Báo cáo khoa học: Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters pdf

Synthesis and characterization of a new and radiolabeled
high-affinity substrate for H
+
/peptide cotransporters
Ilka Knu
¨
tter
1
, Bianka Hartrodt
2
,Ge
´
za To
´
th
3
, Attila Keresztes
3
, Gabor Kottra
4
,
Carmen Mrestani-Klaus
2
, Ilona Born
2
, Hannelore Daniel
4
, Klaus Neubert
2
and Matthias Brandsch
1

1 Biozentrum of the Martin-Luther-University Halle-Wittenberg, Halle, Germany
2 Institute of Biochemistry ⁄ Biotechnology, Faculty of Sciences I, Martin-Luther-University Halle-Wittenberg, Halle, Germany
3 Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
4 Molecular Nutrition Unit, Technical University of Munich, Freising-Weihenstephan, Germany
The peptide transporters peptide cotransporter 1
(PEPT1) (SLC15A1) and peptide cotransporter 2
(PEPT2) (SLC15A2) are presently under intense inves-
tigation because of their physiological importance and
their pharmaceutical relevance as drug carriers [1–6].
Both transporters catalyse the uptake of most dipep-
tides and tripeptides and a variety of peptidomimetic
drugs, such as selected b-lactam antibiotics, some
angiotensin-converting enzyme inhibitors and pro-
drugs such as valaciclovir. H
+
-coupled peptide and
drug transport across cell membranes by PEPT1
and PEPT2, respectively, have been demonstrated at
Keywords
Caco-2; H
+
⁄ peptide cotransporter 1;
H
+
⁄ peptide cotransporter 2; SKPT; Xenopus
laevis oocytes
Correspondence
M. Brandsch, Biozentrum of the Martin-
Luther-University Halle-Wittenberg,
Membrane Transport Group,

Weinbergweg 22, D-06120 Halle, Germany
Fax: +49 345 5527258
Tel: +49 345 5521630
E-mail: matthias.brandsch@
biozentrum.uni-halle.de
(Received 15 August 2007, revised 19 Sep-
tember 2007, accepted 20 September 2007)
doi:10.1111/j.1742-4658.2007.06113.x
In this study we described the design, rational synthesis and functional
characterization of a novel radiolabeled hydrolysis-resistant high-affinity
substrate for H
+
⁄ peptide cotransporters. l-4,4¢-Biphenylalanyl–l-Proline
(Bip-Pro) was synthesized according to standard procedures in peptide
chemistry. The interaction of Bip-Pro with H
+
⁄ peptide cotransporters was
determined in intestinal Caco-2 cells constitutively expressing human
H
+
⁄ peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutively
expressing rat H
+
⁄ peptide cotransporter 2 (PEPT2). Bip-Pro inhibited the
[
14
C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity
(K
i
¼ 24 lm and 3.4 lm, respectively) in a competitive manner. By employ-

ing the two-electrode voltage clamp technique in Xenopus laevis oocytes
expressing PEPT1 or PEPT2 it was found that Bip-Pro was transported by
both peptide transporters although to a much lower extent than the refer-
ence substrate, Gly-Gln. Bip-Pro remained intact to > 98% for at least 8 h
when incubated with intact cell monolayers. Bip-[
3
H]Pro uptake into SKPT
cells was linear for up to 30 min and pH dependent with a maximum at
extracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeled
Bip-Pro but also by known peptide transporter substrates such as dipep-
tides, cefadroxil, Ala-4-nitroanilide and d-aminolevulinic acid, but not by
glycine. Bip-Pro uptake in SKPT cells was saturable with a Michaelis–
Menten constant (K
t
) of 7.6 lm and a maximal velocity (V
max
) of 1.1 nmo-
lÆ30 min
)1
Æmg of protein
)1
. Hence, the uptake of Bip-Pro by PEPT2 is a
high-affinity, low-capacity process in comparison to the uptake of Gly-Sar.
We conclude that Bip-[
3
H]Pro is a valuable substrate for both mechanistic
and structural studies of H
+
⁄ peptide transporter proteins.
Abbreviations

Boc, tert. butyloxycarbonyl; Bip,
L-4,4¢-biphenylalanine; Bip-Pro, L-4,4¢-biphenylalanyl–L-Proline; PEPT1, H
+
⁄ peptide cotransporter 1; PEPT2,
H
+
⁄ peptide cotransporter 2; DPro, L-3,4-dehydro-Proline.
FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5905
intestinal and renal cells [1–3] but also in lung [7],
extrahepatic biliary duct [8], choroid plexus [9] and
other tissues [1–3].
Essentially nothing is known about the location and
structure of the substrate binding domains within the
carrier proteins. Available data are restricted to results
obtained in experiments with chimeric mammalian
peptide transporters derived from the intestinal and
renal isoforms [10,11], site-directed mutagenesis experi-
ments [12–14] and from extensive studies on substrate
specificity combined with molecular modeling
[4,5,15,16].
The most commonly used and best known reference
substrate of H
+
⁄ peptide cotransporters is [
14
C]glycine-
sarcosine ([
14
C]Gly-Sar). This substrate is relatively
stable against intracellular and extracellular enzymatic

hydrolysis, but its affinity constants for peptide trans-
porters are only in the medium range, with K
t
values
of  1.3 mm for PEPT1 [17] and  108 lm for PEPT2
[18]. New high-affinity labeled probes are required for
further studies on the mechanism of transport func-
tion and the structure of the carrier proteins. For
example, the rate limiting step of peptide transporters
has still not yet been determined, and the number of
transporters per cell and their turnover rate in epithe-
lial cells is not known. With regard to transporter
structure, despite the fact that techniques such as
intrinsic tryptophan fluorescence measurement have
been shown to be useful to study the expression and
conformation of recombinant membrane transporters
[19], labeled substrates and inhibitors with a broad
range of affinity to the respective protein are also
essential tools. In the course of our work on
high-affinity inhibitors of PEPT1 and PEPT2, on the
structural modifications that convert a transported
compound into a nontranslocated inhibitor as well as
studies on the structural requirements for a high affin-
ity of substrates [17,18,20], it became evident that a
large aromatic hydrophobic group in the side chain of
the N-terminal amino acid of dipeptides could
enhance the affinity of many derivatives for binding
to the transporters. Besides high affinity, a second
important requirement for any peptide transporter
substrate is a sufficient stability against enzymatic

hydrolysis. Hence, we decided to synthesize a dipep-
tide where l-4,4¢-biphenylalanine (Bip), with its large
aromatic side chain in a short intramolecular distance
from the a-carbon atom, is the N-terminal amino acid
and l-Proline (l-Pro) is the C-terminal amino acid.
The resulting compound, Bip-Pro, was tested with
regard to its interaction with peptide transporters, its
affinity and its stability in a biological system. More-
over, after radioactive labeling we determined the
kinetic parameters and transport characteristics of
Bip-[
3
H]Pro.
Results and Discussion
Synthesis, chemical characterization and stability
of Bip-Pro
Figure 1 shows the structure (Fig. 1A) and the synthe-
sis strategy (Fig. 1B) of Bip-Pro. The purity of the
compound was assessed by TLC, analytical RP-
HPLC, MS and NMR, and was found to exceed
98%. As expected for an Xaa-Pro peptide derivative,
Bip-Pro exists as a mixture of cis and trans conform-
ers in aqueous solution (pH 6.0). In the
1
H
NMR spectrum, Bip-Pro exhibited two sets of NMR
signals indicating the existence of two conformations.
Fig. 1. Structure and synthesis of Bip-Pro. (A) Bip-Pro structure. (B)
Bip-Pro synthesis: mixed anhydride (MA) method. (C) Synthesis of
Bip-[

3
H]Pro. EDC, N-ethyl-N¢-(3-dimethyl aminopropyl)-carbodiimide;
HOSu, N-hydroxysuccinimide.
Labeled high-affinity substrate for peptide transporters I. Knu
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5906 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS
Subsequent analysis of the ROESY spectrum revealed
characteristic strong ROEs between Bip-C
a
H and both
Pro-C
d
H
A
and Pro-C
d
H
B
of the major isomer identi-
fied as a trans isomer. As strong ROEs (or NOEs)
between a protons of adjacent residues C
a
H(i)-C
a
H(i
+1) allow the resonance assignment of populations
containing cis amide linkages [21], the strong ROE
between Bip-C
a

H and Pro-C
a
H of the minor isomer
was used as evidence for its cis conformation. The rel-
ative populations of the cis ⁄ trans isomers were deter-
mined by integration of well-resolved signals in the
1D proton spectrum such as the two Bip-C
a
H signals
[21,22]. In equilibrium, Bip-Pro shows a trans content
of 22%, whereas 78% were in cis conformation. These
values are in agreement with the cis ⁄ trans ratios of
Xaa-Pro dipeptides containing aromatic amino acids
(24–34% trans content) obtained in a previous study
of our group [23].
To determine the stability of Bip-Pro, buffer samples
were analyzed after incubating Caco-2 and SKPT cell
monolayers (surface area 9.62 cm
2
) with the compound
(1 mL, 1 mm) for 10 min up to 8 h. After a 2 h incu-
bation of SKPT monolayers with Bip-Pro containing
buffer, 100% Bip-Pro was found. After 8 h, 99.9% of
Bip-Pro was intact, with the remaining 0.1% being
Bip. At all time points, Bip-Pro was recovered from
monolayers of both cell types intact to > 99% (HPLC
data not shown).
Interaction of Bip-Pro with PEPT1 and PEPT2
We next determined the interaction of Bip-Pro with
PEPT1 and PEPT2 in competition assays, with

[
14
C]Gly-Sar serving as a reference compound. The
intestinal cell line Caco-2, constitutively expressing
PEPT1 [5,17,22], and the renal cell line SKPT, con-
stitutively expressing PEPT2 [18,24], were used as
models. Bip-Pro competed with [
14
C]Gly-Sar uptake
in a dose-dependent manner (Fig. 2). The apparent
K
i
values for substrate uptake inhibition were
24 ± 0.6 lm in Caco-2 cells (PEPT1, Fig. 2A) and
3.4 ± 0.1 lm in SKPT cells (PEPT2, Fig. 2B). It has
been shown that peptide transporters are specific for
the trans conformers of their substrates [22,23]. Tak-
ing the cis⁄ trans content of Bip-Pro (78% ⁄ 22%) into
account, we obtained K
i trans
values for Bip-Pro of
5.2 lm in Caco-2 cells and of 0.75 lm in SKPT cells.
We also determined the inhibition constants (K
i
) for
Bip-Pro by measuring Gly-Sar uptake at two differ-
ent Gly-Sar concentrations (50 and 500 lm in Caco-2
cells and 10 and 100 lm in SKPT cells) in the
presence of increasing concentrations of Bip-Pro (0–
100 lm and 0–50 lm, respectively). The results are

presented as Dixon plots in Fig. 2 (insets). The plots
reveal linearity at both Gly-Sar concentrations with
lines intersecting above the abscissas in the fourth
quadrant, as expected for a competitive inhibitor.
Apparent K
i
values of 34.1 lm (K
i trans
¼ 7.5 lm)
and 1.3 lm (K
i trans
¼ 0.29 lm) were calculated from
the points of intersection of data obtained in Caco-2
cells (Fig. 2A, inset) and SKPT cells (Fig. 2B, inset),
respectively.
Fig. 2. Interaction of Bip-Pro with PEPT1 and PEPT2. Uptake of
[
14
C]Gly-Sar was measured in Caco-2 cells (A) (10 lM [
14
C]Gly-Sar,
pH 6.0, 10 min, n ¼ 4) and in SKPT cells (B) (10 l
M [
14
C]Gly-Sar,
pH 6.0, 10 min, n ¼ 4) in the presence of increasing concentrations
of Bip-Pro (0–0.316 m
M). Uptake rates measured in the absence of
Bip-Pro were taken as 100%. Insets: determination of the inhibition
constants by Dixon type experiments. Uptake of Gly-Sar was mea-

sured at pH 6.0 for 10 min at two Gly-Sar concentrations and at
increasing Bip-Pro concentrations. The diffusional component of
[
14
C]Gly-Sar uptake, of 8% in Caco-2 cells and of 4% in SKPT cells,
measured in the presence of excess of Gly-Sar (30 m
M and 20 mM,
respectively), was subtracted from the total uptake to calculate
the carrier-mediated uptake (n ¼ 4, v ¼ uptake rate in nmolÆ
10 min
)1
Æmg protein
)1
).
I. Knu
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FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5907
Interaction of Bip-Pro with PEPT1 and PEPT2
expressed in Xenopus laevis oocytes
Interaction with Gly-Sar uptake does not necessarily
allow the conclusion that Bip-Pro is indeed trans-
ported. Therefore, the two-electrode voltage clamp
technique that determines transport currents was
applied in X. laevis oocytes expressing either PEPT1 or
PEPT2 [17,18,20,25]. In contrast to the reference
dipeptide glycine-glutamine (Gly-Gln), for Bip-Pro
only a low substrate-evoked inward transport current
was recorded (Fig. 3). At a membrane potential of
)60 mV, PEPT1-mediated transport currents were

21 ± 6% of that generated by saturating Gly-Gln con-
centrations (Fig. 3A). In the case of PEPT2 at a mem-
brane potential of )160 mV, the maximal current was
11 ± 1% of that generated by Gly-Gln (Fig. 3C).
However, Bip-Pro at a concentration of 0.5 mm was
able to inhibit the inward current evoked by 0.5 mm
Gly-Gln at PEPT1 by 44 ± 1% (Fig. 3B). In the case
of PEPT2, a concentration of 0.1 mm Bip-Pro was able
to inhibit the inward current evoked by 0.1 mm Gly-
Gln remarkably by 94 ± 6% (Fig. 3D). The inhibition
was found to be dose dependent and reversible, sug-
gesting a competitive mode of action.
Uptake of Bip-[
3
H]Pro by SKPT cells
After characterization of Bip-Pro as a very high-affin-
ity and enzymatically stable substrate of PEPT1 and
PEPT2, the compound was synthesized in radiolabeled
form according to Fig. 1C [26]. We then characterized
the Bip-[
3
H]Pro uptake across the apical membrane of
SKPT cells. Time-dependent uptake of Bip-[
3
H]Pro
Fig. 3. Characterization of the interaction of Bip-Pro with PEPT1 and PEPT2 in Xenopus laevis oocytes by electrophysiology. Steady-state I–V
relationships were measured by the two-electrode voltage clamp technique in oocytes expressing PEPT1 (A, B) or PEPT2 (C, D) superfused
with modified Barth solution at pH 6.5 and 0.5, or with 0.1 m
M Gly–Gln, in the absence or the presence of increasing concentrations (PEPT1,
0–1 m

M; PEPT2, 0–0.1 mM) of Bip-Pro. The membrane potential was stepped symmetrically to the test potentials shown, and substrate-
dependent currents were recorded as the difference measured in the absence and in the presence of substrates.
Labeled high-affinity substrate for peptide transporters I. Knu
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5908 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS
(18 nm) at pH 6.0 was linear for up to 1 h and reached
a plateau after 2 h of incubation (Fig. 4). The uptake
was found to be saturable: unlabeled Bip-Pro at a con-
centration of 1 mm strongly inhibited Bip-[
3
H]Pro
uptake at all time points. Bip-[
3
H]Pro (4 nm) uptake
was also strongly pH dependent. Maximal uptake was
observed at an extracellular pH of 6.0 (Fig. 4, inset).
The same pH optimum has been observed for the
uptake of [
14
C]Gly-Sar, both in SKPT cells [24] and in
Caco-2 cells [27]. We also studied the time and pH
dependency of Bip-[
3
H]Pro uptake in Caco-2 cells. Sur-
prisingly, in this cell line the uptake was found to be
stimulated by external pH 6.0 only modestly, by 26%
in comparison to pH 7.5. Moreover, unlabeled Bip-Pro
in an excess concentration of 3 mm inhibited the
uptake of the tracer Bip-[

3
H]Pro (4 nm) during 30 min
of incubation by only 21% (data not shown). We con-
clude that the nonspecific binding of the hydrophobic
Bip-[
3
H]Pro to Caco-2 cells is very much higher than
in SKPT cells. Alternatively, a specific intestinal apical
efflux system might mediate strong outward-directed
Bip-[
3
H]Pro transport after its uptake into the cells.
For further functional characterization of Bip-[
3
H]Pro
uptake we therefore used SKPT cells.
Saturation kinetics of Bip-Pro uptake at PEPT2
Bip-Pro uptake as a function of substrate concentra-
tion was measured to determine the kinetic parameters
of the transport process. Uptake rates of Bip-Pro were
determined over a substrate concentration range of
4nm to 100 lm (Fig. 5A) and compared with the
uptake rates of Gly-Sar at a concentration range of
5 lm to 5 mm (Fig. 5B). For each compound, the non-
specific, linear uptake component, which represents
simple diffusion plus binding, was determined by
measuring the uptake in the presence of excess
Fig. 5. Substrate saturation kinetics of Bip-Pro and Gly-Sar transport
in SKPT cells. (A) Uptake of Bip-[
3

H]Pro (4 nM, 30 min, pH 6.0) was
measured over a Bip-Pro concentration range of 0–0.1 m
M. Unspe-
cific uptake ⁄ binding was determined by measuring uptake in the
presence of an excess amount (1 m
M) of unlabeled Bip-Pro. This
component (24%) was subtracted from the total uptake to calculate
the specific uptake. Inset: Eadie–Hofstee transformation of the spe-
cific Bip-Pro uptake data [S, Bip-Pro concentration (l
M); v, uptake
(nmolÆ30 min
)1
Æmg of protein
)1
)]. (B) Uptake of [
14
C]Gly-Sar (5–
20 l
M, 10 min, pH 6.0) was measured over a Gly-Sar concentration
range of 0–5 m
M. Nonspecific uptake ⁄ binding was determined by
measuring uptake in the presence of an excess amount (20 m
M)
of unlabeled Gly-Sar. This component (4%) was subtracted from
the total uptake to calculate the carrier-mediated uptake. Inset:
Eadie–Hofstee transformation of the specific Gly-Sar uptake data
[S, Gly-Sar concentration (m
M); v, uptake rate (nmolÆ10 min
)1
Æmg

protein
)1
)]. Values represent the means ± standard error (SE) for
four determinations.
Fig. 4. Time and pH dependence of the uptake of Bip-[
3
H]Pro in
SKPT cells. Uptake of Bip-[
3
H]Pro (18 nM, n ¼ 4) in SKPT cells was
measured at pH 6.0 for 10 min to 4 h in the absence (d) or in the
presence (s) of unlabeled Bip-Pro (1 m
M). Inset: uptake of Bip-
[
3
H]Pro (4 nM, 2 h) measured at different pH values (n ¼ 4).
I. Knu
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FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5909
amounts of substrate (1 or 20 mm, respectively) and
subtracted from the total uptake rates. For both
substrates, the relationship between carrier-mediated
uptake and substrate concentration was found to fol-
low Michaelis–Menten kinetics (Fig. 5). Eadie–Hofstee
transformation (uptake rate versus uptake rate ⁄ sub-
strate concentration) revealed linearity with a single
component (Fig. 5 insets). The apparent K
t
for

Gly-Sar uptake was 91.3 ± 4.1 lm and the V
max
was
5.6 ± 0.1 nmolÆ10 min
)1
Æmg of protein
)1
. These
parameters agree very well with those of previous
reports [18]. For Bip-Pro uptake, an apparent K
t
of
7.6 ± 1.8 lm and a V
max
of 1.1 ± 0.1 nmolÆ30
min
)1
Æmg of protein
)1
was determined. Hence, the
maximal velocity of Bip-Pro uptake is 16-fold lower
than the maximal velocity of Gly-Sar uptake, whereas
the affinity constant of Bip-Pro uptake is 12-fold
lower. Bip-Pro uptake represents a high-affinity, low-
capacity process, whereas the Gly-Sar uptake occurs
with low affinity but high transport capacity. The
lower V
max
of Bip-Pro uptake, and the higher V
max

of
Gly-Sar uptake, correspond well with the currents
obtained at PEPT2-expressing X. laevis oocytes. The
mean value of the apparent Michaelis–Menten con-
stant calculated from the currents measured at
)160 mV with Bip-Pro concentrations between 20 and
500 lm was 26 lm and the maximal current amounted
to 8% of the current evoked by Gly-Sar at saturating
concentration. In comparison, the inward current elic-
ited by Gly-Sar is 90% of that generated by Gly-Gln
and the affinity of PEPT2 was slightly lower for Gly-
Sar (K
t
¼ 0.3 mm) than for Gly-Gln (K
t
¼ 0.1 m m).
The situation is very similar for PEPT1, where Bip-Pro
elicited 21% and Gly-Sar elicited 101% of the Gly-Gln
current. Thus, the transport of Bip-Pro was also, in
PEPT2-expressing oocytes, a high-affinity, low-capacity
process. These findings suggest that the conformational
change of the carrier protein following H
+
binding
and substrate binding represents the rate limiting step
in the substrate translocation cycle. Differences in the
maximal transport currents of peptide transporters
under saturating substrate concentrations have been
reported before, suggesting that not only apparent K
t

values but also turnover rates may differ between sub-
strates [28].
Substrate specificity of Bip-[
3
H]Pro uptake
In the next series of experiments, the specificity of Bip-
[
3
H]Pro uptake was investigated using fixed concentra-
tions of competitors. The uptake of Bip-[
3
H]Pro (4 nm,
pH 6.0) into SKPT cells was inhibited not only by
unlabeled Bip-Pro itself, but also by well known sub-
strates of H
+
⁄ peptide cotransporters, such as Gly-Sar,
Ala-Ala, Lys-Lys, Ala-Asp, d-Phe-Ala, Ala-Ala-Ala,
d-aminolevulinic acid, cefadroxil and Ala-4-nitroanilide
(all 100 lm, Table 1). Glycine, which is not a substrate
of peptide transporters, did not inhibit Bip-[
3
H]Pro
uptake. The PEPT1 and PEPT2 inhibitor Lys(4-nitrob-
enzyloxycarbonyl)-Val [18,20], which is not transported
itself but interacts with both transporters with very
high affinity, displayed the strongest inhibitory effect
of all compounds tested in this study (Table 1). Pro-
Ala, at a concentration of 100 lm, did not inhibit Bip-
[

3
H]Pro uptake because it is a low-affinity substrate of
PEPT2 with an apparent K
i
value of 2.6 mm [4]. In
contrast, cefadroxil strongly inhibited Bip-[
3
H]Pro
uptake by 85%, corresponding very well with its
apparent K
i
value for PEPT2 of 3 lm [4]. Finally, 8-
aminooctanoic acid, which is no substrate for PEPT2
[2–4], also did not inhibit uptake.
We then determined the apparent K
i
values of five
compounds that tested positive for inhibition of Bip-
[
3
H]Pro uptake. The apparent K
i
values (Table 2) were
calculated by nonlinear regression from data obtained
in competition experiments such as those shown in
Fig. 2. For Bip-Pro, the self-inhibition K
i
was 7.8 ±
0.1 lm (K
i trans

¼ 1.7). Cefadroxil (K
i
¼ 5.2 ± 0.4 lm)
displayed the highest affinity for inhibition followed
by Gly-Sar, Lys-Lys and d-aminolevulinic acid
with apparent K
i
values between 75 and 230 lm. For
comparison, in Table 2 we also present the respective
inhibition constants (K
i
) of these five substrates for the
inhibition of [
14
C]Gly-Sar uptake in SKPT cells. This
Table 1. Specificity of Bip-[
3
H]Pro uptake. Uptake of Bip-[
3
H]Pro
(4 n
M) into SKPT cells was measured at pH 6.0 for 2 h at room
temperature in the absence (control) or presence of inhibitors (all
100 l
M). Data are shown as means ± standard error, n ¼ 4.
Lys[Z(NO
2
)]-Val, Lys(4-nitrobenzyloxycarbonyl)-Val.
Compound Bip-[
3

H]Pro uptake (%)
Control 100 ± 2
Gly 118 ± 8
Gly-Sar 64 ± 5
Bip-Pro 14 ± 1
Ala-Ala 15 ± 1
Pro-Ala 105 ± 3
Lys-Lys 46 ± 2
Ala-Asp 19 ± 1
D-Phe-Ala 65 ± 2
Ala-Ala-Ala 21 ± 1
d-Aminolevulinic acid 78 ± 3
Cefadroxil 15 ± 1
Lys[Z(NO
2
)]-Val 10 ± 1
8-Aminooctanoic acid 111 ± 3
Ala-4-nitroanilide 38 ± 1
Labeled high-affinity substrate for peptide transporters I. Knu
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5910 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS
so-called ABC test shows that the affinity constants
are very similar. Bip-Pro (A) and Gly-Sar (B) were
inhibited to the same extent by the other compounds
(C). Hence, Bip-Pro and Gly-Sar are transported by
the same system.
In conclusion, the results of the present study on the
mechanism and specificity of Bip-Pro uptake in SKPT
cells, together with the electrophysiological data

obtained in X. laevis oocytes expressing PEPT2, pro-
vide unequivocal evidence that Bip-Pro is transported
by PEPT2. Its enzymatic stability allows it to be used
in complex biological systems and its very high affinity
should make it particularly useful as a probe for the
analysis of the structure of the PEPT2 protein. More-
over, via detailed kinetic analyses with the now avail-
able two labeled transporter substrates, Bip-Pro and
Gly-Sar, which differ markedly in maximal transport
rates, the identification of the rate limiting step in the
transport cycle of PEPT1 and PEPT2 became feasible.
Experimental procedures
Materials
The renal cell line SKPT-0193 CL.2, established from iso-
lated cells of rat proximal tubules [24], was provided by U.
Hopfer (Case Western Reserve University, Cleveland, OH,
USA). The human colon carcinoma cell line Caco-2 was
obtained from the German Collection of Microorganisms
and Cell Cultures (Braunschweig, Germany). [Gly-
1-
14
C]Gly-Sar (specific radioactivity 53 mCiÆ mmol
)1
) was
custom synthesized by Amersham International (Little Chal-
font, UK). Dexamethasone, apotransferrin, Gly-Gln, Ala-
Ala, Ala-Ala-Ala, Lys-Lys, d-aminolevulinic acid, cefadroxil,
Gly, Pro-Ala, 8-aminooctanoic acid and Gly-Sar were from
Sigma-Aldrich (Deisenhofen, Germany). Tert. butyloxycar-
bonyl (Boc)–Bip, l-3,4-dehydro-Proline (DPro), d-Phe-Ala

and Ala-Asp were purchased from Bachem (Heidelberg,
Germany). Culture media, media supplements and trypsin
solution were purchased from Invitrogen (Karlsruhe,
Germany) or PAA (Pasching, Austria). Fetal bovine serum
was from Biochrom (Berlin, Germany) and collagenase A
from Roche (Mannheim, Germany). Ala-4-nitroanilide and
Lys(4-nitrobenzyloxycarbonyl)-Val were synthesized accord-
ing to peptide synthesis standard procedures [18,29]. All
other chemicals were of analytical grade.
Synthesis of Bip-Pro and Bip-[
3
H]Pro
Boc-Bip-Pro-OtBu was prepared from Boc–Bip-OH and H-
Pro–OtBuÆHCl using the mixed anhydride coupling method
with isobutylchloroformiate. After purification of the crude
product by flash chromatography (ethyl acetate ⁄ petroleum
ether: 1 : 2, v ⁄ v) the oily, protected dipeptide was depro-
tected with trifluoroacetic acid for 3 h to obtain the dipep-
tide as trifluoroacetate. Purity was measured with TLC,
RP-HPLC and MS and was found to exceed 98%. H-Bip-
Pro–OHÆtrifluoroacetic acid was a cis ⁄ trans isomere mixture
according to the HPLC chromatograms. At room tempera-
ture two peaks were observed, whereas there was only one
peak at temperatures of ‡ 45 °C.
The precursor peptide H-Bip–l-3,4-dehydro-Pro-OH
(H-Bip–DPro-OH) for
3
H-labeling was synthesized as
follows. Boc–Bip-OH was converted to Boc–Bip– N-hy-
droxysuccinimide ester using the water-soluble N-ethyl-

N¢-(3-dimethyl aminopropyl)-carbodiimide as a coupling
reagent. The resulting active ester derivative then reacted
with DPro and triethylamine in acetonitrile to give Boc–Bip–
DPro-OH. After purification of the crude product by flash
chromotography with ethyl acetic acid (5 : 0.1, v ⁄ v) the Boc-
Protected dipeptide was recrystallized from ethyl acetate.
Deprotection was carried out with 4 m HCl ⁄ dioxane to give
H-Bip–DPro-OH as its hydrochloride. Precipitation from
isopropanol ⁄ ethyl ether gave the H-Bip–DPro–OHÆHCl in
high purity (‡ 98%, checked by TLC, RP-HPLC and MS).
The tritium labeling was carried out by catalytic satura-
tion of 2 mg of the precursor peptide in N,N-dimethylfor-
mamide (room temperature, 30 min) using Pd ⁄ C as the
catalyst and carrier-free tritium gas [26]. After tritiation,
the crude peptide product was purified by HPLC (Jasco,
Budapest, Hungary) on a Vydac (Budapest, Hungary) 218
TP 54 column (250 · 4.6 mm) using linear gradient elution
(from 15 to 40%) of acetonitrile (0.08% trifluoroacetic
acid) in water (0.1% trifluoroacetic acid) within 25 min at
a flow rate of 1 mLÆmin
)1
with UV detection at 265 nm.
H-Bip-[
3
H]Pro-OH existed as a mixture of cis ⁄ trans con-
formers, according to the chromatograms. Radioactive pur-
ity of the final product was > 98% according to TLC
[silicagel 60 F254 plate, Merck, Darmstadt, Germany; sol-
vent system n-butanol-acetic acid-water (4 : 1 : 1, v ⁄ v ⁄ v) –
retention factor 0.41] and analytical HPLC (retention time

17.27 min, k¢ ¼ 4.57). Specific radioactivity of Bip-[
3
H]Pro,
Table 2. Inhibition constants (K
i
) of different substrates for the
inhibition of Bip-[
3
H]Pro and [
14
C]Gly-Sar uptake in SKPT cells.
Uptake of Bip-[
3
H]Pro (4 nM, 2 h) or of [
14
C]Gly-Sar (10 lM, 10 min)
was measured at pH 6.0 at increasing concentrations of unlabeled
substrates or inhibitors of PEPT2. Constants were derived from
competition curves such as those shown in Fig. 2 for Bip-Pro. Para-
meters are shown ± standard error (n ¼ 4).
Compound
K
i
(lM)
Bip-[
3
H]Pro uptake [
14
C]Gly-Sar uptake
Gly-Sar 102 ± 9 61 ± 8 [24]

Bip-Pro 7.8 ± 0.1 3.4 ± 0.1
Cefadroxil 5.2 ± 0.4 3 ± 1 [4]
d-Aminolevulinic acid 230 ± 20 231 ± 90 [4]
Lys-Lys 75 ± 9 12 ± 0.3 [4]
I. Knu
¨
tter et al. Labeled high-affinity substrate for peptide transporters
FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5911
estimated by a calibration curve prepared with a standard
dipeptide, was 1.853 TBqÆmmol
)1
(50.1 Ci mmol
)1
).
NMR analysis
The relative populations of the cis ⁄ trans isomers were
determined by NMR measurements [21,22].
1
H NMR spec-
tra of 5.3 mg of Bip-Pro dissolved in 0.7 mL of H
2
O ⁄ D
2
O
(90 : 10, v ⁄ v) were recorded on a Bruker Avance 400 spec-
trometer (Rheinstetten, Germany). All measurements were
carried out at pH 6.0 and 300 K. The pH of the solution
was adjusted by the addition of diluted solutions of DCl
and NaOD. Chemical shifts were calibrated with respect to
internal DSS. Selective water resonance suppression was

achieved by using presaturation during relaxation delay or
by using the 3-9-19 pulse sequence with gradients. Stan-
dard methods were used to perform 1D and 2D experi-
ments, pulse programs being taken from the Bruker
software library. Resonance assignments were made by the
combined analysis of H,H-COSY, ROESY and
13
C-HSQC
spectra. The ROESY spectra were recorded at a mixing
time of 300 ms in the phase-sensitive mode using baseline
correction in both dimensions.
HPLC analysis
The stability of Bip-Pro in the extracellular medium was
analyzed over incubation periods from 10 min up to 8 h.
The amount of Bip-Pro in the extracellular uptake medium
was quantified according to the laboratory standard HPLC
(La-Chrom
Ò
; Merck-Hitachi, Darmstadt, Germany) with a
diode array detector and a Polar-RP-80-A Synergi column
(150 · 4.6 mm; 4 lm; Phenomenex, Aschaffenburg, Ger-
many). The eluent was 30% acetonitrile ⁄ 0.1% trifluoroace-
tic acid in water. UV detection was performed at 220 nm.
The injection volume was 20 lL and the flow rate was
1mLÆmin
)1
.
Cell culture and uptake studies
SKPT cells were cultured in Dulbecco’s modified Eagle’s
medium ⁄ F12 Nutrient Mixture (Ham) (1 : 1, v ⁄ v) and

2mml-glutamine, 10% fetal bovine serum, recombinant
insulin (4 lgÆmL
)1
), epidermal growth factor (10 ng ÆmL
)1
),
apotransferrin (5 lgÆmL
)1
), dexamethasone (5 lgÆ mL
)1
)
and gentamicin (45 lgÆmL
)1
), as described previously
[18,24]. The human colon carcinoma cell line Caco-2 was
routinely cultured with Minimum Essential Medium with
Earle’s salts and l-glutamine (2 mm) supplemented with
10% fetal bovine serum, 1% nonessential amino acid
solution and gentamicin (45 lgÆmL
)1
) [17,20]. Both cell
lines were subcultured in 35-mm disposable Petri dishes
(Sarstedt, Nu
¨
mbrecht, Germany) at a seeding density of
0.8 · 10
6
cells per dish. The cultures of both cell types
reached confluence within 20 h.
Uptake of [

14
C]Gly-Sar or Bip-[
3
H]Pro was measured
4 days (SKPT) or 7 days (Caco-2) after seeding at 22 °C,
as described previously [17,18,20]. The uptake buffer 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
2
,
0.8 mm MgSO
4
and 5 mm glucose. Uptake was initiated
after washing the cells for 30 s in uptake buffer by adding
1 mL of uptake medium containing [
14
C]Gly-Sar (10 lm)or
Bip-[
3
H]Pro (4 nm) with increasing concentrations of the
test compounds (0–31.6 mm). If necessary, the pH of the
solutions was corrected before preparing the required dilu-
tions. After incubation for the desired time periods, the
cells were quickly washed four times with ice-cold buffer,
solubilized in 1 mL of Igepal
Ò
Ca-630 (0.5% v ⁄ v; Sigma
Aldrich, Deisenhofen, Germany) in buffer (50 mm
Tris ⁄ HCl, pH 9.0, 140 mm NaCl, 1.5 mm MgSO
4

) and pre-
pared for liquid scintillation spectrometry. For each experi-
ment, the samples for the protein measurements were
prepared and measured as described previously [20].
X. laevis oocytes expressing PEPT1 and PEPT2
and electrophysiology
Female X. laevis were purchased from the African Xeno-
pus Facility (Kynsa, South Africa). Surgically removed oo-
cytes were separated by collagenase treatment and handled
as described previously [17,18,20,25]. Individual oocytes
were injected with 30 nL of RNA solution containing
30 ng of rabbit PEPT1 or rabbit PEPT2 cRNA. All elec-
trophysiological measurements were performed after 3–
6 days by incubation of oocytes in a buffer composed of
88 mm NaCl, 1 mm KCl, 0.82 mm CaCl
2
, 0.41 mm MgCl
2
,
0.33 mm Ca(NO
3
)
2
, 2.4 mm NaHCO
3
and 10 mm
Mes ⁄ Tris at pH 6.5 (modified Barth solution). The
two-electrode voltage clamp technique was applied to
characterize responses in current (I) and transmembrane
potential (V

m
) to substrate addition in oocytes expressing
PEPT1 or PEPT2 [17,18,20,25]. In short, oocytes were
placed in an open chamber in a volume of 0.5 mL and
continuously superfused with modified Barth solution or
with solutions of Gly-Gln, Gly-Sar and ⁄ or Bip-Pro. Elec-
trodes with resistances between 1 and 10 MW were
connected to a TEC-05 amplifier (NPI Electronic, Tamm,
Germany). Current–voltage (I–V
m
) relationships were mea-
sured using short (100 ms) pulses separated by 200 ms
pauses in the potential range from )160 to +80 mV.
I–V
m
measurements were made immediately before and
30 s after substrate application when current flow reached
steady state. Currents evoked by PEPT1 or PEPT2 at a
given membrane potential were calculated as the difference
of the currents measured in the presence and the absence
of substrate.
Labeled high-affinity substrate for peptide transporters I. Knu
¨
tter et al.
5912 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS
Calculations and statistics
All data are given as the mean ± standard error of three
to four independent experiments. The kinetic parameters
were calculated by nonlinear regression methods (sigma-
plot program; Systat, Erkrath, Germany) and confirmed

by linear regression of the respective Eadie–Hofstee Plots.
The concentration of the unlabeled compound necessary to
inhibit 50% of radiolabeled dipeptide carrier-mediated
uptake (IC
50
) was determined by nonlinear regression using
the logistical equation for an asymmetric sigmoid (allosteric
Hill kinetics): y ¼ Min + (Max–Min) ⁄ (1 + (X ⁄ IC
50
)
–P
),
where Max is the initial Y-value, Min the final Y-value and
the power P represents Hills’ coefficient. Inhibition con-
stants (K
i
) were calculated from IC
50
values.
Acknowledgements
This work was supported by the State Saxony-Anhalt
Life Sciences Excellence Cluster (MB).
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