Abstract

The peptide transporter PEPT2, expressed in a variety of tissues, including kidney, lung, and the central nervous system, mediates the uphill transport of di- and tripeptides, as well as a variety of peptidomimetic drugs. To identify the essential molecular features of substrates that determine affinity and transport by PEPT2, we synthesized a series of amino acid derivatives as well as modified dipeptides. Kinetic constants for the interaction of test compounds with PEPT2 were obtained in a competition assay using Pichia pastoris yeast cells expressing mammalian PEPT2. The two-electrode voltage-clamp technique in Xenopus laevisoocytes was used to assess the substrate's electrogenic transport properties. Whereas ϖ-amino fatty acids showed no affinity for PEPT2, the introduction of a single carbonyl group into the backbone increased both affinity and transport currents more than 30-fold. ϖ-Amino fatty acids, at their amino or carboxyl group coupled to an alanine residue, allowed us to determine the importance of the spatial position of functional groups within the molecule. Affinity and transport function declined by elongating the ϖ-amino acid chain when located in the N-terminal position, whereas the elongation in the carboxyl terminal with an N-terminal alanine caused less pronounced effects. The results clearly establish that a free N terminus, a correctly positioned backbone carbonyl group, and a carboxylic group that is in a suitable distance from the intramolecular carbonyl function and the amino terminal head group are the main features for substrate recognition and transport by PEPT2. This information provides the framework for a rational design of peptidomimetic drugs for delivery via PEPT2.