(Received for publication, October 31, 1996, and in revised form, January 15, 1997)
From the Departments of Molecular Neurobiology
(TANABE), § Anatomy and Neuroscience, and
¶ Neurosurgery, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565, Japan
Here we report the cloning and functional characterization of a rat novel peptide/histidine transporter (PHT1), which was expressed in the brain and the retina. The cDNA encodes the predicted protein of 572 amino acid residues with 12 putative membrane-spanning domains. The amino acid sequence has moderate homology with a nonspecific peptide transporter found in the plant. When expressed in Xenopus laevis oocytes, PHT1 cRNA induced high affinity proton-dependent histidine transport activity. This transport process was inhibited by dipeptides and tripeptides but not by free amino acids such as glutamate, glycine, leucine, methionine, and aspartate. Dipeptide carnosine transport activity was also confirmed by direct uptake measurement. By in situ hybridization analysis, PHT1 mRNA was widely distributed throughout whole brain. Especially, intense hybridization signals were found in the hippocampus, choroid plexus, cerebellum, and pontine nucleus. Signals were located in both the neuronal and small nonneuronal cells in these areas. PHT1 protein could contribute to uptake of oligopeptides, which function as neuromodulators, and clearance of degraded neuropeptides and be a new member in the growing superfamily of proton-coupled peptide and nitrate transporters, although its structure, localization, and pharmacological characteristics are unique among these members.
Peptide transport is a specific biochemical process in which small peptides are transported across a membrane by energy-dependent saturable carriers. The transport systems are reported in bacteria, fungi, plants, and mammalian tissues (1-4). A large number of genes that encode components of oligopeptide transport systems in bacteria have been cloned and sequenced, whereas only a few eukaryotic and no more than three mammalian peptide transport genes have been reported (5-10) to study mechanisms of the transport systems in mammalian kidney and intestine. Cloning of the cDNAs encoding the mammalian oligopeptide transporters (5-10) has offered insight into the molecular mechanism for uptake of oligopeptides. They have been identified as proton-dependent electrogenic transporters, and their physiological roles are to absorb small peptides arising from digestion of dietary protein in the small intestine (11), as well as to absorb filtrated peptides generated by luminal peptidases in the kidney (12, 13). Although these transporter genes are expressed also in the brain, no oligopeptide transporter mainly expressed in the nervous system has been reported previously.
In the mammalian nervous system, various amino acids and neuropeptides fulfill wide variety of functions such as neuronal transmission, cellular metabolism, and cell volume regulation. They should be removed from extracellular space after finishing their roles; for example, excess glutamate is removed from synapses by its transporters. Catabolism cascade and translocation of much of these compounds are unclear yet, although various aminopeptidases have been identified in the central nervous system that might play a role in the catabolism of neuropeptides (14). Peptide transporters in the brain could contribute to clearance of degraded neuropeptides and relate to cell metabolism. They might also contribute to uptake of bioactive peptides.
Here we report the cloning of a novel peptide/histidine transporter expressed in the rat brain. The transporter has been characterized by functional expression in Xenopus laevis oocytes. Tissue distribution and localization have also been studied by Northern blot analysis and in situ hybridization.
A rat brain cDNA
library constructed in the cloning vector ZAP II (primed with
oligo(dT); Stratagene) was screened by means of plaque hybridization.
The probe was a 55-bp1 oligonucleotide
(TCATCCATCCGATGGCTTTGATAGACACCAGTGCCAGCAGTCCAGAACCCACGAA) synthesized
from the human gene in the data base for expressed sequence tags, which
is homologous to rabbit intestinal peptide transporter (PepT1) cDNA
(Homo sapiens cDNA clone 39680-5). The probe was
labeled with [
-32P]ATP using polynucleotide end kinase
supplied by Pharmacia Biotech Inc.. Hybridization was carried out at
42 °C in a solution containing 40% formamide, 5 × saline/sodium phosphate/EDTA, 1 × Denhardt's solution, 0.1%
SDS, and 100 µg/ml salmon sperm DNA. Washing was done under medium
stringency conditions, once in 2 × SSC (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) at room
temperature for 30 min and twice in 1 × SSC and 0.4% SDS at
50 °C for 30 min. Positive clones were identified and plaque
purified by secondary screening, followed by DNA extraction using a
standard method (15). Insert cDNAs in positive phages were
recirculated to the subclone in the pBluescript SK(
) phagemid vector
after superinfection with R408 helper phages as recommended by the
manufacturer (Stratagene). For subsequent sequence analysis, a set of
deletion clones was prepared from the insert cDNAs using a
kilosequencing deletion kit (Takara).
Sequencing was carried out by the dideoxy chain termination method (16) using a Taq dye primer cycle sequencing kit (Applied Biosystems Inc., Tokyo, Japan). The nucleotide sequences were then analyzed with an Applied Biosystems 373A automated DNA sequencer. The final sequence was confirmed from both strands. Sequence homology searches and comparison were performed with the National Center for Biotechnology Information E-mail server FASTA, the BLAST (BLASTX) E-mail server on the Genome Net, and molecular analysis systems in the DNA Data Bank of Japan.
Functional Expression in X. laevis OocytesThis was done as
described previously (17) by microinjecting cRNA (25 ng) derived from
the clone. The plasmid cDNA was linearized with ApaI and
transcribed using T3 RNA polymerase for sense RNA and was also
linearized with EcoRI and transcribed using T7 RNA polymerase for antisense RNA. Capping was done with the cap analog m7G(5)PPP(5
)G (Ambion). Adult female X. laevis were
anesthetized with ice and dissected, and their ovaries were removed and
placed in Ca2+-free ND96 solution (96 mM NaCl,
2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) with 2 mg/ml collagenase at 20 °C for
2 h with gentle agitation. Stage 5-6 oocytes (1~1.2 mm in
diameter; mean value, 1.1 mm) were selected (18) by hand, and oocytes
were injected manually and immediately with 25 ng of a cRNA in
diethylpyrocarbonate-treated H2O by using a positive
displacement pipette (Drummond Scientific Corp., Broomall, PA).
Transport measurements were made with individual oocytes 3 days after
injection. The concentration of [14C]histidine (Amersham
Corp.) was 1 µM with or without inhibitors, and the
uptake medium was ND96 solution (5 mM HEPES, 96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, and 1.8 mM CaCl2), pH 5.5, 6.5, and 7.5. The concentration of [3H]carnosine (Moravek
Biochemicals, Brea, CA) was 1.5 µM. The incubation time
for transport measurements with oocytes was 1 h, at a temperature of 25 °C. After washing the oocytes three times with ND96 at
4 °C, they were homogenized, and the incorporated radioactivity was counted in a Beckman Instruments scintillation counter. Na+
dependence was tested in Na+-free ND96 in which NaCl was
replaced by choline chloride. Nonspecific transport and binding were
determined in parallel experiments with antisense cRNA- and
water-injected and noninjected oocytes. Data represent PHT1
cRNA-induced transport, calculated by subtracting the transport in
control oocytes from the sense cRNA-injected oocytes.
Tissue distribution of mRNA
transcripts coding for this transporter was determined by Northern
blot. Total RNA was isolated by the acid guanidinium
isothiocyanate-phenol-chloroform method as described previously (19).
Aliquots (40 µg) of RNA were separated on 1% agarose-formaldehyde
gels and was transferred onto nylon membranes (Hybond-N, Amersham). A
600-bp rat cDNA (bases 260-860) was labeled by random priming
(Amersham) with [-32P]dCTP (3000 Ci/mmol, Amersham).
Hybridization was carried out at 42 °C overnight in 50% formamide,
5 × SSC, 0.1% SDS, 50 mM sodium phosphate, 5 × Denhardt's solution, and 100 µg/ml salmon sperm DNA. The blots were
washed three times at 42 °C for 30 min in 1 × SSC and 0.1%
SDS. With the aid of intensifying screens, the membranes were then
placed in contact with x-ray film at
80 °C for 5 days. After
quantification of the hybridized probe, it was removed from the
membrane to be hybridized with the glyceraldehyde-3-phosphate dehydrogenase probe. The hybridized probe was removed with a boiling solution of 0.1% SDS poured on the membrane and allowed to cool to
room temperature. After the removal of the probe, hybridization and
washing for the glyceraldehyde-3-phosphate dehydrogenase probe was
carried out in the same manner. Finally, x-ray film was placed on the
membrane at
80 °C for 16 h.
Localization of the transporter mRNA in the brain was determined by in situ hybridization. The antisense and sense probes were synthesized from a 600-bp rat PHT1 cDNA (bases 260-860) insert cloned in the Novagen T vector. To synthesize hybridization riboprobes by in vitro transcription, this sequence was first linearized by digestion with appropriate restriction endonucleases. The linearized cDNA was then incubated at 37 °C for 60 min with a mixture of reagents. This mixture consisted of 2 µl of transcription buffer (5×), 0.5 µl of 100 mM dithiothreitol, 0.5 µl of RNase inhibitor, 0.5 µl of 10 mM ATP, CTP, and GTP, 5 µl of 35S-UTP (NEG-039H, DuPont NEN), and 0.5 µl of DNA template (1 µg/ml) with 1 µl of appropriate RNA polymerase (T7 RNA polymerase for the antisense probe and SP6 RNA polymerase for the sense probe). DNA digestion was attained with the addition of 2 µl of DNase and incubation at 37 °C for 10 min. Efficacy of labeling was estimated by counting radioactivity of the synthesized probes.
In situ hybridization techniques for PHT1 mRNA (RNA probe) were based on those of Wilkinson et al. (20) with some modification. The sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 20 min. After washing with phosphate buffer, the sections were treated with 10 µg/ml of proteinase K in 50 mM Tris-HCl and 5 mM EDTA, pH 8.0, for 5 min at room temperature. They were then fixed again in the same fixative, acetylated with acetic anhydride in 0.1 M triethanolamine, rinsed with phosphate buffer, dehydrated, and air dried. The 35S-labeled RNA probes (sense or antisense) were diluted in hybridization buffer, placed over the sections, and covered with siliconized coverslips. Hybridization was performed overnight in a humid chamber at 55 °C. The hybridization buffer consisted of 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, 10 mM phosphate buffer, 10% dextran sulfate, 1 × Denhardt's solution, 0.2% sarcosyl, 500 µg/ml yeast tRNA, and 200 µg/ml herring sperm DNA (pH 8.0). The probe concentration was 5 × 105 cpm/150 µl/slide. After hybridization, the slides were immersed in 5 × SSC at 55 °C, and the coverslips were allowed to fall off. The sections were then incubated at 65 °C in 50% deionized formamide with 2 × SSC for 30 min. After rinsing with RNase buffer (0.5 M NaCl, 10 mM Tris-HCl, and 5 mM EDTA, pH 8.0) four times for 10 min each at 37 °C, the sections were treated with 1 µg/ml of RNase A in RNase buffer for 30 min at 37 °C. After an additional washing in RNase buffer, the slides were incubated in 50% formamide with 2 × SSC for 30 min at 65 °C, rinsed with 2 and 0.1 × SSC for 10 min each at room temperature, dehydrated in an ascending alcohol series, and air dried.
X-ray film was placed on the uncoated sections for 3 days. Next, the slides were coated with Ilford K-5 emulsion diluted in distilled water containing 2% glycerine (1:1). The slides were exposed for 3 weeks in a tightly sealed dark box at 4 °C, developed in Kodak D-19 emulsion, fixed with photographic fixer, stained with thionine, and coverslipped. After the x-ray macroautoradiogram had been studied, the tissue sections were examined under a regular light microscope.
We isolated 11 positive clones from a rat brain cDNA library using the 55-mer
oligonucleotide probe synthesized from an expression sequence tag clone
that showed homology with the rabbit oligopeptide transporter (PepT1)
cDNA. Restriction enzyme analysis revealed the same digestion
pattern, suggesting that all these clones were originating from a
single gene. It was confirmed by DNA sequence. The longest cDNA
construct was subcloned into pBluescript SK().
The cDNA is 2751 bp long with an open reading frame of 1719 bp
(including the termination codon) encoding a protein of 572 amino acids
(Fig. 1A), of which the gene
was designated PHT1. The open reading frame is flanked by a
24-bp long sequence at the 5-end and by a 1008-bp sequence at the
3
-end. The proposed initiating ATG conforms to a Kozak consensus
sequence (21). A polyadenylation signal, ATAAA, was found in the
3
-noncoding region. The encoded protein is predicted to have a core
molecular mass of 64.9 kDa. There is no N-terminal signal sequence.
Hydropathy analysis (22-25) of the primary amino acid sequence of the
predicted protein shows the presence of 12 putative transmembrane
domains. Four potential N-linked glycosylation sites of PHT1 protein
are located at positions 134, 141, 220, and 435 in the hydrophilic regions that according to a model based on the hydropathy analysis face
the extracellular matrix. There are 11 potential sites for protein
kinase C-dependent phosphorylation but no site for protein kinase A-dependent phosphorylation. The amino acid sequence
of this clone shows significant similarity to the peptide transporter (histidine transport protein) NTR1 from the plant Arabidopsis thaliana (29% identity and 50% similarity) (Fig. 1B).
The consensus sequence with PHT1 and NTR1 proteins was most conserved
in the regions corresponding to putative fifth and sixth membrane
spanning portions (45% identity and 67% similarity). Members of the
mammalian peptide transporters reveal only weak similarity with PHT1
protein such as PepT1 (17% identity and 32% similarity) and PepT2
proteins (12% identity and 27% similarity). However, there is no
significant similarity between PHT1 protein and the hpt-1 protein,
which encodes a protein associated with intestinal peptide transport
(10).
Functional Expression of PHT1
To assess the transport activity of PHT1 protein, uptake of [14C]histidine was monitored by a Xenopus oocyte uptake assay.
The oocytes injected with the sense cRNA (derived from the cDNA
insert cloned into pBluescript SK()) consistently showed more than
5-fold histidine transport activity than did antisense cRNA- or
water-injected or noninjected oocytes. There was no difference among
antisense cRNA- or water-injected or noninjected oocytes.
The histidine uptake into oocytes expressing PHT1 protein during 1 h was linear (Fig. 2A), and an incubation
period of 1 h was used in subsequent experiments to approximate
the initial transport rate. Transport of histidine into oocytes
expressing PHT1 protein was saturable, and the Km
value (pH 5.5) for histidine was 17 µM, showing that PHT1
cDNA encodes a high affinity histidine transporter (Fig. 2,
B and C).
The cation specificity of the induced transport activity was determined by replacing NaCl in the ND96 solution with choline chloride. The replacement did not affect histidine influx, indicating that Na+ would not serve as the cotransported ion (Fig. 2D). Transport activity at 4 °C was almost eliminated, suggesting that this transporter is temperature-dependent (Fig. 2D). The expressed transporter was proton-dependent, because the histidine transport when measured at pH 5.5 was 3-fold more voluminous than at pH 7.5 (Fig. 2E).
Since other mammalian peptide transporters have a broad substrate
specificity, we examined the selectivity of PHT1 protein by inhibition
studies, in which the ability of unlabeled amino acids, dipeptides, and
tripeptides to compete with [14C]histidine (1 µM) was tested. Amino acids such as glutamate, glycine,
leucine, methionine, aspartate, cyclo-(Leu-Gly), and leucine-enkepharine had no effect on the histidine transport at an
inhibitor concentration of 500 µM (Fig. 3;
data not shown as aspartate). On the other hand, the efficient
competitive ability of Gly-Gly, Gly-Leu, Met-Met, carnosine, His-Leu,
and Gly-Gly-Gly was observed even at a concentration of 50 µM (Fig. 3). Various dipeptides and tripeptides thus were
potent inhibitors of histidine transport.
To confirm that PHT1 is an oligopeptide transporter, the transport of
the dipeptide carnosine by the oocytes was measured (Fig.
4). The oocytes injected with the sense cRNA showed more than 10-fold carnosine transport activity than did water-injected oocytes. In addition, 50 µM unlabeled carnosine
efficiently competed with labeled carnosine (1.5 µM) for
the transport process.
Since PHT1 is considered to be an electrogenic transporter, and membrane potential may affect the measured kinetics, determined constants are fundamentally relevant to the experimental conditions, which were not made under voltage-clamped conditions.
Tissue Distribution and Localization of PHT1 mRNA Tissue-specific expression of the mRNA
corresponding to PHT1 was examined by high-stringency Northern analysis
(Fig. 5). PHT1 mRNA (2.9 kilobases) in rat tissues
was abundantly expressed in the brain and eye, whereas faint signals
were observed in the lung and spleen, and no transcripts were detected
in the pancreas, kidney, intestine, liver, heart, and skeletal muscle.
This distribution was significantly different from those of the other
mammalian oligopeptide transporters, PepT1 and PepT2 (5-7), which were found to express abundantly in the small intestine, kidney, and liver.
PHT1 is the first peptide/histidine transporter predominantly expressed
in the nervous system.
To determine the detailed localization of PHT1 gene
expression in the brain, we performed in situ hybridization
using a 35S-labeled cRNA probe. Specificity of
hybridization signals for PHT1 mRNA was confirmed by control study
using the sense cRNA probe. In adult rats, strong expression of PHT1
mRNA was detected in the hippocampus, cerebellum, and pontine
nucleus, and weak to moderate expression was detected in other regions
of the brain, including the cerebral cortex, brain stem, thalamus, and
hypothalamus (Fig. 6, A and B).
The PHT1 mRNA signals were detected in both the neuronal cells and
small nonneuronal cells in the brain.
The amino acid sequence of PHT1, a novel peptide/histidine transporter abundantly expressed in the nervous system, was most similar to that of the peptide transporter (histidine transport protein) NTR1 from the plant A. thaliana (26-28), although weak or no similarity was found between PHT1 and other mammalian peptide transporters.
When expressed in X. laevis oocytes, the PHT1 gene product induced high affinity proton-dependent histidine transport activity. The uptake mediated by the transporter was dependent on proton and temperature and completely independent of extracellular Na+. As PHT1 protein seems to be a member of the peptide and nitrate transporter superfamily, we next determined the substrate specificity of PHT1 protein by competition experiments and direct uptake measurement of dipeptide in the oocytes. Various dipeptides and tripeptides are potent substrates for PHT1. The substrate specificity of transport activities resembles that of the NTR1 protein, which is a high affinity oligopeptide transporter. Among dipeptides and tripeptides used in this study, Met-Met, carnosine, and His-Leu had more potent inhibitory effects, although glycyl peptides (Gly-Gly, Gly-Leu, and Gly-Gly-Gly) were less effective inhibitors of histidine transport. This finding is also similar to the feature of NTR1 protein.
The findings that both neuronal and nonneuronal cells in the brain widely express the mRNA for this transporter suggest that the removal of degraded neuropeptides and oligopeptides as well as the uptake of peptide-derived drugs circulating in the blood might be attained in these cells. In particular, signals in the tuberomamillary nucleus of the posterior hypothalamus suggest that PHT1 protein could uptake histidine as a substrate for histamine synthesis.
There are several oligopeptides that are considered neuromodulators, and some have been found to be eliminated by uptake after being released into the synaptic cleft (29). PHT1 might function as a terminator of these neuromodulators. On the other hand, various neuropeptides, for example, Met-enkephalin, are metabolized to become metabolic fragments, rate of accumulation of which was specific to brain regions due to the difference of peptidases. However, how these degraded oligopeptides are removed from the synapse has not been confirmed (30). PHT1 might be related to the catabolic pathway of neuropeptides. Recent data show that the levels of oligopeptides in serum are manyfold higher than once thought (31). The uptake of small peptides after intravenous administration was observed in the central nervous system as well as in the kidney (32). These oligopeptides might be transported into the cells and be used as nutrients and for the regulation of cellular metabolism in the central nervous system.
To summarize, this report concerns the primary structure and functional characterization of PHT1 protein, a novel proton-coupled histidine and oligopeptide transporter in the nervous system. PHT1 protein could be involved in the clearance of neuromodulators and in the catabolic process of degraded neuropeptides in the brain. Precise mechanisms need elucidation in future studies. This transporter represents a new member in the growing superfamily of proton-coupled peptide and nitrate transporters and shows distinct structural differences and specific localization.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000280[GenBank].
We thank K. Nakano for nucleotide sequencing. We are grateful to Dr. Motohiko Takemura for helpful discussion.