- Top of page
- Materials and methods
- Results and discussion
To elucidate the decisive structural factors relevant for dipeptide–carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15–1.2 mm and 0.08–9.5 mm, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24–70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1–0.16 mm. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion.
Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.
The H+ gradient-dependent symport system PEPT1 actively transports di- and tripeptides and a variety of peptidomimetics across the brush border membrane of intestinal epithelial cells [1–4]. During 1998, various advances were made regarding the elucidation of structural requirements for substrates to be accepted by intestinal and renal peptide transporters. Temple et al.  reported that 4-aminophenylacetic acid, a peptide mimetic lacking a peptide bond, represents a substrate for the intestinal peptide transporter. We have shown recently that replacing the peptide bond in Ala-Pro by a thioxo peptide bond is tolerated by PEPT1 when in trans conformation , and that this carrier transports amino acid aryl amides with high affinity . Daniel's group  discovered that δ-aminolevulinic acid, which is used as an endogenous photosensitizer for photodynamic tumor treatment, represents a high-affinity substrate for PEPT1 and PEPT2. The work of this group also expanded our understanding of the minimal structural requirements of PEPT1 substrates by showing electrogenic transport of ω-amino fatty acids . Several groups have reported that the reason for the higher oral bioavailability of the prodrug valacyclovir compared to acyclovir is its transport via PEPT1 [10,11].
Structural features commonly believed to be crucial for substrate–carrier interaction are molecular size, type of binding, stereo-isomerism, spatial arrangement, hydrophobicity, polarity and charge. Studies on the structural requirements of dipeptides for their recognition by a protein, ideally, should be performed by analyzing the interaction of analogous series of dipeptides with that particular protein. We decided to investigate Xaa-Pro and Pro-Xaa dipeptides and to compare their affinity constants with those of the corresponding Xaa-Ala dipeptides. The role of proline in dipeptides in their transport by peptide carriers has not been studied systematically. Reviewing the available data reveals that at least some Xaa-Pro dipeptides are good substrates for both renal and intestinal type peptide transporters [12–15]. There is almost no information available regarding the effect of N-terminal proline on the interaction of dipeptides with PEPT1. Using brush-border membrane vesicles from rabbit kidney, Miyamoto et al.  and Daniel et al.  found very low or undetectable affinities of Pro-Gly and Pro-Leu to the renal peptide transporter(s). Similarly, in Caco-2 cells, an excess of Pro-Gly (> 20 mm) inhibited the uptake of Gly-Sar by only 59%  and that of cephalexin by only 67% . Such results have led to the prevailing view that Pro-Xaa dipeptides are in general not particularly good substrates for peptide carriers. However, having measured a 10-fold higher affinity of Pro-Pro compared with Pro-Ala in preliminary experiments, we felt compelled to include a series of Pro-Xaa dipeptides in this study.