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Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 851�856 |
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doi:10.1111/j.1745-7270.2007.00354.x |
existence of an endogenous glutamate and aspartate
transporter in Chinese hamster ovary cells
Xunhe Ji#, Yuhua Jin#,
Yaoyue Chen, Chongyong Li, and Lihe Guo*
Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences; Graduate School of Chinese Academy of Sciences,
Shanghai 200031, China
Received: April 11,
2007�������
Accepted: July 26,
2007
# These authors
contributed equally to this work
*corresponding author: Tel,
86-21-54921394; Fax, 86-21-54921392; E-mail, [email protected]
Abstract������� Chinese hamster ovary cells show
endogenous high-affinity Na+-dependent glutamate transport activity.
This transport activity is kinetically similar to a glutamate transporter
family strategically expressed in the central nervous system and is
pharmacologically unlike glutamate transporter-1 or excitatory amino acid
carrier 1. The cDNA of a glutamate/aspartate transporter (GLAST)-like
transporter was obtained and analyzed. The deduced amino acid sequence showed
high similarity to human, mouse, and rat GLAST. We concluded that a GLAST-like
glutamate transporter exists in Chinese hamster ovary cells that might confer
the endogenous high-affinity Na+-dependent glutamate transport activity
evident in these cells.
Keywords������� GLAST; CHO cell; glutamate transporter
L-Glutamate is a major
excitatory neurotransmitter in the mammalian central nervous system (CNS) that
contributes not only to fast synaptic neurotransmission, but also to complex
physiological processes like memory, learning, neuronal plasticity, and
neuronal cell death. To terminate the action of glutamate and maintain its
extracellular� concentration below excitotoxic levels, Na+-dependent
high affinity glutamate transporters [termed excitatory� amino acid
transporters (EAATs)], locating on the plasma membrane of neurons and glial
cells, rapidly remove glutamate from the extracellular space. The family�
consists of five distinct transporters, glutamate/aspartate transporter
(GLAST), glutamate transporter (GLT)-1, excitatory� amino acid carrier (EAAC)
1, EAAT4, and EAAT5, cloned from mammals [1-7].
GLAST is widely expressed in the brain [8] and also shows a broad distribution�
in peripheral organs other than the CNS [9]. Several studies also showed that
there are two alternative splicing variants of GLAST. GLAST-1a, lacking exon 3,
is expressed in rat bone and brain [10]. EAAT1ex9skip, which lacks the entire
exon 9, is expressed in human brain [11].
Chinese hamster ovary (CHO)
cell line constitutes one of the most common cell lines selected for expression
of external genes. To investigate the property of the Na+-dependent
glutamate transporters, the expression of glutamate transporters in CHO cells
has been carried out in some studies. Although the properties of the glutamate
transporters have been made more explicit, these studies also implied that
there existed an endogenous Na+-dependent transport system for
glutamate/aspartate in CHO cells, which brought more difficulties to the
research [1215]. However, no published data are available about the nature of
the endogenous Na+-dependent transport.
The present report shows the
existence of a high-affinity Na+-dependent glutamate transporter in CHO
cells. This transporter was pharmacologically similar to a glial GLAST. The
cDNA sequence for the GLAST-like transporter was also obtained and analyzed.
Materials and Methods
Cell culture
The CHO cell line was obtained
from the China Center for Type Culture Collection (Shanghai, China) then
maintained in RPMI 1640 medium (Gibco, Carlsbad, USA) containing 10% fetal
bovine serum (Gibco).
Transport studies
Measurement of glutamate
uptake was carried out on intact CHO cells plated in 48-well plates. Briefly,
near-confluent cells (approximately 60,000 cells per well) were rinsed three
times with HEPES-buffered saline (137 mM NaCl, 0.7 mM K2HPO4, 1 mM MgCl2, 1 mM
CaCl2,
5.5 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4 with Tris) or with
HEPES-buffered saline in which NaCl was replaced by choline chloride. The cells
were then exposed to 100 ml HEPES-buffered saline
containing 100 nM L-[3H]-glutamate (Amersham Pharmacia Biotech,
Little Chalfont, UK) as necessary with cold substrate for 20 min at 37 �C. The
reaction was terminated by removing the transport medium and washing the cells
three times with ice-cold sodium-free HEPES-buffered saline. Cells were then
solubilized in 2 m KOH solution
and aliquots were measured by a liquid scintillation analyzer (Tri-Carb 2900TR;
Packard, Downers Grove, USA). To estimate the kinetic constants of glutamate
uptake, different concentrations� of L-glutamate were used, varying from 1 to
100 mm. To analyze the sensitivity
of the transport activity� to selected inhibitors dihydrokainate (DHK;
Sigma-Aldrich, St. Louis, USA), L-cysteine (Shanghai Shiyi Chemicals Reagent
Co., Shanghai, China), and DL-threo-b-hydroxyaspartic
acid (THA; Sigma-Aldrich), the uptake for 1 mM
glutamate in the presence of appropriate concentrations� (1 mM to 1 mM) of various compounds was
measured. All tests were replicated between three and five times.
Reverse transcription-polymerase
chain reaction (RT-PCR) and cDNA sequencing
Total RNA from CHO cells was
extracted with TRIzol reagent (Invitrogen, Carlsbad, USA) following the
manufacturer's instructions. The extracts were then treated with DNase for 15
min at room temperature prior to first-strand cDNA synthesis with random 6 mers
and Moloney murine leukemia virus reverse transcriptase (Promega, Madison,
USA). The first-strand cDNA was then subject to 30 cycles of PCR amplification
(denature: 94 �C, 1 min; anneal: 55 �C, 1 min; extension: 72 �C, 1 min) with 2
U Taq DNA polymerase (Takara, Shiga, Japan) and primer pairs as
follows: 5' primer (P1), 5'-GCGGTGATAA�TGT�GGTAT-3'; 3'
primer (P2), 5'-ctacatcttgg�tttcg�ct-3';
5' primer (P3), 5'-atgacNaaaagcaacgga-3';
and 3' primer (P4), 5'-aaagtgatgggtagggtg-3'.
These primers were designed based on the conserved sequences of GLAST cDNA
between human (GenBank accession No. NM_004172, from base pair 245 to 1873),
rat (GenBank accession No. NM_019225, from base pair 204 to 1835), and mouse
(GenBank accession No. NM_148938, from base pair 490 to 2121). As the Cricetulus�
GLAST sequence is not known, we used the following strategy in our study.
First, we used P1 and P2 to obtain product 1 that corresponds to base pair 1336-2121 of NM_148938, or to base pair 1050-1835 of NM_019225 (or to base pair 1091-1873 of NM_004172). Then product� 2 was
obtained by using P3 and P4, which corresponds to base pair 490-1607 of NM_148938, or to base pair 204-1321 of NM_019225 (or to base pair 245-1362 of NM_004172). The above two PCR
products were cloned into the pGEM-T vectors (Promega) and sequenced. The data
were analyzed to obtain the whole CHO-GLAST sequence.
Results
CHO cells showed a robust ability
to uptake [3H]-glutamate
As shown in Fig. 1, [3H]-glutamate
uptake measured in CHO cells showed high affinity with a Km of
approximately 27.1�5.6 mM and a maximum translocation
velocity (Vm) of approximately 67.5�11.5
pmol/well/min. The Km value for glutamate uptake was more
similar to the value for glutamate transporter subtypes isolated from the CNS
[16,17] than to the value for the neutral amino acids transporters [8,18].
Uptake dependent on
extracellular Na+
We further investigated whether
the uptake was dependent on extracellular Na+. As indicated in Fig. 2,
when extracellular NaCl in the incubation medium was replaced by choline
chloride, [3H]-glutamate
uptake by CHO cells was reduced to only approximately 6% of the control, in which
cells were incubated in normal extracellular Na+ concentration.
Uptake inhibited by different
glutamate uptake inhibitors
Cells were incubated with
several glutamate transporter inhibitors at a concentration range from 1 mM to 1 mM. Of the three different
inhibitors tested, GLT-1 inhibitor DHK [16] and EAAC1 inhibitor L-cysteine [19]
showed poorer potential (IC50>1 mM) in inhibiting [3H]-glutamate
accumulation into CHO cells than the non-selective inhibitor THA (IC50=30.59.4
mM) (Fig. 3). Based on these
findings, we surmised that there might be a GLAST-like transporter in CHO
cells.
Analysis of the sequence of
the CHO-GLAST
The cDNA obtained from CHO
cells was a 1632 bp length open reading frame encoding a protein of 543 amino acid
residues (GenBank accession No. EF155647). The deduced amino acid sequence
showed high similarity to human (95%), mouse (98%), and rat (98%) GLASTs (Fig.
4), and significant but much less similarity to the other EAATs (50%-66%).
Discussion
To study the properties of the
glutamate transporters, it would be more useful to have a mammalian cell line
expressing these molecules. CHO cells, which constitute one of the most common
cell lines selected for expression of external genes, displayed endogenous Na+-dependent
glutamate/aspartate uptake activity [15]. It is fortunate that there exist
mutant CHO cell lines lacking Na+-dependent glutamate transport [12]. Levy
et al. successfully established an inducible GLT-1 expression system in the
mutant CHO line Dd-B7 and investigated the stoichiometry of this transporter
[13,14]. Nevertheless, the nature of the endogenous Na+-dependent glutamate transport
activity in CHO cells is unknown.
Our results showed that there
exists a robust, Na+-dependent, high-affinity glutamate
transport activity in CHO cells with a Km value more similar to the
value for glutamate transporter subtypes isolated from the CNS. This transport
activity can be effectively inhibited by the non-selective glutamate
transporter inhibitor THA, but not the selective GLT-1 inhibitor DHK or EAAC1
inhibitor L-cysteine, indicating that it is unlikely the transport activity
involves a GLT-1-like or an EAAC1-like transporter. The total RNA of CHO cells
was then obtained and PCR was carried out using GLAST-specific primer pairs.
The acquired product was sequenced and the deduced amino acid sequence was
analyzed. Results showed that the GLAST-like transporter obtained from CHO
cells was highly similar to the human, mouse, and rat GLAST and that the CHO-GLAST
was not a splicing one of the known GLASTs. It revealed that there are six
amino acid substitutions (60, 105, 140, 200, 269, and 535) in this sequence
that are conserved across human, rat, and mouse. The three N-terminal
substitutions (60, 105, and 140) lie in the first three of the six hydrophobic
regions, proposed to span the plasma membrane in an a-helical
manner [20]. The substitution 200 lies in the putative extracellular loop
between transmembrane domains III and IV, and the substitution 269 lies in the
loop between transmembrane domains IV and V. The substitution 535 lies in the
C-terminal tail. However, amino acid residues that are important for GLAST
functions are still conserved in this sequence, including experimentally
verified N-glycosylation sites of GLAST (N206 and N216) and the residues
pivotal to the binding of glutamate (Y405 and R479) [20]. Thus, the transport
function of this sequence might not be significantly different to those known
GLASTs, and more detailed study concerning CHO-GLAST should be pursued.
We also carried out RT-PCR to
detect whether or not GLT-1, EAAC1, EAAT4, and EAAT5 exist in CHO cells by
using the same strategy as described in "Materials and Methods".
Unfortunately, no PCR product is available. However, this can not exclude the
existence of other glutamate transporters. To clarify this problem, further
work is needed that includes more extensive pharmacological approaches,
knockdown or knockout CHO-GLAST, and immunological approaches.
GLAST/EAAT1 is strategically
localized in retinal Muller cells and cerebellar Bergmann glia [7]. However,
recent research showed that GLAST also localized at tissues other than
synaptical regions, such as bone [21], cartilage [22], the reproductive system
[23], adrenal and pituitary glands [24], and lactating mammary glands [25]. It
is also indicated that GLAST showed a ubiquitous expression in a variety of
peripheral tissues and organs, including ovary [9,26]. Therefore, it is
reasonable that GLAST transporter exists in CHO cells, derived from Chinese
hamster ovary.
Igo and Ash developed a mutant
CHO cell line lacking Na+-dependent glutamate transport [12]. Some
researchers concerned with glutamate transporters, and using the CHO expressing
system, chose the mutant CHO line Dd-B7 to exclude the influence induced by
endogenous glutamate transport activity [13,14]. Others chose to highly express
the particular transporter to diminish the influence of the endogenous
glutamate transport activity [15]. In this study, considering the high overexpression
(20-fold) over the endogenous transport activity, the data concerning EAAT2 is
reasonable. With regard to the data relating to EAAT1 (GLAST), for which the
expression activity was only 4 folds over the endogenous transport activity, it
should be taken into account that benzodiazepines might influence the two
GLASTs differently. However, information regarding the kind of species from
which the GLAST sequence in that study was sourced is not available, making it
difficult to reevaluate the related results. Thus, the fact that there is an
endogenous functional expression of GLAST in CHO cells should be carefully
considered when interpreting functional data obtained from overexpression of
transporters in this system.
In conclusion, this is the first attempt to
uncover the molecular basis of the glutamate transport activity in CHO cells.
The results suggested an existence of a GLAST transporter in CHO cells, and
this transporter might contribute to the observed endogenous Na+-dependent, high-affinity glutamate transport in CHO cells.
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