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/Peptide Cotransporter
(Received for publication, July 18, 1994; and in revised form, December 22, 1994)
From the
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
In mammalian small intestine, a H
-coupled
peptide transporter is responsible for the absorption of small peptides
arising from digestion of dietary proteins. Recently a cDNA clone
encoding a H/peptide cotransporter has been isolated
from a rabbit intestinal cDNA library (Fei, Y. J., Kanai, Y.,
Nussberger, S., Ganapathy, V., Leibach, F. H., Romero, M. F., Singh, S.
K., Boron, W. F., and Hediger, M. A.(1994) Nature 368,
563-566). Screening of a human intestinal cDNA library with a
probe derived from the rabbit H/peptide cotransporter
cDNA resulted in the identification of a cDNA which when expressed in
HeLa cells or in Xenopus laevis oocytes induced
H-dependent peptide transport activity. The predicted
protein consists of 708 amino acids with 12 membrane-spanning domains
and two putative sites for protein kinase C-dependent phosphorylation.
The cDNA-induced transport process accepts dipeptides, tripeptides, and
amino 
-lactam antibiotics but not free amino acids as substrates.
The human H/peptide cotransporter exhibits a high
degree of homology (81% identity and 92% similarity) to the rabbit
H/peptide cotransporter. But surprisingly these
transporters show only a weak homology to the
H-coupled peptide transport proteins present in
bacteria and yeast. Chromosomal assignment studies with somatic cell
hybrid analysis and in situ hybridization have located the
gene encoding the cloned human H/peptide cotransporter
to chromosome 13 q33
q34.
Mammalian small intestine expresses a transport system that is
specific for small peptides consisting of 2-4 amino
acids(1) . Free amino acids do not serve as substrates for this
system. The physiological role of the intestinal peptide transporter is
to absorb small peptides arising from digestion of dietary proteins.
The pharmacological relevance of this transporter has become evident in
recent years because intestinal absorption of orally active amino
-lactam antibiotics and other peptide-like drugs is mediated by
this transporter(1, 2, 3) . It has been
proposed that the transporter has the potential to become an important
drug delivery system(3) . Studies on the energetic aspects of
the transport system have demonstrated that the driving force for the
transporter is an electrochemical H gradient rather
than an electrochemical Na gradient(1, 4, 5, 6) . A
H-dependent peptide transport system is also expressed
in the mammalian kidney(7, 8, 9) , but it is
not known whether the renal transporter is identical to or distinct
from the intestinal transporter.
Microinjection of rabbit intestinal
mRNA into Xenopus laevis oocytes leads to functional
expression of a H-dependent peptide transport system
with characteristics similar to those of the transporter in the native
tissue(10) . Recently we have isolated a cDNA encoding a
H-dependent peptide transporter from a rabbit small
intestinal cDNA library using this Xenopus oocyte expression
system as the screening procedure(11) . We report in this paper
the cloning of a human intestinal H/peptide
cotransporter. The cloned transporter has been characterized by
functionally expressing the transporter in HeLa cells as well as in X. laevis oocytes. Chromosomal localization studies
have revealed that the gene for the transporter is present on
chromosome 13 q33q34.
gt10) used here was prepared with mRNA
from the ileum(12) . The library was screened by plaque
hybridization using VCS 257 cells. The probe was a 0.6-kb (
)fragment arising from the 5` end of the rabbit intestinal
H/peptide cotransporter cDNA(11) . The
fragment was released from the full-length cDNA by EcoRI
digestion and radiolabeled with [
-
P]dCTP
using the oligolabeling kit from Pharmacia LKB Biotechnol.
Hybridization was carried out at 42 °C in a solution containing 50%
formamide, 6 
SSC, 5 Denhardt's solution, 0.1%
SDS, and 100 µg/ml of salmon sperm DNA. Washing was done under
medium stringency conditions, two times in 3 SSC, 0.5% SDS at
55 °C for 30 min and one time in 1 SSC, 0.1% SDS at 60
°C for 30 min. Positive clones were identified and plaque purified
by secondary and tertiary screening. On the tertiary autoradiographs,
100% of the plaques showed positive hybridization signal, confirming
the purity of the clones.
Lambda Prep DNA Purification
System (Promega). Digestion of the DNA with EcoRI yielded
three DNA fragments, 1.2, 0.6 and 0.4 kb in size, in addition to the
two fragments (11 and 40 kb in size) arising from the vector. All three
fragments of the cDNA insert hybridized to the full-length (2.7 kb)
rabbit intestinal H/peptide cotransporter cDNA probe.
The digestion pattern indicated that there are two internal sites for EcoRI in the insert and that the size of the full-length
insert is approximately 2.2 kb. In order to obtain the full-length cDNA
for functional expression, partial digestion with EcoRI was
used. The phage DNA was digested with increasing concentrations of EcoRI, and the digestion fragments were analyzed by Southern
hybridization. Partial digestion yielded three new fragments (2.2, 1.8,
and 1.6 kb) in addition to the above described three and all six
fragments hybridized to the full-length rabbit intestinal
H/peptide cotransporter cDNA probe. The concentration
of EcoRI which generated the maximal amount of the 2.2-kb
fragment was chosen for digestion in a large scale. The full-length
cDNA was isolated, gene cleaned, and subcloned.
Sequencing was done by the dideoxy chain termination method(13) . Synthetic oligonucleotide primers were used whenever necessary to complete the sequencing of both the sense and the antisense strands. Sequence analysis was performed by the software package GCG version 7.B (Genetics Computer Groups, Inc. Madison, WI). Multiple sequence alignment was done using the Genbank Program PILEUP.
) vaccinia
virus encoding T7 RNA polymerase and then transfected with the plasmid
carrying the full-length cDNA. The strategy behind this approach is
that the virus-encoded T7 RNA polymerase catalyzes the transcription of
the cDNA under the control of the T7 promotor allowing transient
expression of the cDNA-encoded protein in the HeLa cell plasma
membrane. After 8-10 h post-transfection, transport measurements
were made at room temperature with
[2-
C]glycyl-[1-C]sarcosine
(specific radioactivity 109 mCi/mmol; Cambridge Research Biochemicals,
United Kingdom). The uptake medium was 25 mM Mes/Tris (pH 6.0)
or 25 mM Hepes/Tris (pH 7.5), containing 140 mM NaCl,
5.4 mM KCl, 1.8 mM CaCl
, 0.8 mM MgSO
, and 5 mM glucose. In most experiments,
the time of incubation for transport measurements was 2 min.
Nonspecific transport was determined in parallel experiments with the
plasmid vector. This transport was negligible and represented only
3-5% of the transport measured in cells which were transfected
with the vector carrying the cDNA insert. Moreover, this value was not
due to carrier-mediated transport as concluded from the lack of
inhibition by excess unlabeled glycylsarcosine. Thus there was no
detectable endogenous glycylsarcosine transport activity in HeLa cells.
Therefore, the transport values measured in cells transfected with the
vector-cDNA construct were analyzed directly without correcting for the
values obtained in cells which were transfected with the vector alone.
G(5`)PPP(5`)G (Pharmacia).
Water-injected oocytes served as controls. Transport measurements were
made with individual oocytes 4 days after injection. The concentration
of
[2-C]glycyl-[1-C]sarcosine
was 30 µM, and the uptake medium was 3 mM Hepes/Mes/Tris (pH 5.5) or 3 mM Hepes/Tris (pH 7.5),
containing 100 mM NaCl, 2 mM KCl, 1 mM MgCl, and 1 mM CaCl. The
incubation time for transport measurements with oocytes was 1 h.
/peptide cotransporter was
determined by Northern blot. Poly(A) mRNA was isolated
from human ileum and two cell lines of human intestinal origin (Caco-2
and HT-29) using the FastTrack mRNA isolation kit (Invitrogen).
Poly(A) mRNA samples (2.5-4 µg) from these
sources were denatured and size-fractionated on an agarose gel. The
fractionated RNA was transferred onto a nylon membrane (Hybond
N, Amersham Corp.) and probed with the full-length
human intestinal H/peptide cotransporter (hPEPT 1)
cDNA under high stringency conditions (hybridization: overnight
incubation at 42 °C in 50% formamide and 10% dextran sulfate;
washing: once with 3 SSC-0.5% SDS at room temperature for 30
min, twice with 3 SSC, 0.5% SDS at 55 °C for 30 min and
once with 0.1 SSC, 0.1% SDS at 55 °C for 30 min). Following
stripping of the blot, similar hybridization was conducted with human
-actin cDNA (Clontech) to assess RNA loading and transfer
efficiency.Northern analysis with hPEPT 1 cDNA was also done using
a commercially available membrane blot containing size-fractionated
poly(A) mRNA from several tissues of human origin
(Clontech). This blot was also reprobed with human -actin cDNA for
assessment of RNA loading and transfer efficiency.
/peptide cotransporter. This
was done by somatic cell hybrid analysis and by in situ hybridization to human metaphase chromosomes. A mapping panel
(panel no. 1) consisting of 17 mouse-human and one Chinese
hamster-human hybrids was obtained from the National Institute of
General Medical Sciences' Human Genetic Mutant Cell Repository
and used in somatic cell hybrid analysis. DNA samples obtained from
these cells were digested with EcoRI, separated by
electrophoresis on agarose gels, transferred onto a nylon membrane, and
hybridized to [P]hPEPT 1 cDNA as described
previously(17) . In situ hybridization of
[
H]hPEPT 1 cDNA to human chromosomes and emulsion
autoradiography were carried out by the method of Harper and Saunders (18) . Chromosomes were G-banded using Wright's stain,
and G-banded chromosomes were analyzed for silver grain localization.
The hPEPT 1 cDNA was labeled by nick translation either in the presence
of [-P]dCTP (Southern blot hybridization)
or in the presence of [H]dCTP and
[H]dTTP (in situ hybridization) and used
in these experiments.
/Peptide
Cotransporter cDNA 10
plaques from a human intestinal cDNA library with a 5` end
fragment (0.6 kb) of the rabbit intestinal H/peptide
cotransporter cDNA as a probe yielded four positive clones. All of
these clones were related as judged from EcoRI digestion
pattern. One of them was characterized in detail in terms of its
structure and function. Digestion of the phage DNA with EcoRI
gave three fragments indicating the presence of two internal EcoRI sites in the insert. The full-length cDNA was prepared
from the phage DNA by partial digestion with EcoRI. A pilot
experiment was run to determine the optimal concentration of the enzyme
which would give the maximal amount of the full-length cDNA insert.
After digesting the phage DNA with increasing concentrations of EcoRI, the digestion products were size-fractionated and then
probed with the full-length rabbit intestinal
H/peptide cotransporter cDNA by Southern blot (Fig. 1). Complete digestion with the maximal amount of the
enzyme used generated three hybridizing signals (1.2, 0.6, and 0.4 kb
in size). With lesser concentrations of the enzyme, the amounts of
these three fragments decreased while larger size fragments appeared.
The size of the largest fragment was 2.2 kb. Two other partial
digestion products (1.8 and 1.6 kb) were also generated. This digestion
pattern indicated that the 2.2-kb full-length insert consisted of the
1.2-kb fragment flanked on one side by the 0.6-kb fragment and on the
other side by the 0.4-kb fragment. The concentration of EcoRI
giving the maximal amount of the 2.2-kb fragment was chosen for a large
scale preparation. The full-length fragment was then isolated and
subcloned into pBluescript SK II.
Figure 1:
EcoRI digestion and Southern
blot analysis of the phage DNA containing the hPEPT 1 cDNA insert. The
phage DNA was digested with increasing concentrations of EcoRI, and the digestion products were electrophoresed and
probed with P-labeled full-length rabbit intestinal
H/peptide cotransporter cDNA. The arrow at
the top indicates the lane containing the maximal amount of the
full-length (2.2 kb in size) hPEPT 1 cDNA. The concentration of EcoRI corresponding to this lane was chosen for a large scale
preparation of the full-length hPEPT 1
cDNA.
The cDNA is 2,263 bp long with an
open reading frame of 2,127 bp (including termination codon) encoding a
protein of 708 amino acids (Fig. 2). The open reading frame is
flanked by a 56-bp long sequence on the 5` end and by a 80-bp long
sequence on the 3` end. The predicted initiation codon is preceded by a
Kozak consensus sequence (GCC GCC)(19) . The encoded protein is
predicted to have a core molecular size of 78,810 Da and an isoelectric
point of 8.6. Hydropathy analysis of the primary amino acid sequence of
the predicted protein shows the presence of 12 putative transmembrane
domains with a long (
200 amino acids) hydrophilic segment between
the transmembrane domains 9 and 10. This hydrophilic segment contains
seven putative N-linked glycosylation sites. When modeled to
accommodate all the transmembrane domains and to allow the long
hydrophilic loop on the extracellular side, the model places both the
amino terminus and the carboxyl terminus on the cytoplasmic side. There
are two potential sites for protein kinase C-dependent phosphorylation
(Ser-357 and Ser-704) but no site for protein kinase A-dependent
phosphorylation. Comparison of the amino acid sequence between this
clone and the rabbit intestinal H/peptide
cotransporter reveals a high degree of homology (81% identity and 92%
similarity). Most of the conserved sequences occur within the putative
transmembrane domains. The human homolog is 1 amino acid residue longer
than the rabbit counterpart. An important difference between these two
proteins is that the rabbit intestinal H/peptide
cotransporter possesses a site for protein kinase A-dependent
phosphorylation (Thr-362) whereas the human homolog does not. The
threonine residue present at this site in the rabbit transporter is
replaced by alanine in the human homolog.
Figure 2: hPEPT 1 cDNA and predicted primary amino acid sequence.
/peptide
cotransporter cDNA (hPEPT 1) insert cloned into pBluescript SK II in
such a way that its transcription is under the control of the T7
promotor. When this plasmid was transfected into HeLa cells expressing
a recombinant vaccinia virus T7 RNA polymerase, the cells were able to
transport glycylsarcosine in a H-dependent manner (Fig. 3). The H dependence was evident from the
4- to 5-fold greater transport when measured at an extracellular pH of
6.0 instead of 7.5. Control cells transfected with the empty vector
showed negligible transport when measured at pH 6.0. An incubation
period of 2 min and an extracellular pH of 6.0 were used in subsequent
experiments with hPEPT 1-transfected cells to approximate the initial
transport rate.
Figure 3:
Transport of glycylsarcosine in HeLa cells
transfected with pBluescript SK II alone or with hPEPT 1 cDNA. Cells
were transfected with either vector alone &cjs1260; or with vector
carrying the hPEPT 1 cDNA (
, 
). Transport of
[C]glycylsarcosine (20 µM) was
measured for different time periods either at pH 6.0 (,
&cjs1260;) or at pH 7.5 (). Values represent means ± S.E.
for three determinations.
Table 1describes the results of competition
experiments performed to determine the substrate specificity of the
hPEPT 1. In these experiments, the ability of unlabeled amino acids,
dipeptides, tripeptides, and amino -lactam antibiotics to compete
with [C]glycylsarcosine (20 µM) for
the transport process induced by hPEPT 1 was studied. At a
concentration of 10 mM, free amino acids had no effect on the
transport. On the other hand, dipeptides, tripeptides, as well as the
three amino -lactam antibiotics (cyclacillin, cefadroxil, and
cephalexin) were found to be potent inhibitors of the transport. The
hPEPT 1-induced transport system thus appears to be specific for small
peptides and peptide-like compounds with no affinity toward free amino
acids.
The induced transport process is saturable as evident from
transport measurements done at varying concentrations of
glycylsarcosine in the range of 0.05-5 mM (Fig. 4). Under these conditions, the transport of
radiolabel from 20 µM [C]glycylsarcosine in hPEPT 1-transfected
cells was reduced in the presence of 25 mM unlabeled
glycylsarcosine to a level observed in vector-transfected cells in the
absence of unlabeled glycylsarcosine, indicting that the transport
induced by hPEPT 1 was almost completely carrier-mediated. The
experimental data were found to fit best for a model describing the
uptake as the result of a single carrier plus a diffusional component.
The diffusional coefficient was 0.35 ± 0.06. The Eadie-Hofstee
transformation of the data for the carrier-mediated uptake yielded a
linear plot (r = -0.98) (Fig. 4). The
kinetic parameters for the carrier-mediated uptake, K
(Michaelis-Menten constant) and V
(maximal
velocity), were 0.29 ± 0.04 mM and 4.7 ± 0.3
nmol/2 min/10
cells. The diffusional component represented
2 and 28% of total uptake measured at 0.05 and 5 mM glycylsarcosine, respectively.
Figure 4: Kinetics of glycylsarcosine transport induced by the hPEPT 1 cDNA in HeLa cells. Transport of glycylsarcosine in hPEPT 1 cDNA-transfected HeLa cells was measured over a concentration range of 0.05-5 mM. The pH of the medium was 6.0, and the incubation time was 2 min. Values represent means ± S.E. for four determinations. Inset, Eadie-Hofstee transformation of the data for carrier-mediated uptake.
The transport function of hPEPT
1 was also assessed by expressing the protein in X. laevis oocytes (Fig. 5). The oocytes injected with cRNA derived
from the hPEPT 1 cDNA showed 8-fold greater activity for
glycylsarcosine transport than water-injected oocytes. The expressed
transporter was H dependent because the dipeptide
transport was significantly higher when measured at pH 5.5 than at pH
7.5. In addition, unlabeled glycylsarcosine was able to compete with
[C]glycylsarcosine for the transport process
induced by hPEPT 1.
Figure 5:
Transport of glycylsarcosine in X.
laevis oocytes injected either with hPEPT 1 cRNA or with water.
Oocytes were microinjected with 25 ng of hPEPT 1 cRNA and transport of
[C]glycylsarcosine (30 µM) was
measured with individual oocytes on day 4 post-injection.
Water-injected oocytes served as controls. Transport in water-injected
oocytes was measured at pH 5.5 (A) whereas transport in
cRNA-injected oocytes was measured at either pH 5.5 (B and D) or pH 7.5 (C). When present (D),
concentration of unlabeled glycylsarcosine was 10 mM. Values
represent means ± S.E. for 12-16 oocytes from two separate
experiments.
The magnitude of stimulation of glycylsarcosine
transport induced in X. laevis oocytes by hPEPT 1 is five to
six times smaller than the corresponding value observed with the rabbit
H/peptide cotransporter cDNA, although the amino acid
sequences in the coding region of these two clones exhibit 81% identity
and 92% similarity. This difference is most likely due to the fact that
the rabbit clone was complete with its poly(A) tail whereas the human
clone has a truncated 3`-noncoding region comprising of only 80 bp with
no poly(A) sequence. The poly(A) tail is known to stabilize mRNA.
Therefore, it is likely that the cRNA derived from the rabbit clone is
more stable in X. laevis oocytes than the cRNA derived from
the human clone.
mRNA from eight tissues:
heart, brain, placenta, lung, liver, skeletal muscle, kidney, and
pancreas (Fig. 6). Among these tissues, a 3.3-kb hybridizing
signal was present in placenta, liver, kidney, and pancreas. The
transcript was absent in other tissues. The functional characteristics
of the H/peptide cotransporter have been investigated
in detail only in intestine and kidney. Human placenta possesses
peptide transport activity (20) but whether this activity is
mediated by the H-coupled peptide transporter is not
known. Therefore, it is interesting that the placenta expresses the
PEPT 1 mRNA though at very low levels. The presence of 3.3-kb mRNA
transcript in kidney is not unexpected, but the observation that the
levels of the transcript in this tissue are manyfold lower than the
levels in intestine is surprising. Brush border membrane vesicles
prepared from kidney exhibit robust H/peptide
cotransport activity(21, 22, 23) . It is
therefore possible that a major portion of the
H/peptide cotransport activity expressed in kidney is
not due to PEPT 1. The existence of multiple forms of
H/peptide cotransporter in mammalian tissues thus
seems very likely. The levels of the 3.3-kb mRNA transcript in liver
and pancreas are significantly higher than in kidney. Interestingly, it
has been reported that liver does not have the ability to take up
peptides from the circulation(24) . Liver contains
heterogeneous population of cells, and therefore the possibility that
the peptide transport activity is expressed in cells other than
hepatocytes cannot be excluded. Whether pancreas expresses
H/peptide cotransport activity is not known.
Figure 6:
Northern blot analysis of PEPT 1 mRNA
transcripts in human tissues. A commercially available
hybridization-ready blot containing poly(A) mRNA from
different human tissues (Multiple Tissue Northern blot; Clontech) was
used to hybridize with the hPEPT 1 cDNA probe. In addition,
poly(A) mRNA samples isolated from human ileum and the
human colon carcinoma cell line Caco-2 were also analyzed in a similar
manner. Each lane contained poly(A) mRNA and lanes
1-10 represent heart, brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas (2 µg each), ileum (2.5 µg), and
Caco-2 cells (4 µg), respectively. The same blot was stripped and
reprobed with the -actin cDNA. The sizes of hybridizing bands were
determined using RNA standards run in parallel in an adjacent
lane.
Northern analysis of poly(A) mRNA isolated from
human ileum revealed the presence of a major RNA species, 3.3 kb in
size, which hybridized to the hPEPT 1 cDNA (Fig. 6). There are
several minor hybridizing RNA species in the human intestine. We also
analyzed poly(A) mRNA from two cell lines of human
intestinal origin for the presence of PEPT 1 mRNA. Caco-2 cells which
are known to possess H/peptide cotransporter activity (25) contains the primary 3.3-kb transcript as well as the
other minor transcripts. Thus, the distribution of the different PEPT 1
mRNA transcripts in this cell line is similar to that in the human
intestine. In contrast, HT-29 cells which are also commonly used as a
model for intestinal transport studies do not contain any detectable
hybridizing signal (data not shown). There was a report by Dantzig and
Bergin (26) describing expression of peptide transport activity
in HT-29 cells. We investigated the transport of glycylsarcosine in
HT-29 cells in our laboratory and found no evidence for the expression
of the H/peptide cotransporter in these cells. ()It is not known at this time whether this discrepancy is
due to the possibility that different clones of the HT-29 cell line
might have been used in these two studies or due to the possibility
that the transport activity measured in the study by Dantzig and Bergin (26) is catalyzed by a carrier other than the
H/peptide cotransporter.
/peptide cotransporter. Southern blot hybridization
with the hPEPT 1 cDNA probe detected six human-specific fragments of
24, 17, 5.4, 3.8 and 2.6 kb, two mouse-specific fragments of 20.5 and
5.6 kb, and three Chinese hamster-specific fragments of 26, 23, and 17
kb. All human-specific fragments showed concordant segregation with
chromosome 13 (Table 2). Regional localization of the hPEPT 1
gene was carried out by in situ hybridization with H-labeled hPEPT 1 cDNA. Of 133 grains over 50 metaphases
analyzed, 25 (18.8%) were located at chromosome 13 q33q24 (Fig. 7). No other chromosomal site was labeled above
background.
Figure 7:
Chromosomal localization of the hPEPT 1
gene. Position and relative abundance of silver grains on chromosome 13
as determined by in situ hybridization using H-labeled hPEPT 1 cDNA as a probe are
indicated.
/peptide cotransporter(11) .
Interestingly, the protein sequences of these transporters do not show
strong homology with other known classes of transport proteins.
However, they do show weak but definite homology with certain transport
proteins from nonmammalian sources (Fig. 8). These proteins are
the peptide transporter from Saccharomyces cerevisiae (Yeast Ptr 2)(27) , a protein encoded by S.
cerevisiae chromosome X1 DNA (Yeast X1 DNA), the
chlorate/nitrate transporter from Arabidopsis thaliana (CHL 1) (28) and the di- and tripeptide
transporter from Lactococcus Lactis (L. Lact.
Ptr)(29) .
Figure 8:
Comparison of primary amino acid sequences
among the cloned human H/peptide cotransproter (hPEPT 1), a peptide transporter from S. cerevisiae (yeast Ptr 2), the chromosome XI DNA from S.
cerevisiae (yeast XI DNA), a peptide transporter from L. lactis (L. lact. Ptr), and a chlorate/nitrate
transporter from A. thaliana (CHL
1).
The yeast Ptr 2 and the L. lactis Ptr catalyze transport of small peptides via a mechanism energized
by an electrochemical H gradient(27, 29) . It is interesting to note
that even though there is a high degree of similarity in the nature of
the driving force and the transported substrates among the yeast Ptr 2, L. lactis Ptr, and hPEPT 1, the homology of the primary amino
acid sequence of hPEPT 1 to the other two proteins is not very high.
Transport of nitrate in A. thaliana catalyzed by CHL 1 occurs
via a H-dependent mechanism(28, 30) .
The significant, even though weak, homology in the amino acid sequence
among these transport systems is indicative of the similarity in the
nature of the substrate and/or driving force involved in transport
processes mediated by these systems. There are other known transport
systems which either utilize an electrochemical H gradient as the energy source or mediate the transport of
peptides, but there is no significant homology between these proteins
and the hPEPT 1. Examples of these transport systems include the
prokaryotic H-coupled lactose permease (31) and the peptide transport systems described in several
Gram-positive and Gram-negative bacteria (32, 33, 34, 35, 36) . With
the exception of L. lactis Ptr, the peptide transport systems
described thus far in bacteria belong to the super family of ABC
transporters or traffic ATPases. This class of transport systems
directly utilizes the energy derived from ATP hydrolysis to energize
active transport of solutes. Another transport system which
surprisingly shows no structural similarity to hPEPT 1 is the
transporter associated with antigen processing(37) . This
transporter is a heterodimer consisting of two proteins TAP 1 and TAP 2
and is involved in the transport of peptide antigens, normally
consisting of about 9 amino acids, across the membrane of endoplasmic
reticulum to be subsequently presented to the major histocompatibility
complex class I molecules. This system, sometimes referred to as a
peptide transporter, is not found in the plasma membrane and is not
coupled to an electrochemical H gradient. It is a
member of the ABC transporter family.
Recently, Dantzig et al.(38) reported on the isolation of a cDNA encoding a
protein associated with intestinal peptide transport. There is no
significant structural similarity between hPEPT 1 and this protein
designated as hpt-1 (16% identity; 41% similarity). This protein
contains a single putative transmembrane domain in contrast to hPEPT 1
which contains 12 putative transmembrane domains. Tissue distribution
of hpt-1 and hPEPT 1 is also different. hpt-1 is expressed in the
gastrointestinal tract and pancreas but is absent in kidney and liver
whereas hPEPT 1 is expressed in all of these tissues. Similarly, hpt-1
is present in Caco-2 as well as HT-29 cells whereas hPEPT 1 is present
in Caco-2 cells but not in HT-29 cells. Transfection of hpt-1 cDNA into
mammalian cells leads to induction of H-dependent
transport of aminocephalosporins. The induced transport is inhibitable
by glycyl-D-proline. Whether there is a functional
relationship between hpt-1 and hPEPT 1 remains to be determined.
The
observation that hPEPT 1 contains two putative sites for protein kinase
C-mediated phosphorylation may be relevant to our recent studies (25) which showed that the activity of the
H/peptide cotransporter expressed in the human
intestinal cell line Caco-2 is regulated by protein kinase C.
Site-directed mutagenesis studies are required to determine the role of
these two sites in this regulation. The findings that hPEPT 1 does not
possess any site for potential phosphorylation by protein kinase A are
interesting and may have physiological relevance. cAMP is an important
modulator of intestinal function and its role in clinical disorders
caused by pathogens such as vibrio cholerae and
enterotoxigenic strains of Escherichia coli is well known. The
importance, if any, of the absence of potential sites in hPEPT 1 for
protein kinase A-mediated phosphorylation in physiological and
pathological conditions remains to be determined.
In summary, we
have isolated a cDNA (hPEPT 1) from a human intestinal cDNA library
which when expressed in X. laevis oocytes and in HeLa cells
induces H gradient-dependent peptide transport
activity. The functional characteristics of the induced activity are
similar to those of the peptide transport activity described in
mammalian small intestine. The deduced primary amino acid sequence of
hPEPT 1 is highly homologous to the rabbit intestinal peptide
transporter but is only distantly related to the peptide transporters
present in yeast and L. lactis. It has no significant homology
to hpt-1, a recently reported protein apparently associated with
intestinal peptide transport. The gene which codes for hPEPT 1 has been
localized to human chromosome 13 q33q34.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U13173[GenBank].
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 I. Ronnestad, P. J. Gavaia, C. S. B. Viegas, T. Verri, A. Romano, T. O. Nilsen, A.-E. O. Jordal, Y. Kamisaka, and M. L. Cancela Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects J. Exp. Biol., November 15, 2007; 210(22): 3883 - 3896. [Abstract] [Full Text] [PDF]
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 A. Vernaleken, M. Veyhl, V. Gorboulev, G. Kottra, D. Palm, B.-C. Burckhardt, G. Burckhardt, R. Pipkorn, N. Beier, C. van Amsterdam, et al. Tripeptides of RS1 (RSC1A1) Inhibit a Monosaccharide-dependent Exocytotic Pathway of Na+-D-Glucose Cotransporter SGLT1 with High Affinity J. Biol. Chem., September 28, 2007; 282(39): 28501 - 28513. [Abstract] [Full Text] [PDF]
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 D. T. Thwaites and C. M. H. Anderson H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine Exp Physiol, July 1, 2007; 92(4): 603 - 619. [Abstract] [Full Text] [PDF]
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 K. Mitsuoka, Y. Kato, Y. Kubo, and A. Tsuji Functional Expression of Stereoselective Metabolism of Cephalexin by Exogenous Transfection of Oligopeptide Transporter PEPT1 Drug Metab. Dispos., March 1, 2007; 35(3): 356 - 362. [Abstract] [Full Text] [PDF]
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V. Nduati, Y. Yan, G. Dalmasso, A. Driss, S. Sitaraman, and D. Merlin Leptin Transcriptionally Enhances Peptide Transporter (hPepT1) Expression and Activity via the cAMP-response Element-binding Protein and Cdx2 Transcription Factors J. Biol. Chem., January 12, 2007; 282(2): 1359 - 1373. [Abstract] [Full Text] [PDF]
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 M. Sala-Rabanal, D. D. F. Loo, B. A. Hirayama, E. Turk, and E. M. Wright Molecular interactions between dipeptides, drugs and the human intestinal H+-oligopeptide cotransporter hPEPT1 J. Physiol., July 1, 2006; 574(1): 149 - 166. [Abstract] [Full Text] [PDF]
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 H. Daniel, B. Spanier, G. Kottra, and D. Weitz From Bacteria to Man: Archaic Proton-Dependent Peptide Transporters at Work Physiology, April 1, 2006; 21(2): 93 - 102. [Abstract] [Full Text] [PDF]
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M. Li, G. D. Anderson, B. R. Phillips, W. Kong, D. D. Shen, and J. Wang INTERACTIONS OF AMOXICILLIN AND CEFACLOR WITH HUMAN RENAL ORGANIC ANION AND PEPTIDE TRANSPORTERS Drug Metab. Dispos., April 1, 2006; 34(4): 547 - 555. [Abstract] [Full Text] [PDF]
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 A. Romano, G. Kottra, A. Barca, N. Tiso, M. Maffia, F. Argenton, H. Daniel, C. Storelli, and T. Verri High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis Physiol Genomics, February 23, 2006; 24(3): 207 - 217. [Abstract] [Full Text] [PDF]
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 E. Romeo, M. H. Dave, D. Bacic, Z. Ristic, S. M. R. Camargo, J. Loffing, C. A. Wagner, and F. Verrey Luminal kidney and intestine SLC6 amino acid transporters of B0AT-cluster and their tissue distribution in Mus musculus Am J Physiol Renal Physiol, February 1, 2006; 290(2): F376 - F383. [Abstract] [Full Text] [PDF]
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 R. K. Bhardwaj, D. Herrera-Ruiz, P. J. Sinko, O. S. Gudmundsson, and G. Knipp Delineation of Human Peptide Transporter 1 (hPepT1)-Mediated Uptake and Transport of Substrates with Varying Transporter Affinities Utilizing Stably Transfected hPepT1/Madin-Darby Canine Kidney Clones and Caco-2 Cells J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1093 - 1100. [Abstract] [Full Text] [PDF]
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P. Gaildrat, M. Moller, S. Mukda, A. Humphries, D. A. Carter, V. Ganapathy, and D. C. Klein A Novel Pineal-specific Product of the Oligopeptide Transporter PepT1 Gene: CIRCADIAN EXPRESSION MEDIATED BY cAMP ACTIVATION OF AN INTRONIC PROMOTER J. Biol. Chem., April 29, 2005; 280(17): 16851 - 16860. [Abstract] [Full Text] [PDF]
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S. Itagaki, Y. Saito, S. Kubo, Y. Otsuka, Y. Yamamoto, M. Kobayashi, T. Hirano, and K. Iseki H+-Dependent Transport Mechanism of Nateglinide in the Brush-Border Membrane of the Rat Intestine J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 77 - 82. [Abstract] [Full Text] [PDF]
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 J. E. Klang, L. A. Burnworth, Y. X. Pan, K. E. Webb Jr., and E. A. Wong Functional characterization of a cloned pig intestinal peptide transporter (pPepT1) J Anim Sci, January 1, 2005; 83(1): 172 - 181. [Abstract] [Full Text] [PDF]
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 B. Alteheld, M. E. Evans, L. H. Gu, V. Ganapathy, F. H. Leibach, D. P. Jones, and T. R. Ziegler Alanylglutamine Dipeptide and Growth Hormone Maintain PepT1-Mediated Transport in Oxidatively Stressed Caco-2 Cells J. Nutr., January 1, 2005; 135(1): 19 - 26. [Abstract] [Full Text] [PDF]
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B. S. Anand, S. Katragadda, and A. K. Mitra Pharmacokinetics of Novel Dipeptide Ester Prodrugs of Acycl |
