The Role of N-Glycosylation in Maintaining the Transporter Activity

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The role of N-glycosylation in maintaining the transporter activity and expression of human Oligopeptide transporter 1 (hPepT1) Ting Chan, Xiaoxi Lu, Tahiatul Shams, Ling Zhu, Michael Murray, and Fanfan Zhou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00462 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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Molecular Pharmaceutics

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The role of N-glycosylation in maintaining the transporter activity and expression of

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human Oligopeptide transporter 1 (hPepT1)

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Ting Chan a, Xiaoxi Lu a, Tahiatul Shams a, Ling Zhu b, Michael Murray c and Fanfan Zhou a,*

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a

Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia

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b

Retinal Therapeutics Research Group, Save Sight Institute, The University of Sydney, Sydney,

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NSW 2000, Australia

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c

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Medical Sciences, The University of Sydney, NSW 2006, Australia

Pharmacogenomics and Drug Development Group, Discipline of Pharmacology, School of

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Keywords: Human oligopeptide transporter 1; N-glycosylation; transport activity; transporter

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expression; post-translational modification.

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Abbreviations: Gly-Sar: glycylsarcosine; HEK: human embryonic kidney; human Oligopeptide

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transporter 1: hPepT1; PBS: phosphate-buffered saline; PNGase F: peptide-N-glycosidase F;

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SLC: solute carrier; TMD: trans-membrane domain.

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ACS Paragon Plus Environment


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ABSTRACT

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Human Oligopeptide transporter 1 (hPepT1) mediates the absorption of dietary peptides

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and a range of clinically relevant drugs. According to the predicted topological structure hPepT1

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contains multiple asparagine residues in putative N-glycosylation sites. This study investigated

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the influence of the six putative N-glycosylation sites within the extracellular region between

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transmembrane domains 9 and 10 on hPepT1 transporter function and expression in HEK-293T

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cells. Our study confirmed that hPepT1 is N-glycosylated in HEK-293T cells, with the

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glycosylated and fully deglycosylated isoforms exhibiting apparent molecular masses of ~78 and

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~55 kDa, respectively. Transport uptake of Glycylsarcosine (Gly-sar) by the hPepT1-N562Q

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variant, but not by other single mutants, was moderately impaired. We also constructed multiple

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N-glycosylation mutants based on the hPepT1-N562Q mutant by mutagenizing the additional

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asparagine residues N404Q, N408Q, N439Q, N509Q and N514Q. Transport function showed a

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graded decrease as the number of mutagenized residues increased and simultaneous removal of

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all six asparagine residues essentially abolished transport activity. Kinetic studies indicated that

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the Vmax values for Gly-sar transport by low activity mutants were decreased compared to wild

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type, which suggested that the cell surface expression and/or turn-over rate of hPepT1 mutants

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was impaired; Km values were unchanged in most cases. Using immunoblotting and

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immunofluorescence, the plasma membrane and total cellular expression of the mutant

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transporters were decreased in accordance with functional impairments. In summary, we provide

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the first molecular evidence that hPepT1 is modified by N-glycosylation and that all six

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asparagine residues in the large extracellular loop between transmembrane domains 9 and 10 are

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subject to N-glycosylation. This information enhances our understanding of the role of the large

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extracellular loop in hPepT1 regulation and could facilitate the development of new hPepT1

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substrate drugs with improved bioavailability.

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INTRODUCTION

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Solute Carrier transporters (SLCs) are essential membrane proteins that mediate the

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cellular influx of endogenous and exogenous substances. Several SLC subfamilies have been

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shown to be physiologically and pharmacologically important. Human Oligopeptide transporters

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(PepTs) encoded by SLC15A genes belong to the Proton-coupled Oligopeptide Transporter

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subfamily1. PepTs regulate the cellular uptake of di- and tri-peptides and peptide-like drugs.

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Therefore, PepTs play significant roles in maintaining homeostasis and the pharmacokinetic

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performance of peptide drugs 2.

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To the present, two human PepTs have been identified - hPepT1 (SLC15A1) and hPepT2

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(SLC15A2). Both have been extensively studied for their roles in drug disposition. hPepT1 is

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primarily expressed at the intestinal epithelium, where it mediates the uptake of dietary nitrogen

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across the apical membrane into enterocytes 3. hPepT1 is also expressed in the renal tubule,

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where it facilitates the reabsorption of nitrogen from the glomerular filtrate 4. In addition,

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hPepT1 facilities the absorption of a wide range of clinically important drugs and pro-drugs,

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such as β-lactam antibiotics, angiotensin-converting enzyme inhibitors, anti-viral agents and anti-

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cancer agents 3. It has been suggested that peptidomimetic chemicals that structurally resemble

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di- or tri-peptides could be potential substrates for hPepT1. Accordingly, understanding the

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regulation and functional importance of hPepT1 is important for optimizing the pharmacokinetic

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profiles of a range of drugs.

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Previous topological predictions and crystal structure studies have suggested that hPepT1

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possesses 12 trans-membrane domains (TMDs) with two major extracellular loops situated

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between TMDs 3 and 4 and TMDs 9 and 10

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post-translational modifications for eukaryotic membrane proteins and disrupting N-

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glycosylation of specific proteins has been reported to be associated with disease development 12-

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localization and/or substrate binding of SLC transporters

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between TMDs 9 and 10 contains six asparagine residues located within putative N-

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glycosylation sites, but the impact of N-glycosylation on hPepT1 function and expression has not

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yet been evaluated. Based on the predicted topology of hPepT1, we performed site-directed

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mutagenesis to disrupt the putative N-glycosylation sites of hPepT1, individually and in

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. N-Glycosylation is one of the most important

. A number of studies have indicated that N-glycosylation is critical for the folding, stability, 15-19

. The extracellular loop in hPepT1



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combination, so as to assess the influence of N-glycosylation on the function and the expression

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of hPepT1.

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MATERIALS AND METHODS

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Materials

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[14C] Glycylsarcosine (Gly-Sar; specific activity 66 mCi/mmol) was purchased from

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American Radiolabeled Chemicals (St. Louis, MO). Culture media was obtained from Thermo

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Scientific (Lidcombe, NSW, Australia). Unless otherwise stated, all other chemicals and

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biochemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

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Site-directed Mutagenesis and over-expression of hPepT1 and its mutants in HEK-293T

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cells

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The SLC15A1 cDNA was kindly provided by Professor Peter J. Meier-Abt, Department

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of Biomedicine, University Hospital Basel, Switzerland. Mutant transporters were generated

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using the Phusion High-Fidelity PCR Kit (New England Biolabs, Arundel, QLD, Australia),

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following the manufacturer’s instructions and using the primers listed in Table 1. All PCR

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reactions were conducted using GC buffer provided by the PCR kit with the following cycling

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conditions: 98 °C, 30 sec; 98 °C, 10 sec, 72 °C, 30 seconds repeated for 25 cycles; 72 °C 10 min

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and 4 °C overnight. Multiple mutants were generated by mutating one additional asparagine

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residue each time to the existing mutant. All multiple mutants were generated using the same

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reagents and protocol as mentioned above. All sequences were confirmed by the dideoxy chain-

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termination method (Ramaciotti Centre for Gene Function Analysis, University of New South

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Wales, Kensington, NSW, Australia).

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Human embryonic kidney (HEK)-293T cells were maintained at 37 °C and 5% CO2 in

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Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Cells were

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transfected with plasmid DNA using Lipofectamine 3000 Reagent (Invitrogen, Mount Waverley,

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VIC, Australia). Twenty-four h after transfection, substrate uptake activities were measured.

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Transport Studies

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Cellular uptake of [14C] Gly-Sar (final concentration 20 µM, 16 nCi/well) in HEK-293 20-22

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cells was conducted as described previously

. Uptake was initiated in phosphate-buffered

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saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 5.0)

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containing 1 mM CaCl2 and 1 mM MgCl2, and was terminated by rapidly washing the cells in



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PBS at 4°C. Uptake studies were conducted within 8 min of initiation because uptake in HEK-

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293T cells was linear over this period. The cells were then solubilized in 0.2 M NaOH and

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neutralized with 0.2 M HCl, and aliquoted for liquid scintillation counting. Uptake was

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standardized to the amount of protein in each well. Kinetic studies were performed with varying

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concentrations of Gly-Sar (10-5000 µM) and a 4 min incubation; apparent Km and Vmax values for

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transport activity were then calculated (GraphPad Prism 6.0; GraphPad Inc, LaJolla, CA).

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Cell surface biotinylation

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The membrane-impermeable biotinylation reagent NHS-SS-biotin (Campbell Science,

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Rockford, IL) was used to determine the cell surface expression of hPepT1 and its variants.

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Twenty-hours after transfection, the medium was removed and the cells were washed with ice-

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cold PBS (pH 8.0; 3mL). Cells were incubated on ice with 1 mL of freshly prepared NHS-SS-

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biotin (0.5 mg in PBS) for 30 min with gentle shaking. After biotinylation, unreacted NHS-SS-

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biotin was quenched by washing the cells with PBS containing 100 mM glycine (3 mL). Cells

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were then lysed with a 30 min incubation with lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM

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EDTA, 0.1% SDS and 1% Triton X-100 that contained the protease inhibitors

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phenylmethylsulfonyl fluoride (200 mg/mL) and leupeptin (3 mg/mL), pH 7.4; 400 µL). Unlysed

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cells were removed by centrifugation at 14,000 g at 4°C. Streptavidin agarose beads (50 µL;

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Genscript, Piscataway, NJ) were added to the supernatant to isolate biotinylated cell membrane

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proteins.

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Tunicamycin and PNGase F treatment

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Twenty-four hours after transfection, HEK-293T cells that over-expressed hPepT1 were

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treated with tunicamycin (50 µg/mL) and incubated at 37°C for 16 h. Uptake and biotinylation

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assays were then conducted to assess the effects of tunicamycin on the function and expression

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of hPepT1. Twenty-four hours after transfection, total lysates from HEK-293T cells that over-

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expressed hPepT1 were treated with PNGase F at 37°C for 1 h and then subjected to Western

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immunoblotting.

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Electrophoresis and immunoblotting


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Denatured samples were loaded onto 7.5% polyacrylamide minigels and electrophoresed

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using a mini cell (Bio-Rad, Gladesville, NSW, Australia). Proteins were transferred to

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polyvinylidene fluoride membranes in an electroelution cell (Bio-Rad) and blocked for 1 h with

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5% dry skim milk in PBS-Tween (80 mM Na2HPO4, 20 mM KH2PO4, 100 mM NaCl, and 0.05%

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Tween 20; pH 7.5), washed, and then incubated overnight at 4°C with an anti-hPepT1 antibody

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(1 µg/mL; Santa Cruz Biotchnology, Dallas, TX; Cat. No. sc-20653). The membranes were

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washed, incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10000;

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Sigma, Cat. No. A0545), and signals were detected using the Clarity ECL Western Blotting

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Substrate (Bio-Rad).

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In some experiments, transfected HEK-293T cells were treated with the protein synthesis

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inhibitor cycloheximide (100 µg/mL). Cells were collected and lysed with lysis buffer at

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different time points as described above. Equal quantities of proteins were denatured and loaded

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onto minigels for immunoblotting.

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Immunofluorescence Analysis

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HEK-293T cells were grown on coverslips in culture plates and transfected with either

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the wild-type or mutant hPepT1 plasmids. Cells were washed twice with PBS (pH 7.4) and fixed

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with 4% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized with

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PBS-glycine-saponin (0.5% glycine and 0.05% saponin in PBS) for 5 min and blocked with 5%

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goat serum in PBS-glycine-saponin for 20 min at room temperature. Cells were then incubated

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with the anti-hPepT1 antibody at room temperature for 1 h, washed three times with PBS (pH 7.4)

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and then incubated with Alexa Fluor 594-conjugated anti-rabbit IgG (Invitrogen) for 45 min.

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Following that, cells were washed three times with PBS (pH 7.4) and incubated with Hoechst

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33342 in PBS (pH 7.4) for 5 min in order to stain nuclei. After that, cells were washed four times

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with PBS (pH 7.4) and mounted with Fluoro-gel MOUNT (ProSciTech, Townsville City, QLD,

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Australia) and visualized using a Leica DMI3000B epifluorescence microscope (Leica

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Microsystems, North Ryde, NSW, Australia).

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Statistics

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Data are expressed throughout as mean ± SEM with p < 0.05 considered as significant.

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The Student’s t-test was used to test for differences between two groups of normally distributed



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data. Differences in transport function of hPepT1 and multiple variants were detected by one-

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way analysis of variance and Dunnett’s testing.

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RESULTS

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N-glycosylation of hPepT1

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It has been predicted that hPepT1 contains several asparagine residues in putative N-

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glycosylation sites in extracellular loop regions 2. A recent study showed that the murine pept1

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isoform is N-glycosylated in the jejunum and colon,23 but there is little information regarding the

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possible N-glycosylation of the human PepT1 isoform - hPepT1. In this study, hPepT1 was

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found to be extensively N-glycosylated when over-expressed in HEK-293T cells. Tunicamycin

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was used to assess the overall impact of N-glycosylation on hPepT1 function and expression.

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After 16 h of tunicamycin treatment (50 µg/mL), the uptake of Gly-Sar through hPepT1 was

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decreased by 50% (p < 0.05; Figure 1A). On immunoblots, the corresponding molecular mass of

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hPepT1 was decreased from ~78 kDa to ~55 kDa following tunicamycin treatment, consistent

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with extensive N-glycosylation in the mature hPepT1 transporter (Figure 1B). In confirmatory

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experiments, lysates from HEK-293T cells that over-expressed hPepT1 were treated with

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peptide-N-glycosidase F (PNGase F), which removes high mannose, hybrid and complex

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oligosaccharides from N-linked glycoproteins; this also decreased the apparent molecular mass

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of hPepT1 to ~55 kDa (Figure 1C).

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Functional analysis of single and multiple N-glycosylation mutants of hPepT1

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According to the proposed topology, six asparagine residues in putative N-glycosylation

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sites (consensus motif N-X-(S/T)) were identified within the extracellular loop between TMDs 9

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and 10 of hPepT1 (N404, N408, N439, N509, N514 and N562). To assess the importance of

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these residues in hPepT1 function, as reflected by Gly-Sar uptake, the SLC15A1 wild-type

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sequence was mutagenized so that one or more asparagine residues were replaced with

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glutamines. As shown in Figure 2A, of the six mutant transporters produced by mutagenesis of

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single asparagines, transport function was only decreased in the case of the hPepT1-N562Q

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mutant (to 78% of wild-type control), while other single-asparagine mutant transporters retained

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full function.

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Next, we constructed a series of multiple mutants based on the hPepT1-N562Q mutant so

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as to assess the potential interplay between putative N-glycosylation sites (N404/562Q;

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N404/408/562Q; N404/408/439/562Q; N404/408/439/509/562Q; N404/408/439/509/514/562Q).

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As shown in Fig. 2B, Gly-sar uptake by the variants that contained three or more mutagenized

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asparagines was significantly impaired (~20%−70% of wild-type hPepT1). The magnitude of the



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impairment was related to the number of mutated residues with the simultaneous removal of all

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six asparagines in putative N-glycosylation sites producing the most pronounced decrease in

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transport activity (~20% of wild-type; Figure 2B).

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Kinetic analysis of single and multiple N-glycosylation mutants of hPepT1

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Kinetic analyses were performed to further evaluate the underlying mechanisms of the

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impaired transport activity of the single and multiple hPepT1 mutants. Kinetic studies were not

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performed for the sextuple mutant (hPepT1-N404/408/439/509/514/562Q), because its activity

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was too low for the reliable determination of kinetic parameters.

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The affinity of the hPepT1-N509Q mutant for Gly-sar uptake was somewhat lower than

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wild type (Km value 589±74 µM vs. 820±64 µM for wild-type hPepT1; p Lys substitution in saposin B involving a conserved amino acidic residue and

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Cloning, functional expression, and chromosomal localization. J Biol Chem 1995, 270, (12),

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glycoprotein by statins enhances doxorubicin cytotoxicity in human neuroblastoma cells. Int J

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glycosylation of MRPs in ovarian carcinoma cells resistant to oxaliplatin. Biochem Pharmacol

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specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat

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Glycosylation increases

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Feng, M.; Fang, Y.; Han, B.; Xu, X.; Fan, P.; Hao, Y.; Qi, Y.; Hu, H.; Huo, X.; Meng, L.;


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of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana). J

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structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1

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oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked

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the electrophoretic mobility and function of the human proton-coupled folate transporter

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458 459 460



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461

FOOTNOTE

462

*

463

Dr. Fanfan Zhou

464

Faculty of Pharmacy, University of Sydney, NSW 2006, Australia

465

Tel: 61-2-93517461, Fax: 61-2-93514391, email: [email protected]

Corresponding Author:

466 467


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468

TABLES

469

Table 1. Primer sequences used to engineer hPepT1 N-glycosylation mutants mutants

Forward Primer (5’ to 3’)

Reverse Primer (5’ to 3’)

N404Q

TAAAGTTTTGAATATAGGACAG

GGAAGAGATATATTCATGGTATTC

AATACCATGAATATATCTCTTCC

TGTCCTATATTCAAAACTTTA

GGAAACAATACCATGCAGATAT

CTCTCCAGGAAGAGATATCTGCAT

CTCTTCCTGGAGAG

GGTATTGTTTCC

CAAACTGACAAGGATACAGATT

GGTGATCCAGGAGAAGAAATCTGT

TCTTCTCCTGGATCACC

ATCCTTGTCAGTTTG

GGGAAAGTTTATGCACAGATCA

GGCATTGTAGCTGCTGATCTGTGC

GCAGCTACAATGCC

ATAAACTTTCCC

CATCAGCAGCTACCAGGCCAGC

CTGGTATGTGCTGGCCTGGTAGCT

ACATACCAG

GCTGATG

GTCCAAAGGAAGCAGGACAGCT

CTTCAGGGCAGCTGTCCTGCTTCCT

GCCCTGAAG

TTGGAC

N509/514

GATCAGCAGCTACCAGGCCAGC

CTGGTATGTGCTGGCCTGGTAGCT

Q

ACATACCAG

GCTGATC

N408Q

N439Q

N509Q

N514Q

N562Q

470 471



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472

Table 2. Kinetic analysis of

473

mutants.

14

C-Gly-Sar uptake by wild type hPepT1 and its N-glycosylation

Construct

Km (µM)

Vmax (pmol/µg/4 min)

hPepT1 WT

820 ± 64

847 ± 26

hPepT1-N509Q

589 ± 73*

602 ± 24**

hPepT1-N562Q

898 ± 87

722 ± 26*

hPepT1-N404/562Q

917 ± 70

736 ± 21*

hPepT1-N404/408/562Q

711 ± 104

517 ±26 **

hPepT1-N404/408/439/562Q

739 ± 105

522 ± 26**

hPepT1-

866 ± 124

342.2 ± 18***

N404/408/439/509/562Q 474

Initial uptake (4 min) of [14C] Gly-sar by hPepT1 and its mutants was estimated at various

475

concentrations. Km and Vmax data were calculated using GraphPad Prism. Values are mean ± SE

476

(n=3). *p

The Role of N-Glycosylation in Maintaining the Transporter Activity

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