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 c
Trang 1high-affinity substrate for H+/peptide cotransporters
Ilka Knu¨tter1, Bianka Hartrodt2, Ge´za To´th3, Attila Keresztes3, Gabor Kottra4,
Carmen Mrestani-Klaus2, Ilona Born2, Hannelore Daniel4, Klaus Neubert2and Matthias Brandsch1
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 tripepdipep-tides 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 SepSep-tember 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 [14C]Gly-Sar uptake via PEPT1 and PEPT2 with exceptional high affinity (Ki¼ 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-[3H]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 (Kt) of 7.6 lm and a maximal velocity (Vmax) of 1.1 nmo-lÆ30 min)1Æmg 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-[3H]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.
Trang 2intestinal 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 ([14C]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 Kt 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-[3H]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 1H 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.
Trang 3Subsequent analysis of the ROESY spectrum revealed
characteristic strong ROEs between Bip-CaH and both
Pro-CdHA and Pro-CdHB of the major isomer
identi-fied as a trans isomer As strong ROEs (or NOEs)
between a protons of adjacent residues CaH(i)-CaH(i
+1) allow the resonance assignment of populations
containing cis amide linkages [21], the strong ROE
between Bip-CaH and Pro-CaH 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-CaH 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 cm2) 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
[14C]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 [14C]Gly-Sar uptake
in a dose-dependent manner (Fig 2) The apparent
Ki 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 Ki 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 (Ki) for
Bip-Pro by measuring Gly-Sar uptake at two
differ-ent Gly-Sar concdiffer-entrations (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 Ki values of 34.1 lm (Ki trans¼ 7.5 lm) and 1.3 lm (Ki trans¼ 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 l M [ 14 C]Gly-Sar,
pH 6.0, 10 min, n ¼ 4) and in SKPT cells (B) (10 l M [14C]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 m M , respectively), was subtracted from the total uptake to calculate the carrier-mediated uptake (n ¼ 4, v ¼ uptake rate in nmolÆ
10 min)1Æmg protein)1).
Trang 4Interaction 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-[3H]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-[3H]Pro uptake across the apical membrane of SKPT cells Time-dependent uptake of Bip-[3H]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 m M ) 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.
Trang 5(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-[3H]Pro
uptake at all time points Bip-[3H]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 [14C]Gly-Sar, both in SKPT cells [24] and in
Caco-2 cells [27] We also studied the time and pH
dependency of Bip-[3H]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-[3H]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-[3H]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-[3H]Pro transport after its uptake into the cells
For further functional characterization of Bip-[3H]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
4 nm 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 n M , 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)1Æmg 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)1Æmg 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 n M , 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 n M , 2 h) measured at different pH values (n ¼ 4).
Trang 6amounts 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 Kt for
Gly-Sar uptake was 91.3 ± 4.1 lm and the Vmax was
5.6 ± 0.1 nmolÆ10 min)1Æmg of protein)1 These
parameters agree very well with those of previous
reports [18] For Bip-Pro uptake, an apparent Kt of
7.6 ± 1.8 lm and a Vmax of 1.1 ± 0.1 nmolÆ30
min)1Æmg 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 Vmax of Bip-Pro uptake, and the higher Vmax 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 (Kt¼ 0.3 mm) than for Gly-Gln (Kt¼ 0.1 mm)
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 Kt
values but also turnover rates may differ between
sub-strates [28]
Substrate specificity of Bip-[3H]Pro uptake
In the next series of experiments, the specificity of
Bip-[3H]Pro uptake was investigated using fixed
concentra-tions of competitors The uptake of Bip-[3H]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-[3H]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-[3H]Pro uptake because it is a low-affinity substrate of PEPT2 with an apparent Ki value of 2.6 mm [4] In contrast, cefadroxil strongly inhibited Bip-[3H]Pro uptake by 85%, corresponding very well with its apparent Ki 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 Ki values of five compounds that tested positive for inhibition of Bip-[3H]Pro uptake The apparent Kivalues (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 Ki was 7.8 ± 0.1 lm (Ki trans¼ 1.7) Cefadroxil (Ki¼ 5.2 ± 0.4 lm) displayed the highest affinity for inhibition followed
by Gly-Sar, Lys-Lys and d-aminolevulinic acid with apparent Ki values between 75 and 230 lm For comparison, in Table 2 we also present the respective inhibition constants (Ki) of these five substrates for the inhibition of [14C]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.
Trang 7so-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 epro-vidence 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-1-14C]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-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-[3H]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 3H-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-[3H]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-[3H]Pro,
Table 2 Inhibition constants (K i ) of different substrates for the
inhibition of Bip-[3H]Pro and [14C]Gly-Sar uptake in SKPT cells.
Uptake of Bip-[ 3 H]Pro (4 n M , 2 h) or of [ 14 C]Gly-Sar (10 l M , 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
Ki(l M ) Bip-[ 3 H]Pro uptake [ 14 C]Gly-Sar uptake
d-Aminolevulinic acid 230 ± 20 231 ± 90 [4]
Trang 8estimated 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].1H NMR
spec-tra of 5.3 mg of Bip-Pro dissolved in 0.7 mL of H2O⁄ D2O
(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 13C-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; 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
1 mLÆmin)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
2 mm l-glutamine, 10% fetal bovine serum, recombinant
insulin (4 lgÆmL)1), epidermal growth factor (10 ngÆmL)1),
apotransferrin (5 lgÆmL)1), dexamethasone (5 lgÆmL)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· 106
cells per dish The cultures of both cell types reached confluence within 20 h
Uptake of [14C]Gly-Sar or Bip-[3H]Pro was measured
4 days (SKPT) or 7 days (Caco-2) after seeding at 22C,
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 CaCl2, 0.8 mm MgSO4 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 [14C]Gly-Sar (10 lm) or Bip-[3H]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 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 MgSO4) 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 CaCl2, 0.41 mm MgCl2,
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 (Vm) 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–Vm) relationships were mea-sured using short (100 ms) pulses separated by 200 ms pauses in the potential range from )160 to +80 mV I–Vm 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
Trang 9Calculations 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 (IC50) was determined by nonlinear regression using
the logistical equation for an asymmetric sigmoid (allosteric
Hill kinetics): y¼ Min + (Max–Min) ⁄ (1 + (X ⁄ IC50)–P),
where Max is the initial Y-value, Min the final Y-value and
the power P represents Hills’ coefficient Inhibition
con-stants (Ki) were calculated from IC50values
Acknowledgements
This work was supported by the State Saxony-Anhalt
Life Sciences Excellence Cluster (MB)
References
1 Ganapathy V, Ganapathy ME & Leibach FH (2001)
Intestinal transport of peptides and amino acids In
Current Topics in Membranes(Barrett KE & Donowitz
M, eds), Vol 50, pp 379–412 Academic Press, San
Diego, CA
2 Daniel H & Kottra G (2004) The proton oligopeptide
cotransporter family SLC15 in physiology and
pharma-cology Pflugers Arch 447, 610–618
3 Daniel H (2004) Molecular and integrative physiology
of intestinal peptide transport Annu Rev Physiol 66,
361–384
4 Biegel A, Knu¨tter I, Hartrodt B, Gebauer S, Theis S,
Luckner P, Kottra G, Rastetter M, Zebisch K,
Thon-dorf I et al (2006) The renal type H+⁄ peptide
symport-er PEPT2: structure-affinity relationships Amino Acids
31, 137–156
5 Brandsch M, Knu¨tter I & Leibach FH (2004) The
intes-tinal H+⁄ peptide symporter PEPT1: structure-affinity
relationships Eur J Pharm Sci 21, 53–60
6 Steffansen B, Nielsen CU, Brodin B, Eriksson AH,
Andersen R & Frokjaer S (2004) Intestinal solute
carri-ers: an overview of trends and strategies for improving
oral drug absorption Eur J Pharm Sci 21, 3–16
7 Groneberg DA, Fischer A, Chung KF & Daniel H
(2004) Molecular mechanisms of pulmonary
peptidomi-metic drug and peptide transport Am J Respir Cell Mol
Biol 30, 251–260
8 Knu¨tter I, Rubio-Aliaga I, Boll M, Hause G, Daniel H,
Neubert K & Brandsch M (2002) H+-peptide
cotrans-port in the human bile duct epithelium cell line
SK-ChA-1 Am J Physiol 283, G222–G229
9 Teuscher NS, Novotny A, Keep RF & Smith DE (2000) Functional evidence for presence of PEPT2 in rat cho-roid plexus: studies with glycylsarcosine J Pharmacol Exp Ther 294, 494–499
10 Do¨ring F, Dorn D, Bachfischer U, Amasheh S, Herget
M & Daniel H (1996) Functional analysis of a chimeric mammalian peptide transporter derived from the intesti-nal and reintesti-nal isoforms J Physiol 497, 773–779
11 Fei YJ, Liu JC, Fujita T, Liang R, Ganapathy V & Lei-bach FH (1998) Identification of a potential substrate binding domain in the mammalian peptide transporters PEPT1 and PEPT2 using PEPT1-PEPT2 and PEPT2-PEPT1 chimeras Biochem Biophys Res Commun 246, 39–44
12 Fei YJ, Liu W, Prasad PD, Kekuda R, Oblak TG, Ganapathy V & Leibach FH (1997) Identification of the histidyl residue obligatory for the catalytic activity of the human H+⁄ peptide cotransporters PEPT1 and PEPT2 Biochemistry 36, 452–460
13 Bolger MB, Haworth IS, Yeung AK, Ann D, von Gra-fenstein H, Hamm-Alvarez S, Okamoto CT, Kim KJ, Basu SK, Wu S et al (1998) Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1 J Pharm Sci 87, 1286–1291
14 Terada T, Irie M, Okuda M & Inui K (2004) Genetic variant Arg57His in human H+⁄ peptide cotransporter 2 causes a complete loss of transport function Biochem Biophys Res Commun 316, 416–420
15 Biegel A, Gebauer S, Hartrodt B, Brandsch M, Neubert
K & Thondorf I (2005) Three-dimensional quantitative structure-activity relationship analyses of b-lactam anti-biotics and tripeptides as substrates of the mammalian
H+⁄ peptide cotransporter PEPT1 J Med Chem 48, 4410–4419
16 Biegel A, Gebauer S, Brandsch M, Neubert K & Thon-dorf I (2006) Structural requirements for the substrates
of the H+⁄ peptide cotransporter PEPT2 determined by three-dimensional quantitative structure-activity rela-tionship analysis J Med Chem 49, 4286–42961
17 Knu¨tter I, Theis S, Hartrodt B, Born I, Brandsch M, Daniel H & Neubert K (2001) A novel inhibitor of the mammalian peptide transporter PEPT1 Biochemistry
40, 4454–4458
18 Theis S, Knu¨tter I, Hartrodt B, Brandsch M, Kottra G, Neubert K & Daniel H (2002) Synthesis and character-ization of high-affinity inhibitors of the H+⁄ peptide transporter PEPT2 J Biol Chem 277, 7287–7292
19 Tyagi NK, Goyal P, Kumar A, Pandey D, Siess W & Kinne RK (2005) High-yield functional expression of human sodium⁄ d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced confor-mational changes as studied by tryptophan fluorescence Biochemistry 44, 15514–15524
Trang 1020 Knu¨tter I, Hartrodt B, Theis S, Foltz M, Rastetter M,
Daniel H, Neubert K & Brandsch M (2004) Analysis of
the transport properties of side chain modified
dipep-tides at the mammalian peptide transporter PEPT1 Eur
J Pharm Sci 21, 61–67
21 Wu¨thrich K, Billeter M & Braun W (1984) Polypeptide
secondary structure determination by nuclear magnetic
resonance observation of short proton-Proton distances
J Mol Biol 180, 715–740
22 Brandsch M, Thunecke F, Ku¨llertz G, Schutkowski M,
Fischer G & Neubert K (1998) Evidence for the
abso-lute conformational specificity of the intestinal H+⁄
pep-tide symporter, PEPT1 J Biol Chem 273, 3861–3864
23 Brandsch M, Knu¨tter I, Thunecke F, Hartrodt B, Born
I, Bo¨rner V, Hirche F, Fischer G & Neubert K (1999)
Decisive structural determinants for the interaction of
proline derivatives with the intestinal H+⁄ peptide
sym-porter Eur J Biochem 266, 502–508
24 Brandsch M, Brandsch C, Prasad PD, Ganapathy V,
Hopfer U & Leibach FH (1995) Identification of a renal
cell line that constitutively expresses the kidney-specific
high-affinity H+⁄ peptide cotransporter FASEB J 9, 1489–1496
25 Boll M, Herget M, Wagener M, Weber WM, Marko-vich D, Biber J, Clauss W, Murer H & Daniel H (1996) Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter Proc Natl Acad Sci USA 93, 284–289
26 To´th G, Lovas S & Otvo¨s F (1997) Tritium labeling of neuropeptides Methods Mol Biol 73, 219–230
27 Brandsch M, Miyamoto Y, Ganapathy V & Leibach
FH (1994) Expression and protein kinase C-dependent regulation of peptide⁄ H+
cotransport system in the Caco-2 human colon carcinoma cell line Biochem J
299, 253–260
28 Sala-Rabanal M, Loo DD, Hirayama BA, Turk E & Wright EM (2006) Molecular interactions between di-peptides, drugs and the human intestinal H+ -oligopep-tide cotransporter hPEPT1 J Physiol 574, 149–166
29 Goodman M, Felix A, Moroder L & Toniolo C (2002) Houben-Weyl Methods of Organic Chemistry, Vol E22a, 4th edn Georg Thieme Verlag, Stuttgart, New York