Báo cáo Y học: Novel fish hypothalamic neuropeptide Cloning of a cDNA encoding the precursor polypeptide and identification and localization of the mature peptide pptx

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 from 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-NH 2 ) 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 lateralis tuberis pars posterioris and pituitary. Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C-terminal -LPQRF-NH 2 sequence, 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-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 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-NH 2 ) 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) 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- 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. MATERIALS AND METHODS RNA preparation Adults goldfish (Carassius auratus)werekeptinordinary water aquariums at 20 ± 2 °C 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 m M dNTP 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 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¢, 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 94 °C, 30 s at 55 °C and 1 min at 72 °C (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 PRISM TM Dye terminator cycle sequencing ready reaction kit (PE-Biosystems, Foster, CA, USA) and a model 373A automated DNA sequencer (PE-Biosystems), then analysed using DNASIS - MAC software (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 (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- 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 mL each) 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 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 i until they settled. Sections (10-lm thick) of the brain were made using acryostatat)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 DIG-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-fGRP serum was confirmed to recognize specifically three putative goldfish LPXRFamide peptides, as well as fGRP, by a competitive ELISA. The IC 50 values (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. 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. 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 4 °C 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]. RESULTS 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 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 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 RFRP-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-NH 2 (goldfish LPXRFamide peptide-1), AKS NINLPQRF-NH 2 (goldfish LPXRFamide peptide-2), and SGTGLSATLPQRF-NH 2 (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 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- 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-NH 2 (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 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+ ) 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. 6004 K. Sawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (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 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 As summarized in Table 1, all of the identified novel peptides in the brains of mammalian, avian and amphibian species include a -LPXRF-NH 2 sequence (X is L or Q) at their C-termini (LPXRFamide peptides) [9–11,14,15]. To determinewhetherthepresenceofLPXRFamidepeptides 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- NH 2 ) 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. Sequence Animal Name Reference SLTFEEVKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH 2 Bovine RFRP-1 [15] ANMEAGTMSHFPSLPQRF-NH 2 Rat RFRP-2 [14] SIKPSAYLPLRF-NH 2 Quail GnIH [9] SSIQSLLNLPQRF-NH 2 Quail GnIH-RP-2 [10] SLKPAANLPLRF-NH 2 Bullfrog fGRP [11] SGTGLSATLPQRF-NH 2 Goldfish Goldfish LPXRFa peptide-3 This study. 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. 6006 K. Sawada et al. (Eur. J. Biochem. 269) Ó FEBS 2002 localized 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 diencephalon, 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. ACKNOWLEDGEMENTS 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. 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