Molecular evolution of the neuropeptide Y (NPY) family of peptides: cloning of three NPY-related peptides from the sea bass (Dicentrarchus labrax)
Introduction
Neuropeptide Y (NPY) belongs to a family of 36-amino-acid peptides that also includes peptide YY (PYY), tetrapod pancreatic polypeptide (PP), and the fish pancreatic peptide Y (PY) [1], [2], [3]. All four peptides possess the necessary residues to adopt the so-called PP-fold, a hairpin-like structure characterized by a type II proline helix (residues 1–8) with three prolines and an alpha helix (residues 15–32) with two tyrosines interdigitating with the three prolines [4]. Like other low molecular-weight secreted peptides, the NPY-related peptides are synthesized as larger peptide precursors. The release of the active hormone or mature peptide requires endoproteolytic processing at cleavage sites which flank the hormone sequence [1], [3], [5], [39]. The overall organization of the known cDNA sequences encoding different NPY-family peptides is strikingly similar. Each cDNA includes a 5′- and a 3′-untranslated region and a sequence coding for the peptide precursor which consist of a hydrophobic signal peptide, the mature peptide, the amidation-proteolytic site and the carboxyterminal extension [3].
NPY was originally discovered in porcine brain [6] and subsequently isolated from neural tissue in both tetrapod and non-tetrapod species [3]. It is one of the most highly conserved neuroendocrine peptides [3], [7] and is present in the central nervous system (CNS) and the peripheral nervous system (PNS) showing both pre- and post-synaptic actions [8], [9]. This neuropeptide has been shown to influence a diverse range of physiological functions including feeding and sexual behaviour [10], [11] as well as blood pressure [12]. PYY was discovered in pig intestine [13]. In mammals, PYY is mainly expressed in the endocrine cells from ileum and colon [8]. In contrast, PP was first purified from chicken pancreas [14] and is exclusively localized in pancreatic endocrine cells [8]. Both PYY and PP are released in response to food intake and inhibit gastric and pancreatic secretion, as well as gastric and intestinal motility [8]. PYY mRNA has further been found in the CNS and pancreas of rat [15], [16].
Pancreatic and gastrointestinal peptides from cartilaginous and bony fishes have also been isolated [17]. However, peptides from non-tetrapod species were found to be equally similar to mammalian NPY as to mammalian PYY. Both a pancreatic peptide and a CNS peptide have been isolated and sequenced in the European dogfish [18], [19]. Comparisons using sequence identity suggest that the CNS peptide is the orthologue (gene homology by speciation) of tetrapod NPY whereas the pancreatic peptide should be the orthologue of tetrapod PYY [17]. Two distinct precursors for putative NPY and PYY have been further demonstrated in the river lamprey [20] and zebrafish [21]. River lamprey PYY transcripts were demonstrated to occur in both CNS and gut but NPY mRNA was only detected in CNS extracts [20]. Recently, an additional PYY peptide has been purified from intestinal extracts in river lamprey [22]. Therefore, the extracted peptide has been given the name PYY1 and the cloned peptide is now called PYY2.
The fish pancreatic peptide PY has been isolated from the pancreas of three species belonging to the acanthomorph fishes, i.e. anglerfish (Lophius americanus) [23], [24], daddy sculpin (Cottus scorpius) [25], [26] and tilapia (Tilapia nilotica) [27], which consist of the two superorders Paracanthopterygii and Acanthopterygii. All these peptides are more closely related to tetrapod NPY and PYY than to any tetrapod PP [17]. It was first proposed that PY corresponds to the ancestor of mammalian NPY and PYY. However, the subsequent identification of highly conserved orthologues of NPY as well as PYY in many vertebrates including bony fishes suggested that the more divergent PY is the fish orthologue of PYY. In fact, only two NPY-related peptides have often been found in non-tetrapod species, i.e. NPY–PYY or NPY–PY [1], [2], [3], [17]. However, it is also plausible that PY is the result of a separate gene duplication event in fishes [2], [3]. The possibility also exists that PY is the fish orthologue of tetrapod PP although the great diversity of PP sequences seem to have eradicated all traces of orthology with PY.
The relationship of fish PY to mammalian PYY and PP has remained unclear despite extensive sequence comparisons [3], [17]. Therefore, we decided to characterize NPY-related peptides by molecular cloning from a member of the acanthomorph lineage, the sea bass (Dicentrarchus labrax) which is included in the family Moronidae. We describe here molecular clones for the three peptides NPY, PYY, and PY, thus demonstrating that this acanthomorph fish has true orthologues of NPY and PYY that are distinct from PY. Furthermore, we show that all three peptides are expressed in the brain, which may indicate that PY is the result of a gene duplication distinct from the one that generated PP in tetrapods.
Section snippets
RT-PCR with degenerate primers
Total RNA was purified from the sea bass forebrain with Trizol (Gibco-BRL) and treated twice with RQ1-DNAse (Promega). Superscript II reverse transcriptase (Gibco-BRL) was used for cDNA synthesis by priming with oligo (dT)12–18 (Gibco-BRL). The cDNA was subsequently used as template for PCR with degenerate primers based on NPY sequences using Vent DNA polymerase (NEB). The following conditions were used: 10 min at 95°C for one cycle, then 1 min at 94°C, 2 min at 48°C and 1 min at 72°C for 35
Results
Degenerate primers based upon the flanks of a large number of NPY-family sequences [2], [3] were used for PCR on cDNA generated from sea bass forebrain mRNA. PCR products of the expected size (108 bp; see Fig. 1) were obtained and cloned. Eighteen PCR clones hybridizing to a goldfish NPY cDNA probe were sequenced. Five clones were found to encode NPY, 12 clones contained PYY, and one clone displayed a sequence similar to the peptide PY previously isolated from the pancreas of three other
Discussion
By RT-PCR with degenerate NPY-family primers in the sea bass, followed by RACE-PCR to obtain full-length clones, we were able to identify and sequence clones for three distinct NPY-family peptide precursors. Furthermore, our results show that all three peptides are expressed in the sea bass brain, as the cloning was performed with forebrain mRNA as starting material. The evolutionary relationships of peptides belonging to the NPY family have been difficult to resolve in teleost fishes.
Acknowledgements
We are grateful to Dr. L. González-Candelas and Dr. D. Ramón for providing excellent working facilities at Instituto de Agroquı́mica y Tecnologı́a de Alimentos (IATA-CSIC) and Dr. F. González-Candelas (Instituto Cabanilles) for his inestimable help when constructing phylogenetic trees. This work has been supported by a CICYT project no.: Mar 95-1888-C03-01 to S.Z. and EU FAIR grant no: CT96-1410 to M.C. During part of this work, J.M.C-R was the recipient of a fellowship from Balaguer-Gonel
References (39)
- et al.
Evolution of neuropeptide Y and its related peptides
Comp Biochem Physiol
(1993) Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide
Regul Pept
(1996)- et al.
New model for the regulation of energy balance and adiposity by the central nervous system
Am J Clin Nutr
(1995) - et al.
Structural characterization of neuropeptide Y from the brain of the dogfish, Scyliorhinus canicula
Peptides
(1992) - et al.
Molecular evolution of peptide tyrosine–tyrosine: primary structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, bichir, python and desert tortoise
Regul Pept
(1999) - et al.
Glycine-extended anglerfish peptide YG (aPY) a neuropeptide Y (NPY) homologue may be a precursor of a biologically active peptide
Peptides
(1989) - et al.
Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius)
Regul Pept
(1986) - et al.
The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY
FEBS Lett.
(1987) - et al.
Characterization of the pancreatic hormones from the Brockmann body of the tilapia: implications for islet xenograft studies
Comp Biochem Physiol
(1995) - et al.
Assignment of the human neuropeptide Y gene to chromosome 7p15.1 by non-isotopic in situ hybridization
Genomics
(1995)
Gene duplication of the human peptide YY gene (PYY) generated the pancreatic polypeptide gene (PPY) on chromosome 17q21.1
Genomics
Aminoacid sequence diversity of pancreatic polypeptide among the Amphibia
Gen Comp Endocrinol
Is it possible to construct phylogenetic tress using polypeptide hormone sequences?
Gen Comp Endocrinol
Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor
Mol Brain Res
Multiplicity of neuropeptide Y receptors: cloning of a third distinct subtype in the zebrafish
Biochem Biophys Res Commun
Cloning of a neuropeptide Y/peptide YY receptor from Atlantic cod: the Yb receptor
Regul Pept
Evolution of neuropeptide Y family of peptides
X-ray analysis (1.4-A resolution) of avian pancreatic polypeptide: small globular protein hormone
Proc Natl Acad Sci USA
Structure–activity relationship of neuropeptide Y analogues with respect to Y1 and Y2 receptors
Biopolymers
Cited by (69)
Crystal structures of human neuropeptide Y (NPY) and peptide YY (PYY)
2022, NeuropeptidesCentral regulation of food intake is not affected by inclusion of defatted Tenebrio molitor larvae meal in diets for European sea bass (Dicentrarchus labrax)
2021, AquacultureCitation Excerpt :Analyses were performed on 1 μL cDNA using MAXIMA SYBR Green qPCR Mastermix (Life Technologies, USA), in a total PCR reaction volume of 15 μL, containing 50–500 nM of each primer. Expression of agrp2, npy and pomca was measured using previously described primers in the same fish species (Tsalafouta et al., 2017; Brandts et al., 2018; Cerdá-Reverter et al., 2000). For cartpt2, new primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) from sequences available based on those obtained from a European sea bass genome database (http://seabass.mpipz.mpg.de/cgi-bin/hgGateway).
New immunomodulatory role of neuropeptide Y (NPY) in Salmo salar leucocytes
2017, Developmental and Comparative ImmunologyPostpyloric Gastrointestinal Peptides
2012, Physiology of the Gastrointestinal Tract