Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis (original) (raw)
Related papers
Effect of N-Glycosylation on the Transport Activity of the Peptide Transporter PEPT1
American journal of physiology. Gastrointestinal and liver physiology, 2015
The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high capacity and low affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ~95 kDa in the small intestine but ~105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515 and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofol...
Prediction of glycylsarcosine transport in Caco-2 cell lines expressing PEPT1 at different levels
Pflügers Archiv - European Journal of Physiology, 2006
H + -coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter mediate the absorption of small peptides and peptide-like drugs in the small intestine. Recently, we constructed a mathematical model to simulate glycylsarcosine (Gly-Sar) transport in Caco-2 cells. In this study, we attempted to adjust our model to a change in the expression level of PEPT1. To obtain cell lines expressing PEPT1 at different levels, recloning of Caco-2 cells was performed, and nine clones were isolated. Compared with parental cells, clones 1 and 9 exhibited the lowest and the highest levels of [ 14 C]Gly-Sar uptake from the apical side, respectively, whereas activities of the basolateral peptide transporter were comparable. Kinetic analysis demonstrated that the difference in the activity of PEPT1 was accounted by variations in V max . Moreover, PEPT1 mRNA level was positively related to the activity of [ 14 C]Gly-Sar uptake (r=0.55). Based on these findings, the V max value of PEPT1 was defined as a variable using the amount of PEPT1 mRNA as an index of the expression level. With this improved model, Gly-Sar transport in clones 1 and 9 was well-predicted, suggesting that our model can simulate Gly-Sar transport in cells expressing PEPT1 at different levels.
Journal of Pharmaceutical Sciences, 1998
0 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 [ 3 H]-(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-GFPlabeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced V max .
Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1
American journal of physiology. Gastrointestinal and liver physiology, 2001
The nature of protein breakdown products and peptidomimetic drugs such as beta-lactams is crucial for their transmembrane transport across apical enterocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-mediated uptake of oligopeptides, an ex vivo assay using the fluorophore-conjugated dipeptide derivative D-Ala-Lys-N(epsilon)-7-amino-4-methylcoumarin-3-acetic acid (D-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small intestine. D-Ala-Lys-AMCA was accumulated by enterocytes throughout all segments of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibition studies revealed competitive inhibition by the beta-lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide glycyl-glutamine. Control...
The International Journal of Biochemistry & Cell Biology, 2009
The mammalian proton-coupled peptide transporter PepT1 is widely accepted as the major route of uptake for dietary nitrogen, as well as being responsible for the oral absorption of a number of classes of drugs, including -lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors. Using sitedirected mutagenesis and zero-trans transport assays, we investigated the role of conserved tyrosines in the transmembrane domains (TMDs) of rabbit PepT1 as predicted by hydropathy plots. All the individual TMD tyrosines were substituted with phenylalanine and shown to retain the ability to traffic to the plasma membrane of Xenopus laevis oocytes. These single substitutions of TMD tyrosines by phenylalanine residues did not affect the proton dependence of peptide uptake, with all retaining wildtype PepT1-like pH dependence. Individual mutations of four of the nine TMD residue tyrosines (Y64, Y287, Y345 and Y587) were without measurable effect on PepT1 function, whereas the other five (Y12, Y56, Y91, Y167 and Y345) were shown to result in altered transport function compared to the wild-type PepT1. Intriguingly, the affinity of Y56F-PepT1 was found to be dramatically increased (approximately 100fold) in comparison to that of the wild-type rabbit PepT1. Y91 mutations also affected the substrate affinity of the transporter, which increased in line with the hydrophilicity of the substituted amino acid (F > Y > Q > R). Y167 was demonstrated to play a pivotal role in rabbit PepT1 function since Y167F, Y167R and Y167Q demonstrated very little transport function. These results are discussed with regard to a proposed mechanism for PepT1 substrate binding.
Cell and Tissue Research, 2002
Immunocytochemical distribution in rat small intestine of PepT1, the oligopeptide transporter responsible for nutritionally important peptide uptake from the adult small intestinal lumen, has been measured during development using an antibody to the C-terminal sequence of PepT1. Distribution is exclusively in the apical brush border of enterocytes from both prenatal and mature animals. However, immediately after birth immunolocalisation of PepT1 extends to the subapical cytoplasm and to the basolateral membrane of enterocytes. No staining is found in crypts or over Goblet cells. Our results imply that the peptide transporter at the basolateral membrane in adult rats must be distinct from PepT1. They also shed new light on the trafficking of PepT1 in enterocytes.
Substrate-induced changes in the density of peptide transporter PEPT1 expressed inXenopusoocytes
American Journal of Physiology-Cell Physiology, 2008
The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of ∼5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocyt...
2003
The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V max , while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.
N-glycosylation dictates proper processing of organic anion transporting polypeptide 1B1.
PLoS One, 2012
Organic anion transporting polypeptides (OATPs) have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion (ADME) of various drugs, xenobiotics and toxins. Putative N-glycosylation sites located in the extracellular loops 2 and 5 is considered a common feature of all OATPs and some members have been demonstrated to be glycosylated proteins. However, experimental evidence is still lacking on how such a post-translational modification affect the transport activity of OATPs and which of the putative glycosylation sites are utilized in these transporter proteins. In the present study, we substituted asparagine residues that are possibly involved in N-glycosylation with glutamine residues and identified three glycosylation sites (Asn134, Asn503 and Asn516) within the structure of OATP1B1, an OATP member that is mainly expressed in the human liver. Our results showed that Asn134 and Asn516 are used for glycosylation under normal conditions; however, when Asn134 was mutagenized, an additional asparagine at position 503 is involved in the glycosylation process. Simultaneously replacement of all three asparagines with glutamines led to significantly reduced protein level as well as loss of transport activity. Further studies revealed that glycosylation affected stability of the transporter protein and the unglycosylated mutant was retained within endoplasmic reticulum.