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

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Synthesis and characterization of a new and radiolabeledhigh-affinity substrate for H+/peptide cotransportersIlka Knu¨tter1, Bianka Hartrodt2,Ge´za To´th3, Attila Keresztes3, Gabor Kottra4,Carmen Mrestani-Klaus2, Ilona Born2, Hannelore Daniel4, Klaus Neubert2and Matthias Brandsch11 Biozentrum of the Martin-Luther-University Halle-Wittenberg, Halle, Germany2 Institute of Biochemistry ⁄ Biotechnology, Faculty of Sciences I, Martin-Luther-University Halle-Wittenberg, Halle, Germany3 Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary4 Molecular Nutrition Unit, Technical University of Munich, Freising-Weihenstephan, GermanyThe peptide transporters peptide cotransporter 1(PEPT1) (SLC15A1) and peptide cotransporter 2(PEPT2) (SLC15A2) are presently under intense inves-tigation because of their physiological importance andtheir pharmaceutical relevance as drug carriers [1–6].Both transporters catalyse the uptake of most dipep-tides and tripeptides and a variety of peptidomimeticdrugs, such as selected b-lactam antibiotics, someangiotensin-converting enzyme inhibitors and pro-drugs such as valaciclovir. H+-coupled peptide anddrug transport across cell membranes by PEPT1and PEPT2, respectively, have been demonstrated atKeywordsCaco-2; H+⁄ peptide cotransporter 1;H+⁄ peptide cotransporter 2; SKPT; Xenopuslaevis oocytesCorrespondenceM. Brandsch, Biozentrum of the Martin-Luther-University Halle-Wittenberg,Membrane Transport Group,Weinbergweg 22, D-06120 Halle, GermanyFax: +49 345 5527258Tel: +49 345 5521630E-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.xIn this study we described the design, rational synthesis and functionalcharacterization of a novel radiolabeled hydrolysis-resistant high-affinitysubstrate for H+⁄ peptide cotransporters. l-4,4¢-Biphenylalanyl–l-Proline(Bip-Pro) was synthesized according to standard procedures in peptidechemistry. The interaction of Bip-Pro with H+⁄ peptide cotransporters wasdetermined in intestinal Caco-2 cells constitutively expressing humanH+⁄ peptide cotransporter 1 (PEPT1) and in renal SKPT cells constitutivelyexpressing 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 oocytesexpressing PEPT1 or PEPT2 it was found that Bip-Pro was transported byboth 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 hwhen incubated with intact cell monolayers. Bip-[3H]Pro uptake into SKPTcells was linear for up to 30 min and pH dependent with a maximum atextracellular pH 6.0. Uptake was strongly inhibited, not only by unlabeledBip-Pro but also by known peptide transporter substrates such as dipep-tides, cefadroxil, Ala-4-nitroanilide and d-aminolevulinic acid, but not byglycine. 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 ahigh-affinity, low-capacity process in comparison to the uptake of Gly-Sar.We conclude that Bip-[3H]Pro is a valuable substrate for both mechanisticand structural studies of H+⁄ peptide transporter proteins.AbbreviationsBoc, 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 5905intestinal and renal cells [1–3] but also in lung [7],extrahepatic biliary duct [8], choroid plexus [9] andother tissues [1–3].Essentially nothing is known about the location andstructure of the substrate binding domains within thecarrier proteins. Available data are restricted to resultsobtained in experiments with chimeric mammalianpeptide transporters derived from the intestinal andrenal isoforms [10,11], site-directed mutagenesis experi-ments [12–14] and from extensive studies on substratespecificity combined with molecular modeling[4,5,15,16].The most commonly used and best known referencesubstrate of H+⁄ peptide cotransporters is [14C]glycine-sarcosine ([14C]Gly-Sar). This substrate is relativelystable against intracellular and extracellular enzymatichydrolysis, but its affinity constants for peptide trans-porters are only in the medium range, with Ktvaluesof  1.3 mm for PEPT1 [17] and  108 lm for PEPT2[18]. New high-affinity labeled probes are required forfurther studies on the mechanism of transport func-tion and the structure of the carrier proteins. Forexample, the rate limiting step of peptide transportershas still not yet been determined, and the number oftransporters per cell and their turnover rate in epithe-lial cells is not known. With regard to transporterstructure, despite the fact that techniques such asintrinsic tryptophan fluorescence measurement havebeen shown to be useful to study the expression andconformation of recombinant membrane transporters[19], labeled substrates and inhibitors with a broadrange of affinity to the respective protein are alsoessential tools. In the course of our work onhigh-affinity inhibitors of PEPT1 and PEPT2, on thestructural modifications that convert a transportedcompound into a nontranslocated inhibitor as well asstudies on the structural requirements for a high affin-ity of substrates [17,18,20], it became evident that alarge aromatic hydrophobic group in the side chain ofthe N-terminal amino acid of dipeptides couldenhance the affinity of many derivatives for bindingto the transporters. Besides high affinity, a secondimportant requirement for any peptide transportersubstrate is a sufficient stability against enzymatichydrolysis. Hence, we decided to synthesize a dipep-tide where l-4,4¢-biphenylalanine (Bip), with its largearomatic side chain in a short intramolecular distancefrom the a-carbon atom, is the N-terminal amino acidand l-Proline (l-Pro) is the C-terminal amino acid.The resulting compound, Bip-Pro, was tested withregard to its interaction with peptide transporters, itsaffinity and its stability in a biological system. More-over, after radioactive labeling we determined thekinetic parameters and transport characteristics ofBip-[3H]Pro.Results and DiscussionSynthesis, chemical characterization and stabilityof Bip-ProFigure 1 shows the structure (Fig. 1A) and the synthe-sis strategy (Fig. 1B) of Bip-Pro. The purity of thecompound was assessed by TLC, analytical RP-HPLC, MS and NMR, and was found to exceed98%. 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 the1HNMR spectrum, Bip-Pro exhibited two sets of NMRsignals 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 ofBip-[3H]Pro. EDC, N-ethyl-N¢-(3-dimethyl aminopropyl)-carbodiimide;HOSu, N-hydroxysuccinimide.Labeled high-affinity substrate for peptide transporters I. Knu¨tter et al.5906 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBSSubsequent analysis of the ROESY spectrum revealedcharacteristic strong ROEs between Bip-CaH and bothPro-CdHAand Pro-CdHBof 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 populationscontaining cis amide linkages [21], the strong ROEbetween Bip-CaH and Pro-CaH of the minor isomerwas 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 the1D proton spectrum such as the two Bip-CaH signals[21,22]. In equilibrium, Bip-Pro shows a trans contentof 22%, whereas 78% were in cis conformation. Thesevalues are in agreement with the cis ⁄ trans ratios ofXaa-Pro dipeptides containing aromatic amino acids(24–34% trans content) obtained in a previous studyof our group [23].To determine the stability of Bip-Pro, buffer sampleswere analyzed after incubating Caco-2 and SKPT cellmonolayers (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 containingbuffer, 100% Bip-Pro was found. After 8 h, 99.9% ofBip-Pro was intact, with the remaining 0.1% beingBip. At all time points, Bip-Pro was recovered frommonolayers of both cell types intact to > 99% (HPLCdata not shown).Interaction of Bip-Pro with PEPT1 and PEPT2We next determined the interaction of Bip-Pro withPEPT1 and PEPT2 in competition assays, with[14C]Gly-Sar serving as a reference compound. Theintestinal cell line Caco-2, constitutively expressingPEPT1 [5,17,22], and the renal cell line SKPT, con-stitutively expressing PEPT2 [18,24], were used asmodels. Bip-Pro competed with [14C]Gly-Sar uptakein a dose-dependent manner (Fig. 2). The apparentKivalues for substrate uptake inhibition were24 ± 0.6 lm in Caco-2 cells (PEPT1, Fig. 2A) and3.4 ± 0.1 lm in SKPT cells (PEPT2, Fig. 2B). It hasbeen shown that peptide transporters are specific forthe trans conformers of their substrates [22,23]. Tak-ing the cis⁄ trans content of Bip-Pro (78% ⁄ 22%) intoaccount, we obtained Ki transvalues for Bip-Pro of5.2 lm in Caco-2 cells and of 0.75 lm in SKPT cells.We also determined the inhibition constants (Ki) forBip-Pro by measuring Gly-Sar uptake at two differ-ent Gly-Sar concentrations (50 and 500 lm in Caco-2cells and 10 and 100 lm in SKPT cells) in thepresence of increasing concentrations of Bip-Pro (0–100 lm and 0–50 lm, respectively). The results arepresented as Dixon plots in Fig. 2 (insets). The plotsreveal linearity at both Gly-Sar concentrations withlines intersecting above the abscissas in the fourthquadrant, as expected for a competitive inhibitor.Apparent Kivalues of 34.1 lm (Ki trans¼ 7.5 lm)and 1.3 lm (Ki trans¼ 0.29 lm) were calculated fromthe points of intersection of data obtained in Caco-2cells (Fig. 2A, inset) and SKPT cells (Fig. 2B, inset),respectively.Fig. 2. Interaction of Bip-Pro with PEPT1 and PEPT2. Uptake of[14C]Gly-Sar was measured in Caco-2 cells (A) (10 lM [14C]Gly-Sar,pH 6.0, 10 min, n ¼ 4) and in SKPT cells (B) (10 lM [14C]Gly-Sar,pH 6.0, 10 min, n ¼ 4) in the presence of increasing concentrationsof Bip-Pro (0–0.316 mM). Uptake rates measured in the absence ofBip-Pro were taken as 100%. Insets: determination of the inhibitionconstants by Dixon type experiments. Uptake of Gly-Sar was mea-sured at pH 6.0 for 10 min at two Gly-Sar concentrations and atincreasing Bip-Pro concentrations. The diffusional component of[14C]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 mM and 20 mM,respectively), was subtracted from the total uptake to calculatethe carrier-mediated uptake (n ¼ 4, v ¼ uptake rate in nmolÆ10 min)1Æmg protein)1).I. Knu¨tter et al. Labeled high-affinity substrate for peptide transportersFEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5907Interaction of Bip-Pro with PEPT1 and PEPT2expressed in Xenopus laevis oocytesInteraction with Gly-Sar uptake does not necessarilyallow the conclusion that Bip-Pro is indeed trans-ported. Therefore, the two-electrode voltage clamptechnique that determines transport currents wasapplied in X. laevis oocytes expressing either PEPT1 orPEPT2 [17,18,20,25]. In contrast to the referencedipeptide glycine-glutamine (Gly-Gln), for Bip-Proonly a low substrate-evoked inward transport currentwas recorded (Fig. 3). At a membrane potential of)60 mV, PEPT1-mediated transport currents were21 ± 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 was11 ± 1% of that generated by Gly-Gln (Fig. 3C).However, Bip-Pro at a concentration of 0.5 mm wasable to inhibit the inward current evoked by 0.5 mmGly-Gln at PEPT1 by 44 ± 1% (Fig. 3B). In the caseof PEPT2, a concentration of 0.1 mm Bip-Pro was ableto inhibit the inward current evoked by 0.1 mm Gly-Gln remarkably by 94 ± 6% (Fig. 3D). The inhibitionwas found to be dose dependent and reversible, sug-gesting a competitive mode of action.Uptake of Bip-[3H]Pro by SKPT cellsAfter characterization of Bip-Pro as a very high-affin-ity and enzymatically stable substrate of PEPT1 andPEPT2, the compound was synthesized in radiolabeledform according to Fig. 1C [26]. We then characterizedthe Bip-[3H]Pro uptake across the apical membrane ofSKPT cells. Time-dependent uptake of Bip-[3H]ProFig. 3. Characterization of the interaction of Bip-Pro with PEPT1 and PEPT2 in Xenopus laevis oocytes by electrophysiology. Steady-state I–Vrelationships were measured by the two-electrode voltage clamp technique in oocytes expressing PEPT1 (A, B) or PEPT2 (C, D) superfusedwith modified Barth solution at pH 6.5 and 0.5, or with 0.1 mM Gly–Gln, in the absence or the presence of increasing concentrations (PEPT1,0–1 mM; 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¨tter et al.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 reacheda plateau after 2 h of incubation (Fig. 4). The uptakewas found to be saturable: unlabeled Bip-Pro at a con-centration of 1 mm strongly inhibited Bip-[3H]Prouptake at all time points. Bip-[3H]Pro (4 nm) uptakewas also strongly pH dependent. Maximal uptake wasobserved at an extracellular pH of 6.0 (Fig. 4, inset).The same pH optimum has been observed for theuptake of [14C]Gly-Sar, both in SKPT cells [24] and inCaco-2 cells [27]. We also studied the time and pHdependency of Bip-[3H]Pro uptake in Caco-2 cells. Sur-prisingly, in this cell line the uptake was found to bestimulated by external pH 6.0 only modestly, by 26%in comparison to pH 7.5. Moreover, unlabeled Bip-Proin an excess concentration of 3 mm inhibited theuptake of the tracer Bip-[3H]Pro (4 nm) during 30 minof incubation by only 21% (data not shown). We con-clude that the nonspecific binding of the hydrophobicBip-[3H]Pro to Caco-2 cells is very much higher thanin SKPT cells. Alternatively, a specific intestinal apicalefflux system might mediate strong outward-directedBip-[3H]Pro transport after its uptake into the cells.For further functional characterization of Bip-[3H]Prouptake we therefore used SKPT cells.Saturation kinetics of Bip-Pro uptake at PEPT2Bip-Pro uptake as a function of substrate concentra-tion was measured to determine the kinetic parametersof the transport process. Uptake rates of Bip-Pro weredetermined over a substrate concentration range of4nm to 100 lm (Fig. 5A) and compared with theuptake rates of Gly-Sar at a concentration range of5 lm to 5 mm (Fig. 5B). For each compound, the non-specific, linear uptake component, which representssimple diffusion plus binding, was determined bymeasuring the uptake in the presence of excessFig. 5. Substrate saturation kinetics of Bip-Pro and Gly-Sar transportin SKPT cells. (A) Uptake of Bip-[3H]Pro (4 nM, 30 min, pH 6.0) wasmeasured over a Bip-Pro concentration range of 0–0.1 mM. Unspe-cific uptake ⁄ binding was determined by measuring uptake in thepresence of an excess amount (1 mM) of unlabeled Bip-Pro. Thiscomponent (24%) was subtracted from the total uptake to calculatethe specific uptake. Inset: Eadie–Hofstee transformation of the spe-cific Bip-Pro uptake data [S, Bip-Pro concentration (lM); v, uptake(nmolÆ30 min)1Æmg of protein)1)]. (B) Uptake of [14C]Gly-Sar (5–20 lM, 10 min, pH 6.0) was measured over a Gly-Sar concentrationrange of 0–5 mM. Nonspecific uptake ⁄ binding was determined bymeasuring uptake in the presence of an excess amount (20 mM)of unlabeled Gly-Sar. This component (4%) was subtracted fromthe total uptake to calculate the carrier-mediated uptake. Inset:Eadie–Hofstee transformation of the specific Gly-Sar uptake data[S, Gly-Sar concentration (mM); v, uptake rate (nmolÆ10 min)1Æmgprotein)1)]. Values represent the means ± standard error (SE) forfour determinations.Fig. 4. Time and pH dependence of the uptake of Bip-[3H]Pro inSKPT cells. Uptake of Bip-[3H]Pro (18 nM, n ¼ 4) in SKPT cells wasmeasured at pH 6.0 for 10 min to 4 h in the absence (d) or in thepresence (s) of unlabeled Bip-Pro (1 mM). Inset: uptake of Bip-[3H]Pro (4 nM, 2 h) measured at different pH values (n ¼ 4).I. Knu¨tter et al. Labeled high-affinity substrate for peptide transportersFEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5909amounts of substrate (1 or 20 mm, respectively) andsubtracted from the total uptake rates. For bothsubstrates, the relationship between carrier-mediateduptake and substrate concentration was found to fol-low Michaelis–Menten kinetics (Fig. 5). Eadie–Hofsteetransformation (uptake rate versus uptake rate ⁄ sub-strate concentration) revealed linearity with a singlecomponent (Fig. 5 insets). The apparent KtforGly-Sar uptake was 91.3 ± 4.1 lm and the Vmaxwas5.6 ± 0.1 nmolÆ10 min)1Æmg of protein)1. Theseparameters agree very well with those of previousreports [18]. For Bip-Pro uptake, an apparent Ktof7.6 ± 1.8 lm and a Vmaxof 1.1 ± 0.1 nmolÆ30min)1Æmg of protein)1was determined. Hence, themaximal velocity of Bip-Pro uptake is 16-fold lowerthan the maximal velocity of Gly-Sar uptake, whereasthe affinity constant of Bip-Pro uptake is 12-foldlower. Bip-Pro uptake represents a high-affinity, low-capacity process, whereas the Gly-Sar uptake occurswith low affinity but high transport capacity. Thelower Vmaxof Bip-Pro uptake, and the higher VmaxofGly-Sar uptake, correspond well with the currentsobtained at PEPT2-expressing X. laevis oocytes. Themean value of the apparent Michaelis–Menten con-stant calculated from the currents measured at)160 mV with Bip-Pro concentrations between 20 and500 lm was 26 lm and the maximal current amountedto 8% of the current evoked by Gly-Sar at saturatingconcentration. In comparison, the inward current elic-ited by Gly-Sar is 90% of that generated by Gly-Glnand the affinity of PEPT2 was slightly lower for Gly-Sar (Kt¼ 0.3 mm) than for Gly-Gln (Kt¼ 0.1 m m).The situation is very similar for PEPT1, where Bip-Proelicited 21% and Gly-Sar elicited 101% of the Gly-Glncurrent. Thus, the transport of Bip-Pro was also, inPEPT2-expressing oocytes, a high-affinity, low-capacityprocess. These findings suggest that the conformationalchange of the carrier protein following H+bindingand substrate binding represents the rate limiting stepin the substrate translocation cycle. Differences in themaximal transport currents of peptide transportersunder saturating substrate concentrations have beenreported before, suggesting that not only apparent Ktvalues but also turnover rates may differ between sub-strates [28].Substrate specificity of Bip-[3H]Pro uptakeIn 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 byunlabeled 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 substrateof peptide transporters, did not inhibit Bip-[3H]Prouptake. The PEPT1 and PEPT2 inhibitor Lys(4-nitrob-enzyloxycarbonyl)-Val [18,20], which is not transporteditself but interacts with both transporters with veryhigh affinity, displayed the strongest inhibitory effectof 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 ofPEPT2 with an apparent Kivalue of 2.6 mm [4]. Incontrast, cefadroxil strongly inhibited Bip-[3H]Prouptake by 85%, corresponding very well with itsapparent Kivalue 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 Kivalues of fivecompounds that tested positive for inhibition of Bip-[3H]Pro uptake. The apparent Kivalues (Table 2) werecalculated by nonlinear regression from data obtainedin competition experiments such as those shown inFig. 2. For Bip-Pro, the self-inhibition Kiwas 7.8 ±0.1 lm (Ki trans¼ 1.7). Cefadroxil (Ki¼ 5.2 ± 0.4 lm)displayed the highest affinity for inhibition followedby Gly-Sar, Lys-Lys and d-aminolevulinic acidwith apparent Kivalues between 75 and 230 lm. Forcomparison, in Table 2 we also present the respectiveinhibition constants (Ki) of these five substrates for theinhibition of [14C]Gly-Sar uptake in SKPT cells. ThisTable 1. Specificity of Bip-[3H]Pro uptake. Uptake of Bip-[3H]Pro(4 nM) into SKPT cells was measured at pH 6.0 for 2 h at roomtemperature in the absence (control) or presence of inhibitors (all100 lM). Data are shown as means ± standard error, n ¼ 4.Lys[Z(NO2)]-Val, Lys(4-nitrobenzyloxycarbonyl)-Val.Compound Bip-[3H]Pro uptake (%)Control 100 ± 2Gly 118 ± 8Gly-Sar 64 ± 5Bip-Pro 14 ± 1Ala-Ala 15 ± 1Pro-Ala 105 ± 3Lys-Lys 46 ± 2Ala-Asp 19 ± 1D-Phe-Ala 65 ± 2Ala-Ala-Ala 21 ± 1d-Aminolevulinic acid 78 ± 3Cefadroxil 15 ± 1Lys[Z(NO2)]-Val 10 ± 18-Aminooctanoic acid 111 ± 3Ala-4-nitroanilide 38 ± 1Labeled high-affinity substrate for peptide transporters I. Knu¨tter et al.5910 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBSso-called ABC test shows that the affinity constantsare very similar. Bip-Pro (A) and Gly-Sar (B) wereinhibited to the same extent by the other compounds(C). Hence, Bip-Pro and Gly-Sar are transported bythe same system.In conclusion, the results of the present study on themechanism and specificity of Bip-Pro uptake in SKPTcells, together with the electrophysiological dataobtained in X. laevis oocytes expressing PEPT2, pro-vide unequivocal evidence that Bip-Pro is transportedby PEPT2. Its enzymatic stability allows it to be usedin complex biological systems and its very high affinityshould make it particularly useful as a probe for theanalysis of the structure of the PEPT2 protein. More-over, via detailed kinetic analyses with the now avail-able two labeled transporter substrates, Bip-Pro andGly-Sar, which differ markedly in maximal transportrates, the identification of the rate limiting step in thetransport cycle of PEPT1 and PEPT2 became feasible.Experimental proceduresMaterialsThe 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 wasobtained from the German Collection of Microorganismsand Cell Cultures (Braunschweig, Germany). [Gly-1-14C]Gly-Sar (specific radioactivity 53 mCiÆ mmol)1) wascustom 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 fromSigma-Aldrich (Deisenhofen, Germany). Tert. butyloxycar-bonyl (Boc)–Bip, l-3,4-dehydro-Proline (DPro), d-Phe-Alaand Ala-Asp were purchased from Bachem (Heidelberg,Germany). Culture media, media supplements and trypsinsolution were purchased from Invitrogen (Karlsruhe,Germany) or PAA (Pasching, Austria). Fetal bovine serumwas from Biochrom (Berlin, Germany) and collagenase Afrom Roche (Mannheim, Germany). Ala-4-nitroanilide andLys(4-nitrobenzyloxycarbonyl)-Val were synthesized accord-ing to peptide synthesis standard procedures [18,29]. Allother chemicals were of analytical grade.Synthesis of Bip-Pro and Bip-[3H]ProBoc-Bip-Pro-OtBu was prepared from Boc–Bip-OH and H-Pro–OtBuÆHCl using the mixed anhydride coupling methodwith isobutylchloroformiate. After purification of the crudeproduct by flash chromatography (ethyl acetate ⁄ petroleumether: 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 mixtureaccording to the HPLC chromatograms. At room tempera-ture two peaks were observed, whereas there was only onepeak at temperatures of ‡ 45 °C.The precursor peptide H-Bip–l-3,4-dehydro-Pro-OH(H-Bip–DPro-OH) for3H-labeling was synthesized asfollows. 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 couplingreagent. The resulting active ester derivative then reactedwith DPro and triethylamine in acetonitrile to give Boc–Bip–DPro-OH. After purification of the crude product by flashchromotography 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 giveH-Bip–DPro-OH as its hydrochloride. Precipitation fromisopropanol ⁄ ethyl ether gave the H-Bip–DPro–OHÆHCl inhigh 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 thecatalyst and carrier-free tritium gas [26]. After tritiation,the crude peptide product was purified by HPLC (Jasco,Budapest, Hungary) on a Vydac (Budapest, Hungary) 218TP 54 column (250 · 4.6 mm) using linear gradient elution(from 15 to 40%) of acetonitrile (0.08% trifluoroaceticacid) in water (0.1% trifluoroacetic acid) within 25 min ata flow rate of 1 mLÆmin)1with 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 time17.27 min, k¢ ¼ 4.57). Specific radioactivity of Bip-[3H]Pro,Table 2. Inhibition constants (Ki) of different substrates for theinhibition of Bip-[3H]Pro and [14C]Gly-Sar uptake in SKPT cells.Uptake of Bip-[3H]Pro (4 nM, 2 h) or of [14C]Gly-Sar (10 lM, 10 min)was measured at pH 6.0 at increasing concentrations of unlabeledsubstrates or inhibitors of PEPT2. Constants were derived fromcompetition curves such as those shown in Fig. 2 for Bip-Pro. Para-meters are shown ± standard error (n ¼ 4).CompoundKi(lM)Bip-[3H]Pro uptake [14C]Gly-Sar uptakeGly-Sar 102 ± 9 61 ± 8 [24]Bip-Pro 7.8 ± 0.1 3.4 ± 0.1Cefadroxil 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 transportersFEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS 5911estimated by a calibration curve prepared with a standarddipeptide, was 1.853 TBqÆmmol)1(50.1 Ci mmol)1).NMR analysisThe relative populations of the cis ⁄ trans isomers weredetermined 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 werecarried out at pH 6.0 and 300 K. The pH of the solutionwas adjusted by the addition of diluted solutions of DCland NaOD. Chemical shifts were calibrated with respect tointernal DSS. Selective water resonance suppression wasachieved by using presaturation during relaxation delay orby 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 Brukersoftware library. Resonance assignments were made by thecombined analysis of H,H-COSY, ROESY and13C-HSQCspectra. The ROESY spectra were recorded at a mixingtime of 300 ms in the phase-sensitive mode using baselinecorrection in both dimensions.HPLC analysisThe stability of Bip-Pro in the extracellular medium wasanalyzed over incubation periods from 10 min up to 8 h.The amount of Bip-Pro in the extracellular uptake mediumwas quantified according to the laboratory standard HPLC(La-ChromÒ; Merck-Hitachi, Darmstadt, Germany) with adiode 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 was1mLÆmin)1.Cell culture and uptake studiesSKPT cells were cultured in Dulbecco’s modified Eagle’smedium ⁄ F12 Nutrient Mixture (Ham) (1 : 1, v ⁄ v) and2mml-glutamine, 10% fetal bovine serum, recombinantinsulin (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 wasroutinely cultured with Minimum Essential Medium withEarle’s salts and l-glutamine (2 mm) supplemented with10% fetal bovine serum, 1% nonessential amino acidsolution and gentamicin (45 lgÆmL)1) [17,20]. Both celllines were subcultured in 35-mm disposable Petri dishes(Sarstedt, Nu¨mbrecht, Germany) at a seeding density of0.8 · 106cells per dish. The cultures of both cell typesreached confluence within 20 h.Uptake of [14C]Gly-Sar or Bip-[3H]Pro was measured4 days (SKPT) or 7 days (Caco-2) after seeding at 22 °C,as described previously [17,18,20]. The uptake buffer was25 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 MgSO4and 5 mm glucose. Uptake was initiatedafter washing the cells for 30 s in uptake buffer by adding1 mL of uptake medium containing [14C]Gly-Sar (10 lm)orBip-[3H]Pro (4 nm) with increasing concentrations of thetest compounds (0–31.6 mm). If necessary, the pH of thesolutions was corrected before preparing the required dilu-tions. After incubation for the desired time periods, thecells were quickly washed four times with ice-cold buffer,solubilized in 1 mL of IgepalÒCa-630 (0.5% v ⁄ v; SigmaAldrich, Deisenhofen, Germany) in buffer (50 mmTris ⁄ 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 wereprepared and measured as described previously [20].X. laevis oocytes expressing PEPT1 and PEPT2and electrophysiologyFemale X. laevis were purchased from the African Xeno-pus Facility (Kynsa, South Africa). Surgically removed oo-cytes were separated by collagenase treatment and handledas described previously [17,18,20,25]. Individual oocyteswere injected with 30 nL of RNA solution containing30 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 of88 mm NaCl, 1 mm KCl, 0.82 mm CaCl2, 0.41 mm MgCl2,0.33 mm Ca(NO3)2, 2.4 mm NaHCO3and 10 mmMes ⁄ Tris at pH 6.5 (modified Barth solution). Thetwo-electrode voltage clamp technique was applied tocharacterize responses in current (I) and transmembranepotential (Vm) to substrate addition in oocytes expressingPEPT1 or PEPT2 [17,18,20,25]. In short, oocytes wereplaced in an open chamber in a volume of 0.5 mL andcontinuously superfused with modified Barth solution orwith solutions of Gly-Gln, Gly-Sar and ⁄ or Bip-Pro. Elec-trodes with resistances between 1 and 10 MW wereconnected 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 mspauses in the potential range from )160 to +80 mV.I–Vmmeasurements were made immediately before and30 s after substrate application when current flow reachedsteady state. Currents evoked by PEPT1 or PEPT2 at agiven membrane potential were calculated as the differenceof the currents measured in the presence and the absenceof 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 FEBSCalculations and statisticsAll data are given as the mean ± standard error of threeto four independent experiments. The kinetic parameterswere calculated by nonlinear regression methods (sigma-plot program; Systat, Erkrath, Germany) and confirmedby linear regression of the respective Eadie–Hofstee Plots.The concentration of the unlabeled compound necessary toinhibit 50% of radiolabeled dipeptide carrier-mediateduptake (IC50) was determined by nonlinear regression usingthe logistical equation for an asymmetric sigmoid (allostericHill kinetics): y ¼ Min + (Max–Min) ⁄ (1 + (X ⁄ IC50)–P),where Max is the initial Y-value, Min the final Y-value andthe power P represents Hills’ coefficient. Inhibition con-stants (Ki) were calculated from IC50values.AcknowledgementsThis work was supported by the State Saxony-AnhaltLife Sciences Excellence Cluster (MB).References1 Ganapathy V, Ganapathy ME & Leibach FH (2001)Intestinal transport of peptides and amino acids. InCurrent Topics in Membranes (Barrett KE & DonowitzM, eds), Vol. 50, pp. 379–412. Academic Press, SanDiego, CA.2 Daniel H & Kottra G (2004) The proton oligopeptidecotransporter family SLC15 in physiology and pharma-cology. Pflugers Arch 447, 610–618.3 Daniel H (2004) Molecular and integrative physiologyof 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 Acids31, 137–156.5 Brandsch M, Knu¨tter I & Leibach FH (2004) The intes-tinal H+⁄ peptide symporter PEPT1: structure-affinityrelationships. 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Georg Thieme Verlag, Stuttgart, New York.Labeled high-affinity substrate for peptide transporters I. Knu¨tter et al.5914 FEBS Journal 274 (2007) 5905–5914 ª 2007 The Authors Journal compilation ª 2007 FEBS . 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. Synthesis and characterization of a new and radiolabeled high-affinity substrate for H+/peptide cotransporters Ilka Knu¨tter1, Bianka Hartrodt2,Ge´za
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