The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

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Bowman-Birk protease inhibitor (BBI), an 8000 mol. wt polypeptide with anti-carcinogenic activity, was coupled to poly(D-lysine) (BBI-SS-PDL) and poly(L-glutamate) (BBI-SS-PLG) with a disulfide-cross-linking agent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). In vitro transformation assays showed that BBI-SS-PDL, but not BBI-SS-PLG, retained the full anticarcinogenic activity of BBI. When administered i.v. to Balb/c mice, a selective localization in the lungs was found with BBI-SS-PDL but not with BBI or BBI-SS-PLG. At 30 min, 3 h and 24 h after the injection of BBI-SS-PDL, the amounts of BBI in the lungs were 118%, 74% and 19% of the injected dose/g of tissue respectively. At the same time points, the amount of radioactivity in the lungs of mice injected with BBI was 5%, 3% and 1% of the injected dose/g of tissue respectively. Therefore, higher amounts of BBI could be targeted to the lungs by injecting BBI as a PDL conjugate. BBI-SS-PDL was also retained in the lungs for a longer period of time than free BBI. In previous studies, BBI has been shown to suppress lung carcinogenesis. The results presented here suggest that BBI-SS-PDL could be more effective than BBI as an anti-carcinogenic agent for the lung.

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