see Results and Fig. 1)]. Brains (n ¼ 200) were
boiled for 7 min and homogenized in 5% acetic acid as
described previously [9,11,14]. The homogenate was cen-
trifuged at 15 000 g for 20 min at 4 °C and the superna-
tant was collected. After precipitation with 75% acetone,
Ó FEBS 2002 Novel fish hypothalamic neuropeptide (Eur. J. Biochem. 269) 6001
the supernatant was passed through a disposable C-18
cartridge column (Mega Bond-Elut; Varian, Harbor, CA,
USA) and the retained material eluted with 60% methanol
was loaded onto an immunoaffinity column. The affinity
chromatography was carried out as described elsewhere
[3,14]. The antibodies against fGRP were conjugated to
CNBr-activated Sepharose 4B as an affinity ligand. The
brain extract was applied to the immunoaffinity column at
4 °C and the adsorbed materials were eluted with 0.3
M
acetic acid containing 0.1% 2-mercaptoethanol. The eluted
fractions were concentrated and subjected to a reversed-

nucleotides 234–252) and 5¢-GGTCTAAAGGAAATAT
GTTC-3¢ (complementary to nucleotides 444–463); primers
for the amplification of b-actin cDNA fragments were
5¢-CTACAACGAGCTGCGTGTTG-3¢ (identical with
nucleotides 296–315 in the goldfish b-actin gene, gb
AB039726) and 5¢-TGCCAATGGTGATGACCTGC-3¢
(complementary to nucleotides 761–780 in the goldfish
b-actin gene). PCR was performed for 30 cycles consisting
of 1 min at 94 °C, 1 min at 55 °C and 1 min at 72 °Cinthe
PCR reaction as described above. PCR products were
resolved on a 1.5% (w/v) agarose gel followed by transfer to
a Hybond N
+
membrane (Amersham Pharmacia Life
Science). The membrane was hybridized with DIG-labelled
oligonucleotide probe (5¢-AAACCTTTGCGGTAGGG
TGG-3¢, complementary to nucleotides 416–435). DIG-
DNA labelling and detection were performed in accordance
with the DIG system protocol (Roche Diagnostics).
In situ
hybridization
In the present study, the site of the peptide mRNA
expression in the brain was further localized by in situ
hybridization. Adult goldfish were killed by decapitation
and brains were immediately immersion-fixed in 4%
paraformaldehyde in phosphate-buffered saline (NaCl/P
i
;
pH 7.3) overnight at 4 °C. Subsequently, brain tissues were
placed in refrigerated 30% sucrose in NaCl/P

goldfish LPXRFamide peptide precursor cDNA. The sequences of
putative goldfish LPXRFamide peptides are boxed. Single or pairs of
basic amino acids as cleavage sites are shown in bold. The poly(A)
adenylation signal AGTAAA is underlined.
6002 K. Sawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002
LPXRFamide peptide-3, 0.74 pmol for fGRP, 20.96 pmol
for chicken RFamide (LPLRFamide) and more than
1000 pmol for other RFamide peptides, e.g. Carassius
RFamide (SPEIDPFWYVGRGVRPIGRFamide) and
molluscan RFamide (FMRFamide).
Acetic acid extracts derived from different brain regions
of the adult goldfish were passed through disposable C-18
cartridges (Sep-pak; Waters, Milford, MA, USA) and the
retained material was subjected to the competitive ELISA as
described previously [9,22,23]. In brief, different concentra-
tions of the standard peptide, fGRP (0.01–100 pmolÆmL
)1
),
or adjusted tissue extracts were added together with the
antiserum (1 : 1000 dilution) to each well of a 96-well
microplate and incubated for 1 h at 37 °C. After the
reaction with alkaline phosphatase-labelled goat anti-rabbit
IgG, immunoreactive products were obtained in substrate
solution of p-nitrophenyl-phosphate and the absorbance
was measured at 415 nm on a microtiter plate reader (MTP-
120, CORONA, Ibaragi, Japan).
Immunohistochemistry
Immunohistochemical analysis was performed as described
previously [9,11,22,23]. In brief, adult goldfish were killed by
decapitation, and brains were fixed as described above.

mixture revealed a major product of  0.5 kb (results not
shown). The predicted amino acid sequence included two
copies of the potential peptide sequence, LPQRFG, down-
stream of the partial fGRP sequence derived from the
second-round PCR primer, implying that this cDNA clone
encoded also a peptide including a C-terminal sequence
similartothatoffGRP(LPLRF-NH
2
). To determine the
5¢-end sequence, we performed 5¢ RACE with specific
primers for the clone. A single product of  0.45 kb (results
not shown) was obtained and sequenced, revealing that
these cDNA clones contained a LPLRFG sequence. The
entire novel goldfish LPXRFamide peptide precursor
cDNA was identified by combining nucleotide sequences
determined by these RACE experiments. As shown in
Fig. 1, the peptide precursor cDNA was composed of 742
nucleotides containing a short 5¢-untranslated sequence of
15 bp, a single open reading frame of 591 bp, and a
3¢-untranslated sequence of 136 bp with the addition of
various lengths of poly(A) tail. The open reading frame
region began with a start codon at position 16 and
terminated with a TAA stop codon at position 607. A
single polyadenylation signal (AGTAAA) was found in the
3¢-untranslated region at position 721. We predicted that the
goldfish LPXRFamide peptide transcript would be trans-
lated with Met1, because a hydropathy plot analysis of the
precursor demonstrated that the most hydrophobic moiety,
which is typical in a signal peptide region, followed Met1.
The cleavage site of the signal peptide was the Gly12-Thr13

with the estimated length of the cDNA, 742 bp. This result
indicates that the cDNA clone includes a full-length
nucleotide sequence encoding the precursor of novel gold-
fish LPXRFamide peptides.
Detection of a novel goldfish LPXRFamide peptide
in the brain
As shown in Fig. 1, three LPXRFamide peptides (goldfish
LPXRFamide peptide-1, -2 and -3) were predicted to be
encoded in the cDNA. In the present study, we further
investigated naturally occurring LPXRFamide peptides in
the brain by immunoaffinity purification combined with
mass spectrometry. Acetic acid extracts of goldfish brains
were passed through a disposable C-18 reversed-phase
cartridge column. The retained material, eluted with 60%
methanol, was then subjected to an affinity chromatography
with the anti-fGRP serum which cross-reacted with three
deduced goldfish LPXRFamide peptides as well as fGRP
(see Materials and methods). The eluted fractions were
Ó FEBS 2002 Novel fish hypothalamic neuropeptide (Eur. J. Biochem. 269) 6003
subjected to the reversed-phase HPLC purification, and the
eluate was fractionated every 2 min.
Each isolated substance was then examined by mass
spectrometry. The mass values of predicted peptides were
calculated on the basis of the sequence of goldfish prepro-
protein. On the nano ESI-TOF-MS, a molecular ion peak in
the spectrum of the substance eluted at 36–38 min was
667.35 m/z ([M + 2H]
2+
). This value was identical to the
mass number calculated for goldfish LPXRFamide peptide-

peptide mRNA in the diencephalon
In situ hybridization of the goldfish LPXRFamide peptide
mRNA was examined in the brain using RNA probe with
sequences complementary to the precursor mRNA.
Expression was detected finally by enzyme immunohisto-
chemistry. An intense expression of goldfish LPXRFamide
peptide mRNA was detected only in the nucleus posterioris
periventricularis (NPPv) in the hypothalamus (Fig. 5A and
C). The control study using sense RNA probe resulted in a
complete absence of the expression of goldfish LPXRF-
amide peptide mRNA in the NPPv (Fig. 5B), suggesting
that the reaction was specific for goldfish LPXRFamide
peptide mRNA. Furthermore, in the serial section the NPPv
cells expressing goldfish LPXRFamide peptide mRNA
(Fig. 5C) were also stained by the anti-fGRP serum cross-
reacted with three deduced peptides including the identified
one, goldfish LPXRFamide peptide-3 (Fig. 5D).
Distribution of novel goldfish LPXRFamide peptide(s)
in the brain
In the present study, goldfish LPXRFamide peptide(s) was
further localized in the brain. As measured using ELISA,
the peptide concentration was greater in the diencephalon
and telencephalon than in other brain regions (Fig. 6A).
The peptide content per region was maximal in the
diencephalon and minimal in the rhombencephalon
Fig. 3. Detection of a goldfish LPXRFamide peptide in the goldfish
brain by tandem MS. (A) Fragmentation patterns of the purified
substance with the observed mass number of 667.35 m/z
([M + 2H]
2+

Fig. 5. Cellular localization of goldfish LPXRFamide peptide mRNA in
the goldfish brain. The expression of goldfish LPXRFamide peptide
mRNA was localized by in situ hybridization. Cellular localization of
goldfish LPXRFamide peptide mRNA expression in the NPPv on
transverse (A) or sagittal (C) hypothalamic sections of goldfish.
Hybridization of goldfish LPXRFamide peptide mRNA by the sense
probe (control) on transverse (B) brain sections. Immunohistochemical
staining on sagittal brain sections (D) of goldfish with the antifGRP
serum cross-reacted with deduced goldfish LPXRFamide peptides.
Scale bars (A–D), 50 lm.
Fig. 4. Localized expression of goldfish LPXRFamide peptide mRNA.
RT-PCR analysis together with Southern hybridization of goldfish
LPXRFamide peptide mRNA in different brain regions of the gold-
fish. (A) Gel electrophoresis of RT-PCR products for goldfish
LPXRFamide peptide mRNA. (B) Identification of the band by
Southern hybridization using digoxigenin-labelled oligonucleotide
probe for goldfish LPXRFamide peptide cDNA corresponding to
1 lg total RNA extracted from the brain was used for a PCR reaction.
(C) RT-PCR for goldfish b-actin as the internal control, in which
cDNA corresponding to 10 ng total RNA was used as template.
Ó FEBS 2002 Novel fish hypothalamic neuropeptide (Eur. J. Biochem. 269) 6005
and the optic tectum (OTec) in the mesencephalon (Fig. 8B)
as well as the NLTp (Fig. 8C) and the pituitary (Fig. 8D). A
few immunoreactive fibers were also scattered in other brain
regions. As shown in the olfactory bulb (Fig. 7B), a
complete absence of such an immunoreaction in all of the
positively stained cell bodies and fibers was observed by
preabsorbing the antiserum with an excess of synthetic
goldfish LPXRFamide peptide-3.
DISCUSSION

we cannot rule out the possibility that premature goldfish
LPXRFamide peptide-1 and -2 are subjected to further
processing and modification or that these two predicted
peptides are present below the detectable levels for the
present mass spectrometric analysis.
The present RT-PCR analysis together with Southern
hybridization indicated a specific expression of the goldfish
LPXRFamide peptide gene in the diencephalon, suggesting
a regional difference in the expression. Identification of the
cells expressing goldfish LPXRFamide peptide mRNA in
the brain must be taken into account when studying the
neuropeptide action. We therefore characterized the site
showing the expression of goldfish LPXRFamide peptide
mRNA by in situ hybridization. The expression was
Fig. 8. Immunohistochemically labelled fibers in the goldfish brain.
Immunohistochemical staining of transverse telencephalic (A),
mesencephalic (B) and diencephalic (C) or sagittal pituitary (D) sec-
tions of goldfish with the antifGRP serum cross-reacted with deduced
goldfish LPXRFamide peptides. VT, ventral telencephalon; Otec,
optic tectum; NLTp, nucleus lateralis tuberis pars posterioris; C,
cerebellum. Scale bars (A–D), 200 lm. Arrows show immunoreactive
fibers.
Table 1. Novel neuropeptides including the C-terminal LPXRF-NH
2
motif in vertebrate brains.
Sequence Animal Name Reference
SLTFEEVKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH
2
Bovine RFRP-1 [15]
ANMEAGTMSHFPSLPQRF-NH

amide peptide. Because preadsorption of the antiserum with
the synthetic goldfish LPXRFamide peptide-3, which was
identified by the mass spectrometric analysis, resulted in a
complete disappearance of the reaction product, the immu-
nohistochemical staining was considered to be specific for
the peptide. A striking observation in the immunohisto-
chemical experiment was the distribution of stained cell
bodies and fibers in the diencephalic region. Immunoreac-
tive cell bodies and fibers were localized in the NPPv and the
NLTp, respectively. In addition, some of immunoreactive
fibers projected to the pituitary gland. These immunohisto-
chemical findings are in good agreement with the previous
findings, indicating that FMRFamide-like immunoreactive
cells project to an area close to or within the pituitary of fish
[16–18]. It has been demonstrated that the paraventricular
organ (PVO) including the NPPv is a source of pituitary
afferents in the goldfish [28]. The NLTp is known to be
involved in the control of pituitary functions in the teleost
[29]. Taken together, these results suggest that goldfish
LPXRFamide peptide-3 identified here acts at least partly
on the pituitary to regulate pituitary hormone secretion, like
GnIH [9], fGRP [11] and RFRP [12].
In addition to the NPPv, we found immunoreactive cell
bodies in the NT. However, the goldfish LPXRFamide
peptide mRNA signal was detected only in the NPPv. The
present in situ hybridization did not detect the signal in the
NT, which may be due to the low expression of goldfish
LPXRFamide peptide mRNA. Otherwise, the localization
of immunoreactive cell bodies in the NT may suggest the
presence of other undiscovered peptide(s) which cross-react

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