Genetic Polymorphisms in Human Proton-Dependent Dipeptide Transporter PEPT1: Implications for the Functional Role of Pro⁵⁸⁶

Abstract

The human proton-dependent dipeptide transporter (PEPT1, gene SLC15A1) is important for intestinal absorption of di-and tripeptides and a variety of peptidomimetic compounds. Using a DNA polymorphism discovery panel of 44 ethnically diverse individuals, nine nonsynonymous and four synonymous coding-region single-nucleotide polymorphisms (SNPs) were identified in PEPT1. HeLa cells were transiently transfected with plasmids constructed by site-directed mutagenesis for each of the nine nonsynonymous variants. Quantitative polymerase chain reaction showed that the mRNA transcription level of all of the mutants was comparable with the mRNA transcription level of the reference sequence in transfected HeLa cells. Functional analysis in transiently transfected HeLa cells revealed that all nonsynonymous variants retained similar pH-dependent activity and K t values for [glycyl-1,2-14 C]glycylsarcosine (GlySar) uptake as the reference PEPT1. In addition, a group of seven peptide-like drugs showed inhibitory effect on Gly-Sar uptake by these variants comparable with the reference, suggesting conserved drug recognition. Of the nine nonsynonymous SNPs, a single SNP (P586L) demonstrated significantly reduced transport capacity as evidenced by a much lower V max value. This was consistent with lower immunoactive protein level (Western analysis) and lower plasma membrane expression (immunocytochemical analysis). Therefore, Pro 586 may have profound effect on PEPT1 translation, degradation, and/or membrane insertion.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

“…Nucleotide position was confirmed by PharmGKB (Hewett et al, 2002). PEPT1 data from Zhang et al (2004b) and Anderle et al (2006). PEPT2 data from Pinsonneault et al (2004) and Terada et al (2004).…”

Genetic Variants of the Human Dipeptide Transporter PEPT1

“…Allelic diversity and haplotype distributions were determined, and nonsynonymous SNPs evaluated for functional effects, extending an earlier study (Zhang et al, 2004). Moreover, we determined allelic expression imbalance and alternative splicing to regulatory variants of PEPT1 in human intestinal samples.…”

“…For the PEPT1 substrate VAC, a significantly larger interindividual than intraindividual variability in intestinal absorption suggests the presence of genetic factors, potentially involving PEPT1 (Phan et al, 2003). In a previous sequencing study of PEPT1 involving 44 ethnically diverse individuals, 13 SNPs had been located in the coding region of PEPT1 (Zhang et al, 2004). Functional characterizations of the nine nonsynonymous SNPs revealed that only one rare variant had reduced transport activity as a result of reduced protein expression.…”

A Single Nucleotide Polymorphism in MGEA5 Encoding O-GlcNAc–selective N-Acetyl-β-d Glucosaminidase Is Associated With Type 2 Diabetes in Mexican Americans

“…The primers used for amplification and sequencing are provided in online appendix Table 1 (available at http:// diabetes.diabetesjournals.org). The PCR amplification conditions and genetic variation discovery process have been described (33).…”