Physiol Genomics 24: 207–217, 2006. First published November 29, 2005; doi:10.1152/physiolgenomics.00227.2005. High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis Alessandro Romano,1 Gabor Kottra,2 Amilcare Barca,1 Natascia Tiso,3 Michele Maffia,1 Francesco Argenton,3 Hannelore Daniel,2 Carlo Storelli,1 and Tiziano Verri1 1 Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Lecce, Lecce, Italy; 2Institute of Nutritional Sciences, Technical University of Munich, Freising-Weihenstephan, Germany; and 3Department of Biology, University of Padua, Padua, Italy Submitted 9 September 2005; accepted in final form 23 November 2005 Romano, Alessandro, Gabor Kottra, Amilcare Barca, Natascia Tiso, Michele Maffia, Francesco Argenton, Hannelore Daniel, Carlo Storelli, and Tiziano Verri. High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis. Physiol Genomics 24: 207–217, 2006. First published November 29, 2005; doi:10.1152/physiolgenomics.00227.2005.—Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K0.5 for glycyl-L-glutamine ⬃18 ␮M at ⫺120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development. otic vesicle; pept2; peptide transport; two-electrode voltage clamp; whole mount in situ hybridization; Xenopus laevis oocytes is due to plasma membrane transport proteins belonging to the peptide transporter (PTR) family (41). Recently, PTR proteins among vertebrates were integrated into the solute carrier 15 (SLC15) proton oligopeptide cotransporter family (10). In higher vertebrates, two members of this family, namely PEPT1 (SLC15A1; Refs. 6, 14, 15, CELLULAR UPTAKE OF SMALL PEPTIDES Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org). Address for reprint requests and other correspondence: T. Verri, Laboratory of General Physiology, Dept. of Biological and Environmental Sciences and Technologies, Univ. of Lecce, via Provinciale Lecce-Monteroni, I-73100 Lecce, Italy (e-mail: physiol@ultra5.unile.it). 21, 23, 28) and PEPT2 (SLC15A2; Refs. 4, 22, 32, 35) have been characterized extensively. Both proteins translocate diand tripeptides across the plasma membrane even against a concentration gradient by utilizing an inwardly directed electrochemical H⫹ gradient. Also, they transport a variety of peptidomimetics such as ␤-lactam antibiotics, aminopeptidase and angiotensin-converting enzyme inhibitors, ␦-aminolevulinic acid, and selected prodrugs (31). PEPT1 is a low-affinity/ high-capacity system, with apparent affinity constants ranging between 0.2 and 10 mM depending on the substrate. In mammals, it is predominantly expressed in the epithelial cells of small intestine, but also of kidney (proximal tubule S1 segment) and bile duct, and in the acinar cells of pancreas. On the other hand, PEPT2 is a high-affinity/low-capacity system, with apparent affinity constants of 0.005– 0.5 mM. In mammals, it is expressed in kidney (epithelial cells of the proximal tubule S2 and S3 segments), peripheral nervous system (satellite glial cells), central nervous system (astrocytes, ependymal and subependymal cells, epithelial cells of the choroid plexus), lung (type II pneumocytes, tracheal and bronchial epithelial cells), and lactating mammary gland (epithelial cells of the terminal duct and glandules as well as the main segmental ducts). Expression of PEPT2 mRNA has also been reported in spleen, colon, and pancreas (11, 31). The cyprinid teleost Danio rerio (zebrafish) represents a valuable model in experimental biology, embryology, developmental genetics, and toxicology as well as in regulatory and integrative physiology and comparative functional genomics (see, for instance, Ref. 5). Numerous zebrafish models of human disease have been characterized (see, for instance, Ref. 12). Furthermore, substantial genomic data are deposited in public databases, which is allowing both construction of zebrafish microarrays and global gene expression analysis. Because of the permeability of its embryos to small molecules, zebrafish is also a suitable model for chemical genomics (see, for instance, Ref. 29). We aimed at characterizing peptide transporters in zebrafish, and, in this respect, the molecular and functional description of the zebrafish low-affinity/high-capacity peptide transporter PEPT1 has been carried out (48). In the present paper, we report the functional characterization of the zebrafish highaffinity/low-capacity PEPT2(SLC15A2)-type peptide transporter, as well as information on its gene (pept2) structure and chromosomal localization, and distribution of pept2 mRNA in the adult fish and during larval development. Interestingly, expression of zebrafish pept2 mRNA was detected in the otic vesicle, the embryonic structure from which the auditory and vestibular organ of the fish (that is homolog to the inner ear of 1094-8341/06 $8.00 Copyright © 2006 the American Physiological Society Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. 207 208 MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH mammals) will arise. To our knowledge, this is the first experimental evidence that associates pept2 expression to inner ear in vertebrates. MATERIALS AND METHODS All experiments with Xenopus laevis were approved by the Bavarian State Ethics Committee and followed the German Guidelines for Care and Handling of Laboratory Animals. All protocols for zebrafish care and use were approved and authorized by the Italian Ministry of Health (Animal Food and Health-Office X). Fish breeding and embryo collection. Adult zebrafish were bred by natural crosses in a male-to-female ratio of 2:1 (51). Immediately after spawning, the bottoms of the aquariums were siphoned. The fertilized eggs were harvested, washed, and placed in 9-cm-diameter petri dishes in 0.6 mg/l Instant Ocean sea salts (Aquarium Systems, Sarrebourg, France). The developing embryos were incubated at 28.5°C until use. Developmental stages of zebrafish embryos were expressed as days postfertilization (dpf) at 28.5°C (18). Plasmids. An expressed sequence tag (EST) cDNA clone (GenBank accession no. AW153469) putatively encoding for zebrafish PEPT2 was purchased from the Integrated Molecular Analysis of Gene Expression (IMAGE) Consortium (IMAGE clone no. 2601882). The insert was cut out of its original plasmid pME18S-FL3 by XhoI digestion and subcloned into pBluescript II SK⫺ (Stratagene, La Jolla, CA) for further use (zfPepT2Put-pBSII-SK⫺). Preliminary sequencing performed on both strands using M13 forward and reverse primers and 20-mer synthetic oligonucleotides (Proligo France, Paris, France) confirmed that the IMAGE clone effectively encoded for zebrafish PEPT2 but revealed a 75-bp deletion in the cDNA insert (corresponding to nucleotides 835–909 of the complete zebrafish pept2 cDNA sequence reported in Fig. 1), most probably due to an erroneous splicing event involving exon 11 (see Fig. 2 and Table 1). Restoration of the deletion was achieved as follows. Briefly, RT-PCR-based amplification of a 2,020-bp fragment was obtained starting from adult zebrafish total RNA (TRIzol Reagent; Invitrogen, Carlsbad, CA). Reverse transcription was performed (50°C/60 min) using SuperScript III RT (Invitrogen) in the presence of oligo(dT)18 and PCR using high-fidelity Platinum Taq DNA polymerase (Invitrogen) in the presence of zebrafish pept2-specific primers (forward: 5⬘-ATCTGTCTACAGCTATTTAC-3⬘, starting at nucleotide 197; reverse: 5⬘-AAGTCGATTAGCTTATTCAC-3⬘, starting at nucleotide 2216; Fig. 1). The amplification product was digested by NsiI and BglII, purified by 1% agarose gel, and directionally cloned into NsiI/BglII-cut zfPepT2PutpBSII-SK⫺ instead of the correspondent deleted fragment. The recombinant clone (zfPepT2-pBSII-SK⫺; Fig. 1) containing the complete zebrafish pept2 cDNA sequence was used for all further analyses. Both strands of the zfPepT2-pBSII-SK⫺ insert were sequenced to confirm restoring procedure and nucleotide identity. The resulting sequence was deposited in the GenBank database (GenBank accession no. DQ192597).1 In silico analysis. Zebrafish PEPT2 amino acid sequence was deduced using the open reading frame (ORF) finder program at http://www.ncbi.nlm.nih.gov. Putative transmembrane domains and potential N-glycosylation and protein kinase C recognition sequences were defined using the PROSITE 17.0 computational tools. Sequence 1 Sequence data submission and accession numbers: sequence data reported are available in the DNA DataBank of Japan (DDBJ)/European Molecular Biology Laboratory (EMBL)/GenBank databases under the accession no. DQ192597 (zebrafish pept2). PEPT2 similarity search was performed by FASTA 3.39. Multiple sequence alignments were obtained by Clustal W 1.82 (46). The phylogenetic reconstruction was generated by the neighbor-joining (NJ) method (36), as implemented in MEGA 2.1 (20). GenBank accession numbers for sequence comparisons were AAA17721 (rabbit PEPT1) (14), AAB61693 (human PEPT1) (21), BAA09318 (rat PEPT1) (23), AAG29092 (mouse PEPT1) (15), AAK14788 (sheep PEPT1) (28), AAO43094 (pig PEPT1), AAL67837 [dog (Madin-Darby canine kidney cell) PEPT1], AAK39954 (chicken PEPT1) (6), AAO16604 (turkey PEPT1) (47), AAQ65244 (zebrafish PEPT1) (48), AAC48495 (rabbit PEPT2) (4), AAB34388 (human PEPT2) (22), BAA09631 (rat PEPT2) (35), and NP_067276 (mouse PEPT2) (32). The genomic structure of zebrafish pept2 was obtained by comparing the zebrafish pept2 cDNA sequence (see Fig. 1) with the genomic clones BX511220.3 (D. rerio clone CH211-127P7, working draft sequence, 4 unordered pieces) and BX548071.2 (D. rerio clone DKEY-25L23, working draft sequence, 5 unordered pieces). Annotation was carried out by the genome viewer/annotation software Artemis (release 4) (34). Information on the chromosomal localization of zebrafish pept2 was found at http://www.ensembl.org. Identification of zebrafish pept2 transcription start site. To establish the position of the transcription start site of the zebrafish pept2 mRNA, 5⬘-RACE was performed as described (8). Briefly, the singlestranded cDNA was reverse transcribed from zebrafish gut poly(A)⫹ RNA using the antisense pept2-specific primer zfPepT2-RACE-I (5⬘-AATAGACAGATAGATGATGG-3⬘). The same primer was also used to perform the first round of PCR amplification. Following nested amplification using the pept2-specific primer zfPepT2RACE-II (5⬘-CCATGAGTCTGCTATCAAGG-3⬘), the resulting PCR fragment was subcloned into pCRII-TOPO vector (TOPO TA Cloning, Invitrogen) and sequenced in both directions. Expression in X. laevis oocytes and electrophysiology. Female clawed frogs (X. laevis) were purchased from Nasco (Fort Atkinson, WI). To collect oocytes, the animals were anesthetized by immersion in 0.7 g/l MS-222 (3-aminobenzoic acid ethyl ester; Sigma, St. Louis, MO). Surgical procedures were performed as described (30). After the final collection, the frogs were killed with an anesthetic overdose. Oocytes were treated with 2.5 mg/ml collagenase for 90 min, separated manually, and incubated in Barth’s solution containing (in mM) 88 NaCl, 1 KCl, 0.8 MgSO4, 0.4 CaCl2, 0.3 Ca(NO3)2, 2.4 NaHCO3, and 10 HEPES (pH 7.5) at 17°C overnight. Stage V/VI oocytes were injected with 14 ng (in 14 nl) of in vitro-synthesized zebrafish pept2-specific cRNA (Ambion mMessage mMachine T7 kit; AMS, Wiesbaden, Germany) and incubated for 3– 6 days at 17°C. Two-electrode voltage clamp (TEVC) experiments were performed as described (19). Briefly, the oocyte was placed in an open chamber (⬃0.5-ml total volume) and continuously superfused (⬃3 ml/min) with the Barth’s solution or with solutions containing the substrate dipeptide. Electrodes with resistance between 1 and 10 M⍀ were connected to a TEC-05 amplifier (npi electronic, Tamm, Germany). Oocytes were voltage clamped at ⫺60 mV, and current-voltage (I-V) relations were measured using short (100 ms) pulses separated by 200-ms pauses in the potential range ⫺160 to ⫹80 mV. I-V measurements were made immediately before and 30 – 40 s after substrate application when current flow reached steady state. The zebrafish PEPT2-evoked current at a given membrane potential was calculated as the difference between the currents measured in the presence and absence of substrate. I-V relations were calculated with a Visual Basic (VBA) routine written in Microsoft Excel. Positive currents denote positive charges flowing out of the oocyte. Fig. 1. Nucleotide and predicted amino acid sequence. Nos. at left refer to the nucleotide (top row) and amino acid (bottom row) positions. Nucleotides are numbered, starting from the first ATG initiation codon within a strong Kozak consensus sequence. ***Stop codon. A polyadenylation signal is double underlined. In the amino acid sequence, putative transmembrane domains are underlined and named I–XII. Potential extracellular N-glycosylation sites (open boxes) and potential protein kinase C phosphorylation sites at the cytoplasmic surface (shaded boxes) are indicated. Physiol Genomics • VOL 24 • www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH Physiol Genomics • VOL 24 • PEPT2 www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. 209 210 MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH PEPT2 Fig. 2. Unrooted phylogenetic tree depicting the evolutionary relationship of vertebrate PEPT1 and PEPT2 transporters. The unrooted tree was constructed using the neighbor-joining method (36) based on the alignment of the complete amino acid sequences of known vertebrate PEPT1 and PEPT2 transporters. Bootstrap values (1,000 replicates) indicating the occurrence of nodes are reported above each branch in the figure. Gly-L-Gln (Sigma) was added to the solutions in concentrations as indicated in the text. After addition of the dipeptide, the pH was adjusted if necessary. The percentage of the zwitterionic form at a given pH was calculated with dissociation constant (pK) values taken from Ref. 40 (for Gly-L-Gln, pK1 ⫽ 2.88 and pK2 ⫽ 8.29). Transport parameters, i.e., apparent Gly-L-Gln affinity (K0.5; ␮M) and maximal transport current (Imax; nA), were calculated on the basis of the Michaelis-Menten equation from four data points. pH dependence of Imax was determined in paired experiments, i.e., each oocyte was perfused with solutions having pH values of 8.5, 7.5, 6.5, or, in another series, 7.5 and 5.5. The sequence of pH values was varied to avoid systematic errors. At each pH value, in addition to the substratefree control solution, four substrate concentrations were applied in the concentration range 20 –500 ␮M (pH 5.5–7.5) or 50 –500 ␮M (pH 8.5). Data are means ⫾ SE of n no. of experiments. Statistically significant differences (P ⬍ 0.05) were determined using the Student’s t-test for paired or nonpaired data as appropriate. RT-PCR. Total RNA was extracted from either tissues of adult animals or 1- to 7-dpf embryos (TRIzol Reagent, Invitrogen). Adult fish were anesthetized by immersion in 0.2 g/l MS-222 and then killed by decapitation before organ removal. Embryos were killed by anesthetic overdose. RT-PCR was performed using the GeneAmp Gold RNA PCR Reagent kit (Applied Biosystems, Foster City, CA). Reverse transcription was performed (42°C/12 min) in the presence of oligo(dT)16 and PCR using zebrafish pept2-specific primers (forward: 5⬘-GACTCATGGTTGGGGAAGTT-3⬘, starting at nucleotide 271; reverse: 5⬘-TGAAGCCTCCACCAAAATCC-3⬘, starting at nucleotide 970; Fig. 1). Amplification was performed for 35 cycles (95°C denaturation/30 s, 65°C annealing/1 min, 72°C extension/45 s; final synthesis: 72°C/7 min). Analogously, for ␤-actin-specific RNA, reverse transcription was performed (42°C/12 min) in the presence of oligo(dT)16 and PCR using zebrafish ␤-actin-specific primers (GenBank accession no. NM_131031; forward: 5⬘-CGTGACATCAAGGAGAAGCT-3⬘, starting at nucleotide 681; reverse: 5⬘-ATCCA- Table 1. DNA sequences at the exon/intron boundaries of the zebrafish pept2 gene No. Exon Size, bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 58 47 88 142 93 100 91 78 83 84 90 45 36 89 82 126 171 47 108 97 144 105 1,170 Donor Exon/Intron* AAAATGAAGG GCGCTCACCG GGAATGAAAG GGAAGTTCAA CTGTTCATAT TGAAGACAAT ATACTAAGAG ATCGCCTTAG ATGTATCGGG GAAGTACTCG TGACCAGCAG TATGGATTTT TCAAATGCAG TCAAACTCAC TTACGTCATT ACCATATGAG ATGCAATCTG CGTATATAAG CAAGGGGAGA ACTCTCAGAG CTACTCACAG CATGGAACAG Intron Size, bp† gtaactgttt gtgagttcaa gtaatgaaaa gtaagtattg gtaagcttct gtaagtcaat gtcagtctca gtgagggtct gtatgtaaat gtacgtttca gtaaaaggtt gtaagtgttt gtgagccaaa gtgagtgctg gtatgtacat gtaacatcac gtgagtgagt gtacatttaa gtgagtattt gtgtgtgatc gtacagtaag gtacattttg 531 854 415 374 641 879 109 3,450 106 95 365 270 680 282 121 3,565 79 161 88 2,979 76 86 Acceptor Intron/Exon* actgttttag ttgtgcacag tttttttaag ttttttgcag ttcctcatag tcttattcag ttgtttatag gctcatctag ttttcaccag tctttttcag tctgtgcaag tgttttgcag tgttgttaag gcatttttag ccttggccag aaatctacag tgttaaccag tccatcacag tgtctttcag atttccacag gtgcacgcag caatcaccag ACAAAGATGT AAATTATGTG CGGTGCTGAC AACCATCATC AGCTCTGTCA ATAGACGAGC GTGATGTGCA TGGTGTTTAT TTTGCTATAA AAACGACTCA GGGTCACGCT GGTGGAGGCT ATGCTGAATG TCCGCTAAAG AAAACCGTGG GAACCTTCGG GTAAAAGATC GTTTATCAAT ATATAATAAA GCGGGCAATA GCACCGGCGA TGGGTGGAGT *Exon sequences are indicated by uppercase letters and intron sequences by lowercase letters. †Intron sizes were determined from sequence analysis of contigs CH211-127P7 (BX511220.3) and DKEY-25L23 (BX548071.2). Physiol Genomics • VOL 24 • www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH CATCTGCTGGAAGGT-3⬘, starting at nucleotide 1123). Amplification was performed for 35 cycles (95°C denaturation/30 s, 50°C annealing/1 min, and 72°C extension/1 min; final synthesis: 72°C/7 min). RT-PCR products were separated on a 1% agarose gel and stained by ethidium bromide, and their identity was confirmed by cloning and sequencing. Whole mount in situ hybridization. A 244-bp fragment corresponding to a portion of zebrafish PEPT2 large extracellular loop between the transmembrane domains IX and X was amplified by PCR from zfPepT2-pBSII-SK⫺ using the following pept2-specific primers (forward: 5⬘-CTCTTACCGGAGAATCTCAG-3⬘, starting at nucleotide 1316; reverse: 5⬘-TCCTCAGTGCCAACTGTTAC-3⬘, starting at nucleotide 1559; Fig. 1). The amplification product was subcloned into the pCRII-TOPO vector and the recombinant clone (zfP2OL-pCRIITOPO) sequenced to confirm the identity of the insert. The 244-bp digoxigenin (DIG)-11-UTP (Roche, Mannheim, Germany)-labeled antisense riboprobe was synthesized in vitro with T7 RNA polymerase (Roche) using the KpnI-cleaved zfP2OL-pCRII-TOPO. The corresponding sense riboprobe was synthesized with SP6 RNA polymerase (Roche) using the XhoI-cleaved zfP2OL-pCRII-TOPO. Whole mount in situ hybridization was performed as described (45). Stained embryos were dehydrated with methanol, clarified in benzyl benzoatebenzyl alcohol (2:1, vol/vol), and mounted in glycerol. Embryos were observed on a Leica DMR microscope equipped with Nomarski optics and a DC500 Leica digital camera. RESULTS Sequence analysis. The zebrafish pept2 cDNA was obtained using a combination of bioinformatics, RT-PCR, and 5⬘-RACE techniques, as detailed in MATERIALS AND METHODS. Zebrafish pept2 cDNA was 3,238-bp long, with 88 bases of 5⬘-untranslated region (5⬘-UTR), an ORF of 2,160 bp, and 970 bases of 3⬘-UTR. The 3⬘-UTR contained a polyadenylation signal (AATAAA) starting at position 3067. The open reading frame of zebrafish pept2 encoded a putative protein of 719 amino acids (Fig. 1). Hydropathy analysis predicted at least 12 po- PEPT2 211 tential membrane-spanning domains with a large extracellular loop between transmembrane domains IX and X (Fig. 1). Three putative extracellular N-glycosylation sites and eight putative intracellular recognition sites for protein kinase C were identified (Fig. 1). Compared with PEPT1 and PEPT2 members of the PTR family already known from other vertebrates, the predicted zebrafish PEPT2 amino acid sequence exhibited a higher percentage of identity with the PEPT2-type (51–53%) than with the PEPT1-type (47–50%) transporters. The phylogenetic reconstruction of vertebrate proteins clustered zebrafish PEPT2 to the PEPT2 branch of the phylogenetic tree and indicated early divergence of the fish sequence with respect to those of the tetrapod group (Fig. 2). A search of the National Center for Biotechnology Information (NCBI) zebrafish genome nucleotide database (Sept. 2003) revealed two contigs, CH211-127P7 (accession no. BX511220.3) and DKEY-25L23 (accession no. BX548071.2), sharing identity with the zebrafish pept2 cDNA sequence. By sequence comparison, approximately nine-tenths of the zebrafish pept2 cDNA (nucleotides 152–3131; see Fig. 1) was present on contig CH211-127P7, in the first of four unordered pieces, and within this region, the last 20 exons and 19 introns of the pept2 gene were identified (Table 1). The remaining pept2 gene (corresponding to nucleotides ⫺88 to 151 on the zebrafish pept2 cDNA; see Fig. 1) was obtained by analysis of contig DKEY-25L23. Within this contig, in the first (reversecomplemented sequence) of five unordered pieces partially overlapping contig CH211-127P7, the first three exons and three introns of the pept2 gene were identified (Table 1). A diagram summarizing the organization of the zebrafish pept2 gene and showing its relationships to mRNA and protein is drawn in Fig. 3. The complete pept2 gene spans ⬃19 kb, with 23 exons and 22 introns (see Fig. 3 and Table 1). The translation initiation site is present on exon 1. Exon sizes vary from Fig. 3. Structure of the zebrafish pept2 gene and its relationships to the mRNA and the predicted secondary structure of the PEPT2 protein. Exons of the zebrafish pept2 gene are depicted as black vertical bars, widths drawn to scale. In the middle section, the relative contribution of each exon to the spliced mRNA transcript is shown. Gray regions indicate the untranslated 5⬘ and 3⬘ sequences. Marked base pair position of the intron/exon boundaries refers to the translation start. Superimposition of the exon boundaries with the predicted secondary structure of the PEPT2 protein is shown at bottom. Triangles indicate the exon/intron boundaries, and the numbered gray boxes represent the predicted 12 membrane-spanning domains. Physiol Genomics • VOL 24 • www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. 212 MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH 36 to 171 bp with the exception of exon 23, which is 1,170 bp (containing the TAG stop codon and the full segment of the 3⬘-UTR). Intron sizes vary from 76 to 3,565 bp (Table 1). No introns are present within zebrafish pept2 UTRs. Nucleotide sequences at the intron/exon boundaries of zebrafish pept2 conform to the GT/AG rule for intron donor and acceptor splice sites (Table 1). Zebrafish pept2 gene was localized on linkage group 9 (chromosome 9), closest to the GLI-Kruppel family member GLI2a gene (gli2a) and to the putative locus zgc:100975 encoding a predicted protein of unknown function containing a JmjC domain (9), as assessed by consulting the Ensembl web site featuring the zebrafish assembly version 4 (Zv4; released on July 12, 2004), which was produced by integrating the whole genome shotgun assembly with data from the physical map. Function. The zwitterionic dipeptide Gly-L-Gln, a wellknown high-affinity substrate for mammalian peptide transporters, was used as a test compound in TEVC experiments to establish the basic kinetic properties of the zebrafish PEPT2 transporter (Fig. 4). I-V relations were initially measured 30 – 40 s after the start of the superfusion of oocytes (clamped at ⫺60 mV) with 0.05– 0.5 mM Gly-L-Gln at the extracellular pH value of 7.5 (as shown in Fig. 4A) and did not differ substantially from those reported for mammalian PEPT2 using the same substrate and under similar experimental conditions (see, for instance, Ref. 1). In fact, in the voltage range ⫹80 to ⫺40 mV, inward currents were only minimal, whereas a weak potential dependence was observed in the physiological range ⫺20 to ⫺60 mV. At hyperpolarizing membrane potentials (⫺60 to ⫺160 mV), however, inward currents steeply increased and showed a nearly linear dependence on membrane potential (Fig. 4A). Inward currents increased as a function of Gly-L-Gln concentration according to Michaelis-Menten kinetics. K0.5 and Imax values at pH 7.5 and two different membrane potentials (⫺160 and ⫺120 mV), as determined in the extracellular Gly-L-Gln concentration range of 0.02– 0.5 mM, are shown in Table 2. These data clearly indicate that zebrafish PEPT2 operates as a high-affinity/low-capacity transport system, and a comparison with previously published results shows that the mammalian and zebrafish transporters exhibit comparable K0.5 for Gly-L-Gln under similar experimental conditions (see, for instance, Ref. 1). Steady-state I-V relations obtained during perfusion of oocytes with different substrate concentrations allowed K0.5 and Imax to be determined as a function of membrane potential and external pH. The dependencies of K0.5 and Imax on membrane potential and extracellular pH are presented in Fig. 4, B and C, respectively. K0.5 values were affected by both membrane potential and pH. K0.5 values steadily decreased, passing from ⫺160 mV to less negative potentials (Fig. 4B), this being observed at each external pH tested (i.e., pH 5.5, 6.5, 7.5, and 8.5) and in the potential range ⫺160 to ⫺100 mV. At less negative membrane potential values, kinetic analysis of the experimental data could not reasonably be performed due to extremely low transport currents. The observed behavior for zebrafish PEPT2 was similar to that found with the mammalian PEPT2 in the same membrane potential range (see, for instance, Ref. 1). Moreover, at each membrane potential, apparent affinity for Gly-L-Gln at pH 7.5 was always higher than that measured at pH 6.5 and 5.5 and at pH 8.5 (Fig. 4B). However, the actual affinity at each pH value might be different than the measured one, if only the Physiol Genomics • VOL 24 • PEPT2 Fig. 4. Electrophysiological characterization of zebrafish PEPT2-expressing Xenopus laevis oocytes. A: steady-state current-voltage (I-V) relationships for Gly-L-Gln. Zebrafish PEPT2-expressing oocytes were perfused at pH 7.5 in the presence of increasing (0.05– 0.5 mM) Gly-L-Gln concentrations. Apparent Gly-L-Gln affinity (K0.5; B) and maximal transport current (Imax; C) of inwardly directed Gly-L-Gln transport as a function of membrane potential and pH. Zebrafish PEPT2-expressing oocytes were perfused at pH 5.5 (n ⫽ 4), 6.5 (n ⫽ 10), 7.5 (n ⫽ 11), or 8.5 (n ⫽ 11) in the presence of increasing Gly-L-Gln concentrations (20 –500 ␮M). At each substrate concentration, steady-state I-V relationships were recorded, and currents obtained were replotted as a function of Gly-L-Gln concentration. Data were fitted to a Michaelis-Menten equation by nonlinear regression analysis using the least-squares method and K0.5 and Imax values of Gly-L-Gln calculated for individual membrane potentials. www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH 213 PEPT2 Table 2. pH dependence of the kinetic parameters of inwardly directed Gly-L-Gln transport via zebrafish PEPT2 by two-electrode voltage clamp experiments Oocytes Clamped at ⫺160 mV Oocytes Clamped at ⫺120 mV No. of Experiments pH Neutral form, % K0.5 ␮M Imax % K0.5 ␮M Imax % n/N (K0.5) n/N (Imax) 5.5 6.5 7.5 8.5 99.6 98.4 86.0 38.1 97⫾25 79⫾12 48⫾5 163⫾14 122⫾18 84⫾6 100 89⫾4 82⫾16 38⫾8 18⫾4 118⫾27 233⫾42 108⫾6 100 74⫾8 4/4 20/3 11/2 11/2 4/4 10/2 11/2 11/2 Kinetic parameters (apparent Gly-L-Gln affinity, K0.5; and maximal transport current, Imax) were calculated at ⫺160 and ⫺120 mV by least-square fit to the Michaelis-Menten equation (see Fig. 4). Maximal transport current (Imax) values are expressed as the percentage of Imax calculated at pH 7.5 in the same experiment. Percentage of the dipeptide present in its neutral (zwitterionic) form was calculated as described in MATERIALS and METHODS. Due to the decreasing concentration of zwitterionic species at increasing pH values, K0.5 could be overestimated at pH 8.5 (see RESULTS). n and N specify the no. of oocytes and frogs, respectively. neutral form of Gly-L-Gln (99% at pH 5.5, 98% at pH 6.5, 86% at pH 7.5, and only 38% at pH 8.5; Table 2) is transported. In this case, the measured K0.5 values would be overestimated, only slightly at pH 5.5, 6.5, and 7.5 (by 1, 2, and 16%, respectively) but highly at pH 8.5 (by 163%). On the other side, as expected from an electrogenic transporter, Imax also exhibited a clear dependence on membrane potential, and its value steadily decreased, passing from ⫺160 mV to less negative potentials at each external pH (Fig. 4C). Varying extracellular pH from 8.5 to 5.5 monotonically reduced the potential dependence of Imax, which was less pronounced at pH 5.5 than at the other pH values tested, thus suggesting that a steeper transmembrane pH gradient significantly contributes to the transport when membrane potential changes from ⫺160 mV to less negative potentials (Fig. 4C). Kinetic parameters as a function of extracellular pH at ⫺160 and ⫺120 mV are reported in Table 2. Tissue distribution of zebrafish pept2 in adult fish. Using zebrafish pept2-specific primers, a 700-bp RT-PCR product was amplified from total RNA isolated from the kidney of adult zebrafish as well as from the brain and the gut (Fig. 5). Under our experimental conditions, a faint signal was also obtained in samples of gill, eye, and skeletal muscle, while no signal was obtained in samples of spleen (Fig. 5). As a control to assess RNA quality, ␤-actin RNA amplification was performed using zebrafish ␤-actin-specific primers, which invariably gave comparable 443-bp amplification products for all tested tissues (Fig. 5). Expression of zebrafish pept2 during embryonic and early larval development. Zebrafish pept2 expression (Fig. 6) was temporally analyzed during the embryonic (day 1 to day 3 postfertilization) and the early larval (day 4 to day 7 postfertilization) development (18) by RT-PCR using the same primers and similar experimental conditions as those described above, with ␤-actin RNA amplification serving as control. A signal for zebrafish pept2-specific mRNA could be detected already at day 1 postfertilization, and its expression did not change up to day 7 postfertilization. Zebrafish pept2 expression was also analyzed during the embryonic and early larval periods (starting 1 and up to 5 dpf) by whole mount in situ hybridization, using a specific antisense riboprobe (Fig. 7, A–F). As a negative control, the corresponding sense riboprobe was used (Fig. 7, G–I). Interestingly, in situ analysis revealed abundant expression of zebrafish pept2 at the level of the brain from day 1 postfertilization. In particular, specific labeling was Physiol Genomics • VOL 24 • clearly detected at the ependymal cell layer surrounding the ventricular cavities of the forebrain and the midbrain (Fig. 7, A and B), which highly correlates with the distribution of pept2 mRNA in the mammalian nervous system (see, for instance, Ref. 3). Furthermore, from day 2 to day 5 postfertilization, zebrafish pept2 mRNA was also detected in the epithelium of the developing otic vesicle (Fig. 7, C–F), the embryonic structure that will grow into the auditory and vestibular organ (the inner ear) of the adult fish (see, for instance, Ref. 52). In particular, pept2 expression appeared to associate to the epithelium of the developing semicircular canals, a system of Fig. 5. Tissue distribution of zebrafish pept2 in adult fish. A: RT-PCR performed on equal amounts of total RNA (1 ␮g) isolated from adult fish tissues using either zebrafish pept2- or zebrafish ␤-actin-specific primers (see MATERIALS AND METHODS). H2O indicates no RNA in RT (negative control). B: PCR on control plasmids to assess primer specificity. No plasmid, H2O; zebrafish pept1 cDNA, zfPepT1-pSPORT1 (5 ng, negative control) (48); zebrafish pept2 cDNA, zfPepT2-pBSII-SK⫺ (5 ng, positive control). www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. 214 MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH PEPT2 Fig. 6. Expression of zebrafish pept2 in zebrafish embryos. RTPCR performed on equal amounts of total RNA (2 ␮g) isolated from 1–7 days postfertilization (dpf) embryos using either zebrafish pept2- or zebrafish ␤-actin-specific primers (see MATERIALS AND METHODS). H2O indicates no RNA in RT (negative control). toroidal spaces arranged orthogonally to each other in the inner ear that detect angular accelerations in the fish and are a homolog to the semicircular canals of the higher vertebrates (humans included) (52). To our knowledge, this is the first experimental evidence that pept2 is expressed at inner ear structures in vertebrates. DISCUSSION By screening EST databases, we have identified a cDNA (GenBank accession no. AW153469) encoding for a novel zebrafish peptide transporter that was made functional by restoring a 75-bp deletion in the cDNA insert. The encoded protein, designated as zebrafish PEPT2, represents the ortholog of the mammalian PEPT2, as its predicted amino acid sequence shares a significantly high overall identity to PEPT2-type transporters compared with other known members of the PTR family in vertebrates and clusters to the PEPT2 monophyletic group of the reconstructed phylogenetic tree, giving rise to the more basal branch. At present, zebrafish PEPT2 is the second recognized zebrafish peptide transporter after zebrafish PEPT1 (48). Extensive functional analysis of the cloned mammalian PEPT2-type peptide transporters by TEVC has previously allowed a detailed characterization of the electrophysiological properties of such proteins when expressed in X. laevis oocytes (see, for instance, Refs. 1, 7, 49, and 53). In this study, the same experimental approach has been used to determine the basic properties of zebrafish PEPT2 and the kinetic constants of substrate binding and translocation. In general terms, zebrafish PEPT2 is a classical high-affinity/low-capacity system that operates in an electrogenic mode and with similar affinities for the model dipeptide Gly-L-Gln as the mammalian counterparts (see, for instance, Ref. 1). Moreover, zebrafish PEPT2 shows the same I-V characteristics as the corresponding mammalian transporters, i.e., the transport rate is in the membrane potential range up to ⫺60 mV low but increases steeply at more negative potentials. At the highest negative membrane potential tested (⫺160 mV), the Imax depends only slightly on the extracellular pH, suggesting that the K0.5 value for the cotransported proton is ⬍3 nM (⬃pH 8.5), whereas at ⫺120 mV, the pH dependency of Imax is slightly more pronounced, and Imax increases markedly when the pH is lowered from 6.5 to 5.5, suggesting that a high transmembrane pH gradient may provide an additional driving force, especially at submaximal membrane potentials. The complete zebrafish pept2 gene (which is located on chromosome 9) is only ⬃19-kb long, thus representing the shortest pept2 gene fully characterized so far in vertebrates, given that the human and mouse pept2 genes are ⬃47 kb (see human chromosome 3 genomic sequence NC_000003.9, position 123095977–123143148) and ⬃35 kb (see mouse Physiol Genomics • VOL 24 • chromosome 16 genomic sequence NC_000082.1, position c36633116 –36598335, and Ref. 32), respectively, while the rat pept2 gene seems to be ⬃30 kb (see rat chromosome 11 WGS supercontig NW_047356.1, position Rn11_1876:19214841– 19245214; although this region covering the rat pept2 gene has not been fully resolved yet). Also, zebrafish pept2 gene contains 23 exons and 22 introns, the same number as mammalian and zebrafish pept1, whereas human, mouse, and rat pept2 genes all contain 22 exons and 21 introns only (see Supplemental Fig. S1; available at the Physiological Genomics web site).2 In particular, when comparing the structure of zebrafish pept2 to that of the mammalian counterparts, a remarkable conservation is evident, with exons 3–23 of the zebrafish invariably corresponding to exons 2–22 of the mammalian pept2, while no important sequence correlation apparently exists between exons 1 and/or 2 of the zebrafish and exon 1 of the mammalian pept2 (see Supplemental Fig. S2). Furthermore, in all pept2 genes studied, exon 1 is the one containing 5⬘-UTR and initiator methionine. No additional obvious differences in the genomic organization of mammalian and zebrafish pept2 genes can be observed, except the differences in length of the corresponding introns. In this respect, homology searches between the pept2 sequences of such evolutionary distant species as zebrafish and mammalians might offer a fast and reliable method of phylogenetic footprinting (see, for instance, Ref. 24) for detection and functional analysis of novel, putative, conserved regulatory sequences (cis-regulatory elements) and/or enhancers. In mammals, expression of pept2 has been observed in kidney, nervous system, lung, lactating mammary gland, spleen, colon, and pancreas (for a review, see Refs. 11 and 31). Similarly, the results from our study demonstrate that pept2 is expressed in different tissues/organs of the zebrafish, with unambiguous RT-PCR amplification products being obtained starting from RNA isolated from kidney, brain, and gut of the adult fish. While expression of pept2 in kidney and brain is a widely accepted fact, expression in the gut is still controversial. In fact, it is known that pept2 is not expressed by the epithelial cells of the gastrointestinal tract, with the possible exception of the colon (as proposed for the rabbit) (13). However, it has been shown recently that mammalian pept2 is highly expressed at the intestinal level, where it strongly associates to the enteric glial cells and tissue-resident macrophages in the neuromuscular layer of the gastrointestinal tract (33). In this respect, the RT-PCR signal detected starting from RNA of the whole gut of the zebrafish might be suggestive of (a) similar location(s) of zebrafish pept2 in the gastrointestinal tract of the adult fish. 2 The Supplemental Material for this article (Supplemental Figs. S1 and S2 and Supplemental Table S1) is available online at http://physiolgenomics. physiology.org/cgi/content/full/00227.2005/DC1. www.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH PEPT2 215 Fig. 7. In situ expression patterns of zebrafish pept2 in embryos at different developmental stages. At 1 dpf, pept2 is widely expressed in the ventricular zone of the embryonic brain (A), and specific staining associates to the ependymal cell layer (B). Strong pept2 expression occurs in zebrafish embryonic ear starting at 2 dpf (C), and staining of otic epithelial structures persists at 3 (D), 4 (E), and 5 (F) dpf. Sense riboprobe controls are shown at 1 (G), 2 (H), and 4 (I) dpf. ac, Anterior crista; am, anterior macula; asc, anterior semicircular canal projection; di, diencephalon; dls, dorsolateral septum; dv, diencephalic ventricle; ep, ependymal cell layer; ey, eye; hb, hindbrain; hv, hindbrain (4th) ventricle; lc, lateral crista; lsc, lateral semicircular canal projection; mb, midbrain; mhb, midbrain-hindbrain boundary; mv, midbrain ventricle; ov, otic vesicle; pc, posterior crista; psc, posterior semicircular canal projection; scp, semicircular canal projections; te, telencephalon. In each picture, anterior is to the left. Dorsal views are shown in A and B, ventral view is shown in H, and lateral views with dorsal to the top are shown in C–G and I. Zebrafish embryos and early larvae are optically transparent. This trait approximately lasts up to day 5 postfertilization and enables easy visualization and inspection of many different tissues/organs, which can be complemented with gene expression analysis at the transcript and/or protein level. In this respect, analysis of gene expression patterns by whole mount in situ hybridization during the embryonic and early larval stages of the fish offers an invaluable support for immediate association of highly expressed genes at the different growing anatomic structures. Physiol Genomics • VOL 24 • In this study, analysis in zebrafish embryos clearly indicates that, with respect to pept1, the expression of which starts at day 2 postfertilization and reaches high levels by day 4 postfertilization (48), zebrafish pept2 is already expressed at day 1 postfertilization. This time corresponds to the start of the “pharyngula” period (18), that is, the evolutionarily conserved (phylotypic) stage when the vertebrate embryo acquires the classic vertebrate bauplan, and the morphologies of the embryos of the diverse vertebrates are most comparable with one another (2). For the zebrafish, this stage approximately dewww.physiolgenomics.org Downloaded from journals.physiology.org/journal/physiolgenomics (054.091.047.013) on November 28, 2021. 216 MOLECULAR AND FUNCTIONAL ANALYSIS OF ZEBRAFISH scribes the 2nd (24 – 48 h postfertilization) of the 3 days of the embryonic development. At the start of the pharyngula period (⬃24 h postfertilization), zebrafish pept2 mRNA can already be detected at the ependymal (and possibly the subependymal) layer of cells surrounding the forebrain and midbrain ventricular cavities, where its expression is particularly abundant, as visualized by whole mount in situ hybridization. Our findings are in close agreement with data on the distribution of pept2 mRNA in mammalian brain, obtained by a similar experimental approach, that report expression by ependymal cells, subependymal cells, and epithelial cells of the choroid plexus as well as by astrocytic glial cells (3). At the ependymal layer and choroid plexus, PEPT2 would play a pivotal role in the removal of many various neuropeptides, peptide fragments, and peptidomimetics from the cerebrospinal fluid, as assessed in recent years by a number of functional studies in cellular, tissue, and animal experimental systems (see, for instance, Refs. 16, 25–27, 38, 39, 42– 44). Interestingly, it has recently been shown that pept2 expression in rat brain is maximal in the fetus and declines rapidly with advancing age (37). Analogously, pept2 transcription declines at the brain level during zebrafish development (A. Romano, unpublished observations). Also, we show that pept2 is highly expressed at the otic vesicle of the developing zebrafish, starting at day 2 postfertilization (i.e., the latest stage of the pharyngula period) and continuing through the subsequent embryonic (i.e., the “hatching” period, up to day 3 postfertilization) and early larval stages (from the “protruding mouth” period up to day 5 postfertilization) (18). This is the embryonic structure that will develop into the auditory/vestibular organ (the inner ear) of the adult fish (see, for instance, Ref. 52). In particular, we suggest the possible association of zebrafish pept2 mRNA with the epithelium of the maturing semicircular canals, i.e., the system that detects angular accelerations. In adult zebrafish, there are three semicircular canals in each ear, anterior, lateral (or horizontal), and posterior, arranged roughly orthogonally to one another. They form as a result of the union between the tips of three cylindrical protrusions from the wall of the otic capsule, which grow toward corresponding bulges of a projection from the lateral wall (50). Development of the semicircular canal system is first visualized at ⬃45 h postfertilization (i.e., the “high pec” stage) with the outgrowth of the epithelial protrusions from the opposite walls of the otic vesicle. Such protrusions project and eventually fuse in the center of the otic vesicle between 52 (i.e., the “long pec” stage) and 64 h postfertilization (i.e., the “pec fin” stage) and serve as hubs of the semicircular canals (17, 50). The canals are the fluid-filled toroidal spaces surrounding each hub. The topology of the canals is complete by 64 h postfertilization, but further outgrowth is required for the semicircular canals to achieve their characteristic curved, canal-like shape. Our in situ data represent the first experimental indication that pept2 is expressed in the vertebrate inner ear. However, it is not possible now to assign any explicit functional role to pept2 at the inner ear, although a clearing role for small peptides and peptide-like molecules similar to that reported for pept2 at the interface between the epithelium and the brain cavities cannot be excluded in principle. In summary, the zebrafish PEPT2-type peptide transporter has been identified and characterized with respect to its transport function and spatio-temporal expression in adult fish and Physiol Genomics • VOL 24 • PEPT2 embryos. Information on pept2 gene structure and chromosomal localization has also been presented. As a novel finding, expression of zebrafish pept2 mRNA has been reported in the otic vesicle, which opens the option that pept2 might be expressed in the inner ear of higher vertebrates and eventually involved in the vestibular and/or auditory functions in humans. 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