Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1

Abstract

The binding affinities of a number of amino-acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N-acetyl-Phe (Ac-Phe), phe-amide (Phe-NH2), N-acetyl-Phe-amide (Ac- Phe-NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]-v-Phe-L-Gln. In an equivalent set of experiments, the blocked peptides Ac-Phe-Tyr, Phe-Tyr-NH2 and Ac-Phe-Tyr- NH2 were compared with the parent compound Phe-Tyr. Comparing amino acids and derivatives, only Ac-Phe was an effective inhibitor of peptide uptake (K(i) = 1.81 ± 0.37 mu). Ac-Phe-NH2 had a very weak interaction with PepT1 (K(i) = 16.8 ± 5.64 mM); neither Phe nor Phe-NH2 interacted with PepTl with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe-Tyr (K(i) = 0.10 ± 0.04 mM). The blocked C-terminal peptide Phe-Tyr-NH2 also interacted with PepT1 with a relatively high affinity (K(i) = 0.94 ± 0.38 mM). Both Ac-Phe-Tyr and Ac- Phe-Tyr-NH2 interacted weakly with PepT1 (K(i) = 8.41 ± 0.11 and 9.97 ± 4.01 mm, respectively). The results suggest that the N-terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala-Ala-Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C- terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.