From the Biozentrum and the
Institute of
Biochemistry, Department of Biochemistry/Biotechnology,
Martin-Luther-University Halle-Wittenberg, and the ¶ Max-Planck
Research Unit "Enzymology of Protein Folding," D-06120 Halle,
Germany
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ABSTRACT |
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This study was initiated to determine whether the
intestinal H+/peptide symporter PEPT1 differentiates
between the peptide bond conformers of substrates. We synthesized a
modified dipeptide where the peptide bond is replaced by the isosteric
thioxo peptide bond. The Ala-Pro derivative Ala-[CS-N]-Pro exists
as a mixture of cis and trans conformation in
aqueous solution and is characterized by a low cis/trans
isomerization rate. The compound was recognized by PEPT1 with high
affinity. The Ki value of Ala-
[CS-N]-Pro for
the inhibition of the uptake of radiolabeled glycylsarcosine in Caco-2
cells was 0.30 ± 0.02 mM, determined in solution with 96% trans conformation. In contrast, the
Ki value was 0.51 ± 0.02 mM when
uptake media with 62% trans conformer were used. We
conclude that only the trans conformer interacts with the
transport system. From our data, a significant affinity of the
cis conformer at PEPT1 cannot be derived. In a second
approach, conformer-specific uptake of Ala-
[CS-N]-Pro was studied
by analyzing the intracellular content of Caco-2 cells following
transport as well as the composition of the extracellular medium using
capillary electrophoresis. The percentage of trans
conformer that was 62% in the uptake medium increased to 92% inside
the cells. This is the first direct evidence that an
H+/peptide cotransport system selectively binds and
transports the trans conformer of a peptide derivative.
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INTRODUCTION |
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The peptide transporters expressed in the brush border membrane of the intestinal and renal epithelial cells are responsible for the absorption of oligopeptides that consist of two or three amino acids. These transporters are driven by a transmembrane proton gradient and mediate the symport of their substrates with H+ (1-4). In addition to their natural substrates, peptide transporters are also capable of transporting many pharmacologically active peptidomimetics (5, 6). The structural requirements for substrates to be accepted are not completely understood. However, all of the chemically diverse substrates studied so far seem to bear interacting sites correctly aligned to the transporter by sterical resemblance to the backbone of physiologically occurring di- and tripeptides (5, 6). Therefore, in the design of pharmacologically active peptidomimetics that interact with peptide carriers, modification of the backbone will become increasingly important.
In the present investigation, we studied the influence of the substrate
backbone dynamics caused by peptide bond cis/trans isomerization on the intestinal peptide transport. Both cis
and trans conformers can be found in peptides and proteins
due to the partial double bond character of the peptide bond. Yet, the free energy difference between cis and trans
isomers is lower in the imide than in the amide bond. Therefore, among
peptides made of gene-coded amino acids, a significant population of
the energetically disfavored cis conformation has been
described so far only for Xaa-Pro peptide bonds (7, 8). For proteases, it has been shown that they prefer the trans conformation at
and near the scissile bond of their substrates (9). On the other hand,
peptidyl prolyl cis/trans isomerases utilize both
cis and trans isomers of peptide and protein
substrates (8). For peptide receptors, a preference either for
cis conformers (10) or for all-trans conformers
(11, 12) has been hypothesized. Whether peptide transporters are able
to differentiate between cis and trans conformers
of di- and tripeptides has not yet been shown. Measuring 4- to 12-fold
lower affinities of Gly-Pro and Gly-Sar compared with Gly-Gly and
Gly-Ala at the renal peptide transporter, Daniel et al.
(13), however, have already discussed that isomerization at the peptide
bond might be responsible. To study cis/trans conformational effects, ideally, conformers of one and the same substrate should be
used. Unfortunately, it has been proven to be difficult to stabilize
pure peptide bond conformers for a period sufficient for transport
experiments because the cis/trans interconversion of natural
Xaa-Pro dipeptides occurs at room temperature within seconds to minutes
(8). A peptide therefore had to be found that is characterized by being
a proteolytically stable substrate for the peptide carrier, by a
sufficiently low cis/trans interconversion rate and by the
possibility to vary its isomer ratio. We have chosen to synthesize a
modified Ala-Pro where the peptide carbonyl oxygen is replaced by
sulfur. Thioxo oligopeptides are known to be isosteric to their
non-thioxylated counterparts (14). However, replacing the peptide bond
by a thioxo peptide bond in Xaa-Pro peptides resulted in an up to
100-fold retardation in the cis/trans isomerization rates
due to a higher barrier of rotation at the C-N bond (14, 15). Using
the dipeptide derivative Ala-[CS-N]-Pro as a substrate in the
present investigation, we provide first direct evidence for the
conformational specificity of peptide transport.
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EXPERIMENTAL PROCEDURES |
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Synthesis of Ala-[CS-N]-Pro--
The thioxo dipeptide was
synthesized by thioxylation of Boc-Ala-Pro-OtBu with Lawesson's
reagent (15), deprotected using 95% trifluoroacetic acid, and
characterized by 1H-, 13C-NMR, mass
spectrometry and HPCE.1
HPCE and NMR Analyses--
HPCE analysis was performed using a
BioFocus 3000 (Bio-Rad, Germany) (14, 16). UV detection was done at 270 nm where Ala-[CS-N]-Pro shows maximal absorption. 1H
and 13C-NMR spectra were recorded on an ARX-500
spectrometer (Bruker, Germany) at 500.13 and 125.76 MHz, respectively.
Assignment of signal sets to cis and trans
conformation was done using the 13C-NMR pattern of
ProC
and ProC
signals as well as the characteristic upfield shift of the cis AlaC
H
signal in the 1H spectra compared with the corresponding
trans signal (7).
Cell Culture and Uptake Measurements--
The human colon
carcinoma cell line Caco-2 was obtained from the German Collection of
Microorganisms and Cell Cultures and routinely cultured as described
(17, 18). Uptake of [glycine-1-14C]Gly-Sar (53 mCi/mmol,
Amersham International, UK) was measured 8 days after seeding (17). The
uptake buffer (1 ml) contained 25 mM Mes/Tris (pH 6.0), 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, 5 mM glucose, 20 µM [14C]Gly-Sar,
and increasing concentrations of unlabeled Ala-[CS-N]-Pro (0 to 5 mM). In a different approach, uptake of Ala-
[CS-N]-Pro was measured by incubating the washed monolayers with 1 ml of uptake
medium containing Ala-
[CS-N]-Pro (1 mM) for 10, 30, or 60 min. After washing the cells, 1 ml of deionized water was added to
each monolayer. The dishes were frozen and thawed twice, and contents
were suspended with the use of a 1-ml syringe and 25-gauge needle.
Centrifugation at 70,000 × g for 10 min yielded a
clear supernatant that was analyzed using HPCE.
Data Analysis-- Results are given as means ± S.E. (n = 6-9). Non-linear regression analysis, calculation of inhibition constants (Ki) from IC50 values and statistical analysis was done as described (17, 18). Areas of HPCE peaks were integrated and divided by migration time.
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RESULTS AND DISCUSSION |
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Cis/trans Ratio of Ala-[CS-N]-Pro--
Both 1H-
and 13C-NMR spectra showed two sets of signals for
Ala-
[CS-N]-Pro, which were assigned to trans and
cis isomers. Using HPCE, we were able to separate the
cis/trans conformers of Ala-
[CS-N]-Pro. A striking
feature of Ala-
[CS-N]-Pro was that immediately after dissolving in
uptake buffer at pH 6.0, this compound had a trans content
of 96%, whereas only 4% were in cis conformation (Fig. 1, solution I). After
dissolving the peptide, interconversion of Ala-
[CS-N]-Pro isomers
proceeded slowly toward cis/trans equilibrium. After 2 days
at room temperature, equilibrium with 62% trans and 38%
cis isomers was reached (Fig. 1, solution II).
Both ratios were verified by NMR analysis (Fig. 1). Such differences in
cis/trans ratios between ad hoc solutions and
equilibrated solutions have already been described for natural Xaa-Pro
dipeptide conformers (16). The evidently low interconversion rate of
Ala-
[CS-N]-Pro isomers, which enabled the following experiments to
be done, was confirmed by monitoring the cis/trans ratio
changes after dissolution using HPCE. These measurements revealed a
half-time of about 12 h for Ala-
[CS-N]-Pro isomers at
30 °C. Storing Ala-
[CS-N]-Pro solution at
20 °C delayed
the isomerization rate further, thereby actually "locking" the
respective cis/trans ratio. Therefore, for all the following
experiments, Ala-
[CS-N]-Pro solutions (pH 6.0) were prepared,
sterilized, and split into solution I that was kept at
20 °C for 2 days and solution II that was kept at 22 °C for 2 days. Before each
experiment, solutions were diluted to the desired substrate
concentration, and the cis/trans ratio was determined by
HPCE.
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Inhibition of [14C]Gly-Sar Uptake by
Ala-[CS-N]-Pro I and II--
Uptake of Gly-Sar into Caco-2 cells
is driven by an inwardly directed H+ gradient (17) and
mediated by a single transport system (Kt = 1.1 ± 0.1 mM), which has been identified as the low affinity, high capacity system PEPT1 present in human small intestine (17-19). We measured the ability of Ala-
[CS-N]-Pro to inhibit
[14C]Gly-Sar uptake in dependence of the
cis/trans ratio. In case of solution I, where the relative
trans content was 96% (Fig. 1), the Ki
value was 0.30 ± 0.02 mM (Fig.
2), which is comparable with those of
Ala-Xaa dipeptides (17). However, the Ki value was
increased by 70% to 0.51 ± 0.02 mM when the
equilibrated Ala-
[CS-N]-Pro solutions containing only 62% trans conformer were used. Shown in Fig. 2 also is the
theoretical displacement curve that results under the presumption that,
at a cis/trans ratio of 38/62%, exclusively the
trans isomer competes with labeled Gly-Sar at the binding
site of the transport system. The measured and the simulated
displacement curves are virtually identical. This provides evidence
that only the trans isomer of Ala-
[CS-N]-Pro interacts
with PEPT1. We cannot completely rule out at this time, however, a very
low affinity (Ki > 40 mM) of the
cis conformer at PEPT1, but this cannot be derived from our
data with any confidence.
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Intracellular Accumulation of
Ala-[CS-N]-Pro--
Demonstrating the competition between
trans Ala-
[CS-N]-Pro and Gly-Sar at the binding site of
PEPT1 does not allow the unambiguous conclusion that trans
Ala-
[CS-N]-Pro is transported into the cell. Therefore, in a
different approach, we analyzed the intracellular content of Caco-2
cells by HPCE after uptake of Ala-
[CS-N]-Pro. The incubation
medium contained Ala-
[CS-N]-Pro (1 mM, pH 6.0) at
cis/trans equilibrium (62% trans, Fig. 1,
solution II). Inside the cells, however, the relative trans
content increased in a time-dependent manner (Fig.
3 B-D) reaching 92 ± 0.7%. As shown in Fig. 3, B-D, the cis conformer
also appeared inside the cells during incubation. Because of the long
half-time for establishing the conformational equilibrium, an increase
of the cis amount due to reequilibration inside the cells is
negligible during the experiment. More likely, the minor increase in
cis species was due to simple diffusion through the cell
membrane. To confirm that, we repeated the experiment in the absence
(outside 7.5) and presence (outside 6.0) of a pH gradient using a
30-min uptake time (data not shown). First, changing the pH had no
effect on the cis/trans ratio in the uptake buffer. Second,
the small intracellular total cis amount was unaffected by
the outside pH, whereas the total trans amount was increased
2-fold by pH 6.0 compared with 7.5.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gerd Scherer for NMR measurements and Ilona Kunze for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Land Sachsen-Anhalt Grant 2217A/0085G and fellowship (to F. T.) and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Martin-Luther-University Halle-Wittenberg, Dept. of Biochemistry/Biotechnology, Institute of Biochemistry, Kurt-Mothes-Str. 3, D-06120 Halle, Germany. Tel.: 49-345-552-4853; Fax: 49-345-552-7011.
1 The abbreviations used are: HPCE, high-performance capillary electrophoresis; Gly-Sar, glycylsarcosine; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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