Novel fish hypothalamic neuropeptideCloning of a cDNA encoding the precursor polypeptide and identification and localization of the mature peptide Kaori Sawada1,2, Kazuyoshi Ukena1,2, Ho
Trang 1Novel fish hypothalamic neuropeptide
Cloning of a cDNA encoding the precursor polypeptide and identification
and localization of the mature peptide
Kaori Sawada1,2, Kazuyoshi Ukena1,2, Honoo Satake3, Eiko Iwakoshi3, Hiroyuki Minakata3
and Kazuyoshi Tsutsui1,2
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-NH2motif 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-NH2from the goldfish brain,
by a combination of 3¢ and 5¢ rapid amplification of cDNA
ends (RACE) The deduced peptide precursor consisted of
197 amino acid residues, encoding three putative peptide
sequences that included -LPXRF (where X is L or Q) at their
C-termini Mass spectrometric analyses revealed that a
tri-decapeptide (SGTGLSATLPQRF-NH2) was derived from
the precursor in the brain as an endogenous ligand Southern
blotting analysis of reverse-transcriptase-mediated PCR
products demonstrated a specific expression of the goldfish peptide gene in the diencephalon In situ hybridization revealed the cellular localization of goldfish peptide mRNA
in the nucleus posterioris periventricularis in the hypotha-lamus Immunoreactive cell bodies were also restricted to the the nucleus posterioris periventricularis and the nervus ter-minalis and immunoreactive fibers were distributed in sev-eral brain regions including the nucleus latsev-eralis tuberis pars posterioris and pituitary Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C-terminal -LPQRF-NH2sequence, which may possess multiple regu-latory functions and act, at least partly, on the pituitary to regulate pituitary hormone release
Keywords: cDNA cloning; goldfish; hypothalamic neuro-peptide; in situ hybridization; mass spectrometry
Since the molluscan neuropeptide Phe-Met-Arg-Phe-NH2
(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-NH2(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-NH2) 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 (RP-1 and GnIH-RP-2), which contained a C-terminal -LPXRF-NH2(X is L
or Q) sequence [10] The chicken pentapeptide LPLRF-amide [2] may be a fragment of GnIH [9] Recently, we have further isolated an amphibian dodecapeptide (SLKPAANLPLRF-NH2) that is closely related to GnIH from the bullfrog hypothalamus [11] This peptide possessed growth hormone (GH)-releasing activity and was designa-ted as frog GH-releasing peptide (fGRP) [11] In addition, a gene database search [12] has determined cDNAs encoding
Correspondence to K Tsutsui, Laboratory of Brain Science,
Faculty of Integrated Arts and Sciences, Hiroshima University,
Higashi-Hiroshima 739–8521, Japan,
Fax: +81 824-24-0759, Tel.: +81 824-24-6571,
E-mail: tsutsui@hiroshima-u.ac.jp
Abbreviations: DIG, digoxigenin; fGRP, frog growth
hormone-releasing peptide; GnIH, gonadotropin-inhibitory hormone;
GnIH-RP, GnIH gene-related peptide; LPXRFamide, a peptide
containing a C-terminal -LPXRF-NH 2 (X is Leu or Gln); nano
ESI-TOF-MS, nanoflow electrospray ionization time-of-flight MS;
NLTp, nucleus lateralis tuberis pars posterioris; NPPv, nucleus
pos-terioris periventricularis; NT, nervus terminalis; PC, precursor
con-vertase; PrRP, prolactin-releasing peptide; PVO, paraventricular
organ; RACE, rapid amplification of cDNA ends; RFamide peptide, a
peptide containing a C-terminal Arg-Phe-NH 2 ; RFRP,
RFamide-related peptide; RT-PCR, reverse-transcriptase-mediated PCR; VT,
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)
Trang 2novel 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-NH2) as one of rodent
RFRPs [14] In addition, a pentatriacontapeptide (SLTFEE
VKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH2)
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-NH2
motif at their C-termini
The presence of novel neuropeptides featuring the
C-terminal -LPXRF-NH2 sequence (LPXRFamide
pep-tides) may be a conserved property of vertebrate brains, in
particular the hypothalamus In view of the
immunohisto-chemical finding indicating that some of FMRFamide-like
immunoreactive neurons project to an area close to or
within the pituitary of fish [16–18], we looked for novel
LPXRFamide peptides from the goldfish brain In the
present study, a cDNA encoding the LPXRFamide peptide
was characterized and subsequently a mature endogenous
peptide was identified in the goldfish brain The localization
of its transcript in the goldfish brain was further
investi-gated
M A T E R I A L S A N D M E T H O D S
RNA preparation
Adults goldfish (Carassius auratus) were kept in ordinary
water aquariums at 20 ± 2C and used in the present
study The experimental protocol was approved in
accord-ance with the Guide for the Care and Use of Laboratory
Animals prepared by Hiroshima University, Japan Total
RNA of the diencephalon was extracted by the guanidium
thiocyanate/phenol/chloroform extraction method followed
by the isolation of poly(A)+RNA with Oligotex-(dT) 30
(Daiichikagaku, Tokyo, Japan) in accordance with the
manufacturer’s instructions
Determination of the cDNA 3¢-end sequence
All PCR amplifications were carried out in a reaction
mixture containing Taq polymerase [Ex Taq polymerase
(Takara Shuzo, Kyoto, Japan) or gene Taq polymerase
(NIPPON GENE, Tokyo, Japan)] and 0.2 mMdNTP on a
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 94C, 30 s at
45C and 2.5 min at 72 C, and of 30 cycles for 30 s at
94C, 30 s at 50 C and 2.5 min at 72 C (5.5 min for last 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¢, complementary to neucleotides 431–450); this was followed
by further amplification of the first-round PCR products with the anchor primer and gene-specific primer 2 (5¢-AAA CCTTTGCGGTAGGGTGG-3¢, complementary to nucle-otides 416–435) Both first-round and second-round PCRs were performed for 35 cycles for 30 s at 94C, 30 s at 55 C and 1 min at 72C (11 min for last cycle) The second-round PCR products were subcloned and the inserts were amplified as described above
DNA sequencing All nucleotide sequences were determined with an ABI PRISMTMDye terminator cycle sequencing ready reaction kit (PE-Biosystems, Foster, CA, USA) and a model 373A automated DNA sequencer (PE-Biosystems), then analysed usingDNASIS-MACsoftware (Hitachi Software Engineering, Kanagawa, Japan) Universal M13 primers or gene-specific primers were used to sequence both strands
Northern blot hybridization
A digoxigenin (DIG)-labelled precursor polypeptide cDNA (complementary to nucleotides 6–623, including all open reading frames) was synthesized with a DNA-labelling kit (Roche Diagnostics) and used as a probe for Northern blot analysis mRNA was separated on a denaturing 1% (w/v) agarose/formaldehyde gel and fixed on a Hybond N+ 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 (n¼ 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 4C and the superna-tant was collected After precipitation with 75% acetone,
Trang 3the 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
4C and the adsorbed materials were eluted with 0.3M
acetic acid containing 0.1% 2-mercaptoethanol The eluted
fractions were concentrated and subjected to a
reversed-phase HPLC column (ODS-80TM; Tosoh, Tokyo, Japan)
with a linear gradient of 16–36% acetonitrile containing
0.1% trifluoroacetic acid for 100 min at a flow rate of
0.5 mLÆmin)1 The isolated immunoreactive substances
(1 mLeach) were then subjected to MS analyses as
described below
After evaporation of the isolated material, the residue was
dissolved in 50% methanol containing 0.1% formic acid
and the molecular mass was analysed by a nanoflow
electrospray ionization time-of-flight MS (nano
ESI-TOF-MS) (Q-TOF, Micromass, Wythenshawe, UK) as described
previously [10,14,19] The expected mass value of each
deduced peptide was calculated using the protein prospector
program (UCSF) and a corresponding peak was further
examined in a tandem MS analysis The needle voltage was
optimized at 1000 V and the cone voltage was set at 50 V
Argon was used as the collision gas and the energy was set at
28 V
Southern blot hybridization of RT-PCR products The first-strand cDNA was synthesized from total RNA (1 lg) prepared from each brain region with M-MLV reverse transcriptase (Promega, Madison, USA) and an oligo(dT) primer in accordance with the manufacturer’s instruction (Promega) The oligonucleotide primer set used for the amplification of peptide cDNA fragments was 5¢-CACCATCCTGCGACTTCAC-3¢ (identical with 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 94C, 1 min at 55 C and 1 min at 72 C in the 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/Pi;
pH 7.3) overnight at 4C Subsequently, brain tissues were placed in refrigerated 30% sucrose in NaCl/Pi until they settled Sections (10-lm thick) of the brain were made using
a cryostat at )20 C and were placed onto 3-amino-propyltriethoxysilane-coated slides In situ hybridization was carried out according to our previous method [20,21] using the labelled antisense RNA probe The DIG-labelled antisense RNA probe was produced with RNA labelling kit (Roche Diagnostics) from a part of the peptide precursor cDNA (complementary to nucleotides 6–623, including all open reading frames) Control for specificity of the in situ hybridization of the peptide mRNA was performed using the DIG-labelled sense RNA probe, which
is complementary to a sequence of antisense probe Competitive enzyme-linked immunosorbent assay (ELISA) Because the deduced peptide precursor encoded three putative LPXRFamide peptides [goldfish LPXRFamide peptide-1, -2 and -3 (see Results and Fig 1)], we synthesized these three peptides In this study, peptide levels in the brain were quantified by a competitive ELISA using the anti-fGRP serum [11] This anti-anti-fGRP serum was confirmed to recognize specifically three putative goldfish LPXRFamide peptides, as well as fGRP, by a competitive ELISA The
IC50values (concentrations yielding 50% displacement) in the competitive ELISA were estimated as follows; 0.46 pmol for goldfish LPXRFamide peptide-1, 3.43 pmol for goldfish LPXRFamide peptide-2, 1.13 pmol for goldfish
Fig 1 Nucleotide sequence and deduced amino acid sequence of the
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.
Trang 4LPXRFamide 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 37C 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
Brains were transversely or sagittally frozen-sectioned at
10 lm thickness on a cryostat at)20 C After blocking
of nonspecific binding components, the sections were
immersed with the anti-fGRP serum at a dilution of
1 : 1000 overnight at 4C and subsequently with
rhodam-ine-conjugated goat antirabbit IgG The specificity of the
staining was assessed by a substitution of the control serum
for the antiserum; in this control serum, the antiserum
(1 : 1000 dilution) was preabsorbed independently by
incubation overnight with the identified peptide, goldfish
LPXRFamide peptide-3, at a saturating concentration
(10 lgÆmL)1) Immunoreactive cell bodies and fibers in the
goldfish brain were studied using a Nikon fluorescence
microscope The structures of goldfish brain were identified
according to the goldfish brain atlas [24]
R E S U L T S
Characterization of a cDNA encoding the novel
goldfish LPXRFamide peptide precursor
To obtain novel LPXRFamide peptide precursor cDNA
fragments from the goldfish diencephalon, we initially
per-formed an RT-PCR experiment with degenerate primers
corresponding to the partial fGRP sequence and the anchor
primer, followed by reamplification of the first-round PCR
products with degenerate primers corresponding to the other
partial fGRP sequence and the same anchor primer Here,
the C-terminal amide group was thought to be derived from
a C-terminal Gly residue, which is known to be a typical
amidation signal [25,26] Electrophoresis of the nested PCR
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
similar to that of fGRP (L PL RF-NH) 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 bond, which is supported by the -3, -1 rule [27] As shown in Fig 1, the deduced precursor polypeptide consisted of 197 amino acid residues, encoding three putative sequences that included -LPXRF (X is L or Q) at their C-termini As the previous characterization of cDNAs encoding avian and mammalian LPXRFamide peptides, i.e GnIH [10], GnIH-RP-2 [10], rat RFGnIH-RP-2 [14] and bovine RFRP-1 [15], has shown that N-terminal cleavage sites of these peptides are between Arg and Ser/Ala, the sequences of mature goldfish LPXRFamide peptides are predicted as follows: SLE IEDFTLNVAPTSGRVSSPTILRLHPKITKPTHLHAN LPLRF-NH2 (goldfish LPXRFamide peptide-1), AKS NINLPQRF-NH2(goldfish LPXRFamide peptide-2), and SGTGLSATLPQRF-NH2 (goldfish LPXRFamide pep-tide-3) These predicted peptides are flanked on both ends
by single or pairs of endoproteolytic residues Arg (Fig 1) Glycine preceding the C-terminal cleavage site may serve as
a C-terminal amidation signal as described above [25,26] Northern blot analysis of poly(A)+ RNA detected a single band of 0.75 kb (Fig 2), suggesting that no alternatively spliced forms were present In addition, the apparent migration of 0.75 kb was in good agreement 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
Trang 5subjected 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-3 Therefore, the sequence was determined by a tandem
mass spectrometric analysis (Fig 3) Assignment of the
observed typical fragment ions, i.e N-terminal (b) and
C-terminal (y) ions, indicated that the amino acid sequence
of this peak was compatible with the sequence SGT
GLSATLPQRF-NH2 (Fig 3) In contrast, mature forms
corresponding to goldfish LPXRFamide peptide-1 and
-2 were not detected by the mass spectrometry which was
conducted twice using different samples
Expression of the novel goldfish LPXRFamide peptide
gene in different brain regions
The expression pattern of the novel goldfish LPXRFamide
peptide gene in four different regions of the brain was
determined by Southern blotting analysis of RT-PCR
products prepared from the telencephalon, diencephalon,
mesencephalon and rhombencephalon As an internal
control, we detected the expression of the gene encoding
goldfish b-actin in each brain region The goldfish b-actin
cDNA fragment with the size of 0.5 kb was amplified
with the primer set based on the goldfish b-actin gene
sequence in all brain tissues at a similar level (Fig 4C) In
contrast, a single hybridized band for the 230 bp RT-PCR
product between nucleotides 234–463 was detected
exclu-sively in the diencephalon (Fig 4A and B) We therefore conclude that goldfish LPXRFamide peptide(s) is biosyn-thesized exclusively in the diencephalon, which includes the hypothalamus
Cellular localization of novel goldfish LPXRFamide peptide mRNA in the diencephalon
In situhybridization 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+ ) by a tandem MS analysis The spectrum shows typical mass values of predicted tridecapeptide fragment ions (B) Observed N-terminal (b) and C-terminal (y) fragmentation ions are assigned in the sequence of the tridecapeptide, goldfish LPXRFamide peptide-3.
Fig 2 Transcript size of goldfish LPXRFamide peptide mRNA
Nor-thern blot analysis of mRNA prepared from the goldfish
diencepha-lon mRNA was purified from 50 lg total RNA of the goldfish
diencephalon and subjected to Northern blot hybridization with a
digoxigenin-labelled goldfish LPXRFamide peptide cDNA probe The
single positive band is indicated by an arrow The positions of RNA
molecular mass markers are shown on the left.
Trang 6(Fig 6B) To examine the precise peptide localization in the brain, we conducted immunohistochemical analysis In the diencephalon, abundant immunoreactive cell bodies were localized in the NPPv in the hypothalamus (Fig 7C and D) Interestingly, some of these immunoreactive cells may project to the nucleus lateralis tuberis pars posterioris (NLTp) in the diencephalon (Fig 8C) and to the pituitary (Fig 8D) In addition to the NPPv, some immunoreactive cell bodies were detected in the nervus terminalis (NT) of the olfactory bulb, which was characterized morphologically in the previous study [17] (Fig 7A) Immunoreactive fibers were present in the ventral telencephalon (VT) (Fig 8A)
Fig 6 Quantitation of goldfish LPXRFamide peptide(s) in different brain regions by ELISA (A) Peptide concentration per unit weight tissue (B) Peptide content per each brain tissue Each column and vertical line represent the mean ± SEM (n ¼ 6 samples, one sample from 10 fish) *P < 0.05, **P < 0.01 (vs rhombencephalon) by Duncan’s multiple range test.
Fig 5 Cellular localization of goldfish LPXRFamide peptide mRNAin
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.
Trang 7and 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
D I S C U S S I O N
As summarized in Table 1, all of the identified novel peptides in the brains of mammalian, avian and amphibian species include a -LPXRF-NH2sequence (X is Lor Q) at their C-termini (LPXRFamide peptides) [9–11,14,15] To determine whether the presence of L PXRFamide peptides
in the brain is a conserved property in vertebrates, we looked for novel fish LPXRFamide peptides having a similar C-terminal structure In the present study we first identified a cDNA encoding the novel fish LPXRFamide peptide from the goldfish diencephalon by a combination of 3¢ and 5¢ RACE We found that the precursor polypeptide encodes three putative LPXRFamide peptide sequences that are flanked on both ends by monobasic or dibasic endoproteolytic residues, Arg (Fig 1) Moreover, a series of mass spectrometric analyses verified the expression of goldfish LPXRFamide peptide-3
(SGTGLSATLPQRF-NH2) as a mature endogenous ligand From the previous [9–11,14,15] and present findings (see Table 1), it may be stated that the presence of LPXRFamide peptides is a generally conserved property in vertebrate brains On the other hand, the mature peptides corresponding to goldfish LPXRFamide peptide-1 and -2 were not detected in this study Because both putative goldfish LPXRFamide pep-tide-1 and -2 lack dibasic C-terminal cleavage sequences in contrast with goldfish LPXRFamide peptide-3, it is unlikely that these peptides are generated as mature forms However,
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.
Fig 7 Immunohistochemically labelled cell bodies in the goldfish brain.
Immunohistochemical staining of sagittal olfactory bulb (A and B) or
transverse diencephalic (C and D) sections of goldfish with the
anti-fGRP serum cross-reacted with deduced goldfish LPXRFamide
pep-tides (A, C and D) or with the antiserum preabsorbed with a saturating
concentration of the identified goldfish LPXRFamide peptide-3 (B).
The inset in (C) is shown magnified in (D) NPPv, nucleus posterioris
periventricularis; NT, nervus terminalis Scale bars (A–D), 200 lm.
Trang 8localized in the NPPv in the hypothalamus The control
study using sense RNA probe resulted in a complete
absence of the expression of goldfish LPXRFamide peptide
mRNA, suggesting the validity of the in situ hybridization
technique Interestingly, the NPPv cells expressing goldfish
LPXRFamide peptide mRNA stained specifically by the
antiserum that cross-reacted with the goldfish
LPXRF-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
with the antiserum used in this study Immunoreactive fibers
were also distributed in other brain regions, such as the VT
and OTec These findings are in harmony with the ELISA
data indicating that the peptide content was maximal in the
diencephalon and high in the telencephalon and
mesen-cephalon Although the telencephalon included many
immunoreactive fibers, the peptide content in the
telen-cephalon was lower than that in the dientelen-cephalon, due to
small tissue mass Judging from such a distribution pattern,
the goldfish LPXRFamide peptide may be multifunctional
as with other LPXRFamide peptides, e.g GnIH [9], fGRP
[11] and RFRPs [12–15,23] and other RFamide peptides,
e.g PrRP [8] and neuropeptide FF [7] Further experiments
are needed to understand the possible multiple regulatory
functions of the goldfish LPXRFamide peptide that was
identified in this study
A C K N O W L E D G E M E N T S
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture, Japan
(12440233, 12894021, 13210101 to K T and 12640669 to H M.) and
the SUNBOR Grant from Suntory Institute for Bioorganic Research,
Osaka, Japan (to K U.) We are grateful to Dr Miki Hisada (Suntory
Institute for Bioorganic Research, Osaka, Japan) for her valuable
discussion.
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