From the Department of Pharmacy, Kyoto University Hospital, Faculty
of Medicine, Kyoto University, Kyoto 606-01, Japan
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INTRODUCTION |
In the small intestine and kidney, epithelial assimilation of
oligopeptides are mediated by H+-coupled peptide transport
systems (1-3). Because there are 20 amino acids that comprise
oligopeptides, there can be 400 dipeptides and 8000 tripeptides with
various charges and molecular sizes. In addition to the native small
peptides, the peptide transporter recognizes a wide variety of
peptide-like drugs such as orally active
-lactam antibiotics (4-6),
bestatin (an antineoplastic drug) (7, 8), and angiotensin converting
enzyme inhibitors (9). The peptide transporter shows such a broad range
of substrate specificity; however, the mechanism that recognizes
substrates by the peptide transporter has been incompletely understood.
Previously, we have cloned rat H+/peptide transporters,
PEPT1 (10) and PEPT2 (11), and constructed PEPT1- and PEPT2-expressing
transfectants (12-14). Using these transfectants, we demonstrated that
both PEPT1 and PEPT2 recognized various orally active
-lactam
antibiotics (14). When PEPT1-expressing cells were treated with
diethylpyrocarbonate (DEPC),1
which is a histidine residue modifier, ceftibuten (anionic
cephalosporin without an
-amino group) uptake was completely
abolished (12). Furthermore, using the site-directed mutagenesis
technique, the histidine residues at positions 57 and 121 of rat PEPT1
were suggested to be involved in substrate recognition and/or
responsible for the intrinsic activity of the transporter
(12).
To elucidate the diversity of the substrate recognition by the peptide
transporters, it is needed to clarify the mechanisms involved in the
interaction of the substrates with the essential residues of PEPT1 and
PEPT2. Because histidine residues of the peptide transporters have been
indicated as the most important key amino acid residues (12, 15, 16),
we investigated the functional role of histidine residues to examine
the preventive effect of various substrates on the DEPC-induced
inactivation of PEPT1 and PEPT2. We report here that the DEPC-sensitive
histidine residue of rat PEPT1 and PEPT2 can serve as the binding site
of the
-amino group of the substrates.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The parental LLC-PK1 cells
obtained from the American Type Culture Collection (ATCC CRL-1392) were
cultured in complete medium, which consisted of Dulbecco's modified
Eagle's medium (Life Technologies, Inc.), supplemented with 10% fetal
bovine serum (Whittaker Bioproducts Inc., Walkersville, MD) without
antibiotics in an atmosphere of 5% CO2, 95% air at
37 °C. The LLC-PK1 cells transfected with rat PEPT1
cDNA (LLC-rPEPT1) and with rat PEPT2 cDNA (LLC-rPEPT2) were used as described, previously (14). In the uptake experiments, the
cells were cultured for 6-7 days in complete medium.
Uptake Studies by Cell Monolayers--
Uptake of
[14C]glycylsarcosine was measured in cells grown in 60-mm
plastic dishes as described previously (14). The protein content of the
cell monolayers solubilized in 1 N NaOH was determined by
the method of Bradford (17) using a Bio-Rad protein assay kit with
bovine
-globulin as the standard.
Materials--
Cefadroxil (Bristol Meyers Co., Tokyo, Japan),
cefixime (Fujisawa Pharmaceutical Co., Osaka, Japan), ceftibuten
(Shionogi and Co., Osaka, Japan), cephradine (Sankyo Co., Tokyo,
Japan), cyclacillin (Takeda Chemical Industries, Osaka, Japan.), and
bestatin ((2R,3S)-3-amino-2-hydroxy-4-phenylbutanoyl-L-leucine)
(Nippon Kayaku Co., Tokyo, Japan) were gifts from the
respective suppliers. [14C]Glycylsarcosine (1.78 GBq/mmol) was obtained from Daiichi Pure Chemicals Co., Ltd. (Ibaraki,
Japan). Glycylsarcosine and lysyl-L-lysine were obtained
from Sigma. Glutamyl-L-glutamic acid was purchased from the
Peptide Institute Inc. (Osaka, Japan). Captopril and DEPC were obtained
from Nacalai Tesque (Kyoto, Japan). All other chemicals used were of
the highest purity available.
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RESULTS |
At first, we examined the concentration dependence for the
inhibitory effect of DEPC on [14C]glycylsarcosine uptake.
When the LLC-rPEPT1 and LLC-rPEPT2 cells were treated with various
concentrations of DEPC, the half-maximal inhibition for
[14C]glycylsarcosine uptake in both transfectants was
observed at about 0.4 mM, and the maximal inhibition was at
1 mM (Table I). Therefore,
the concentration of DEPC for subsequent studies was 1 mM.
Fig. 1 shows the effect of DEPC treatment
on [14C]glycylsarcosine uptake by LLC-rPEPT1 or
LLC-rPEPT2 cells and the preventive effect of glycylsarcosine on the
DEPC-induced inhibition. The uptake of
[14C]glycylsarcosine by the transfectants was inhibited
markedly by the pretreatment with 1 mM DEPC. This
inhibition was abolished mostly by unlabeled 10 mM
glycylsarcosine.
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Table I
Effect of DEPC concentration on [14C]glycylsarcosine
uptake by LLC-rPEPT1 and LLC-rPEPT2 cells
The cells were preincubated at 25 °C for 10 min with various
concentrations of DEPC (pH 6.0). After preincubation, the cells were
rinsed once with the incubation medium and then incubated with
[14C]glycylsarcosine (20 µM, pH 6.0) for 15 min
at 37 °C. The radioactivity of the solubilized cells was determined.
Each value represents the mean ± S.E. of three independent
monolayers. The values in parentheses represent the percentage of
control.
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Fig. 1.
Effect of DEPC pretreatment on
[14C]glycylsarcosine uptake by LLC-rPEPT1 (A)
and LLC-rPEPT2 cells (B). The cells were preincubated
at 25 °C for 10 min with 1 mM DEPC (pH 6.0) in the absence or the presence of glycylsarcosine at 10 mM. After
preincubation, the cells were rinsed once with the incubation medium
and then incubated with [14C]glycylsarcosine (20 µM, pH 6.0) for 15 min at 37 °C. The radioactivity of
the solubilized cells was determined. Each column represents the
mean ± S.E. of three independent monolayers. GLY-SAR,
glycylsarcosine.
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Next, we examined whether the DEPC-induced inactivation of PEPT1 and
PEPT2 was affected by the pH of the incubation medium. As shown in Fig.
2, glycylsarcosine uptake at both pH 6.0 (with a H+ gradient) and pH 7.4 (without a H+
gradient) was blocked by pretreatment with DEPC in the LLC-rPEPT1 and
LLC-rPEPT2 cells.

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Fig. 2.
Effect of DEPC pretreatment on
[14C]glycylsarcosine uptake either at pH 6.0 or 7.4 by
LLC-rPEPT1 (A) and LLC-rPEPT2 cells (B).
The cells were preincubated at 25 °C for 10 min in the absence
(open columns) or the presence (shaded columns)
of 1 mM DEPC (pH 6.0). After preincubation, the cells were
rinsed once with the incubation medium and then incubated with
[14C]glycylsarcosine (20 µM) at pH 6.0 or
pH 7.4 for 15 min at 37 °C. The radioactivity of the solubilized
cells was determined. Each column represents the mean ± S.E. of
three independent monolayers.
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The histidine residues might have been located with the PEPT1 substrate
binding site, and therefore, we examined the effect of two
cephalosporins on the DEPC inactivation of PEPT1. Fig. 3A shows the effect of
cephradine (aminocephalosporin) and ceftibuten (anionic
cephalosporin without an
-amino group) concentration on the
DEPC-induced inhibition of the glycylsarcosine uptake by the LLC-rPEPT1
cells. Rat PEPT1 has a much higher affinity to ceftibuten than to
cephradine (13, 14). Cephradine prevented the DEPC-induced inhibition
of glycylsarcosine uptake at 5-10 mM, but ceftibuten had
no preventive effect even at 10 mM. Similar results were
observed for the LLC-rPEPT2 cells (Fig. 3B).

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Fig. 3.
Effect of cephradine and ceftibuten
concentration on the DEPC-induced inhibition of
[14C]glycylsarcosine uptake by LLC-rPEPT1 (A)
and LLC-rPEPT2 cells (B). The cells were preincubated
at 25 °C for 10 min with 1 mM DEPC (pH 6.0) in the
absence ( ) or the presence of an increasing concentration of
cephradine ( ) and ceftibuten ( ). After preincubation, the cells
were rinsed once with the incubation medium and then incubated with
[14C]glycylsarcosine (20 µM, pH 6.0) for 15 min at 37 °C. The radioactivity of the solubilized cells was
determined. The uptake of [14C]glycylsarcosine in the
absence of DEPC was taken as 100% (827 ± 38 and 243 ± 6.0 pmol/mg protein/15 min in the LLC-rPEPT1 and LLC-rPEPT2 cells,
respectively; mean ± S.E. of three monolayers). Each point
represents the mean of two experiments except for the control.
GLY-SAR, glycylsarcosine.
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To elucidate the effect of ceftibuten, we examined the effect of the
charge of the substrates on the DEPC-induced inactivation of PEPT1. As
shown in Fig. 4A, the
DEPC-induced inhibition of [14C]glycylsarcosine uptake by
LLC-rPEPT1 cells was prevented by various charged substrates but not by
ceftibuten. When the inhibitory effect of these compounds was examined,
all the dipeptides, cephradine, and ceftibuten inhibited
[14C]glycylsarcosine uptake (Fig. 4B).
These results suggested that the preventive effect of substrates was
independent of either their ionic charges or affinity to the
transporters. For the LLC-rPEPT2 cells, these substrates prevented the
DEPC-induced inhibition of the glycylsarcosine uptake in a manner
similar to that used for inhibiting LLC-rPEPT1 cells (data not
shown).

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Fig. 4.
Effect of various dipeptides with different
charge, cephradine, and ceftibuten on DEPC-induced inhibition of
[14C]glycylsarcosine uptake (A) and on
[14C]glycylsarcosine uptake (B) by LLC-rPEPT1
cells. A, LLC-rPEPT1 cells were preincubated at 25 °C for
10 min with 1 mM DEPC (pH 6.0) in the absence or the
presence of 10 mM of various dipeptides with different
charge, cephradine, and ceftibuten. After preincubation, the cells were
rinsed once with the incubation medium and then incubated with
[14C]glycylsarcosine (20 µM, pH 6.0) for 15 min at 37 °C. B, LLC-rPEPT1 cells were incubated with
[14C]glycylsarcosine (20 µM, pH 6.0) for 15 min at 37 °C in the absence or the presence of each inhibitor (10 mM). The radioactivity of the solubilized cells was
determined. Each column represents the mean ± S.E. of three
independent monolayers. GLY-SAR, glycylsarcosine; GLU-GLU, glutamyl-L-glutamic acid;
LYS-LYS, lysyl-L-lysine; CED, cephradine; CETB, ceftibuten.
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We investigated whether the preventive effects depended on the
-amino group of the substrates. As shown in Fig.
5A, the substrates with an
-amino group such as glycylsarcosine, cephradine, and cefadroxil
(aminocephalosporin) prevented the DEPC-induced inhibition of
[14C]glycylsarcosine uptake by LLC-rPEPT1 cells. However,
all peptide-like drugs without an
-amino group such as ceftibuten,
cefixime, bestatin, and captopril (angiotensin converting enzyme
inhibitor) had no effect at a concentration of 10 mM in the
LLC-rPEPT1 cells. Cyclacillin did not have the preventive effect
despite its having an
-amino group. Similar results were obtained in
LLC-rPEPT2 cells except for cefadroxil (Fig. 5B). In the
absence of DEPC, the pretreatment of cefadroxil at 10 mM
had an inhibitory effect on glycylsarcosine uptake (data not shown).
Therefore, cefadroxil at 10 mM might have a cis-inhibitory
effect on the [14C]glycylsarcosine uptake, considering
that cefadroxil had a higher affinity for PEPT2 with an apparent
inhibition constant of 3 µM (14).

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Fig. 5.
Effect of various compounds on DEPC-induced
inhibition of [14C]glycylsarcosine uptake by LLC-rPEPT1
(A) and LLC-rPEPT2 cells (B). The cells
were preincubated at 25 °C for 10 min with 1 mM DEPC (pH
6.0) in the absence or the presence of each compound (10 mM). After preincubation, the cells were rinsed once with the incubation medium and then incubated with
[14C]glycylsarcosine (20 µM, pH 6.0) for 15 min at 37 °C. The radioactivity of the solubilized cells was
determined. Each column represents the mean ± S.E. of three
independent monolayers. GLY-SAR, glycylsarcosine; CED, cephradine; CDX, cefadroxil;
ACPC, cyclacillin; CETB, ceftibuten; CFIX, cefixime; BES, bestatin; CAP,
captopril.
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DISCUSSION |
In the present study, glycylsarcosine uptake by the PEPT1- and
PEPT2-expressing transfectants was inhibited by pretreatment with DEPC
in the absence and the presence of H+ gradient, which
suggests that the histidine residue modified by DEPC at least served as
the substrate binding site. This could be supported by the fact that
the various dipeptides and aminocephalosporins prevented the
DEPC-induced inactivation of PEPT1 and PEPT2. On the other hand, the
peptide-like drugs without an
-amino group had no preventive effect,
although they can be transported by the peptide transporter (5-7, 9).
These findings suggest that the histidine residue located in the
recognition site is involved in the binding site of the
-amino group
of the dipeptides and aminocephalosporins. Because only the
unprotonated imidazole ring reacts with DEPC (18), it is possible that
the imidazole group of the histidine residue located at the recognition
site is protonated by the
-amino group of the dipeptides and
aminocephalosporins but not by the peptide-like drugs that do not have
an
-amino group. The
-amino group of the substrates might
interact with the imidazole ring of the histidine residue of peptide
transporters by proton binding. It is noted that these results were
observed for PEPT1 and PEPT2 in a similar manner, which suggests that
the DEPC-sensitive histidine residue plays the same role in both
transporters.
Among the substrates examined that had an
-amino group, only
cyclacillin did not show the preventive effect against the DEPC inactivation. This may be due to the structure of cyclacillin. Cyclacillin has an
-carbon group as part of its cyclohexane ring; therefore, the cyclohexane ring may interfere with the
-amino group-histidine interaction. Nevertheless, cyclacillin was recognized by PEPT1 at a relatively high affinity (14, 19). A possible explanation
is that the hydrophobic NH2-terminal side chain of cyclacillin interacts with the peptide transporters instead of the
-amino group-histidine interaction. As reported by Daniel et
al. (20), the marked hydrophobicity of the
NH2-terminal side chain of aminopenicillins increased the
affinity to the renal H+/peptide cotransporter. For the
peptide-like drugs without an
-amino group such as ceftibuten and
cefixime, which are very hydrophilic, there might be interactions
between these drugs and the binding site of the peptide transporter
other than the
-amino group-histidine interaction.
We have previously reported that histidines 57 and 121, which are
located at the predicted transmembrane domains 2 and 4 of rat PEPT1,
are involved in substrate binding and/or are responsible for the
intrinsic activity of the transporter (12). In contrast to our results,
Fei et al. (21) demonstrated that histidine 57 of human
PEPT1 was absolutely essential for the catalytic activity but histidine
121 of human PEPT1 did not appear to play an essential role for the
catalytic activity. The reason for this discrepancy regarding the role
of histidine 121 in the rat and human PEPT1 remains unknown. However,
it is possible that at least two histidine residues are involved in the
transport activity of PEPT1. Indeed, Steel et al. (22)
proposed that one histidine residue as a cation site was responsible
for the proton coupling and that a second histidine residue was
adjacent to the peptide binding site in the studies to
determine differently charged dipeptide-H+ flux
coupling ratios. Mackenzie et al. (23)
demonstrated that human PEPT1 bound H+ first and then the
substrate as demonstrated by the biophysical and kinetic analysis of
the human PEPT1. These findings suggest that two histidine residues are
necessary, because the histidine residue with a protonated imidazole
ring cannot bind the
-amino group of the substrates as shown by the
present study. Although we cannot identify the histidine residue of the
binding site from this study, either histidine 57 or 121 of PEPT1 might
be the candidate residue for the binding site of the
-amino group of
the substrates.
In conclusion, this is the first demonstration that the
-amino group
of the dipeptides and aminocephalosporins interacts with the
DEPC-sensitive histidine residue of rat PEPT1 and PEPT2. The present
findings represent the first step for understanding the substrate
recognition mechanisms by peptide transporters.