Interaction of β-Lactam Antibiotics with Histidine Residue of Rat H+/Peptide Cotransporters, PEPT1 and PEPT2*

Peptide transporters mediate the H+-coupled uphill transport of oligopeptides and peptide-like drugs such as β-lactam antibiotics in the intestinal and renal brush-border membranes. Two H+/peptide cotransporters, PEPT1 and PEPT2, have been cloned and functionally characterized. In this study, we examined the interaction of the dipeptides and β-lactam antibiotics with the histidine residue of rat PEPT1 and PEPT2 transfected into the renal epithelial cell line LLC-PK1. Diethylpyrocarbonate (DEPC), which is a histidine residue modifier, abolished the glycylsarcosine uptake by both transfectants. The DEPC-induced inhibition of glycylsarcosine uptakevia PEPT1 or PEPT2 was attenuated by an excess of dipeptide or aminocephalosporin. In contrast, anionic cephalosporins without an α-amino group and bestatin, which is an antineoplastic drug with a β-amino group, did not attenuate the DEPC-induced inactivation of PEPT1 and PEPT2. The DEPC inactivation of PEPT1 was almost prevented by various charged dipeptides, which suggests that the inability of the drugs without an α-amino group to prevent the DEPC inactivation was not due to their ionic charge. These findings suggest that the α-amino group of β-lactam antibiotics interacts with the histidine residue of PEPT1 and PEPT2 and may be involved in the mechanism of substrate recognition by the peptide transporters.

In the small intestine and kidney, epithelial assimilation of oligopeptides are mediated by H ϩ -coupled peptide transport systems (1)(2)(3). Because there are 20 amino acids that comprise oligopeptides, there can be 400 dipeptides and 8000 tripeptides with various charges and molecular sizes. In addition to the native small peptides, the peptide transporter recognizes a wide variety of peptide-like drugs such as orally active ␤-lactam antibiotics (4 -6), bestatin (an antineoplastic drug) (7,8), and angiotensin converting enzyme inhibitors (9). The peptide transporter shows such a broad range of substrate specificity; however, the mechanism that recognizes substrates by the peptide transporter has been incompletely understood. Previously, we have cloned rat H ϩ /peptide transporters, PEPT1 (10) and PEPT2 (11), and constructed PEPT1-and PEPT2-expressing transfectants (12)(13)(14). Using these transfectants, we dem-onstrated that both PEPT1 and PEPT2 recognized various orally active ␤-lactam antibiotics (14). When PEPT1-expressing cells were treated with diethylpyrocarbonate (DEPC), 1 which is a histidine residue modifier, ceftibuten (anionic cephalosporin without an ␣-amino group) uptake was completely abolished (12). Furthermore, using the site-directed mutagenesis technique, the histidine residues at positions 57 and 121 of rat PEPT1 were suggested to be involved in substrate recognition and/or responsible for the intrinsic activity of the transporter (12).
To elucidate the diversity of the substrate recognition by the peptide transporters, it is needed to clarify the mechanisms involved in the interaction of the substrates with the essential residues of PEPT1 and PEPT2. Because histidine residues of the peptide transporters have been indicated as the most important key amino acid residues (12,15,16), we investigated the functional role of histidine residues to examine the preventive effect of various substrates on the DEPC-induced inactivation of PEPT1 and PEPT2. We report here that the DEPCsensitive histidine residue of rat PEPT1 and PEPT2 can serve as the binding site of the ␣-amino group of the substrates.

EXPERIMENTAL PROCEDURES
Cell Culture-The parental LLC-PK 1 cells obtained from the American Type Culture Collection (ATCC CRL-1392) were cultured in complete medium, which consisted of Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Whittaker Bioproducts Inc., Walkersville, MD) without antibiotics in an atmosphere of 5% CO 2 , 95% air at 37°C. The LLC-PK 1 cells transfected with rat PEPT1 cDNA (LLC-rPEPT1) and with rat PEPT2 cDNA (LLC-rPEPT2) were used as described, previously (14). In the uptake experiments, the cells were cultured for 6 -7 days in complete medium.
Uptake Studies by Cell Monolayers-Uptake of [ 14 C]glycylsarcosine was measured in cells grown in 60-mm plastic dishes as described previously (14). The protein content of the cell monolayers solubilized in 1 N NaOH was determined by the method of Bradford (17) using a Bio-Rad protein assay kit with bovine ␥-globulin as the standard.

RESULTS
At first, we examined the concentration dependence for the inhibitory effect of DEPC on [ 14 C]glycylsarcosine uptake. When the LLC-rPEPT1 and LLC-rPEPT2 cells were treated with various concentrations of DEPC, the half-maximal inhibition for [ 14 C]glycylsarcosine uptake in both transfectants was observed at about 0.4 mM, and the maximal inhibition was at 1 mM (Table I). Therefore, the concentration of DEPC for subsequent studies was 1 mM. Fig. 1 shows the effect of DEPC treatment on [ 14 C]glycylsarcosine uptake by LLC-rPEPT1 or LLC-rPEPT2 cells and the preventive effect of glycylsarcosine on the DEPC-induced inhibition. The uptake of [ 14 C]glycylsarcosine by the transfectants was inhibited markedly by the pretreatment with 1 mM DEPC. This inhibition was abolished mostly by unlabeled 10 mM glycylsarcosine.
Next, we examined whether the DEPC-induced inactivation of PEPT1 and PEPT2 was affected by the pH of the incubation medium. As shown in Fig. 2, glycylsarcosine uptake at both pH 6.0 (with a H ϩ gradient) and pH 7.4 (without a H ϩ gradient) was blocked by pretreatment with DEPC in the LLC-rPEPT1 and LLC-rPEPT2 cells.
The histidine residues might have been located with the PEPT1 substrate binding site, and therefore, we examined the effect of two cephalosporins on the DEPC inactivation of PEPT1. Fig. 3A shows the effect of cephradine (aminocephalosporin) and ceftibuten (anionic cephalosporin without an ␣amino group) concentration on the DEPC-induced inhibition of the glycylsarcosine uptake by the LLC-rPEPT1 cells. Rat PEPT1 has a much higher affinity to ceftibuten than to cephradine (13,14). Cephradine prevented the DEPC-induced inhibition of glycylsarcosine uptake at 5-10 mM, but ceftibuten had no preventive effect even at 10 mM. Similar results were observed for the LLC-rPEPT2 cells (Fig. 3B).
To elucidate the effect of ceftibuten, we examined the effect of the charge of the substrates on the DEPC-induced inactivation of PEPT1. As shown in Fig. 4A, the DEPC-induced inhibition of [ 14 C]glycylsarcosine uptake by LLC-rPEPT1 cells was  prevented by various charged substrates but not by ceftibuten.
When the inhibitory effect of these compounds was examined, all the dipeptides, cephradine, and ceftibuten inhibited [ 14 C] glycylsarcosine uptake (Fig. 4B). These results suggested that the preventive effect of substrates was independent of either their ionic charges or affinity to the transporters. For the LLC-rPEPT2 cells, these substrates prevented the DEPC-induced inhibition of the glycylsarcosine uptake in a manner similar to that used for inhibiting LLC-rPEPT1 cells (data not shown). We investigated whether the preventive effects depended on the ␣-amino group of the substrates. As shown in Fig. 5A, the substrates with an ␣-amino group such as glycylsarcosine, cephradine, and cefadroxil (aminocephalosporin) prevented the DEPC-induced inhibition of [ 14 C]glycylsarcosine uptake by LLC-rPEPT1 cells. However, all peptide-like drugs without an ␣-amino group such as ceftibuten, cefixime, bestatin, and captopril (angiotensin converting enzyme inhibitor) had no effect at a concentration of 10 mM in the LLC-rPEPT1 cells. Cyclacillin did not have the preventive effect despite its having an ␣-amino group. Similar results were obtained in LLC-rPEPT2 cells except for cefadroxil (Fig. 5B). In the absence of DEPC, the pretreatment of cefadroxil at 10 mM had an inhibitory effect on glycylsarcosine uptake (data not shown). Therefore, cefadroxil at 10 mM might have a cis-inhibitory effect on the [ 14 C]glycylsarcosine uptake, considering that cefadroxil had a higher affinity for PEPT2 with an apparent inhibition constant of 3 M (14). DISCUSSION In the present study, glycylsarcosine uptake by the PEPT1and PEPT2-expressing transfectants was inhibited by pretreatment with DEPC in the absence and the presence of H ϩ gradient, which suggests that the histidine residue modified by DEPC at least served as the substrate binding site. This could be supported by the fact that the various dipeptides and aminocephalosporins prevented the DEPC-induced inactivation of PEPT1 and PEPT2. On the other hand, the peptide-like drugs without an ␣-amino group had no preventive effect, although they can be transported by the peptide transporter (5)(6)(7)9). These findings suggest that the histidine residue located in the recognition site is involved in the binding site of the ␣-amino group of the dipeptides and aminocephalosporins. Because only the unprotonated imidazole ring reacts with DEPC (18), it is possible that the imidazole group of the histidine residue located at the recognition site is protonated by the ␣-amino group of the dipeptides and aminocephalosporins but not by the peptide-like drugs that do not have an ␣-amino group. The ␣-amino group of the substrates might interact with the imidazole ring of the histidine residue of peptide transporters by proton binding. It is noted that these results were observed for PEPT1 and PEPT2 in a similar manner, which suggests that the DEPCsensitive histidine residue plays the same role in both transporters.
Among the substrates examined that had an ␣-amino group, only cyclacillin did not show the preventive effect against the DEPC inactivation. This may be due to the structure of cyclacillin. Cyclacillin has an ␣-carbon group as part of its cyclohexane ring; therefore, the cyclohexane ring may interfere with the ␣-amino group-histidine interaction. Nevertheless, cyclacillin was recognized by PEPT1 at a relatively high affinity (14,19). A possible explanation is that the hydrophobic NH 2 -terminal side chain of cyclacillin interacts with the peptide transporters instead of the ␣-amino group-histidine interaction. As reported by Daniel et al. (20), the marked hydrophobicity of the NH 2terminal side chain of aminopenicillins increased the affinity to the renal H ϩ /peptide cotransporter. For the peptide-like drugs without an ␣-amino group such as ceftibuten and cefixime, which are very hydrophilic, there might be interactions between these drugs and the binding site of the peptide transporter other than the ␣-amino group-histidine interaction.
We have previously reported that histidines 57 and 121, which are located at the predicted transmembrane domains 2 and 4 of rat PEPT1, are involved in substrate binding and/or are responsible for the intrinsic activity of the transporter (12). In contrast to our results, Fei et al. (21) demonstrated that histidine 57 of human PEPT1 was absolutely essential for the catalytic activity but histidine 121 of human PEPT1 did not appear to play an essential role for the catalytic activity. The reason for this discrepancy regarding the role of histidine 121 in the rat and human PEPT1 remains unknown. However, it is possible that at least two histidine residues are involved in the transport activity of PEPT1. Indeed, Steel et al. (22) proposed that one histidine residue as a cation site was responsible for the proton coupling and that a second histidine residue was adjacent to the peptide binding site in the studies to determine differently charged dipeptide-H ϩ flux coupling ratios. Mackenzie et al. (23) demonstrated that human PEPT1 bound H ϩ first and then the substrate as demonstrated by the biophysical and kinetic analysis of the human PEPT1. These findings suggest that two histidine residues are necessary, because the histidine residue with a protonated imidazole ring cannot bind the ␣-amino group of the substrates as shown by the present study. Although we cannot identify the histidine residue of the binding site from this study, either histidine 57 or 121 of PEPT1 might be the candidate residue for the binding site of the ␣-amino group of the substrates.
In conclusion, this is the first demonstration that the ␣-amino group of the dipeptides and aminocephalosporins interacts with the DEPC-sensitive histidine residue of rat PEPT1 and PEPT2. The present findings represent the first step for understanding the substrate recognition mechanisms by peptide transporters.