Proton Oligopeptide Transporters Pot Slc15A

With the elucidation of the human genome, and advances in molecular biology and cloning, it should be of no surprise that peptidomimetic drugs are increasingly utilized as therapeutic agents for the treatment of numerous disorders. Peptide-like agents have a broad range of clinical applications in the treatment of many disorders including AIDS, hypertension, and cancer. The currently known peptide transporters include the Peptide Transporters 1 and 2, PepT1 (SLC15A1) and PepT2 (SLC15A2); the Peptide/Histidine Transporters 1 and 2, PHT1 (SLC15A4) and PHT2 (SLC15A3); and the Intestinal Peptide Transporter PT1 (CDH17). PT1 is the only protein identified which is not classified as a member of the proton oligopeptide transporter (SLC15) family, also known as the proton-coupled oligopeptide transporter (POT) superfamily (Fei et al., 1998; Meredith and Boyd, 2000). In fact, PT1 is considered a member of the cadherin family. PepT2 expression has not been shown in the GI tract and thus it will not be discussed in this chapter.

Several comprehensive reviews can be found describing the common characteristics of the oligopeptide transporter proteins (Graul and Sadee, 1997; Nussberger et al., 1997a; Yang et al., 1999; Meredith and Boyd, 2000; Rubio-Aliaga and Daniel, 2002; Herrera-Ruiz and Knipp, 2003). Members of the POT superfam-ily are predicted to contain 12 predicted transmembrane a-helical spans, with a majority of the proteins having intracellulary localized N- and C-termini (Covitz et al., 1998; Lee, 2000; Herrera-Ruiz and Knipp, 2003). Two characteristic protein signatures of the POT family members have been identified, known as the PTR2 family signatures (Steiner et al., 1995): (1) [GA] - [GAS] - [LIVM-FYWA] - [LIVM] - [GAS] - D - x - [LIVMFYWT] - [LIVMFYW] - G -x(3) - [TAV] - [IV] - x(3) - [GSTAV] - x - [LIVMF] - x(3) - [GA] and (2) [FYT] - x(2) - [LMFY] - [FYV] - [LIVMFYWA] - x - [IVG] - N - [LIVMAG] -G - [GSA] - [LIMF], and a third consensus proposed has been proposed by Fei et al. (1998) (GTGGIKPXV). Saier et al. (1999) have proposed three different signature sequences associated with the POT superfamily based on their phylogenetic analysis.

The cloned human PepT1 cDNA sequence encodes a 708 amino acid protein with an estimated molecular weight of 79kDa, and an isoelectrical point of 8.6 (Liang et al., 1995). PepT1 has shown expression in several animal species (Fei et al., 1994, 2000; Saito et al., 1995; Chen et al., 1999; Pan et al., 2001; Klang et al., 2005; Van et al., 2005), each exhibiting high homology with other species. PepT1 protein expression has been demonstrated in the human small intestine (Liang et al., 1995; Herrera-Ruiz et al., 2001) and was localized on the apical plasma membrane of enterocytes in rats (Ogihara et al., 1999). Other studies have demonstrated that PepT1 isoforms are localized intracellularly in lysosomes (Zhou et al., 2000 Sun et al., 2001). Apical expression of PepT1 has been established in both prenatal and mature animals (Shen et al., 2001; Rome et al., 2002). Furthermore, PepT1 cellular localization has been demonstrated to vary with the stage of animal development. Hussain et al. (2002) revealed that PepT1 is exclusively expressed in the apical membrane of enterocytes from both prenatal and mature animals; however, immunolocalization studies showed that immediately after birth, PepT1 was also expressed intracellularly in the basal cytoplasm, as well as the basolateral membrane of the intestinal epithelium.

Recently, two putative human peptide/histidine (hPHT) transporters have been identified with expression observed in several human tissues (Botka et al., 2000; Knipp and Herrera-Ruiz, 2004). The hPHT1 mRNA sequence is approximately 2.7 kb long, encoding a translated 577 amino acid protein with an estimated molecular weight of 62 kDa, and a predicted pI of 9.2. Four N-linked glycosylation sites were predicted, along with several protein phosphorylation sites (Herrera-Ruiz and Knipp, 2003). Human PHT2 has not widely been studied, and little is known about its biological significance. PHT2 was first isolated from the human placenta and has an open reading frame of 1.7 kb, encoding a protein with 581 amino acids with an estimated molecular mass of 64.6 kDa. Only the rat isoform of the PHT2 protein has been partially evaluated (Sakata et al., 2001). Three N-linked gly-cosylation sites on the rPHT2 protein are predicted and protein phosphorylation sites (PKA and PKC) were identified (Herrera-Ruiz and Knipp, 2003). Ortholo-gous expression in rat and mouse has been reported for both PHT1 and PHT2 (Yamashita et al., 1997; Botka et al, 2000; Sakata et al., 2001). Both hPHT1 and hPHT2 have shown expression along the entire GI tract, especially in the small intestine and colon (Herrera-Ruiz et al., 2001), an important difference in relation with hPepT1. Expression of hPHT1 has been shown in the plasma membrane of intestinal tissue segments (Bhardwaj et al., 2005a). Studies have demonstrated that PHT1 and PHT2 are expressed in the human and rat GI tracts and in Caco-2 cells (Herrera-Ruiz et al., 2001). Intracellular localization rPHT2 expression has also been demonstrated intracellular localization in lysosomes, autophagosomes, and vacuoles of HEK-293T and baby hamster kidney (BHK) cells (Sakata et al., 2001).

The human Intestinal Peptide Transporter 1 (HPT1) has a cDNA coding region of 2.5 kb long, encoding a 120 kDa protein comprising 832 amino acids. While HPT1 is related to the cadherin family of proteins (Dantzig et al., 1994), it has demonstrated peptide and cephalosporin transport activity (Yang, 1998). Since it is not a POT family member, it will not be discussed further. Peptide Transporter Mediated Transport

Transepithelial peptide transporters use a proton gradient and membrane potential to provide the necessary driving force for substrate translocation (Daniel, 1996; Adibi, 1997; Nussberger et al., 1997a,b). The required proton gradient is generated through the activity of an electroneutral proton/cation exchanger, the Na+/H+ antiporter (Meredith and Boyd, 2000; Theis et al., 2001). Peptide or pep-tidomimetic molecule uptake is commensurate with the translocation of a proton into epithelial cells, making substrate uptake strongly dependent on the extracellular pH, where a pH of 4.5-6.5, depending on the net charge of the substrate, is optimal for transport activity (Temple et al., 1995, 1996; Amasheh et al., 1997; Balimane and Sinko, 2000; Kottra et al., 2002). The optimal extracellular pH for both PepT1 and PHT1 has been estimated to be 6.0 and 5.0, respectively. In addition, this model suggests that peptides which are not appreciably degraded intracellularly are transported out of the cells by an as-yet unidentified basolateral peptide transporters which have lower affinities than the PepT-like transporters (Terada et al., 1999, 2000b; Irie et al., 2001,2004).

It has been established that all PepT1 substrates share the same substrate-binding site due to the fact that uptake strictly conforms to the Michaelis-Menten equation and exhibits the competitive inhibition regardless of substrate charge (Wenzel et al., 1996; Mackenzie et al. 1996; Sawada et al., 1999). Proton coupling occurs in the H+-binding site of PepT1, where a H+ is bound prior to anionic or neutral substrate uptake but is not required for cationic substrates (Nussberger et al., 1997b). This model assumes that anionic substrates cannot access the substrate-binding site lacking a H+, and that the protonated substrate-binding site can accept only negatively charged substrates. Recently, a computational model of the H+-coupled substrate transport of neutral and charged molecules has been published (Irie et al., 2005). The authors established a PepT1 mechanistic model demonstrating the normally observed bell shaped uptake versus pH shaped curves for different charged substrates based on two novel main hypotheses: (1) H+ binds to not only the H+-binding site, but also the substrate-binding site; and (2) H+ at the substrate-binding site inhibits the interaction of neutral and cationic substrates, but is necessary for that of anionic substrates.

Studies have shown the importance of certain amino acid residues in the transport activity of PepT-like proteins (Fei et al., 1997; Bolger et al., 1998; Yeung et al., 1998; Chen et al., 2000; Meredith, 2004). Studies utilizing PepT1/PepT2 chimeras suggest that the first half of the transporters (first 400 residues) contain substrate-binding domain segments as well as defining other functional properties (Doring et al., 1996, 2002; Terada et al., 2000a; Kulkarni, 2003a,b). The significance of the PepT chimeras with respect to these transporters is unknown since similar studies have not been conducted for PHT1/PHT2 peptide transporters. The Substrate Specificity of Peptide Transporters The PepT-Like Transporters

Studies elucidating the substrate specificity of PepT1 indicate that this protein transports almost all possible dipeptides, tripeptides, as well as numerous pep-tidomimetics and that their respective sizes or molecular weights were not significant factors (Leibach and Ganapathy, 1996). Analysis of the binding and transport characteristics of PepT1 have led to the development of several molecular models attempting to establish a PepT-substrate template (Swaan and Tukker, 1997; Bailey et al., 1999; Zhang et al., 2002a,b; Gebauer et al., 2003; Biegel et al., 2005). Brandsch et al. (2004) summarized the substrate structural characteristics required for high PepT1 affinity (<0.5mM) which include (a) L-amino acids, (b) the presence of an acidic or hydrophobic function at the C-terminus, (c) the presence of a weakly basic group in a-position at the N-terminus, (d) exhibiting a ketomethylene moiety or acid amide bond, and in the case of having a peptide bond (e) to present it in a trans configuration. PepT1 has also been shown to mediate the transport of a variety of drugs (Table 7.5) with differing degrees of affinity and capacity, depending on their chemical structure. Generally, PepT1 has a high transport capacity, which makes it highly attractive as a drug target.

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