Articles in PresS. Am J Physiol Gastrointest Liver Physiol (March 23, 2017). doi:10.1152/ajpgi.00343.2016
Glycosylation of PEPT1 at N50
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Glycans in the intestinal peptide transporter PEPT1 contribute to function and
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protect from proteolysis
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Tamara Stelzl, Kerstin E. Geillinger-Kästle, Jürgen Stolz,
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and Hannelore Daniel*
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Nutritional Physiology, Technische Universität München, 85350 Freising, Germany
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Running head: Glycosylation of PEPT1 at N50
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*
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Universität München, Lehrstuhl für Ernährungsphysiologie, Gregor-Mendel-Str. 2, 85350
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Freising,
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[email protected]
To whom correspondence should be addressed: Prof. Dr. Hannelore Daniel, Technische
Germany,
Phone.:
+49-8161-713400;
Fax:
+49-8161-713999;
Email:
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Keywords: peptide transport; glycoprotein; N-linked glycosylation; Xenopus laevis
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____________________________________________________________________________
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Abstract
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Despite the fact that many membrane proteins carry extracellular glycans, little is known about
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whether the glycan chains also affect protein function. We recently demonstrated that the
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proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six
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asparagine residues (N50, N406, N439, N510, N515, N532). Mutagenesis-induced disruption of
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the individual N-glycosylation site N50, which is highly conserved among mammals, was
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detected to significantly enhance the PEPT1 mediated inward transport of peptides. Here, we
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show for the murine protein, that the inhibition of glycosylation at sequon N50 by substituting
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N50 with glutamine, lysine or cysteine, or by replacing S52 with alanine, equally altered PEPT1
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transport kinetics in oocytes. Further, we provide evidence that the uptake of [14C]-glycyl1
Copyright © 2017 by the American Physiological Society.
Glycosylation of PEPT1 at N50
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sarcosine in immortalized murine small intestinal (Mode-K) or colonic epithelial (PTK-6) cells
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stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild
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type protein. By using electrophysiological recordings and tracer flux studies, we further
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demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line
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with these findings, we show that attachment of biotin derivatives, comparable in weight to 2-4
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monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition,
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our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against
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proteolytic cleavage by proteinase K, while a remarkable intrinsic stability against trypsin, even
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in absence of N-linked glycans, was detected.
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New & Noteworthy
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This study highlights the role of N50-linked glycans in modulating the bidirectional transport
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activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux
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measurements in Xenopus oocytes have shown that a removal of the N50 glycans increases the
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maximal peptide transport rate in the inward and outward direction. This effect could be largely
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reversed by replacement of N50 glycans for structural dissimilar biotin derivatives. Besides, N-
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glycans were detected to stabilize PEPT1 against proteolytic cleavage.
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Introduction
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The peptide transporter 1 (PEPT1) is a prototypical member of the solute carrier 15 gene family
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(SLC 15) with high expression in apical membranes of the upper small intestine. PEPT1
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transports in essence all natural di- and tripeptides as well as peptide-like drugs, such as ß-
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lactam antibiotics [6, 48, 65], angiotensin-converting enzyme inhibitors [42, 60], the
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antineoplastic drug bestatin [26, 53] and other nonpeptidic compounds [10, 21]. Peptide
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transport is proton-coupled and driven by an inwardly directed electrochemical gradient with the
64
membrane potential as the main driving force [16, 61, 62, 67]. Crystal structures of prokaryotic
65
homologues [8, 19, 20, 45] and hydropathy analysis [12, 37] indicate the presence of 12 alpha-
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helical transmembrane domains (TMD) in mammalian H+/peptide transporters (Fig. 1).
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According to current knowledge, TMD 1, 3, 4, 7, 8, 9 and 10 participate in the formation of a
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central aqueous channel that constitutes a substrate translocation route across the lipid bilayer
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[5, 13, 15].
2
Glycosylation of PEPT1 at N50
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Murine PEPT1, which is composed of 709 amino acid residues, has a calculated core molecular
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mass of 79 kDa [14], but when glycosylated, a protein of around 105 kDa is detected. As
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recently shown by us [59], six asparagine residues (positions N50, N406, N439, N510, N515 and
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N532) carry glycans (Fig. 1). Disruption of single or multiple sequons in the large extracellular
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loop connecting TMD 9 and 10 (sequons N406, N439, N510, N515, N532) only marginally
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affected the PEPT1 transport characteristics when studied by two-electrode voltage clamp
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technique (TEVC) and tracer flux analyses in Xenopus laevis oocytes. In contrast, elimination of
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N50 significantly altered PEPT1 transport kinetics. Electrophysiological studies disclosed a more
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than 2-fold increase in inward transport currents with glycyl-sarcosine (Gly-Sar) as a substrate,
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which was associated with a similar reduction in substrate affinity. The enhanced transport rates
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were neither attributable to a higher cell-surface expression of N50Q, nor to a higher ion
81
conductance [59], suggesting that it is indeed the transport cycle of the individual protein that is
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altered.
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In the current study, we assessed how N-glycosylation at N50 alters PEPT1 kinetics for both, the
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inward and outward transport mode. Using radiotracer flux measurements, as well as an
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electrophysiological approach, we examined whether the gain of function is simply due to the
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loss of the N-glycan mass at this particular site. To mimic an oligosaccharide chain, we
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exchanged N50 by cysteine and used biotinylation reagents to add various masses to this
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position. In addition, we also analyzed the role of N-glycans in conferring proteolytic stability to
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PEPT1.
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Materials and Methods
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Construction of PEPT1 N-glycosylation defective transporters
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Potential N-glycosylation sites in the PEPT1 sequence (UniProt ID: Q9JIP7) were identified
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using the NetNGlyc 1.0 server. Asparagine in position 50 was exchanged with glutamine,
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cysteine or lysine, respectively serine 52 with alanine. To this end, two mutation-specific
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megaprimers carrying terminal EcoRV and XhoI sites were amplified and subsequently coupled
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with Phusion® polymerase (NEB, Ipswich, USA) at 25°C in a thermocycler (Biometra, Göttingen,
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Germany). Primer combinations used for the synthesis of megaprimers are listed in Table 1.
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Resulting fusion PCR products were digested with XhoI and EcoRV (Thermo Scientific,
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Waltham, USA) and cloned into pCR®II-TOPO-3´end vector (Invitrogen, San Diego, USA).
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Mutations were confirmed by sequencing (GATC Biotech, Constance, Germany).
3
Glycosylation of PEPT1 at N50
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105
Generation of cRNA
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The expression vectors carrying individual PEPT1 constructs were linearized with NotI and
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cRNA synthesis performed with the mMessage machine T7 kit (Ambion, Darmstadt, Germany)
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according to the manufacturer`s specifications. Following phenol extraction and ethanol
109
precipitation, 2 µg linearized plasmid DNA was transcribed into cRNA and adjusted to a final
110
concentration of 1 µg/µl. Prior to Xenopus oocyte microinjection, all cRNA preparations were
111
evaluated for size and integrity on a 1% agarose-formaldehyde gel and stored at -80°C.
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113
Xenopus laevis oocyte preparation and cRNA injection
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X. laevis maintenance and oocyte harvest procedures were approved by the local authority for
115
animal care in research (Regierung von Oberbayern, approval no. 55.2-1-54-2532.3-64-11).
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Oocytes were surgically removed from X. laevis frogs (anaesthetized in a solution of 0.7 g/l 3-
117
aminobenzoic acid ethyl ester; Sigma-Aldrich, Taufkirchen, Germany) and enzymatically
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defolliculated by collagenase A treatment (2.5 mg/ml for 90 min) (Roche, Mannheim, Germany).
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Healthy stage V and IV oocytes were manually sorted and stored in Barth-solution (88 mM NaCl,
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1 mM KCl, 0.8 mM MgSO4, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES;
121
pH 7.4) supplemented with 5 mM pyruvate and 0.2 mM gentamycin at 17°C overnight. Intact
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oocytes were injected with 18.4 nl cRNA or water as a control. For co-expression experiments,
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cRNA´s for the murine sodium glucose transporter 1 (SGLT1; UniProt ID: Q9QXI6) and PEPT1
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variants were microinjected in a ratio of 1:1 to a final volume of 36.8 nl. The N-glycosylation
125
inhibitor tunicamycin was dissolved in 4% dimethyl sulfoxide (DMSO) in water and co-injected
126
with PEPT1 cRNA to a final concentration of 2.5 ng per oocyte. For protein expression, injected
127
oocytes were kept for 3-4 days at 17°C in Barth-solution.
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129
Electrophysiological recordings
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Two-electrode voltage clamp (TEVC) experiments were conducted as described [1, 31]. Briefly,
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oocytes were placed in an open chamber and continuously superfused with Barth-solution or
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Gly-Sar (Sigma-Aldrich, Taufkirchen, Germany) over the concentration range of 0.3-40 mM at a
133
flow rate of 3 ml/min. Microelectrodes backfilled with 0.5 mM KCl and an electrode resistance
134
between 1-3 mΩ were used to clamp oocytes at a holding potential of -60 mV. Inwardly directed
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transport was calculated as the difference in currents registered in the presence or absence of
136
substrate. Current-voltage (I-V) relations were recorded with a Tec-03 amplifier (npi electronic
137
GmbH, Tamm, Germany) for the duration of 100 ms, separated by pauses of 200 ms, in the
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potential range of +80 to -160 mV. Data acquisition was performed with CellWorks (v. 5.1; npi
4
Glycosylation of PEPT1 at N50
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electronic GmbH, Tamm, Germany). The substrate-dependent maximal inward currents of
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PEPT1 were normalized to currents evoked by 1 mM alpha-MDG (pH 6.5) as substrate for the
141
co-expressed SGLT1 transporter. This correction was performed to compensate for fluctuations
142
in gene expression levels between individual oocytes and oocyte batches. The kinetic
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parameters Km (mM) and Imax (nA) were calculated by least-squares fits to the Michaelis-Menten
144
equation. Transient outward current recordings were performed according to Kottra et al. [31]. In
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short, oocytes were injected with rising volumes (9.2-59.8 nl) of 1 M Gly-Sar (pH 7.5) dissolved
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in 1 mM ethylene glycol-bis(2-aminoethylether)-N′,N′,N′,N′-tetraacetic acid in water (EGTA;
147
Sigma-Aldrich, Taufkirchen, Germany). 30 sec post-injection, I-V relations were recorded over a
148
period of 6 min in intervals of 30 sec. Outward currents are denoted with a positive sign, while
149
negative values represent inward currents. For current recordings without normalization to co-
150
expressed SGLT1, an outlier analysis was performed. According to the formula zi = (xi - M)/SD,
151
a z-score was computed for individual datasets (xi is the original value, M is the mean and SD
152
the standard deviation). Values with a z-score ± 1.5 or beyond were considered as outlier.
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Electrophysiological data are presented as mean ± SEM or median ± min/max (+ inside bars
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marks mean values) of 10-30 oocytes from at least two oocytes batches. Statistical significant
155
differences were determined by one-way ANOVA followed by Dunnett's posttest analysis or
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Student´s t-test using GraphPad Prism v. 4.01. Significance levels are indicated with * P < 0.05,
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** P < 0.01, *** P < 0.001.
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Tracer efflux studies with Xenopus laevis oocytes
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Radiotracer efflux experiments were performed after initial loading of X. laevis oocytes with [14C]-
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Gly-Sar solution (56 Ci/mol, 17.8 mM; custom-synthesized by GE Healthcare, Munich, Germany;
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ratio of labeled to unlabeled substrate 1:10) at pH 6.5 for 10 min at 23°C. After briefly rinsing
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with Barth-solution (pH 7.4), oocytes were transferred into new reaction vessels devoid of
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radioactivity and excess liquid removed. Following addition of 100 µl Barth-solution per 5 pre-
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incubated oocytes, radioactive efflux was determined over a period of 20 min in the supernatant.
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For measurement of the residual radioactivity in oocytes, samples were dissolved in 20% SDS in
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water for 2-3 h at 50°C with shaking (500 rpm; Heidolph® Titramax 1000). After addition of a
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scintillation cocktail (Rotiszint eco plus; Roth, Karlsruhe, Germany) radioactivity was counted in
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a Tri-Carb 2810 TR instrument (PerkinElmer, Waltham, MA). The total radioactivity in oocytes
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was calculated at time point zero and corrected for radiation of water-injected oocytes. Export
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rate constants (Kexp) were calculated as the slope of the linear regression analysis applying first
172
order kinetics and efflux half-life´s determined by the formula t1/2=ln2/Kexp.
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5
Glycosylation of PEPT1 at N50
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Protein preparation from oocytes or mammalian cultured cells
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3-4 days after cRNA injection, 20 oocytes expressing the different transporter genes were
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transferred into 200 µl lysis-buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM
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dithiothreitol) and homogenized. Following centrifugation for 1 min at 15300 rcf and 4°C,
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supernatants were collected and protein contents determined by Bradford protein assay (Bio-
179
Rad, Munich, Germany). In case of mammalian cells, confluent cells (75 cm2 culture flask) were
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aspirated from media and washed with cold phosphate buffered saline (PBS; pH 7.4). Adhering
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cells were scraped off with PBS containing 1 mM PMSF and spun at 2300 rcf for 2 min at 4°C.
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Sedimented cells were resuspended in 200 µl PBS containing 1 mM PMSF and lysed by
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passing through a 2 ml syringe with a 24G´ cannula. Following centrifugation at 425 rcf for 3 min
184
at 4°C, the total protein fraction was separated by high speed centrifugation at 42500 rcf at 4°C
185
for 1 h. Total membranes were resuspended in PBS containing PMSF and protein yields
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quantified by Bradford assay.
187
188
Immunoblotting of protein extracts
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SDS-PAGE was performed with 10% SDS-acrylamide gels in a mini-protean 3 system from Bio-
190
Rad (Bio-Rad, Munich, Germany). Separated proteins were transferred to a nitrocellulose
191
membrane (Whatman, Maidstone, UK) in a wet tank blotting system at 0.36 A for 25 min. After
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blocking for 1 h in 1% bovine albumin (AppliChem, Darmstadt, Germany), the membrane was
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probed with a polyclonal rabbit antiserum against PEPT1 (custom-made Pineda, Berlin,
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Germany;
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CVGKENPYSSLEPVSQTNM corresponding to the C-terminus of rat PEPT1) and ß-actin as
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loading control (Santa Cruz, Dallas, USA; dilution 1:2000, polyclonal goat IgG C-11 sc-1615)
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overnight at 4°C. Following three washings with PBS containing 0.05% Tween-20, the
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membrane was stained with IRDye®-labeled secondary antibodies (LI-COR Biosciences, Bad
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Homburg, Germany; dilution 1:12000; C50113-03; C40415-02) at room temperature for 2 h.
200
Fluorescence signals were detected with an infrared fluorescence Odyssey scanner and
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quantified with the Image StudioTM Lite software (v. 3.1) supplied from LI-COR.
dilution
1:5000,
polyclonal
rabbit
IgG
raised
against
the
peptide
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203
Surface biotinylation of oocytes
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Oocytes were biotinylated with either 2-((biotinoyl)amino)ethyl-methanethiosulfonate (MTSEA-
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biotin; Biotium, Hayward, USA) or MTSEA-biotin capped with ethylenediamine (MTSEA-biotin-X
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and MTSEA-biotin-XX). These reagents are impermeable to the cell membrane and covalently
207
react with surface exposed sulfhydryl groups in proteins. Oocytes expressing individual PEPT1
208
versions were incubated in 2 mM MTSEA-biotin solution dissolved in PBS (MTSEA-biotin stock
6
Glycosylation of PEPT1 at N50
209
solutions: 100 mM in DMSO) for 15 min at room temperature. Following five times washing with
210
Barth-solution (pH 7.4), oocytes were assayed electrophysiologically.
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Proteolysis assay
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To assess the effect of glycosylation on the proteolytic stability of PEPT1, wild type and N-
214
glycosylation deficient transporters were exposed to the proteases trypsin and proteinase K.
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Total protein extracts from oocytes (15 µg protein) were digested with 20 units of trypsin (Sigma-
216
Aldrich, Taufkirchen, Germany) freshly dissolved in 1 M HCl and 2 mM CaCl2 (pH 3). After 10
217
min, trypsin-inhibitor (Sigma-Aldrich, Taufkirchen, Germany) was added to a final concentration
218
of 2 mM. Alternatively, 2.5 ng/µl proteinase K (VWR, Darmstadt, Germany) was used. The
219
reaction was stopped after 6 min at ambient temperature with 5 mM PMSF. After incubation with
220
the proteases, proteins were separated by SDS-PAGE, followed by Western blotting with the
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PEPT1 serum.
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223
Retroviral transduction of PEPT1 in Mode-K and PTK-6 cells
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Expression of PEPT1 in the murine intestinal epithelial cell lines Mode-K [66] and PTK-6 [68]
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was accomplished by retroviral transduction. Virus production was achieved by transfection of
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an ecotropic Platinum-E (Plat-E) packaging cell line (Cell Biolabs, San Diego, USA) with the
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retroviral transfection vector pMXs (Cell Biolabs, San Diego, USA). For generation of the pMXs
228
expression vector, PEPT1 was amplified with gene-specific primers incorporating a C-terminal
229
HA
230
TATTTCGAAATGGGGATGTCCAA
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GTCTCGGGG 3´; Reverse primer: 5´ ATAGTCGACTTAAGCATAATCTGGAACATCATATGGAT
232
ACATATTTGTCTGTGAGACTGGT3´). PCR products were digested with SalI and Bsp119I and
233
ligated into the pMXs vector using the same restriction sites. Prior to Plat-E cell transfection, cell
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culture plates were coated with collagen A (Biochrom, Berlin, Germany), thoroughly washed with
235
PBS and Plat-E cells (passage P34) seeded in a density of 9.99x105 cells per well of a 6-well
236
tissue culture plate. Plat-E cells were cultivated in DMEM medium (Sigma-Aldrich, Taufkirchen,
237
Germany) supplemented with 1 µg/ml puromycin and 10 µg/ml blasticidin. After transfection,
238
these antibiotics were replaced by 1% penicillin-streptomycin. Six hours after Plat-E seeding,
239
cells were transfected with pMXs vector constructs. Transfection was performed with the
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ProFection Mammalian Transfection System (Promega, Mannheim, Germany) according to the
241
manufacturer’s specifications. Sixteen hours post-transfection the cell medium containing the
242
retroviruses was removed and filtered using a cellulose-acetate filter (pore size 0.45 µm). For
epitope
tag
and
the
restriction
sites
SalI
and
Bsp119I
(Forward
primer:
5´
7
Glycosylation of PEPT1 at N50
243
retroviral transduction, 7.5x104 Mode-K (P33) or PTK-6 cells (P44) per well of a six well culture
244
plate were seeded and recovered overnight. The next day, the culture medium was renewed and
245
supplemented with 3 µg/ml polybrene (Santa Cruz Biotechnology, Heidelberg, Germany).
246
Following 1 h incubation at 37°C, 1 ml of retroviral lysate was added per well. After 20 hours, the
247
culture medium was replaced by media containing 10 µg/ml blasticidin for positive transformants
248
selection.
249
250
Uptake measurements of [14C]-Gly-Sar in Mode-K and PTK-6 cells
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Following retroviral transduction, Mode-K (P30-35) and PTK-6 cells (P40-45) were grown to
252
confluence in six well culture plates. A single well was used for protein content determination.
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Therefore, cells were scraped off in 500 µl PBS (pH 7.4) and sonicated for 12 cycles with
254
amplitude 50 (UP200S; Hielscher Ultrasonics GmbH, Teltow, Germany). Cell debris was
255
removed by centrifugation and protein content of supernatants quantified by Bradford assay. The
256
remaining cells were washed with 1 ml MES-Tris buffer (MTB: 140 mM NaCl, 5.40 mM KCl, 1.77
257
mM CaCl2, 0.80 mM MgSO4, 5 mM glucose, 27 mM MES; pH 6.0) and incubated with 500 µl
258
[14C]-Gly-Sar solution (final concentration of 6 µM) for 10 min at 37°C while shaking (200 rpm;
259
Heidolph® Titramax 1000). At the end of the incubation, the uptake solution was removed and
260
the cells washed twice with 1 ml MTB buffer. After lysis in 1 ml Igepal buffer (50 mM Tris, 140
261
mM NaCl, 1.50 mM MgSO4, 0.50% Igepal CA-630; pH 8.0), detached cells were transferred to
262
scintillation vials and measured in 3 ml Rotiszint eco plus in a Tri-Carb 2810 liquid scintillation
263
counter.
264
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266
Results
267
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PEPT1 N50Q has increased substrate import and export rates
269
Previous studies established that PEPT1 can transport in a bidirectional manner with inward and
270
outward transport currents following Michaelis-Menten kinetics [31]. Since inwardly-directed
271
transport currents for the N50Q mutant were increased [59], we here examined whether this is
272
also found for outward-transport with radiotracer efflux measurements using the non-
273
hydrolysable dipeptide [14C]-Gly-Sar (Fig. 2). We observed a three times higher Gly-Sar uptake
274
in oocytes expressing the N50Q transporter than for wild type (16.73 ± 0.30 pmol/20 min/oocyte
275
for N50Q, versus 5.66 ± 0.43 pmol/20 min/oocyte for wild type; data not shown). Efflux by the
276
N50Q mutant protein expressed as initial [14C]-Gly-Sar influx, proved to be increased as well 1.58
Glycosylation of PEPT1 at N50
277
fold. The radioactivity remaining in oocytes at the end of incubation was 68% of the preloaded
278
amount for N50Q and 78% for wild type. Kinetic analysis demonstrated that tracer export
279
followed first order reaction kinetics with rate constants of 0.26 min-1 for N50Q and 0.11 min-1 for
280
wild type. The calculated t1/2 for export from oocytes was 2.63 min for N50Q, and 6.58 min for
281
the wild type transporter. Thus, both substrate uptake and export are accelerated in N50Q.
282
TEVC analysis of oocytes clamped to a membrane potential of -60 mV and superfused with 40
283
mM Gly-Sar (at pH 7.5) revealed average inward currents of -513.49 ± 152.90 nA for PEPT1
284
wild type and of -1311.99 ± 341.57 nA for N50Q (Fig. 3). With hyperpolarization of the
285
membrane potential to -160 mV, a gradual increase in inward currents to -1840.97 ± 432.94 nA
286
in wild type and -3434.58 ± 720.86 nA in N50Q was observed with a steady-state I-V relationship
287
of similar shape for both proteins types. Electrogenic Gly-Sar efflux was measured by loading
288
oocytes with increasing amounts of substrate. Since stage VI oocytes have a cytosolic pH
289
between 7.4 and 7.7 [7, 55, 58], there is no significant proton gradient when an extracellular pH
290
of 7.5 is used. Under these conditions, the outwardly directed PEPT1 transport primarily
291
depends on the membrane potential and occurs at a membrane potential more positive than +20
292
mV [31]. The mean aqueous volume of oocytes amounts to ~410 nl [32, 70]. Therefore, injection
293
of 9.2 nl of a 1 M Gly-Sar solution brings the cytosolic concentration to about 22 mM, injection of
294
59.8 nl rises the intracellular Gly-Sar concentration to about 127 mM. Average outward transport
295
currents detected at +60 mV in PEPT1 increased after loading of 22 mM Gly-Sar within 6 min to
296
302.77 ± 72.37 nA (inset Fig. 3A) while at 127 mM Gly-Sar currents of 781.86 ± 86.79 nA were
297
recorded. In comparison, outward current recordings for N50Q were on average 1.5-2.5 times
298
higher (713.23 ± 189.50 nA at 22 mM Gly-Sar; 1155.50 ± 135.50 nA at 127 mM Gly-Sar) (inset
299
Fig. 3B). At a cytosolic Gly-Sar concentration of ~43 mM, outward currents for the PEPT1 wild
300
type protein (409.85 ± 153.34 nA) at +60 mV were comparable to corresponding inward currents
301
at -60 mV. At the same time, outward currents detected for N50Q (+60 mV: 779.95 ± 187.41 nA)
302
appeared to be 1.7-times lower than the inward currents. This analysis thus showed that the
303
inward as well as the outward transport currents of N50Q are increased by a factor of 2-2.5
304
relative to the wild type protein. Albeit all electrophysiological data were corrected for basal
305
transport in water-injected control oocytes, superfusion of oocytes expressing the PEPT1 N50Q
306
transporter with Gly-Sar generated, as opposed to the wild type protein, also outward transport
307
currents at membrane potentials more positive than +30 mV (Figs. 3A-B). This suggests the
308
presence of small leakage currents, although the exact nature of ions translocating, respectively
309
the underlying causes, are unknown thus far.
310
To test whether asparagine 50 itself is a critical residue for PEPT1 function or whether the lack
311
of the glycan causes the observed effects, we changed S52 to an alanine (Fig. 4). This
9
Glycosylation of PEPT1 at N50
312
disruption of the sequon abolishes glycosylation, but leaves N50 intact. TEVC analysis revealed
313
similar changes in the kinetic parameters for S52A (Km(S52A) = 1.27 ± 0.22 mM; Imax(S52A) = 2.45 ±
314
0.50 nA) and N50Q (Km(N50Q) = 1.33 ± 0.23 mM; Imax(N50Q) = 1.95 ± 0.55 nA). Both protein variants
315
thus possessed a reduction in substrate affinity and increase in maximal inward transport
316
relative to the wild type protein (Km(WT) = 0.66 ± 0.12 mM, Imax(WT) = 0.85 ± 0.21 nA).
317
Immunoblotting demonstrated decreased protein masses for both, N50Q and S52A in
318
comparison to wild type PEPT1 (Fig. 5). This implies that the absence of glycans attached to
319
N50 causes the increased transport velocity. This observation is further corroborated by the fact
320
that the exchange of N50 for lysine generated a similar effect (Km(N50K) = 1.03 ± 0.15 mM;
321
Imax(N50K) = 4.06 ± 0.61 nA) as shown in Fig. 4. In summary, the disruption of the sequon around
322
N50 resulting in a lack of glycosylation causes the increased transport velocity, irrespective of
323
the nature of the amino acid exchange.
324
325
Changes in PEPT1 N50Q transport activity are conserved among species and cell types
326
Protein glycosylation varies considerably between species [49] and it is therefore very unlikely
327
that Xenopus laevis oocytes produce the same glycans as mammalian cells. There is good
328
evidence for differences in oligosaccharide trimming and glycan modification between
329
mammalian and amphibian expression systems [42, 43]. We therefore expressed the mutant
330
proteins also in the murine intestinal epithelium cell lines Mode-K and PTK-6. Western blot
331
analysis of whole-cell extracts revealed a slightly lower PEPT1 molecular mass in Mode-K cells
332
derived from the small intestine (~95 kDa) compared to the colonic cell line PTK-6 (~100 kDa)
333
(Figs. 6A-B). We have previously shown that the PEPT1 mass is different between jejunum and
334
colon of mouse [69] by different glycosylation. Studies of Gly-Sar uptake in Mode-K and PTK-6
335
cells clearly revealed an increased substrate uptake for the N50Q transporter (Figs. 6C-D). With
336
10.59 ± 0.18 nmol/g protein/min, the [14C]-Gly-Sar uptake of N50Q expressed in Mode-K cells
337
was 60% higher than for wild type. Also N50Q expressed in PTK-6 cells exhibited a 50% higher
338
uptake rate than the wild type (6.21 ± 0.22 nmol/g protein/min). Non-transduced Mode-K and
339
PTK-6 cells did not show significant [14C]-Gly-Sar uptake (Figs. 6C-D) nor presence of the
340
PEPT1 protein in a Western blot (data not shown). In summary, these results indicate that the
341
altered transport characteristics by lack of glycosylation at N50 is not specific for Xenopus
342
oocytes and can be reproduced in other cell types. This also indicates that the nature of the
343
glycan - which may differ by cell type - seems less important for the reduced velocity of the
344
glycosylated PEPT1 variants.
345
346
Site-specific biotinylation mimics glycosylation
10
Glycosylation of PEPT1 at N50
347
A possible explanation that the loss of glycosylation at N50 causes an increase in bidirectional
348
transport velocity could be that the glycans add additional molecular mass to a protein domain
349
that undergoes a conformational change in the transport cycle. To test this concept, we created
350
proteins with masses added at position N50 by site-specific biotinylation with membrane-
351
impermeable cysteine-reactive reagents that could mimic the effects of the glycan. The reagents
352
used were various forms of MTSEA-biotin that attached masses of 380, 500 or 600 Daltons,
353
which is comparable in weight to a glycan structure containing 2, 3 or 4 hexoses.
354
Due to the observation that cysteine residues identified as exposed to the extracellular space in
355
the human PEPT1 protein - which shares 85% sequence identity with murine PEPT1 and carries
356
cysteine residues at identical positions - could not be modified with sulfhydryl-reactive reagents
357
[34], we decided to use a PEPT1 version containing all naturally occurring cysteine residues and
358
solely replaced N50 by cysteine. Voltage-clamp recordings in oocytes revealed a reduction in
359
Gly-Sar affinity for N50C (Km(N50C) = 1.20 ± 0.23 mM; -60 mV) which was similar to N50Q
360
(Km(N50Q) = 1.40 ± 0.29 mM) (Fig. 7A). The exchange of PEPT1 N50 caused an increase in
361
transport velocity relative to wild type (Imax(WT) = -528.38 ± 108.67 nA), and this effect was more
362
pronounced for N50Q (Imax(N50Q) = -1452.28 ± 247.21 nA) than for N50C (Imax(N50C) = -1080.70 ±
363
215.60 nA) (Fig. 7B). Biotinylation of surface cysteine residues did not significantly alter the
364
overall transport characteristics of wild type and N50Q transporters in oocytes. However,
365
biotinylation of oocytes expressing N50C significantly changed transport characteristics.
366
Labeling with MTSEA-biotin (~0.38 kDa molecular mass) increased the Gly-Sar affinity by 13%
367
compared to the untreated control (Fig. 7A), whereas treatment with MTSEA-biotin-X (~0.5 kDa)
368
or MTSEA-biotin-XX (~0.6 kDa) increased the binding affinity by 22%, and 24%, respectively.
369
The corresponding inward currents declined with a higher biotin mass attached (Fig. 7B). I-V
370
relationships for wild type, N50Q and N50C were found to be voltage dependent within a
371
membrane potential range of 0 to -160 mV, regardless of the biotin treatment (Figs. 7C-E).
372
373
N-glycans prevent PEPT1 from proteolytic degradation
374
A recent analysis of the crystal structure of the large extracellular loop of the mammalian peptide
375
transporter provided evidence that this protein domain acts as trypsin binding site [4] with the
376
hypothesis that this anchoring mechanism could increase the local concentration of peptides to
377
facilitate their uptake by PEPT1. Since the large loop of PEPT1 contains five of the six N-glycans
378
and glycans are generally considered to protect from proteolytic degradation, we examined to
379
which extent glycosylation protects PEPT1 from cleavage by proteases. Proteolytic stability of
380
PEPT1 was assessed in oocyte membrane extracts which were treated with different proteases,
11
Glycosylation of PEPT1 at N50
381
followed by gel separation, Western transfer and quantification of PEPT1. Although trypsin was
382
found to have 54 predicted cleavage sites in murine PEPT1 [17], in our experiments, proteolysis
383
occurred very slowly and degradation rates were almost identical in all glycan-variants tested
384
(Figs. 8A-B). Within 10 min of incubation, PEPT1 protein levels declined by ~47% for wild type,
385
with similar rates also for the N50Q, N406Q/N439Q/N510Q/N515Q/N532Q and the
386
N50Q/N406Q/N439Q/N510Q/N515Q/N532Q versions. This degradation occurred almost
387
completely within the first minute after trypsin addition, without any differences between protein
388
variants. This might be an indication for the presence of different types of membrane fractions
389
that are formed during protein preparation. It is possible that PEPT1 in inside-out vesicles or
390
open lamellar membranes is more susceptible to tryptic cleavage. In contrast, PEPT1 carriers in
391
right-side-out vesicles might be protected from proteolytic attack.
392
For proteinase K, 384 putative cleavage sites in PEPT1 were predicted [17]. The wild type
393
protein displayed a high stability against proteinase K, with only 33% protein loss over 10 min,
394
whereas the non-glycosylated PEPT1 variant was lost to 94% (Figs. 8C-D). Intermediate values
395
were
396
N406/N439/N510/N515/N532
397
expression of the PEPT1 gene in the presence of tunicamycin also significantly increased the
398
sensitivity of the PEPT1 protein towards proteinase K cleavage accompanied by a loss of any
399
detectable protein within 2 min of incubation.
400
____________________________________________________________________________
found
for
the
N50Q
mutant
and
and
PEPT1
transporters
N50/N406/N439/N510/N515/N532.
lacking
In
the
addition,
sequons
oocyte
401
402
Discussion
403
404
Glycosylation of secretory proteins occurs at asparagine residues within conserved acceptor
405
motifs [3] with N-glycans transferred en bloc by the oligosaccharyltransferase from dolichol to
406
the nascent polypeptide chain [39, 56]. For integral membrane proteins, only a few studies have
407
assessed the functional role of glycans. These show that protein folding, protein stability,
408
trafficking, secretion and function can be impaired by a lack of glycosylation [22, 41]. However, a
409
gain of function, such as the drastic increase in transport velocity described here for PEPT1
410
lacking glycosylation at N50, is a rare finding. Other examples where deglycosylation resulted in
411
increased activity include the lecithin-cholesterol acyltransferase (LCAT). LCAT which forms
412
cholesteryl esters from cholesterol [51] has four N-glycosylation sites (N20, N84, N272, N384)
413
[36], with a lack of glycosylation at N384 causing a twofold increase in enzyme activity [30].
414
Another example where removal of a single glycan considerably changed the enzyme activity is
12
Glycosylation of PEPT1 at N50
415
the endothelial lipase with four sites carrying complex-type N-glycans [27, 28]. Elimination of the
416
glycan at N62 markedly increased phospholipase activity in reconstituted HDL particles [40, 57].
417
The increase was up to 7-fold for apolipoprotein E (apoE2) and 26-fold for apolipoprotein A-I
418
containing HDL particles. A similar effect has also been described for bovine pancreatic
419
ribonuclease. This enzyme occurs as a mixture of an unglycosylated form called RNAse A and
420
various glycoforms, collectively called RNAse B [50]. In RNAse B, the single glycosylation site
421
(N34) is modified by oligomannose type N-glycans containing two residues of N-
422
acetylglucosamine and 5-9 residues of mannose. Treatment of RNase B with exoglycosidase
423
permits the generation of glycoforms with fewer mannose residues which provide an ideal tool to
424
study the relationship of glycosylation to activity [52]. Rudd and coworkers demonstrated a more
425
than three times higher hydrolytic activity for RNAse A relative to RNAse B [52]. Moreover, it was
426
shown that the enzymatic activity of RNAse B decreased continuously as the mannose content
427
and glycan mass increased [29, 52]. Here we describe for the first time a similar change of
428
protein function in a nutrient transport protein. The removal of the glycan at N50 in the intestinal
429
peptide transporter PEPT1 caused a 2- to 3-fold rise in maximal transport velocity (Figs. 2, 3
430
and 4). The gain in transport velocity was always associated with a significant reduction in the
431
affinity for the substrate Gly-Sar. As demonstrated by both, electrophysiological analyses and
432
tracer flux experiments, the reduction in affinity occurs for the inward and outward transport
433
direction. This suggests that the release of the substrate on the trans-side occurs much faster,
434
which in turn can promote the more rapid conformational reorientation of the empty transporter.
435
Based on structural features of the bacterial homologues [8, 45, 46], N50 is located within the
436
extracellular region connecting TMD 1 and 2 (Fig. 1). From functional studies of mutant and
437
chimeric PEPT1 proteins, TMD´s one to four and seven to nine contribute to substrate binding
438
[9, 13, 34, 64]. It is thus well conceivable that a glycan attached to N50 with an estimated mass
439
of 5-10 kDa in the oocyte expression system (Fig. 5) can affect the transport characteristics. Our
440
biotinylation experiments demonstrated that the “gain of function for maximal transport” can be
441
reversed to wild type levels by attaching biotin derivatives (Fig. 7). Unlike PEPT1 wild type and
442
N50Q, MTSEA-biotinylation of N50C resulted in a significant increase in the apparent Gly-Sar
443
affinity and a decrease in maximal transport velocity. This effect was more pronounced with a
444
MTSEA-biotin derivative of a higher molecular weight. Although the reagents used to modify
445
cysteine at position 50 are chemically and structurally dissimilar to glycans, they add a mass
446
comparable to 2-4 monosaccharides contained in a glycan. It thus appears that the effect of the
447
glycan on the transport activity is solely or mainly exerted by the molecular mass added,
448
whereas structure and composition of this modification appears not important. It goes without
449
saying that biotin derivatives with higher mass also occupy more space. Thus, it is unclear if it is
13
Glycosylation of PEPT1 at N50
450
due to the added mass or due to steric hindrance that PEPT1 modified at N50 by a glycan or by
451
MTSEA-biotin is significantly slowed in its transport activity.
452
For other transporters such as the intestinal Na+-dependent glucose cotransporter [24], the
453
cystic fibrosis transmembrane conductance regulator [18] or the cardiac Na+/Ca+ exchanger [25]
454
it was shown that the N-linked glycans are not required for protein function. The same applies for
455
N-glycans attached to the large extracellular loop in PEPT1 that connects TMD 9 and 10 [59].
456
Although the glycosylation sites in mouse PEPT1 bring about ~35 kDa of total extra mass, 25 to
457
30 kDa thereof is found attached to residues in the large loop that by current knowledge does
458
not contribute to the transport process.
459
There is ample evidence that protein glycosylation can increase the thermal and dynamic
460
stability [47, 52], solubility [63] and resistance to proteolytic cleavage [11, 35]. In the
461
gastrointestinal tract, PEPT1 has highest expression levels in duodenum and jejunum and these
462
are the tissues facing the highest proteolytic activities in the body [2] with the different proteases
463
derived from pancreatic secretion. Extracellular domains of transport proteins in the apical
464
membrane are thus particularly prone to rapid degradation with loss of functionality. Indeed, N-
465
linked glycosylation of the intestinal anion exchanger SLC26A3 was shown to confer resistance
466
to trypsin [23]. Within PEPT1, the large extracellular loop connecting TMD 9 and 10 has recently
467
been shown to possess a trypsin binding site with residues N550 and E573 identified as
468
important for transporter-enzyme interaction [4]. This association links the degradation of dietary
469
proteins to the transport of the peptides released that could make the overall process more
470
efficient. However, this protein association requires that PEPT1 is intrinsically stable against
471
trypsin.
472
Our study now provides evidence that this is the case and moreover, we demonstrate that this
473
stability is an intrinsic property of the PEPT1 protein backbone and is not conferred by N-linked
474
glycans (Fig. 8). In contrast to trypsin, the resistance of PEPT1 to the fungal enzyme proteinase
475
K was dependent on glycosylation and even correlated with the number of N-glycosylation sites
476
in the transporter. Moreover, treatment of oocytes with the N-glycosylation inhibitor tunicamycin
477
also rendered PEPT1 susceptible to very rapid proteolytic degradation. We previously
478
demonstrated that tunicamycin treatment of oocytes neither inhibited PEPT1 trafficking to the
479
cell membrane nor its expression [59], which suggests that glycosylation is not required for
480
protein folding.
481
We are aware that all processes related to glycosylation in the oocyte expression system -
482
despite its wide use - may differ substantially from those in intestinal epithelial cells as the major
483
site of PEPT1 expression. That may apply N-glycosylation site occupancy, glycan type, glycan
484
size and the chemical nature of the monomers comprising the glycans. However, we here
14
Glycosylation of PEPT1 at N50
485
demonstrate that the changes in the kinetics found for N50 are phenotypically conserved when
486
PEPT1 is expressed in intestinal cells (Fig. 6) and we also showed before that glycosylation of
487
N50 has this effect in the human PEPT1 transporter [59].
488
At present, the precise function and structure of the N-glycan attached to N50 remains unknown.
489
Nonetheless, our experiments show that the removal of this glycan - unlike any of the other
490
glycans - changes markedly the transport characteristics of PEPT1. These changes can be
491
reverted by the covalent attachment of chemically different molecules such as biotin containing
492
structures used here. It thus appears that the exact nature of the glycan is irrelevant for the
493
observed effects on substrate affinity and maximal transport velocity of PEPT1. Since
494
biotinylation reagents with higher masses appear to produce a stronger effect, it seems plausible
495
to assume that the protein domain around N50 is intrinsically mobile and has to undergo
496
structural changes during the transport cycle with a restrained freedom of movement by the
497
mass attached to N50 or steric hindrance. Yet, this model does not readily explain the higher
498
substrate affinity of the PEPT1 variants that have either a glycan or a biotin moiety attached.
499
However, our findings challenge the proposed kinetic model of PEPT1 in which the return of the
500
unloaded carrier to the outside facing conformation determines overall velocity [38, 54].
501
____________________________________________________________________________
502
503
Acknowledgements
504
We thank Helene Prunkl for taking care of the frogs and support in oocyte handling and
505
preparation. We also acknowledge Margot Siebler for excellent technical assistance. We are
506
also very grateful to Prof. D. Haller for providing the Mode-K and PTK-6 cell lines and Maren
507
Ludwig for the support in retroviral transduction experiments.
508
509
510
Grants
511
This work was funded by the Deutsche Forschungsgemeinschaft (DFG) as part of the Research
512
Training Group GRK1482.
513
514
515
Conflict of Interest
516
The authors declare that they have no conflict of interest with the contents of this article.
517
518
15
Glycosylation of PEPT1 at N50
519
Footnotes
520
1
521
Universität München, Lehrstuhl für Ernährungsphysiologie, Gregor-Mendel-Str. 2, 85350
522
Freising,
523
[email protected]
To whom correspondence may be addressed: Chair of Nutritional Physiology, Technische
Germany,
Tel.:
(+49)-8161-713400;
Fax:
(+49)-8161-713999;
E-mail:
524
525
2
526
ApoE, apolipoprotein E; DMSO, dimethyl sulfoxide; Gly-Sar, glycyl-sarcosine; Imax, maximal
527
velocity; I-V, current-voltage relationship; Km, Michaelis constant; NT, non-transduced cells;
528
LCAT, lecithin-cholesterol acyltransferase; Plat-E, Platinum-E cells; SGLT1, sodium-glucose
529
transporter 1; MTSEA-biotin, 2-((biotinoyl)amino)ethyl-methanethiosulfonate; P, cell passage;
530
PEPT1, peptide transporter 1; TEVC, two-electrode voltage clamp; TMD, transmembrane
531
domain; WT, wild type; X. laevis, Xenopus laevis
532
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533
534
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a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and
665
PepT2. EMBO J 30: 417-426, 2011.
666
667
46. Newstead S. Molecular insights into proton coupled peptide transport in the PTR family
of oligopeptide transporters. Biochim Biophys Acta 1850: 488-499, 2015.
668
47. Öberg F, Sjöhamn J, Fischer G, Moberg A, Pedersen A, Neutze R, Hedfalk K.
669
Glycosylation increases the thermostability of human aquaporin 10 protein. J Biol Chem
670
286: 31915-31923, 2011.
671
48. Okano T, Inui K, Maegawa H, Takano M, Hori R. H+ coupled uphill transport of
672
aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border
673
membranes. J Biol Chem 261: 14130-14134, 1986.
674
675
49. Rademacher TW, Parekh RB, Dwek RA. Glycobiology. Ann Rev Biochem 57: 785-838,
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676
50. Raines RT. Ribonuclease A. Chem Rev 98: 1045-1066, 1998.
677
51. Rousset X, Vaisman B, Amar M, Sethi AA, Remaley AT. Lecithin:cholesterol
678
acyltransferase - from biochemistry to role in cardiovascular disease. Curr Opin
679
Endocrinol Diabetes Obes 16: 163-171, 2009.
680
52. Rudd PM, Joao HC, Coghill E, Fiten P, Saunders MR, Opdenakker G, Dwek RA.
681
Glycoforms modify the dynamic stability and functional activity of an enzyme.
682
Biochemistry 33: 17-22, 1994.
683
684
53. Saito H, Inui K. Dipeptide transporters in apical and basolateral membranes of the
human intestinal cell line Caco-2. Am J Physiol 265: G289-294, 1993.
685
54. Sala-Rabanal M, Loo DD, Hirayama BA, Turk E, Wright EM. Molecular interactions
686
between dipeptides, drugs and the human intestinal H+-oligopeptide cotransporter
687
hPEPT1. J Physiol 574: 149-166, 2006.
688
689
690
691
55. Sasaki S, Ishibashi K, Nagai T, Marumo F. Regulation mechanisms of intracellular pH
of Xenopus laevis oocyte. Biochim Biophys Acta 1137: 45-51, 1992.
56. Schwarz F, Aebi M. Mechanisms and principles of N-linked protein glycosylation. Curr
Opin Struct Biol 21: 576-582, 2011.
20
Glycosylation of PEPT1 at N50
692
57. Skropeta D, Settasatian C, McMahon MR, Shearston K, Caiazza D, McGrath KC, Jin
693
W, Rader DJ, Barter PJ, Rye KA. N-Glycosylation regulates endothelial lipase-mediated
694
phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins. J Lipid
695
Res 48: 2047-2057, 2007.
696
58. Steel A, Nussberger S, Romero MF, Boron WF, Boyd CA, Hediger MA. Stoichiometry
697
and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1. J
698
Physiol 498: 563-569, 1997.
699
59. Stelzl T, Baranov T, Geillinger KE, Kottra G, Daniel H. Effect of N-glycosylation on the
700
transport activity of the peptide transporter PEPT1. Am J Physiol Gastrointest Liver
701
Physiol 310: G128-141, 2016.
702
60. Swaan PW, Stehouwer MC, Tukker JJ. Molecular mechanism for the relative binding
703
affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril,
704
enalaprilat, and lisinopril. Biochim Biophys Acta 1236: 31-38, 1995.
705
61. Takuwa N, Shimada T, Matsumoto H, Himukai M, Hoshi T. Effect of hydrogen ion-
706
gradient on carrier-mediated transport of glycylglycine across brush border membrane
707
vesicles from rabbit small intestine. Jpn J Physiol 35: 629-642, 1985.
708
62. Takuwa N, Shimada T, Matsumoto H, Hoshi T. Proton-coupled transport of
709
glycylglycine in rabbit renal brush-border membrane vesicles. Biochim Biophys Acta 814:
710
186-190, 1985.
711
63. Tams JW, Vind J, Welinder KG. Adapting protein solubility by glycosylation. N-
712
glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions.
713
Biochim Biophys Acta 1432: 214-221, 1999.
714
715
64. Terada T, Saito H, Sawada K, Hashimoto Y, Inui K. N-terminal halves of rat H+/peptide
transporters are responsible for their substrate recognition. Pharm Res 17: 15-20, 2000.
716
65. Tsuji A, Terasaki T, Tamai I, Hirooka H. H+ gradient-dependent and carrier-mediated
717
transport of cefixime, a new cephalosporin antibiotic, across brush-border membrane
718
vesicles from rat small intestine. J Pharmacol Exp Ther 241: 594-601, 1987.
719
66. Vidal K, Grosjean I, Evillard JP, Gespach C, Kaiserlian D. Immortalization of mouse
720
intestinal epithelial cells by the SV40-large T gene: Phenotypic and immune
721
characterization of the MODE-K cell line. J Immunol Methods 166: 63-73, 1993.
722
67. Vig BS, Stouch TR, Timoszyk JK, Quan Y, Wall DA, Smith RL, Faria TN. Human
723
PEPT1 pharmacophore distinguishes between dipeptide transport and binding. J Med
724
Chem 49: 3636-3644, 2006.
21
Glycosylation of PEPT1 at N50
725
68. Whitehead RH, Robinson PS, Williams JA, Bie, W, Tyner AL, Franklin JL. A
726
conditionally immortalized colonic epithelial cell line from a Ptk6 null mouse that polarizes
727
and differentiates in vitro. J Gastroenterol Hepatol 23: 1119-1124, 2008.
728
69. Wuensch T, Schulz S, Ullrich S, Lill N, Stelzl T, Rubio-Aliaga I, Loh G, Chamaillard
729
M, Haller D, Daniel H. The peptide transporter PEPT1 is expressed in distal colon in
730
rodents and humans and contributes to water absorption. Am J Physiol Gastrointest Liver
731
Physiol 305: G66-73, 2013.
732
70. Zeuthen T, Zeuthen E, Klaerke DA. Mobility of ions, sugar, and water in the cytoplasm
733
of Xenopus oocytes expressing Na+-coupled sugar transporters (SGLT1). J Physiol 542:
734
71-87, 2002.
735
736
737
738
739
740
Figure legends
741
742
Fig. 1. Secondary structural model of the murine PEPT1 transporter
743
Based on hydropathy plot analysis with the TMHMM server v. 2.0 [33], PEPT1 is composed of
744
12 alpha-helical transmembrane domains with amino- and carboxy-termini facing the cytosolic
745
side. The murine PEPT1 transporter has six canonical N-glycosylation sites localized in the first
746
(N50) and fifth (N406, N439, N510, N515 and N532) extracellular loops occupied by N-glycans.
747
PEPT1 contains a total of 12 cysteine residues. According to this model, five cysteine residues
748
(C25, C189, C197, C540, C566) are exposed on the extracellular surface, while seven cysteines
749
are located intracellularly (C142, C239, C351) or within transmembrane helices (C9, C63, C594,
750
C661; not shown).
751
752
Fig. 2. [14C]-Gly-Sar efflux studies in oocytes
753
Oocytes expressing PEPT1 wild type (WT) or N50Q were preloaded with [14C]-labeled Gly-Sar
754
by incubation for 10 min. The intracellular radioactivity in oocytes after preloading was set to
755
100% and the efflux determined by counting the increase in radioactivity over 20 min in the
756
extracellular medium. Data are depicted as mean ± SEM of 20 oocytes and corrected for
757
background radioactivity observed in water injected control oocytes (data not shown). Two-tailed
758
unpaired t-test (* P < 0.05, ** P < 0.01).
759
22
Glycosylation of PEPT1 at N50
760
Fig. 3. Electrophysiological recordings of Gly-Sar efflux in oocytes
761
A and B: I-V relations of Gly-Sar induced currents in oocytes expressing PEPT1 wild type (A) or
762
N50Q (B). Dipeptide-evoked inward currents were recorded from oocytes superfused with 40
763
mM Gly-Sar at pH 7.5 (dashed lines, n = 20). Reverse substrate transport was measured after
764
injection of oocytes with 1 M Gly-Sar (pH 7.5) to an intracellular concentration of 22 to 127 mM.
765
The insets show the time course of outwardly directed membrane currents at +60 mV. The solid
766
black lines represent outward currents recorded after 6 min in oocytes (n = 20-30) following
767
microinjection of Gly-Sar to a cytosolic concentration of 22 mM, 43 mM and 127 mM. Data were
768
corrected for transport in water injected control oocytes and depicted as mean ± SEM.
769
770
Fig. 4. TEVC analysis of PEPT1 transporters N50Q, N50K and S52A
771
A and B: Apparent affinity constants (A) and maximal inward currents (B) at -60 mV for PEPT1
772
wild type, N50Q, N50K and S52A. All measurements were performed with Gly-Sar (0.3-40 mM,
773
pH 6.5). The negative inward current recordings were normalized to negative inward currents of
774
1 mM alpha-MDG (pH 6.5), resulting in positive Imax values. Statistical analysis: One-way
775
ANOVA with Dunnett´s posttest, confidence interval 95% (** P < 0.01, *** P < 0.001). Km and Imax
776
values are indicated as median ± min/max (mean marked with +) of 15-20 oocytes.
777
C: Dependence of the maximal Gly-Sar induced inward currents on the membrane potential.
778
Currents recorded over a Gly-Sar concentration range of 0.3-40 mM at pH 6.5 (n = 8) in
779
individual oocytes were fitted to the Michealis-Menten equation by non-linear regression analysis
780
using the least-squares method and average Imax values calculated for oocytes within the
781
membrane potential range of 0 mV to -160 mV. Data were not normalized and are presented as
782
mean ± SEM.
783
784
Fig. 5. Immunoblot analysis of PEPT1 N50Q and S52A
785
A: Western blot analysis of whole cell extracts from oocytes revealed differences in the migration
786
of PEPT1 between the wild type and N-glycosylation defective transporters. In comparison to
787
wild type PEPT1 (molecular mass ~95 kDa), the transporters N50Q and S52A revealed a 5-10
788
kDa lower PEPT1 mass.
789
B: Analysis of the immunoblot (Fig. 5A) with the LI-COR imaging software Image Studio Lite (v.
790
3.1, LI-COR Biosciences, Bad Homburg, Germany). Distinct changes in the location of the
791
maximal fluorescence intensity for the PEPT1 wild type and the N-glycosylation deficient
792
transporters are apparent. The fluorescence maximum for PEPT1 wild type is marked with a
793
dashed line.
794
23
Glycosylation of PEPT1 at N50
795
Fig. 6. N50Q displays increased transport activity in mammalian cells
796
A and B: Immunoblotting of PEPT1 wild type expressed in Mode-K and PTK-6 cells revealed the
797
existence of a distinct mass difference between both cell lines (A). While Mode-K cells (murine
798
epithelial cells derived from the small intestine) exhibited a PEPT1 mass of about ~95 kDa, an
799
increase to ~100 kDa was observed in colonic epithelial PTK-6 cells.
800
High resolution imaging of the Western-Blot with the LI-COR imaging software Image Studio Lite
801
(B) illustrates the localization of the maximal fluorescence intensities for PEPT1 in Mode-K and
802
PTK-6 cells (marked with a dashed line).
803
C and D: [14C]-Gly-Sar uptake experiments performed with Mode-K (C) and PTK-6 (D) cells
804
demonstrated a higher rate of tracer transport for PEPT1 N50Q relative to wild type. Uptake
805
values were standardized for transporter abundance as determined by Western blot (data not
806
shown). Non-transduced cells (NT) neither revealed PEPT1 expression (data not shown) nor
807
uptake of [14C]-Gly-Sar.
808
809
Fig. 7. Characterization of PEPT1 N50C in oocytes: biotin mimics glycan attachment
810
A and B: Apparent affinity constants (A) and maximal inward currents (B) at -60 mV for PEPT1
811
wild type, N50Q or N50C. TEVC experiments were performed with 0.3-40 mM Gly-Sar at pH 6.5.
812
Kinetic parameters of dipeptide transport were determined for individual transporters with and
813
without MTSEA-biotin labeling. Chemical modification was performed by incubating intact
814
oocytes for 15 min in 2 mM MTSEA-biotin, MTSEA-biotin-X, or MTSEA-biotin-XX prior to
815
electrophysiological recordings. Biotinylation of water-injected oocytes did not induce inward
816
transport currents (data not shown). Km and Imax values are indicated as mean ± min/max of four
817
independent measurements of 15-20 oocytes. Statistical analyses were performed by 1-way
818
ANOVA versus the untreated control (* P < 0.05, ** P < 0.01, *** P < 0.001).
819
C-E: Maximal Gly-Sar induced currents as a function of membrane potential. Representative Imax
820
values calculated for biotin-labeled and unlabeled oocytes (n = 6-8) expressing PEPT1 wild type
821
(C), N50Q (D) or N50C (E) at membrane potentials of 0 to -160 mV.
822
823
Fig. 8. N-glycosylation of PEPT1 and resistance towards proteolysis
824
A-D: Total protein extracts (15 µg) prepared from oocytes expressing PEPT1 wild type or N-
825
glycosylation defective versions were incubated with trypsin (A and B) or proteinase K (C and
826
D). After protease treatment, cell extracts were subjected to Western blot analysis and PEPT1
827
abundance quantified from 4-6 individual experiments. Representative Western blots are shown
828
in B and D. The extent of proteolysis was determined by comparing the PEPT1-specific
829
fluorescence intensities between untreated and protease-treated cell extracts. Changes in the
24
Glycosylation of PEPT1 at N50
830
fluorescence of the target protein after protease treatment were expressed as percent with
831
respect to the untreated control. All data are indicated as mean ± SEM of ≥ 15 oocytes out of six
832
oocyte batches. Student's unpaired t-test was used to compare the protease resistance of the
833
PEPT1 N-glycosylation versions to the wild type protein (* P < 0.05, ** P < 0.01, *** P < 0.001).
834
835
836
837
838
839
840
841
842
843
844
845
Tables
846
847
Table 1. Primer sequences used for site-directed mutagenesis of murine PEPT1
Mutation
N50Q
Megaprimer
1
N50K
S52A
Primer Sequence (5´ 3´)
Tm, °C
N50Q_for
CTGGGACGACCAACTCTCCACGG
63.5
WT_reva)
ATAGGGCCCGATATCTCACATATTTGTCTGTGAG
ACTGGTTCCAATG
75.9
WT_forb)
TATCTCGAGATGGGGATGTCCAAGTCTCGGGG
72.1
N50Q_rev
CCGTGGAGAGTTGGTCGTCCCAG
63.5
1
N50C_for
CTGGGACGACTGTCTCTCCACGG
61.7
2
N50C_rev
CCGTGGAGAGACAGTCGTCCCAG
61.7
1
N50K_for
CTGGGACGACAAGCTCTCCACGG
63.7
2
N50K_rev
CCGTGGAGAGCTTGTCGTCCCAG
63.7
1
S52A_for
CTGGGACGACAATCTCGCCACGG
66.2
2
N50C
Primer
25
Glycosylation of PEPT1 at N50
2
S52A_rev
CCGTGGCGAGATTGTCGTCCCAG
848
a/b)
849
mutagenesis are marked in bold letters and restriction sites for XhoI and EcoRV are underlined.
66.2
Flanking primers used for the generation of megaprimers. Amino acids modified by site-directed
850
851
852
26
Figure 1.
Figure 2.
Figure 3.
A
B
Figure 4.
A
B
C
Figure 5.
A
B
Figure 6.
A
C
B
D
Figure 7.
A
B
C
D
E
Figure 8.
A
C
B
D