Functional Characterization of the Peptide Transporter PEPT2 in Primary Cultures of Human Upper Airway Epithelium

Abstract

This study characterizes the expression and function of the peptide transporter hPepT2 (SLC15A2) in differentiated primary cultures of human upper airway lung epithelia obtained from six human donors. Genotype analysis of a SNP in exon 15 of hPepT2 genotypes in six donors revealed an expected distribution of the two main variants present at similar frequency (two AA homozygotes, two BB homozygotes, and two AB heterozygotes). Real-time PCR analysis of the hPepT2 mRNA message revealed no significant differences among genotypes. hPEPT2 was expressed on the apical membrane in all donor specimens, demonstrated by cell surface biotinylation and Western analysis (104 kD). We then compared transepithelial transport of the prototypical substrate (3)H-glycylsarcosine in all donor cultures in the absence and presence of known inhibitors of hPEPT2 to ascertain the phenotype of functionally expressed hPepT2 in the upper airway epithelium. An array of inhibitors included dipeptides, beta-lactam antibiotics, bestatin, and ACE inhibitors. hPEPT2 exhibited saturable Michaelis-Menten-type kinetic parameters for GlySar, corroborating previously reported values for K(T) and J(max). Donor-to-donor variation of transport for different substrates did not correlate with hPepT2 haplotypes in this sample cohort. These findings demonstrate functional hPEPT2 transporter expression in primary cultures of human lung epithelial cells. hPEPT2-mediated transport could serve as a strategy for noninvasive systemic delivery of peptides and peptidomimetics drugs.

Modifications to the Peptidoglycan Backbone Help Bacteria To Establish Infection

“…It has also been reported that bacterial pore-forming toxins, which insert into host membranes, may aid in Nod1 ligand delivery into the host cytosol (64). Additionally it has been shown that host cells express peptide transporters that can transport both Nod1 and Nod2 ligands directly into the host cytosol (2,78).…”

Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

“…Lung cell culture was conducted as previously described (9). and maintained in a 1:1 mixture of Dulbecco's modified Eagle's media (DMEM) and Ham's F12 media (DMEM/F12) (Invitrogen, Carlsbad, CA) supplemented with 2% Ultroser G and antibiotics at 378C, and 5% CO 2 .…”

“…All transport experiments were performed as described previously (9). The culture inserts with hLECs were washed three times and conditioned for 2 hours with 0.2 ml apical (pH 5 6.5) and 0.4 ml basal transport buffer (BL) (pH 5 7.4).…”

“…PEPT2 displays promiscuous ligand affinity and mediates, in addition to its natural di-and tripeptide substrates, the uptake of numerous peptidomimetic drug molecules, such as b-lactam antibiotics and antivirals. Our previous work and that of others characterized PEPT2 expression and function in humans in situ in bronchial, bronchiolar, and alveolar type II epithelial cells (9)(10)(11). We first reported the functional characterization of PEPT2-mediated transport across the apical surface of fully differentiated, polarized, primary human upper airway epithelial cultures (hUAECs, henceforth referred to as human lung epithelial cells [hLECs]).…”

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

“…Pept2 mRNA is expressed in specific cell types of the brain, including astrocytes, subependymal cells, and ependymal cells, and Pept2 protein is detected along the apical membrane of epithelial cells of the choroid plexus (Table 5) (Berger and Hediger, 1999;Shu et al, 2002). Pept2/PEPT2 mRNA and/or protein are also expressed in the enteric nervous system, colon, liver, pancreas, lungs, nasal mucosa, and mammary glands (Groneberg et al, 2001(Groneberg et al, , 2002Zhang et al, 2004a;Bahadduri et al, 2005;Ruhl et al, 2005;Lu and Klaassen, 2006;Quarcoo et al, 2009).…”