To study the significance of N-glycosylation, green fluor-escence protein GFP-tagged wild type GAT1 NNN and N-glycosylation defective mutants DDQ, DGN, DDN and DDG were expressed in CHO ce
Trang 1GABA-uptake of GABA-transporter 1
Terminal N-glycans facilitate efficient GABA-uptake activity
of the GABA transporter
Guoqiang Cai1,2, Petrus S Salonikidis3, Jian Fei1, Wolfgang Schwarz3, Ralf Schu¨lein4,
Werner Reutter2and Hua Fan2
1 Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China
2 Institut fu¨r Molekularbiologie und Biochemie, CBF, Charite´ Universita¨tsmedizin Berlin, Berlin-Dahlem, Germany
3 Max-Planck Institut fu¨r Biophysik, Frankfurt, Germany
4 Forschungsinstitut fu¨r Molekulare Pharmakologie, Berlin-Buch, Germany
The cellular membrane transporter for the inhibitory
neurotransmitter c-aminobutyric acid (GABA) belongs
to a family of secondary active systems that are driven
by electrochemica1 gradients of Na+and Cl– [1] The
main physiological function of the transporter is believed to be the control of the concentration and dwell time of GABA in the synaptic cleft Because the transport of one molecule of GABA is coupled to the
Keywords
GABA transporter; N-glycosylation; N-glycan
trimming; membrane trafficking;
patch-clamp
Correspondence
H Fan, Institut fu¨r Molekularbiologie und
Biochemie, Campus Bejamin Franklin,
Charite´ Universita¨tsmedicin Berlin,
Arnimallee 22, D-14195 Berlin-Dahlem,
Germany
Fax: +49 30 84451541
Tel: +49 30 84451544
E-mail: hua.fan@charite.de
(Received 17 July 2004, revised 24 January
2005, accepted 2 February 2005)
doi:10.1111/j.1742-4658.2005.04595.x
Neurotransmitter transporters play a major role in achieving low concen-trations of their respective transmitter in the synaptic cleft The GABA transporter GAT1 belongs to the family of Na+- and Cl–-coupled trans-port proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4 To study the significance of N-glycosylation, green fluor-escence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM) This indicates that N-glycosylation, but not terminal trimming of the N-glycans
is involved in the attainment of a correctly folded and stable conformation
of GAT1 All N-glycosylation mutants were expressed on the plasma mem-brane, but they displayed markedly reduced GABA-uptake activity Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity Further experiments showed that both N-glycosylation mutations and dMM reduced the Vmax value, while not increasing the Km value for GABA uptake Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extra-cellular Na+and slowed kinetics of the transport cycle This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1 They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding
Abbreviations
CHO, Chinese hamster ovary; dMM, 1-deoxymannojirimycin; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; GABA, c-aminobutyric acid; GAT1, GABA transporter type I; GFP, green fluorescence protein.
Trang 2cotransport of two Na+ ions and one Cl– ion [2–4],
the translocation across the membrane is associated
with a current that can be measured by voltage
clamp-ing In the absence of GABA the transport cycle is
not completed, but transient charge movements can be
detected that reflect partial reactions associated with
extracellular Na+ binding and hence provide kinetic
information about the transport cycle [5–7]
Four subtypes of GABA transporters (GAT1–4)
have been found so far [8,9] GABA transporter type
1 (GAT1) is a single polypeptide of about 67 kDa
with 12 putative transmembrane domains Both
N- and C-termini are located in the cytoplasm The
large extracellular loop between transmembrane
domains 3 and 4 contains three conserved
N-glyco-sylation sites (Asn176, Asn181 and Asn184) It has
been demonstrated that all three N-glycosylation sites
are used in vivo and that no additional sites are
pre-sent [10]
N-glycosylation is a major post-translational
modi-fication in eukaryotic cells Recent results suggest
that this post-translational modification may influence
many of the physicochemical and biological
proper-ties of the proteins, such as protein folding, stability,
targeting, dynamics and ligand binding, as well as
cell-matrix and cell–cell interactions [11–16] It has
been suggested that N-glycosylation is involved in
the regulation of the transport activity and surface
expression of neurotransmitter transporters [10,17]
Functional expression of the GABA transporter is
abolished by tunicamycin, a potent inhibitor of
N-glycosylation [18] Experiments with HeLa
trans-fectants showed that removal of one or two
glycosy-lation sites by site-directed mutagenesis had little
effect on the expression of GABA-uptake activity
However, removal of all three N-glycosylation sites
resulted in a reduction of GABA-uptake activity [10]
Although such experiments indicate that
N-glycosyla-tion mutaN-glycosyla-tions lead to a reducN-glycosyla-tion of GABA-uptake
activity, we do not know how the N-linked
oligosac-charide side chains influence the function of this
trans-porter Liu et al demonstrated in Xenopus oocytes
that mutations of two of the three N-glycosylation
sites led to a reduction in turnover rates and complex
changes in the interaction of external Na+ with the
transport protein as measured by voltage clamping [7]
However, the question remained as to whether the
reduction in function of the mutants was due to a
change in the biochemical properties of this
transpor-ter or to a reduction in the number of GABA
trans-porters per cell In order to clarify the functional
significance of N-glycosylation and N-linked
oligosac-charides in GAT1, green fluorescence protein
(GFP)-tagged wild type GAT1 (NNN) and four glycosylation mutants (DDQ, DGN, DDN, and DND) were stably expressed in CHO cells lacking endogenous GAT1 The influence of N-glycosylation mutations and inhibi-tion of N-glycosylainhibi-tion processing on biochemical properties and function were investigated
In this work, we demonstrated that defective N-gly-cosylation resulted in reduction of the stability and a decrease in the cell surface expression of this protein The GAT1 mutants containing two N-glycosylation mutations showed a delayed intracellular translocation, but they targeted to the plasma membrane and showed reduced GAT1-specific GABA-uptake activity If all three N-glycosylation sites were eliminated, a decreased percentage of DDQ mutants was found on the cell surface However, the GABA-uptake activity could hardly be detected in this mutant Inhibition of N-glycosylation processing by 1-deoxymannojirimycin (dMM) affected neither the cell surface expression nor stability of this protein, but it resulted in marked reduc-tion of GABA-uptake activity This suggests that N-glycans, in particular terminal structures of N-gly-cans, are involved in the GABA-uptake process of GAT1 Finally, we found that deficiency of N-glycosy-lation did not affect the affinity of GAT1 for GABA The observed reduction of GAT1-specific GABA-uptake due to deficiency of N-glycans was attributed to
a reduced apparent affinity for extracellular Na+ions, resulting in a reduction of the kinetics of the transport cycle
Results
Expression of GAT1/GFP fusion proteins
in CHO cells cDNAs of GFP tagged wild type (NNN) and mutants DND, DDN, DGN and DDQ were transfected into CHO cells, which do not express endogenous GAT1 and GFP Stable transfectants were selected by fluores-cence activated cell sorting (FACS) Flow cytometry analysis showed the expression of NNN and the mutants on the surface of transfected CHO cells (Fig 1A) Fluorescence and immunofluorescence micro-scopy showed that both GFP-fluorescence and anti-GAT1 antibodies can be used to detect the expression
of the GAT1⁄ GFP fusion protein (NNN) on surface and interior of CHO cells (Fig 1B,C)
The expression of NNN was determined by West-ern blotting with either anti-GAT1 pAb (Fig 2A) or anti-GFP mAb (Fig 2B) following immunoprecipita-tion with anti-GFP pAb This GAT1⁄ GFP fusion protein showed several bands in SDS⁄ PAGE, two
Trang 3monomeric forms running as a main band of about
108 kDa and a small band of about 96 kDa The
108 kDa polypeptide was resistant to Endo H diges-tion, while the 96 kDa polypeptide was converted into a polypeptide of 90 kDa after digestion with Endo H (Fig 2B) Digestion of both monomeric forms with PNGase F resulted in a single 90 kDa N-glycan-free peptide (Fig 2C, lane 2) This indicates that the 108 kDa peptide contains mature N-glycans
of the complex type, while the 96 kDa peptide con-tains only N-glycans of the mannosidic type The
210 kDa band may represent a dimeric form or a protein aggregate In addition, inhibition of N-glyco-sylation processing of NNN by 1-deoxymannojirimy-cin (dMM) leads to the reduction of NNN molecule mass to 96 kDa (Fig 2C, lanes 3 and 6) After diges-tion with either PNGase F or Endo H, this 96 kDa polypeptide was converted to a 90 kDa N-glycan-free polypeptide (Fig 2C, lanes 4 and 5), indicating that the 96 kDa polypeptide contains only N-glycans of oligomannosidic type
Fig 1 Flow cytometry, fluorescence microscopy and
immunofluo-rescence microscopy of GFP-tagged GAT1 in transfected CHO
cells (A) Flow cytometry of GFP-tagged GAT1 wild type and
mutants The polyclonal anti-GAT1 IgG was used for
immunostain-ing Visualization was performed with R-phycoerythrin-conjugated
goat anti-(rabbit IgG) Ig NNN, GFP-tagged wild type GAT1 DND,
DDN, DGN and DDQ, GFP-tagged N-glycosylation mutants (B)
Fluorescence microscopy of NNN The fluorescence of GFP in
GFP ⁄ GAT-fusion protein (NNN) was detected (C)
Immunofluores-cence microscopy of NNN Anti-GAT1 polyclonal antibodies were
used for immunostaining after cell fixation and permeabilization.
Visualization was performed with R-phycoerythrin-conjugated goat
anti-(rabbit IgG) Ig.
Fig 2 Protein expression and N-glycosylation processing of GFP-tagged GAT1 in CHO cells NNN stable transfected CHO cells were incubated with and without dMM (1 m M ) for 72 h The solubilized protein of transfected cells (1 · 10 7
) was subjected to immunopre-cipitation with anti-GFP Igs Aliquots of each immunoprecipitate were treated either with Endo H or PNGase F The resulting mix-ture and the other aliquots of the immunoprecipitate were analyzed
by SDS ⁄ PAGE (7.5%) and immunoblotting with anti-GAT1 pAb (A)
or anti-GFP mAb (B, C).
Trang 4Expression of N-glycosylation mutants
on the plasma membrane of CHO cells
The expression of NNN and N-glycosylation mutants
DGN, DDN, DND and DDQ on the plasma membrane
of CHO cells was investigated As shown in Fig 3A,
N-glycosylation mutants exhibited a reduced molecular
mass in comparison to that of the wild type NNN
Nevertheless, all N-glycosylation mutants, as well as the
wild type NNN, were expressed on the plasma
mem-brane Although all three N-glycosylation sites are
absent in DDQ, this mutant was also detected on the
plasma membrane of CHO cells, suggesting that
N-gly-cosylation or N-linked oligosaccharides are important,
but not essential for the translocation of GAT1 to the
cell surface All intracellular proteins of wild type as
well as mutants (with the exception of DDQ) gave two
bands, while plasma membrane proteins gave only one
large band (Fig 3A) On the basis of the Endo H
diges-tion (Fig 2B,C), the large band is assigned to proteins
with N-glycans of mature complex type and the small
band to the proteins with N-glycans of mannose-rich
type All bands of the mutants had reduced molecular
mass, compared with that of wild type NNN The
reduced molecular masses of mutants are compatible
with the absence of N-glycans at the two eliminated
N-glycosylation sites, suggesting that the mutants in the
cell interior contain N-glycans of both mannosidic and
complex types, while those in the plasma membrane
contain only N-glycans of the mature complex type
The relative levels of surface vs intracellular GAT1
and mutants in a steady expression state were
quanti-fied The distributions between cell surface and cell
interior of the mutants DGN, DND and DDN were
not significantly different from that of wild type NNN
About 46 ± 4.7% is found on the cell surface
How-ever, the percentage of the cell surface expression in
mutant DDQ which lacks all three N-glycosylation
sites was only 30 ± 4.4% in the steady expressed state
(Fig 3B)
N-Glycosylation mutations result in reduction
of GABA-uptake activity
For quantitative measurement of the specific activity
of GABA-uptake, an aliquot of the stable CHO
trans-fectants was used for the GABA-uptake assay, and
another aliquot was used to determine the amount of
the membrane-expressed wild type or mutant proteins
The GABA-uptake activities were normalized to the
same amount of cell surface proteins of wild type and
mutants Compared with that of the wild type, the
GABA-uptake activities of the N-glycosylation
mutants were reduced significantly Figure 3C shows that the GABA-uptake activities of mutants with double N-glycosylation mutations, DND, DGN and
Fig 3 Determination of expression of GFP-tagged GAT1 mutants
on the surface of transfected CHO cells and measurement of GABA-uptake by GFP-tagged GAT1 wild type and mutants in trans-fected CHO cells (A) Cell surface and intracellular expression of GFP-tagged GAT1 wild type (NNN) and mutants (DGN, DND, DDN, and DDQ) were analyzed by biotin labelling and Western blotting Anti-GAT1 serum or anti-GFP mAb MAB2510 were used for immu-nostaining I, intracellular expression; M, plasma membrane expres-sion (B) The protein bands obtained in western blotting were analyzed by phosphoimager scanning Each value represents the mean ± SEM of three separate experiments The total protein of the cell surface and intracellular bands of each wild type or mutant were set at 100% (C) Measurement of GABA-uptake by GFP-tagged GAT1 wild type and mutants in transfected CHO cells The measured GABA-uptake activity was normalized to the amount of GAT1 or mutant protein expressed on the plasma membrane The activity of GABA-uptake by NNN was set at 100% All other values were expressed relative to this value The values represent the mean ± SEM of four separate experiments.
Trang 5DDN, were reduced to 64% (±5.6%), 42% (±12.4%)
and 32% (±8.2%) of that of NNN, respectively
GAT1-mediated transport could hardly be detected in
the mutant DDQ, although this mutant was expressed
on the plasma membrane Mutant DDQ does not
exhi-bit any N-glycosylation site Because all values were
normalized to the transporter proteins in the plasma
membrane, the reduced specific activities of the
mutants are not due to a reduced number of GABA
transporters per cell, but to a reduced transport rate
This suggests that N-linked oligosaccharide side-chains
are important for the GABA transport activity
1-Deoxymannojirimycin inhibits the GABA-uptake
of GAT1
In order to gain further insight into the role of the
terminal structures of the N-glycans of GAT1,
N-gly-cosylation processing of NNN was inhibited by
1-de-oxymannojirimycin (dMM) Inhibition by dMM leads
to the formation of NNN molecules containing
N-gly-cans of oligomannosidic type Figure 4A shows that
after treatment with dMM (1 mm) for 72 h, the
amount of plasma membrane NNN containing
man-nosidic N-glycans was in the same range as that of
NNN containing mature complex N-glycans without
treatment with dMM However, the activity of
GABA-uptake was reduced to 37% after treatment with dMM
(Fig 4B) This indicates that the terminal trimming of
N-oligosaccharides is not involved in the regulation of
plasma membrane trafficking of GAT1, but in the
regulation of GABA uptake
As well as wild type, mutant DND, DGN and
DDN exhibited only one small band on SDS⁄ PAGE
after treatment with dMM (data not shown),
indica-ting that, like wild type, they contain only mannosidic
N-glycans The level of cell surface expression was
sim-ilar with and without dMM treatment for both wild
type and mutants (Figs 4A and 5A) However, their
GABA-uptake activity was reduced to half after
treat-ment with dMM (1 mm) for 48 h (Fig 5B) Although
mutant DND, DGN and DDN contain only one
N-glycosylation site, deficiency of terminal trimming of
their N-oligosaccharides strongly affected their
GABA-uptake activity These indicate that the terminal
structure of the oligosaccharides facilitate efficient
GABA-uptake activity of the GABA transporter
Defective N-glycosylation results in reduction
of the stability of GAT1
In order to study the influence of the N-glycosylation
and N-linked oligosaccharides on protein stability, the
intracellular decay time of the GAT1, GAT1 treated with dMM, and the mutants was determined by pulse-chase experiments (Fig 6) Figure 6B shows that wild type NNN was very stable with an exponential half-life
of about 22 h, whereas the half-life of mutant DDN containing two N-glycosylation mutations was reduced
to 12 h The half-life of DDQ containing all three N-glycosylation mutations was reduced even more, compared with that of DDN, showing a value of only 5.5 h In contrast, the stability of NNN containing only mannosidic N-glycans after treatment with dMM
Fig 4 Influence of dMM on plasma membrane trafficking and GABA-uptake of GFP-tagged GAT1 wild type NNN stable trans-fected CHO cells were incubated with and without dMM (1 m M ) for 72 h (A) Aliquots of cells were used for membrane biotinyla-tion After solubilization, 300 lg total proteins of cell lysates were precipitated with streptavidin beads The eluates were analyzed by Western blotting using anti-GFP mAb (B) Another aliquot of cells was used for measurement of GABA-uptake as described above The values represent the mean ± SEM of three separate experi-ments.
Trang 6is similar to that of NNN containing N-glycans of the
mature complex type The results suggest that
N-glyco-sylation is important for the stability of this protein,
but the terminal structure of the N-glycans is not
Defective N-glycosylation reduces the trafficking
of GAT1 to the plasma membrane
In order to study the influence of N-glycosylation on
plasma membrane trafficking of GAT1, the
distribu-tion of wild type and mutants on the cell surface and
in the cell interior was kinetically analyzed by
pulse-chase experiments Figure 7 shows that after a 40 min
chase, 34% of total wild type (NNN) proteins, whereas
only 18 and 12% of total mutant DDN and DDQ
proteins, respectively, were expressed on the plasma
membrane After a 120-min chase, the membrane
expression of the NNN was increased to 50%, whereas
that of mutant DDN and DDQ was increased only to
40% and 15%, respectively This result suggests that
deficiency of N-glycosylation impairs the plasma mem-brane trafficking of GAT1
Defective N-glycosylation or dMM treatment did
not increase the KmGABA values of GAT1 The above results show that both N-glycosylation mutations and terminal structures of N-linked oligo-saccharide side chains have a measurable effect on the GABA-uptake activity of GAT1 To determine whe-ther the N-linked oligosaccharide side chains of GAT1 influence the affinity of GAT1 for GABA, concentra-tion dependencies were analyzed on the basis of the Michaelis–Menten equation
KmGABAþ ½GABA
and the parameters for NNN with and without treat-ment with dMM, and for N-glycosylation mutant DDN were determined As shown in Fig 8, the Vmax
Fig 5 Analysis of the cell surface expres-sion and GABA-uptake activity of GFP-tag-ged GAT1 wild type and mutants after treatment with dMM CHO stable transfect-ants were incubated with and without dMM (1 m M ) for 48 h (A) Cell surface expression
by FACS analysis Anti-GAT IgG was used for the immunostaining Visualization was performed with R-phycoerythrin-conjugated goat anti-(rabbit IgG) Ig (B) GABA-uptake activity The GABA-uptake activities were normalized to the amount of GAT1 or
muta-nt protein expressed on the plasma mem-brane The activity of GABA-uptake by NNN was set at 100% The values represent the mean ± SEM of five separate experiments.
Trang 7GABA values of NNN treated with dMM, and of
mutant DDN were reduced significantly The Vmax
pmolÆlgÆprotein)1Æmin)1, whereas the value for mutant
DDN was only 0.29 pmolÆlgÆprotein)1Æmin)1 After
treatment of NNN with dMM, the Vmax GABA value
of NNN containing mannose-rich N-glycans was
strongly reduced to 0.55 pmolÆlgÆprotein)1Æmin)1
Although mutations at N-glycosylation sites, as well as
N-glycans of the oligomannosidic type reduced the
Vmax value of rate of GABA uptake markedly, the Km
GABA values were not affected The data in Fig 8 were fitted with a common KmGABA value of 4.1 lm These results suggest that the defect of N-linked oligo-saccharides did not reduce the binding affinity of GAT1 to GABA As treatment with dMM did not affect GAT1 protein translocation to the plasma mem-brane, the decreased GABA-uptake activity of NNN
Fig 6 Biological stability of GFP-tagged GAT1 wild type and
mutants in transfected CHO cells (A) CHO stable transfectants
(2 · 10 6 cells per dish) were preincubated with and without dMM
(1 m M ) for 72 h, then pulse-labelled with 3.7 · 10 6
Bq per dish [ 35 S]methionine for 1 h and immediately chased for the stated
times Immunoprecipitates of cell lysates obtained at the indicated
chase-times were analyzed by SDS ⁄ PAGE (B) The results of the
pulse-chase experiments were analyzed by phosphoimager
scan-ning The radioactivities obtained by immunoprecipitation of the
pulse-labelled cells without chase were set at 100% All other
val-ues were expressed relative to this value Each time point
repre-sents the mean ± SEM of three separate experiments Solid lines
represent the exponential fit with half-lives of 22 and 23 h for
wild type GAT1 in the absence and presence of dMM, respectively,
and of 12 and 5.5 h for the DDN and DDQ mutants, respectively.
Fig 7 Plasma membrane trafficking of GFP-tagged GAT1 wild type and mutants in transfected CHO cells (A) CHO stable transfectants were pulse-labelled with 3.7 · 10 6 Bq per dish [ 35 S]methionine for
1 h and chased for 0 min, 40 min, 80 min, 120 min and 180 min Membrane biotinylation was performed after chase After cell solu-bilization, total GFP-tagged GAT1 wild type and mutant proteins were immunoprecipitated with anti-GFP pAb and eluted with
100 lL sample buffer containing 0.5% SDS The eluates were dilu-ted with NaCl ⁄ P i buffer to 400 lL The biotin-labelled membrane proteins were isolated from the diluted eluates with streptavidin beads After removal of all membrane proteins, the intracellular pro-teins were immunoprecipitated with anti-GFP antibodies Both M (membrane) and I (intracellular) precipitates were eluted and ana-lyzed by SDS ⁄ PAGE (B) The results of the pulse-chase experi-ments were analyzed by phosphoimager scanning The total radioactivity of membrane and intracellular fractions obtained by immunoprecipitation at each chase time were set at 100% Each value represents the mean ± SEM of membrane fractions derived from three separate experiments.
Trang 8after treatment with dMM may be caused by the
reduction in substrate translocation by GAT1
(turn-over rate)
Defective N-glycosylation results in reduced
GAT1-mediated currents and reduced rate of
external Na+interaction
To obtain additional information on the mechanism of
the reduced rate of GABA-uptake due to the
muta-tions, we performed electrical measurements under
voltage clamp for wild type and mutant DDN and
DGN The number of transporters was calculated
from the transient charge movement in the absence of
extracellular Na+ [5–7] Figure 9A shows the
depend-ence of the GAT1-mediated current, expressed as
charges translocated per functional transporter on the
cell surface per second, on the extracellular Na+
con-centration The results were similar to those for the
GABA uptake, in that the current produced by a
sin-gle transporter was reduced by the mutation to 46 and
57% for DGN and DDN, respectively, which is close
to the reduced GABA uptake seen in the flux
measure-ments A signal from DDQ could hardly be detected
Though treatment with dMM makes the CHO cells
very unstable for the patch-clamp method, we were,
nevertheless, able to obtain evidence for a reduced
GAT1-mediated current (data not shown) The
dependence on Na+ concentration reveals that muta-tion of the two N-glycosylamuta-tion sites reduced the apparent affinity from 24 m)1 to about 8 m)1 The transient currents in the absence of GABA were ana-lyzed for jumps in potential to the holding potential of )30 mV The kinetics of the reaction step associated with the extracellular Na+ binding was slowed down
by both mutations (Fig 9B) All the rate constants slightly increased with increasing Na+ concentration,
Fig 8 Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild
type (NNN) with and without dMM and N-glycosylation mutant
(DDN) Kinetic analysis of GABA-uptake by GFP-tagged GAT1 wild
type (NNN) with and without dMM and N-glycosylation mutant
(DDN) GABA-uptake assays of wild type NNN pre-incubated with
and without dMM (1 mM) and mutant DDN were performed with
different GABA concentrations All values presented were
calcula-ted after subtraction of the mock values The data were fitcalcula-ted by a
Michealis–Menten equation with a common Km value of 4.1 lM
and Vmaxvalues of 1.21, 0.55 and 0.29 pmolÆlg protein)1Æmin)1for
wild type without and with dMM and for DDN, respectively The
values represent the mean ± SEM of three separate experiments.
Fig 9 Electrophysiological characterization of GAT1-mediated steady-state and transient currents CHO transient transfectants were subjected to whole-cell patch clamp (A) Steady-state, GABA-induced currents were determined during voltage pulses from a holding potential of )30 mV to )100 mV at different extracellular
Na+concentrations The solid lines represent fits of the Hill equa-tion with Hill coefficients of n ¼ 1.3 for the wild type GAT1 and
n ¼ 1 for the mutants (as used previously by Liu et al 1998) and
K m Na values of 40 and about 130 mM for wild type and the mutants, respectively (B) The rate constants of the GAT1-mediated current decline in response to a voltage jump from +100 mV to )30 mV were determined at different extracellular Na +
concentra-tions All data are averages of 3 to 9 determinations ± SEM.
Trang 9the value for the wild type NNN being about twice
that for the mutants
Discussion
There is increasing evidence that cotranslational
N-gly-cosylation crucially influences the three-dimensional
structure, the biological half-life and intracellular
traf-ficking of proteins It is also essential for many
recog-nition processes [13,14,16] Previous studies showed
that the mutation of N-glycosylation sites resulted in a
reduction of GABA-uptake activity by GAT1 [7,10]
However, the possibility that this reduction in function
results from a decrease in the number of GABA
trans-porters per cell was not excluded In order to clarify
whether N-glycans are directly involved in the
GABA-uptake process and whether the modulation of
N-gly-cosylation influences the biochemical properties of this
protein, quantitative and kinetic analysis of GABA
transport expression and activity was performed using
stable CHO transfectants Both our own and
commer-cially available anti-GAT1 antibodies were unsuitable
for the quantitative analysis of GAT1, as they bind
very weakly to this protein Therefore, wild type
GAT1 and N-glycosylation defective mutants were
tagged with GFP, which has been reported not to
influence the intracellular distribution of GAT1;
more-over, the tag does not modulate the relevant functions
of GAT1 [20] For the quantitative analysis of GABA
transport activity, the cell surface expression of GAT1
wild type and N-glycosylation mutants was determined
by cell surface biotinylation and the resulting values
were used for normalization This is a well established
method for the quantitative analysis of cell surface
proteins, in which the biotinylation reagent does not
react with intracellular proteins [21]
We found that all N-glycosylation mutants were
expressed on the cell surface, even when all three
N-glycosylation sites have been removed, e.g in
mutant DDQ (Fig 3A), indicating that
N-glycosyla-tion is not essential for the plasma membrane
traffick-ing of GAT1 However, the mutant DDQ was
expressed at lower levels on the cell surface, indicating
that deficiency of N-glycosylation impairs plasma
membrane translocation of GAT1 It was reported that
deficiency of N-glycosylation influenced the
intracellu-lar trafficking of some glycoproteins [22–25] In order
to examine whether the N-glycosylation of GAT1 has
influence on its plasma membrane trafficking, a kinetic
analysis by pulse-chase experiments was performed
These experiments revealed that the plasma membrane
trafficking of N-glycosylation mutant proteins was
reduced (Fig 7), although the distributions between
cell surface and cell interior of the mutants DGN, DND and DDN in the steady expression state were not significantly different from that of wild type NNN (Fig 3B) It has been reported that, in the endoplasmic reticulum (ER) and in the early secretory pathway, the N-glycans play a pivotal role in protein folding, olig-omerization, quality control, sorting, and transport Thus defective N-glycosylation may lead to a misfold-ing of this protein, resultmisfold-ing in its partial retention in the ER and its rapid digestion thereafter [16,19] Accordingly, our results indicate that the intracellular trafficking of N-glycosylation mutants was delayed, and⁄ or partly inhibited due to retention of some mutant protein in the ER, followed by digestion The transfectants of GAT1 wild type (NNN) and mutant DGN, DND and DDN exhibited in SDS⁄ PAGE (Fig 3A) two intracellular bands, whereas only one large band was found in the plasma membrane fraction However, treatment of the transfectants with dMM, which inhibits N-glycosylation processing, resulted in only small band in the SDS⁄ PAGE for both the wild type (NNN) (Figs 2C and 4A) and mutant DNG, DND and DDN (data not shown) The mutant DDQ, which does not possess any N-glycosyla-tion site, expressed only one N-glycan-free band of
90 kDa in both the intracellular and the plasma mem-brane compartments (Fig 3A) The 108 kDa large band of NNN was Endo H resistant, whereas digestion with PNGase F converted it to a 90 kDa N-glycan free polypeptide (Fig 2C), indicating a mature N-glycan of complex type However, the 96 kDa small band of NNN was converted to a 90 kDa polypeptide after either Endo H- or PNGase F-digestion (Fig 2B,C), indicating an N-glycan of the mannosidic type The N-glycosylation mutants DGN, DDN and DND exhibited a reduced molecular mass in accordance with the absence of N-glycans at the two eliminated N-gyl-cosylation sites in those proteins This suggests that the mutants DGN, DND and DDN, as well as wild type NNN, were N-glycosylated in CHO cells and their N-glycans were processed before they arrived at the cell surface
To determine the influence of N-glycosylation on the quality control of GAT1, the half-life of GAT1 wild type and mutants was also investigated by kinetic ana-lysis of pulse-chase experiments We found that the half-life of mutants containing either double (DDN) or triple (DDQ) N-glycosylation mutations was remark-ably reduced (Fig 6) Keynan et al reported that the functional expression of the GABA transporter in HeLa cells was abolished by tunicamycin, a potent inhibitor of N-glycosylation [18] We found that inhibi-tion of N-glycosylainhibi-tion processing by dMM did not
Trang 10affect either the protein stability (Fig 6) or
intracellu-lar trafficking (Figs 4A and 5A) This suggests that
cotranslational N-glycosylation, but not the terminal
trimming of N-glycans is involved in the regulation of
the stability and trafficking of GAT1 It has been
reported that a variety of molecular chaperones and
folding enzymes assist the folding of newly synthesized
proteins in the ER If N-glycosylation is inhibited,
some glycoproteins fail to fold or assemble efficiently,
resulting in a prolonged retention in the ER and an
increased proteolytic breakdown [26–29] Our results
indicate that the impaired plasma membrane
traffick-ing and reduced stability of the N-glycosylation
mutants of GAT1 must be a result of misfolding of
these proteins
The deficiency of N-glycosylation results in a
mark-edly reduced GABA-uptake activity (Fig 3C) as well
as a GAT1-mediated current in CHO cells (Fig 9A)
This is in accordance with our previous work using the
expression system of the Xenopus oocyte [7] In order
to exclude the possibility that the reduction in function
in the mutants could be due to a reduction in the
num-ber of GABA transporters per cell, values for the
transport activity were normalized for the surface
pro-tein of these mutants Double N-glycosylation mutants
showed a marked reduction of GABA-uptake activity
of 60–40% of that of the wild type GAT-mediated
GABA transport activity could hardly be detected in
the mutant lacking all three N-glycosylation sites
(DDQ), despite the fact that this protein was expressed
on the surface of CHO cells (Fig 3A) The
N-glyco-sylation processing inhibitor 1-deoxymannojirimycin
(dMM) also strongly inhibited GABA-uptake (Figs 4B
and 5B), although the amount of cell surface
expres-sion and the intracellular trafficking of GAT1 were
not affected by dMM (Figs 4A and 5A) This indicates
that the observed reduction of GABA-uptake activity
is a result of a deficiency of N-glycans The possibility
that the reduced GAT1 activity could be due to a
gen-eral effect of the inhibitor on other glycoproteins
required for GAT1 activity is very unlikely It has been
demonstrated that GAT transport function can be
reconstructed in liposomes and that no other
pro-teins are needed for GABA-uptake activity [30] Our
results suggest that N-glycans, in particular their
terminal structure, are involved in the GABA-uptake
process of GAT1 However, the GABA-uptake tolerates
the modification of neuraminic acid to N-propanoyl
neuraminic acid, as incubation with
N-propanoylman-nosamine (P-NAP), a synthetic precursor of
N-propa-noyl neuraminic acid [31–33], did not significantly
change the GABA-uptake activity of GAT1 (data not
shown)
How do the oligosaccharides of GAT1 influence GABA-uptake? In order to clarify the functional mech-anism of oligosaccharide side chains in GABA-uptake,
a kinetic analysis was performed Deficient N-glycosy-lation decreased the Vmax values of GABA-uptake by GAT1, while the Km GABA values were not affected Similar results were also obtained after treatment with dMM (Fig 8) Our results indicate that the turnover rate of the transporter is affected, but not the substrate binding process This provides strong evidence that N-glycans, in particular their terminal structures, are involved in regulating the GABA translocation of GAT1, but not in binding of GAT1 to GABA
Transport of GABA by GAT1 across the cell mem-brane is driven by an electrochemical gradient of Na+ and Cl– [1,34] with a stoichiometry that results in an electrogenic substrate transport Voltage-clamp experi-ments suggest that deficient N-glycosylation reduces the affinity of GAT1 for Na+ [7] The present work revealed that the reduced transport activity can at least partially be attributed to a reduced apparent affinity of GAT1 for extracellular Na+ and slowed kinetics of the transport cycle (Fig 9) This was observed in both wild type and mutants after inhibition with dMM As the GABA transport process is driven by the gradient
of Na+, it is reasonable to deduce that the affinity of GAT1 for Na+determines the turnover rate of GABA transport As the data presented in Fig 9 are for a sin-gle, functional transporter expressed on the cell sur-face, the reduced GABA-uptake cannot be due to reduced cell surface expression of transporters In this event the oligosaccharides of GAT1 play a role in the regulation of GABA-uptake by affecting the affinity for sodium ions
In conclusion, cotranslational N-glycosylation is important for the correct folding of GAT1 to a func-tional conformation Defective N-glycosylation leads
to decreased protein stability and disturbed intracellu-lar trafficking N-Linked oligosaccharides, in particuintracellu-lar their terminal structures, are involved in the regulation
of GABA-transport of GAT1 by influence on its affin-ity for sodium ions
Experimental procedures
Construction of N-glycosylation mutants of GAT1 and of GAT1/GFP fusion proteins
The mutants were based on the neuronal wild type GABA transporter type 1 of mouse (mGAT1) The cDNAs of N-glycosylation mutants DGN (N176D, N181G), DDN
were constructed earlier [7] In each mutant, two of three