Identification of a Potential Substrate Binding Domain in the Mammalian Peptide Transporters PEPT1 and PEPT2 Using PEPT1-PEPT2 and PEPT2-PEPT1 Chimeras

doi:10.1006/bbrc.1998.8566

Biochemical and Biophysical Research Communications

Volume 246, Issue 1, 8 May 1998, Pages 39-44

https://doi.org/10.1006/bbrc.1998.8566 Get rights and content

Abstract

The mammalian peptide transporters PEPT1 and PEPT2 are energized by a transmembrane electrochemical H⁺gradient and exhibit similar broad substrate specificity. These transporters however differ in their affinity for substrates, PEPT1 being a low-affinity transporter and PEPT2 being a high-affinity transporter. To identify the substrate binding domain in PEPT1 and PEPT2 which is responsible for the differing affinities, we constructed a series of PEPT1-PEPT2 and PEPT2-PEPT1 chimeras using anin vivorestriction site-independent procedure and determined their substrate affinities. A comparison of these kinetic data for different chimeras with those of the wild-type PEPT1 and PEPT2 in conjunction with the specific structural PEPT1/PEPT2 crossover regions in these chimeras has led to the identification of a putative substrate binding site, which is comprised of the transmembrane domains 7, 8 and 9 of the transporters.

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Variable results were observed from these studies. Indeed, it was shown that the functional characteristics of two peptide transporters seem to be inherent either in H1 to 9 (Doring et al., 1996), or in H7, 8 and 9 (Fei et al., 1998) or H1 to 6 (Terada et al., 2000). Other experiments based on model predictions and molecular modeling suggested for PepT1 that the channel for substrate translocation was formed by H1, 3, 5, 7 and 10 (Bolger et al., 1998).
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Additionally, F. heteroclitus PepT1 transporters (along with other fish PepT1s) exhibit 12 transmembrane domains and a large extracellular loop connecting the ninth and tenth transmembrane segments which is consistent with the SLC15A1 family (reviewed by Daniel., 2004). TMD 7–9 appear to play an essential role in determining substrate affinity (Fei et al., 1998; Terada et al., 2000). The similarity of these TMDs in piscine sequences (Table 2) confirms the importance of this region for SLC15A1 functionality.
Expression and function of the oligopeptide transporter PepT1 in response to changes in environmental salinity have received little study despite the important role that dipeptides play in piscine nutrition. We cloned and sequenced two novel full-length cDNAs that encode Fundulus heteroclitus PepT1-type oligopeptide transporters, and examined their expression and functional properties in freshwater- and seawater-acclimated fish and in response to fasting and re-feeding. Phylogenetic analysis of vertebrate SLC15A1 sequences confirms the presence of two PepT1 isoforms, named SLC15A1a and SLC15A1b, in fish. Similar to other vertebrate SLC15A1s, these isoforms have 12 transmembrane domains, and amino acids essential for PepT1 function are conserved. Expression analysis revealed novel environment-specific expression of the SLC15A1 isoforms in F. heteroclitus, with only SLC15A1b expressed in seawater-acclimated fish, and both isoforms expressed in freshwater-acclimated fish. Fasting and re-feeding induced changes in the expression of SLC15A1a and SLC15A1b mRNA. Short-term fasting resulted in up-regulation of PepT1 mRNA levels, while prolonged fasting resulted in down-regulation. The resumption of feeding resulted in up-regulation of PepT1 above pre-fasted levels. Experiments using the in vitro gut sac technique suggest that the PepT1 isoforms differ in functional characteristics. An increased luminal pH resulted in decreased intestinal dipeptide transport in freshwater-acclimated fish but suggested an increased dipeptide transport in seawater-acclimated fish. Overall, this is the first evidence of multiple isoforms of PepT1 in fish whose expression is environmentally dependent and results in functional differences in intestinal dipeptide transport.
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Regular ArticleIdentification of a Potential Substrate Binding Domain in the Mammalian Peptide Transporters PEPT1 and PEPT2 Using PEPT1-PEPT2 and PEPT2-PEPT1 Chimeras

Abstract

Curr. Opin. Cell Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

FEBS Letters

J. Biol. Chem.

Biochim. Biophys. Acta

Annu. Rev. Nutr.

Curr. Opin. Nephrol. and Hyperten.

Regular Article
Identification of a Potential Substrate Binding Domain in the Mammalian Peptide Transporters PEPT1 and PEPT2 Using PEPT1-PEPT2 and PEPT2-PEPT1 Chimeras