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An Expression System for Mammalian Amino Acid Transporters Using a Stably Maintained Episomal Vector
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
254, 208–214 (1997)
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An Expression System for Mammalian Amino Acid Transporters Using a Stably Maintained Episomal Vector James C. Matthews,*,† Ara M. Aslanian,* Kelly K. McDonald,* Wenbo Yang,* Marc S. Malandro,* Donald A. Novak,† and Michael S. Kilberg*,1 *Department of Biochemistry and Molecular Biology and †Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610
Received August 8, 1997
Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X0 AG, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNAtransfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein– Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.41) and EAAC1 (391) transport activity than does the vaccinia/HeLa expression system (2.61 and 221, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X0AG, or A; and (iii) the endogenous transport velocities of Systems ASC and X0 AG in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism. q 1997 Academic Press
Transient expression of cloned solute transporter proteins in cultured cells is used to characterize trans1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Florida College of Medicine,1600 S.W. Archer Road, Box 100245, Gainesville, FL 32610-0245. Fax: (352) 392-6511.
port properties, identify the apparent size of protein products, study protein trafficking, and investigate the functional significance of conserved amino acid residues through the use of chimera, deletion, and mutagenic analyses. A number of cell transfection/expression regimens have been developed to express transport proteins in model systems. Of these, the vaccinia virus/HeLa cell system is used often to express a wide variety of mammalian transporters (1–3). Protocols for the transient expression of cDNAs in HeLa cells using vaccinia typically consist of infecting HeLa cells with a recombinant vaccinia virus that encodes T7 RNA polymerase, followed by liposome-mediated transfection of the cells with the transporter cDNA vector, for which transcription is under control of the T7 promoter. Despite the usefulness of the vaccinia/HeLa cell expression model, it may not necessarily be optimal for all transport proteins. For example, the amount of System ASCT1 activity (Na/-dependent neutral amino acid transport) expressed after transfection of the ASCT1 cDNA using this regimen is only one- to threefold over background (4, 5), although higher expression in oocytes is possible (6). We have tried without success numerous modifications of the originally reported vaccinia/HeLa cell expression regimen (3) to optimize the vaccinia-induced expression of the ASCT1 protein. Systematic changes have included the use of different transfection reagents, expression times, vectors, and cell lines. Therefore, we decided to evaluate a different expression system to determine whether greater amounts of functional System ASCT1 activity could be achieved. Epstein–Barr virus (EBV)2-based vectors are capa2 Abbreviations used: EBV, Epstein–Barr virus; 293c18 cells, human embryonic kidney (293) cell line stably transfected with EBNA1 cDNA; MeAIB, 2-(methylamino) isobutyric acid; MBP, maltose binding protein; pBKS II, pBluescript II KS { plasmid.
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0003-2697/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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ble of stable transformation of cells that express the transacting EBNA-1 gene (7). When transfected into human embryonic kidney 293 cells (HEK 293) that have been made competent by stable transfection with the EBNA-1 cDNA (293c18 cells), the pDR2 vector has several features that suggest it would be useful for transporter expression studies. First, the cDNA pDR2 vector contains an EBV origin of replication sequence and, therefore, is maintained as an episome for many cell divisions, in the amount of 10 to 20 copies per cell. Second, the transcription of the cDNA insert sequence is driven by the Rous sarcoma viral (RSV) promoter, known to promote high levels of exogenous protein expression. Third, because the pDR2 vector contains the hygromycin resistance gene (hygr), only those cells transfected with the cDNA pDR2 vector are able to survive hygromycin B-resistance selection. Thus, transfection with the pDR2 vector allows differences in transfection efficiencies to be normalized by hygromycin B selection. Fourth, unlike vaccinia virus-based expression systems (8), the transfection of cells with the EBNA-1 gene and pDR2 vector has not been reported to reduce the translation of endogenous cellular proteins. This research evaluates the relative amount of functional amino acid transport activity of System ASCT1, encoded by human hippocampal ASCT1 cDNA, and 0 System XAG , encoded by rat hippocampal EAAC1 cDNA, after expression in either the vaccinia/HeLa cell or EBNA-1/293c18 cell model. The results document that (i) the EBNA-1/293c18 expression system results in a larger increase over background of System ASCT1 transporter than does the vaccinia/HeLa expression system; (ii) the level of cDNA-induced EAAC1 activity over endogenous System X0AG activity in the EBNA-1/ 293c18 model exceeds the relatively high level of expression observed in the vaccinia/HeLa model; (iii) transfection of only the pDR2 vector does not affect the endogenous transport capacities of Systems ASC, 0 , or A; and (iv) exogenous cDNA-mediated induction XAG 0 of either System ASC or XAG activity in 293c18 cells does not adversely affect the endogenous transport capacity of the other system. We conclude that the EBNA1/293c18 cell expression model represents a useful transient expression regimen for the characterization of mammalian amino acid transport proteins, especially for those that exhibit relatively low activity in transient expression systems that lack a selection mechanism for expressing cells. MATERIALS AND METHODS
Preparation of plasmid constructs. A full-length ASCT1 cDNA was isolated and cloned from a human hippocampal cDNA library (4, 9) and a full-length EAAC1 cDNA was isolated from a rat brain hippocam-
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pal library by degenerate PCR followed by cDNA library screening (10). The ASCT1 or EAAC1 cDNAs were subcloned, as previously described (4), into pBluescript KS II (pBKS II) at the XhoI/EcoRI sites of the multiple cloning region for expression driven by either the T7 RNA polymerase or cytomegalovirus (CMV) polymerase promoter sequences. The ASCT1 or EAAC1 cDNAs also were subcloned between the SalI and BamHI sites of the pDR2 plasmid vector (Clonetech Laboratories, Palo Alto, CA). Expression of ASCT1 and EAAC1 transporters in HeLa cells. HeLa cells were purchased from American Tissue Culture Collection (Rockville, MD) and were maintained as described (4). Transient transporter expression in HeLa cells was performed as described (10, 11). Briefly, HeLa cells were plated at a density of 150,000 cells per well on Costar 24-well cluster trays and infected with recombinant vaccinia virus (vTF7-3, 15 pfu/cell) expressing T7 RNA polymerase (1). The cells were infected for 45 min at 377C. The virus-containing medium was then removed and replaced with minimal essential medium (OptiMEM I, GibcoBRL, Gaithersburg, MD) containing 12 ml/ml lipofectamine (GibcoBRL) and 4 mg/ml ASCT1 or EAAC1 cDNA in pBII KS. The cells were incubated for 8 h before assaying for amino acid transport activity. Background transport activity was assayed in cells transfected with the pBII KS vector that lacked a cDNA insert. Expression of ASCT1 or EAAC1 transporter in 293c18 cells. The 293c18 cell line was maintained according to the instructions of the supplier (Invitrogen, San Diego, CA). These cells have been stably transfected with the EBV transformation antigen EBNA-1 to permit the episomal maintenance of the pDR2 plasmid, which contains an EBV origin of replication. The 293c18 cells in passage numbers 2–5 were cultured until 80% confluent at 377C in 5% CO2 and Dulbecco’s minimum essential medium that contained streptomycin sulfate (100,000 mg/l), amphotericin B (250 mg/l), penicillin G sodium (100,000 U/l), and G418 sulfate (250 mg/l) and 10% fetal bovine serum (DMEM/FBS), where indicated. The cells were then transfected in OptiMEM by incubation with 4 mg/ml EAAC1–pDR2, or pDR2 vector lacking a cDNA insert, and 12 ml/ml lipofectamine. After 7 h, the transfection solution was removed and the cells were cultured in DMEM/FBS for 24 h and then for 10 days in DMEM/FBS that contained hygromycin B (400 U/ml) to select for pDR2-transfected cells. During the selection period, the medium was changed every 3 days. After 10 days, the cells were washed three times with PBS (pH 7.4), trypsinized, resuspended in DMEM/FBS that contained 10% DMSO, slow-frozen at 0807C for 1 day, and then stored in liquid N2 . After thawing, the cells were cultured in hygromycin B-containing DMEM/FBS until 80% con-
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fluent for use in whole-cell transport assays or determination of mRNA and protein content. Whole-cell transport assay. Amino acid uptake by HeLa or 293c18 cells was measured by the 24-well cluster tray method of Gazzola et al. (12) with modifications by our laboratory (13). To normalize the intracellular pools of amino acids before the transport assays, cells were incubated at 377C for 30 min in Na/ –Kreb’s– Ringer’s phosphate (NaKRP; 119 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4 , 1.2 mM KHCO3 , 5.6 mM glucose, 0.5 mM CaCl2 , 25 mM Na2HPO4 , pH 7.4). The amount of Na/-dependent uptake was calculated as the difference between uptake in Na/-containing KRP and Na/-free (choline-KRP) buffers. System 0 XAG /EAAC1 activity was calculated as the amount of 1 mM [3H]glutamate Na/-dependent uptake that was inhibitable by unlabeled 500 mM D-aspartate (14). System A activity was taken as the Na/-dependent uptake of 50 mM [3H]2-(methylamino) isobutyric acid (MeAIB) (15, 16). System ASC/ASCT1 activity was calculated as the amount of Na/-dependent 5 mM [3H]serine uptake that remained in the presence of unlabeled 5 mM MeAIB and 5 mM arginine to eliminate serine uptake by transport Systems A and y/L, respectively (5). Northern analysis. Total RNA was isolated (17) from cultured cells grown to 80% confluence. The RNA was size-separated in a 1% agarose gel by electrophoresis in the presence of 0.02 M formaldehyde. Densitometric analysis of ethidium bromide-stained 28S and 18S ribosomal RNA bands was used to confirm that the lanes were equally loaded with RNA. The RNA was transferred by downward capillary action (18) to 0.45mm nylon membranes, covalently cross-linked by UV light, and hybridized for 15 to 18 h at 677C with individual 32P-labeled cDNA probes in solutions that contained 1% BSA, 7% SDS, 0.5 M Na2HPO4 (pH 7.2), and 1 mM EDTA. The radiolabeled cDNA probes were prepared from full-length rat EAAC1 (2.2 kb) (10) and full-length human ASCT1 (4) by random priming extension using [32P]dCTP (Amersham Life Sciences Inc., Arlington Heights, IL) and a kit from GibcoBRL. After hybridization, the blots were washed 4 1 15 min at 677C in 40 mM Na2HPO4 (pH 7.2), 0.1% SDS, and 1 mM EDTA. Immunoblot analysis. Immunoblot analysis for EAAC1 was performed as described (19). Briefly, a plasma membrane-enriched fraction of both transfected cell types was generated by differential centrifugation (20). The membranes were resuspended in 0.25 mM sucrose, 10 mM Hepes–KOH (pH 7.5), 1 mM EGTA, and 2 mg/ml each of N-tosyl-L-phenylalanine chloromethyl-ketone, N-a-p-tosyl-L-lysine ketone, leupeptin hemisulfate, aproptinin, and pepstatin A to prevent proteolysis. Membrane proteins were separated by 7.5% SDS–PAGE (21) and then electrotransferred
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TABLE 1
Serine Uptake by System ASC by HeLa or HEK 293c18 Cells Expressing ASCT1 Vector Cells HeLa 293c18
ASCT1
(pmolrmg01proteinrmin01) 119 { 1.7 697 { 32
305 { 6.9* 4447 { 303*
Note. Each value is the mean { SE for Na/-dependent uptake of 5 mM L-[3H]serine (in the presence of 5 mM MeAIB). HeLa (n Å 3) or HEK 293c18 cells (293c18, n Å 4) were transfected with vector only (Vector) or ASCT1–vector (ASCT1), using pBKS II or pDR2 vectors, respectively. Uptake was measured for 1 min at 377C as described in the text. These values are representative of multiple experiments. * P õ 0.005 versus vector-only transfected cells.
to a 0.45-mm nitrocellulose membrane (Schleicher & Schuell, Keene, NH). For the detection of EAAC1 protein, blots were probed with 43 ng IgG/ml of the EAAC1 polyclonal antibody (19) or preimmune sera in blocking solution (1% nonfat dry milk and 2% casein hydrolysate in 10 mM Tris–Cl, pH 7.5, 300 mM NaCl) for 1 h at room temperature with agitation. Horseradish peroxidase-conjugated protein A (Amersham) was used to detect immunoreactive bands by visualization with a chemiluminescence kit (Amersham). Statistical analysis. Transport assays were performed in quadruplicate within each experiment and most experiments were repeated using independent batches of transfected cells. Differences between mean uptake values were determined by Student’s two-tailed t tests. RESULTS
Expression of human ASCT1 mRNA and activity in HeLa or 293c18 cells. The expression of the human ASCT1 transport protein in HeLa cells transiently transfected with pBSKII–ASCT1 plasmid or in 293c18 cells transfected with stably maintained pDR2–ASCT1 was evaluated. Consistent with previous reports (4, 5, 9), the activity of ASCT1 in HeLa cells transfected with the System ASCT1 cDNA was about 2.6 times that in cells transfected with the pBSKII plasmid only (Table 1). Previous Northern blot analysis by this laboratory has demonstrated that the steady-state ASCT1 mRNA content is increased to a much greater degree than the transport activity in HeLa cells that have been transfected with the pBSKII–ASCT1 construct (5). To determine if the expression of ASCT1 activity could be increased by employing the EBNA-1/293c18 cell expression system, 293c18 cells were transfected with the human ASCT1 cDNA sequence in pDR2. The ASCT1mediated Na/-dependent uptake of 5 mM [3H]serine by
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pDR2/293c18 EXPRESSION OF TRANSPORT PROTEINS TABLE 2
Activities of Amino Acid Transport Systems X0 AG , A, and ASC in pDR2-Transfected or Nontransfected HEK 293c18 Cells Nontransfected System X0 AG A ASC
FIG. 1. Steady-state expression of ASCT1 mRNA in HEK 293c18 cells transfected with ASCT1 transporter cDNA. Northern blot analysis of total cellular RNA (30 mg per lane) was performed as described under Materials and Methods. Replicate blots were hybridized with a 32P-labeled cDNA probe specific for human ASCT1 mRNA. Densitometry of a photographic negative of the ethidium bromide-stained gel was used to document that the amounts of 28S and 18S ribosomal RNA bands were equal between lanes.
the ASCT1-transfected cells was increased 6.4 times over control cells transfected with pDR2 vector only (Table 1). Consistent with the increase in System ASCT1 activity, ASCT1 mRNA was detected readily in ASCT1-transfected 293c18 cells (Fig. 1), whereas ASCT1 mRNA was not detectable in equal amounts of RNA isolated from pDR2-only-transfected cells. Therefore, the endogenous System ASC transport activity is likely to result from the expression of another member of the ASC gene family (22, 23). The expression of ASCT1 protein levels was not investigated in either expression system because antibodies are not available, but expression of activity is taken to represent functional protein residing in the plasma membrane. To determine whether the pDR2 expression system alters the endogenous amino acid transport capacity of 293c18 cells, the transport velocities of Systems ASC, 0 were compared between nontransfected A, and XAG 293c18 cells and pDR2-transfected 293c18 cells subjected to hygromycin B selection. The activity of these endogenous transport systems was not affected by pDR2 transfection/selection (Table 2), documenting that these procedures do not increase or decrease endogenous membrane transport. Expression of rat EAAC1 mRNA and activity in HeLa or 293c18 cells. To further assess the utility of the EBNA-1/293c18 expression model, EAAC1-mediated Na/-dependent anionic amino acid transport activity, mRNA, and protein content were measured in either 293c18 cells transfected with pDR2–EAAC1 vector or HeLa cells transiently transfected with pBKS II–
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pDR2-transfected
(pmolrmg01proteinrmin01) 34.7 { 4.9 149 { 12 847 { 10
27.8 { 5.7 127 { 10 795 { 32
Note. Each value is the mean { SE uptake (pmolrmg01proteinr min01; n Å 5 to 8 observations) by the indicated transport activity in HEK 293c18 cells transfected with the pDR2 plasmid lacking insert and selected in hygromycin B as described in the text. The Na/dependent uptake of 1 mM L-glutamate in the presence of 500 mM 0 D-aspartate (System XAG), 50 mM MeAIB (System A), or 5 mM L[3H]serine in the presence of 5 mM MeAIB and 5 mM L-arginine (System ASC) by cells transfected with vector only (pDR2) or not transfected (293c18) was measured for 1 min at 377C as described in the text. Uptake values between nontransfected and pDR2-transfected cells did not differ when compared with Student’s two-tailed t test (a Å 0.05 level).
EAAC1 cDNA (Fig. 2, Table 3). Two predominant species of mRNA (4.2 and 2.3 kb) were expressed in HeLa cells transfected with the pBKS II–EAAC1 plasmid, whereas the EAAC1 mRNA was below detectable levels in pBKS II-only-transfected cells (Fig. 2A). Immunoblot analysis revealed a similar increase in EAAC1 protein
FIG. 2. Steady-state expression of EAAC1 mRNA and protein following transfection of either HeLa or HEK 293c18 cells. Northern and immunoblot blot analyses were performed as described in the text for HeLa (A) or 293c18 (B) cells transfected with vector only (Vector) or EAAC1 cDNA (EAAC1), using pBKS II (HeLa cells) or pDR2 (293c18 cells) vectors, respectively. Total cellular RNA (7.5 mg per lane, HeLa; or 25 mg/lane, 293c18) was hybridized with a 32Plabeled cDNA probe specific for EAAC1 mRNA. Densitometry of a photographic negative of the ethidium bromide-stained gel was used to document that the amounts of 28S and 18S ribosomal RNA bands were equal between lanes. Plasma membrane proteins (35 mg) were subjected to 7.5% SDS–PAGE and immunoblotted with polyclonal antibodies to EAAC1, as described under Materials and Methods.
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Na/-Dependent Uptake of Aspartate by HeLa or HEK 293c18 Cells Expressing EAAC1 Vector
EAAC1
(pmolrmg01proteinrmin01)
Cells HeLa 293c18
50.2 { 0.2 61.1 { 4.6
1106 { 76* 2396 { 209*
Note. Each value is the mean { SE uptake of 50 mM L-[3H]aspartate (pmolrmg01proteinrmin01) by HeLa cells (n Å 3) or by HEK 293c18 cells (293c18, n Å 4) transfected with vector only (Vector) or EAAC1vector (EAAC1), using pBKS II (HeLa) or pDR2 (293c18 cells), respectively. Uptake was measured for 1 min at 377C as described in the text. These values are representative of multiple experiments. * P õ 0.005 versus vector-only transfected cells.
content in HeLa cells transfected with EAAC1–pBKS II (Fig. 2A). In both transfection treatments, a prominent band of about 60 kDa and a weakly expressed band of about 57 kDa were detected. For the pDR2– EAAC1-transfected 293c18 cells, a single EAAC1 mRNA species of 3.4 kb was detected by Northern analysis of total RNA (Fig. 2B), which is consistent with that detected in human kidney tissues (24). As with HeLa cells, no endogenous EAAC1 mRNA was detectable in 293c18 cells transfected with plasmid only. Expression of the transfected EAAC1 cDNA resulted in a single EAAC1 protein band of about 68 kDa (Fig. 2B). The appearance of multiple protein bands for EAAC1 has been reported in native tissue (19, 25), as well as after cRNA injection into oocytes (25) and cDNA transfection into cultured cells (26, 27). The difference in the apparent molecular size of EAAC1 protein when EAAC1 cDNA is expressed in HeLa versus 293c18 cells also is consistent with the observations of others who have documented protein sizes ranging from 61 to 78 kDa (19, 25–27). It is thought that these differences arise from cell-specific patterns of glycosylation because treatment with the appropriate glycosidase results in generation of a single band corresponding to the predicted core molecular size of 57 kDa (24, 26). The existence of different mRNA species following expression of the 2.2-kb EAAC1 cDNA probably arises from incomplete termination of RNA polymerase, incorrect processing of the nascent transcript, or differences in polyadenylation. The Na/-dependent uptake of 50 mM [3H]aspartate in EAAC1-transfected HeLa cells was about 22 times greater than that in vector-only-transfected controls (Table 3), a result consistent with a previously published report (10). The Na/-dependent uptake of 50 mM [3H]aspartate was also higher (approximately 39 times) in 293c18 cells transfected with the pDR2–EAAC1 plasmid than in vector-only-transfected controls (Table 3). The aspartate uptake was inhibited by either 5 mM
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L-glutamate (96%) or 5 mM D-aspartate (92%) (data not shown), consistent with published reports of EAAC1 0 transporter substrate specificity (28) and System XAG activity. For both expression systems, the increase in EAAC1-mediated Na/-dependent glutamate transport activity paralleled the increase in EAAC1 mRNA and protein content (Fig. 2). Given the lack of detectable endogenous EAAC1 mRNA, the endogenous System 0 XAG activity may be the result of expression of one or more of the other glutamate/aspartate transporter genes (29–33). To determine if the transfected cDNA altered the activity of other endogenous transporters, the endogenous System ASC activity was measured in pDR2EAAC1-transfected 293c18 cells and the endogenous 0 System XAG activity was measured in pDR2–ASCT1transfected 293c18 cells (Table 4). Consistent with the hypothesis that these cDNA sequences encode specific transporter proteins, the expression of either human ASCT1 or rat EAAC1 in 293c18 cells did not alter the activity of alternate endogenous amino acid transport systems within the cell. Furthermore, these observations, along with those in Table 1, indicate that endogenous transport in 293c18 cells is not adversely affected by the transfection process, the subsequent hygromycin B selection, or the expression of exogenous transport proteins.
DISCUSSION
This study demonstrates that the 293c18/pDR2 expression model for transporter expression in mammalian cells is a viable alternative to the often used vaccinia/HeLa cell system. Indeed, the data show that relative to the endogenous rate, the measurable activ-
TABLE 4
Consequence of EAAC1 or ASCT1 cDNA Expression on the Endogenous Transport Activities of Systems ASC and X0 AG in HEK 293c18 Cells Vector only Transfected cDNA
Endogenous system tested
ASCT1 ASCT1 EAAC1 EAAC1
ASC X0 AG ASC X0 AG
ASCT1 or EAAC1
(pmolrmg01proteinrmin01) 1338 32.5 427 13.4
{ 105 { 0.7 { 117 { 0.7
5007 32.6 499 342
{ 170* { 1.6 { 30 { 31*
Note. Each value is the mean { SE for System ASC-mediated uptake (pmolrmg01proteinrmin01) of 5 mM L-[3H]serine or System 3 X0 AG-mediated uptake of 1 mM L-[ H]glutamate by HEK 293c18 cells (n Å 6 to 8) after transfection with pDR2 plasmid-only (vector), ASCT1–pDR2 (ASCT1), or EAAC1–pDR2 (EAAC1). Uptake was measured for 1 min at 377C as described in the text. * P õ 0.05 versus vector-only transfected cells.
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ity of two different amino acid transporters, ASCT1 and EAAC1, is greater when their cDNAs are expressed in selectable 293c18/pDR2 cells than in HeLa/vaccinia cells. Compared to the 293c18 cell/ASCT1–pDR2 transfection protocol, the increase in System ASCT1 transport activity in ASCT1–pBKS II-transfected HeLa cells was relatively small, despite the fact that steady-state ASCT1 mRNA levels are much greater in ASCT1-transfected cells than in plasmid-only-transfected cells (5). These observations suggest that some transporter proteins, when expressed in vaccinia-infected HeLa cells, are produced in significant quantities, but that functional transport capacity may not be realized, possibly compromised by inadequate trafficking or the presence of other required regulatory proteins. As shown for a variety of transport proteins expressed with the vaccinia/HeLa system, the results from the present study show that EAAC1 mRNA, protein content, and activity are increased significantly in vaccinia virus-infected HeLa cells. A number of parameters were altered in an attempt to optimize the conditions for functional ASCT1 expression in vaccinia-infected cells. The transfection of several independent cell lines (each shown to be susceptible to vaccinia infection) with either ASCT1–pBKS II or ASCT1–pcDNA3 plasmid was evaluated, but each of the possible combinations gave similar low levels of increased System ASCT1 activity to that observed in several previous studies (4, 5). Likewise, several combinations of cDNA and lipofectamine concentrations were evaluated, as well as cell density and culture media composition. None of these parameters significantly enhanced the functional expression of System ASCT1 activity. Depending on the experimental goals, an additional limitation of the vaccinia-based transfection system can be the transient nature (usually 8 to 12 h) of the expression, imposed because of virus-induced deterioration of the cells with time of culture. Consequently, the sensitivity and reproducibility of transporter expression are compromised by transfection efficiency and viral infectivity, with little or no opportunity to normalize independently transfected culture dishes or wells. In contrast, one of the strengths of the 293c18/ pDR2 system is the ability to measure cells shortly after transfection or to select only those cells that have been transfected with pDR2 plasmid. Thus, after a selection period of the investigator’s choosing, cells from several large culture flasks can be combined to generate a common pool and then plated onto 24-well cluster trays. In this way, the transfection efficiency across all transport assays will be constant, even if the hygromycin B selection period was shortened and, thus, less than 100%. The maintenance of 10 to 20 copies of pDR2 plasmid as a replicating episome permits cell division without cDNA dilution. In this sense, the 293c18/pDR2
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system has similarities to both transient and stable transfections. The observation that the relative increase in transport activity paralleled the increase in mRNA and protein levels in cells transfected with transporter cDNA suggests that the 293c18/pDR2 expression system will be an effective model to use for the study of (i) amino acid transport protein function/structure, (ii) regulators of transporter expression and function, and (iii) cDNA library screening in mammalian cells. With regard to the later point, the fact that the pDR2 vector does not to integrate into cellular chromosomes, but instead is maintained as an episome, would allow use of the pDR2/293c18 system to screen cDNA expression libraries with subsequent recovery of the cDNA. There are several potential limitations of the 293c18/ pDR2 expression system that should be considered by prospective users of this expression model. First, if one wishes to select for 100% of expressing cells, this process takes up to 10 days. Second, the inability of EBV to replicate in rodent cells (7) eliminates its use in cells from important laboratory animals. Third, our experience indicates that the maximal level of increased transporter activity achieved after the hygromycin B selection period can only be maintained for 12 to 16 subsequent cell divisions (unpublished data). After this number of cell divisions, the activity significantly decreases. Despite these restrictions, as demonstrated by the present research, the expression of mammalian cDNAs with the pDR2 vector into 293c18 cells expressing EBNA-1 has the potential to be a flexible and useful model for the study of amino acid transport proteins in mammalian cells. ACKNOWLEDGMENTS This research was supported by the National Institutes of Health, the National Institute of Child Health and Human Resources (HD29934 to D.A.N.), the Institute for Diabetes, Digestive, and Kidney Diseases (DK-28374 to M.S.K.), and an Institutional National Research Service Award (DK07667 to J.C.M.).
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8. Pow, D. V., and Crook, D. K. (1996) Neuroscience 70, 295–302. 9. Kilberg, M. S., Tamarappoo, B. K., Shafqat, S., and Fremeau, R. T.(1994) in Transport in the Liver (Keppler, D., and Jungerman, K., Eds.), pp. 51–58, Kluwer, Lancaster, UK. 10. Velaz-Faircloth, M., McGraw, T. S., Malandro, M. S., Fremeau, R. T., Kilberg, M. S., and Anderson, K. J. (1996) Am. J. Physiol. 270, C67–C75. 11. Fremeau, R. T., Caron, M. G., and Blakely, R. D. (1992) Neuron 8, 915–926. 12. Gazzola, G. C., Dall’Asta, V., Franchi-Gazzola, R., and White, M. F. (1981) Anal. Biochem. 115, 368–374. 13. Kilberg, M. S. (1989) Methods Enzymol. 173, 564–575. 14. Hoeltzli, S. D., Kelley, L. K., Moe, A. J., and Smith, C. H. (1990) Am. J. Physiol. 259, C47–C55. 15. Christensen, H. N., Oxender, D. L., Liang, M., and Vatz, K. A. (1965) J. Biol. Chem. 240, 3609–3616. 16. Kilberg, M. S., Handlogten, M. E., and Christensen, H. N. (1981) J. Biol. Chem. 256, 3304–3312. 17. Puissant, C., and Houdebine, L.-M. (1990) BioTechniques 8, 148– 149. 18. Zhou, M. Y., Xue, D., Gomez-Sanchez, E. P., and Gomez-Sanchez, C. E. (1994) BioTechniques 16, 58–59. 19. Matthews, J. C., Beveridge, M. J., Malandro, M. S., Rothstein, J. D., Campbell-Thompson, M., Verlander, J., Kilberg, M. S., and Novak, D. A. (1998) Am. J. Physiol., in press. 20. Dudeck, K. L., Dudenhausen, E. E., Chiles, T. C., Fafournoux, P., and Kilberg, M. S. (1987) J. Biol. Chem. 262, 12565–12569.
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21. Laemmli, U. K. (1970) Nature 227, 680–685. 22. Utsunomiya-Tate, N., Endou, H., and Kanai, Y. (1996) J. Biol. Chem. 271, 14883–14890. 23. Malandro, M. S., and Kilberg, M. S. (1996) Annu. Rev. Biochem. 65, 305–336. 24. Kanai, Y., Stelzner, M., Nußberger, S., Khawaja, S., Hebert, S. C., Smith, C. P., and Hediger, M. A. (1994) J. Biol. Chem. 269, 20599–20606. 25. Dowd, L. A., Coyle, A. J., Rothstein, J. D., Pritchett, D. B., and Robinson, M. B. (1996) Mol. Pharmacol. 49, 465–473. 26. Nicholson, B., and McGivan, J. D. (1996) J. Biol. Chem. 271, 12159–12164. 27. Haugeto, O., Ullensvang, K., Levy, L. M., Chaudhry, F. A., Honore, T., Nielsen, M., Lehre, K. P., and Danbolt, N. C. (1996) J. Biol. Chem. 271, 27715–27722. 28. Kanai, Y., Smith, C. P., and Hediger, M. A. (1994) FASEB J. 8, 1450–1459. 29. Pines, G., Danbolt, N. C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., Koepsell, H., Storm-Mathisen, J., Seeberg, E., and Kanner, B. I. (1992) Nature 360, 464–467. 30. Storck, T., Schulte, S., Hofmann, K., and Stoffel, W. (1992) Proc. Natl. Acad. Sci. USA 89, 10955–10959. 31. Kanai, Y., and Hediger, M. A. (1992) Nature 360, 467–471. 32. Fairman, W. A., Vandenberg, R. J., Arriza, J. L., Kavanaugh, M. P., and Amara, S. G. (1995) Nature 375, 599–603. 33. Arriza, J. L., Eliasof, S., Kavanaugh, M. P., and Amara, S. G. (1997) Proc. Natl. Acad. Sci. USA 94, 4155–4160.
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AB972432
An Expression System for Mammalian Amino Acid Transporters Using a Stably Maintained Episomal Vector James C. Matthews,*,† Ara M. Aslanian,* Kelly K. McDonald,* Wenbo Yang,* Marc S. Malandro,* Donald A. Novak,† and Michael S. Kilberg*,1 *Department of Biochemistry and Molecular Biology and †Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32610
Received August 8, 1997
Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X0 AG, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNAtransfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein– Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.41) and EAAC1 (391) transport activity than does the vaccinia/HeLa expression system (2.61 and 221, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X0AG, or A; and (iii) the endogenous transport velocities of Systems ASC and X0 AG in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism. q 1997 Academic Press
Transient expression of cloned solute transporter proteins in cultured cells is used to characterize trans1 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Florida College of Medicine,1600 S.W. Archer Road, Box 100245, Gainesville, FL 32610-0245. Fax: (352) 392-6511.
port properties, identify the apparent size of protein products, study protein trafficking, and investigate the functional significance of conserved amino acid residues through the use of chimera, deletion, and mutagenic analyses. A number of cell transfection/expression regimens have been developed to express transport proteins in model systems. Of these, the vaccinia virus/HeLa cell system is used often to express a wide variety of mammalian transporters (1–3). Protocols for the transient expression of cDNAs in HeLa cells using vaccinia typically consist of infecting HeLa cells with a recombinant vaccinia virus that encodes T7 RNA polymerase, followed by liposome-mediated transfection of the cells with the transporter cDNA vector, for which transcription is under control of the T7 promoter. Despite the usefulness of the vaccinia/HeLa cell expression model, it may not necessarily be optimal for all transport proteins. For example, the amount of System ASCT1 activity (Na/-dependent neutral amino acid transport) expressed after transfection of the ASCT1 cDNA using this regimen is only one- to threefold over background (4, 5), although higher expression in oocytes is possible (6). We have tried without success numerous modifications of the originally reported vaccinia/HeLa cell expression regimen (3) to optimize the vaccinia-induced expression of the ASCT1 protein. Systematic changes have included the use of different transfection reagents, expression times, vectors, and cell lines. Therefore, we decided to evaluate a different expression system to determine whether greater amounts of functional System ASCT1 activity could be achieved. Epstein–Barr virus (EBV)2-based vectors are capa2 Abbreviations used: EBV, Epstein–Barr virus; 293c18 cells, human embryonic kidney (293) cell line stably transfected with EBNA1 cDNA; MeAIB, 2-(methylamino) isobutyric acid; MBP, maltose binding protein; pBKS II, pBluescript II KS { plasmid.
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ble of stable transformation of cells that express the transacting EBNA-1 gene (7). When transfected into human embryonic kidney 293 cells (HEK 293) that have been made competent by stable transfection with the EBNA-1 cDNA (293c18 cells), the pDR2 vector has several features that suggest it would be useful for transporter expression studies. First, the cDNA pDR2 vector contains an EBV origin of replication sequence and, therefore, is maintained as an episome for many cell divisions, in the amount of 10 to 20 copies per cell. Second, the transcription of the cDNA insert sequence is driven by the Rous sarcoma viral (RSV) promoter, known to promote high levels of exogenous protein expression. Third, because the pDR2 vector contains the hygromycin resistance gene (hygr), only those cells transfected with the cDNA pDR2 vector are able to survive hygromycin B-resistance selection. Thus, transfection with the pDR2 vector allows differences in transfection efficiencies to be normalized by hygromycin B selection. Fourth, unlike vaccinia virus-based expression systems (8), the transfection of cells with the EBNA-1 gene and pDR2 vector has not been reported to reduce the translation of endogenous cellular proteins. This research evaluates the relative amount of functional amino acid transport activity of System ASCT1, encoded by human hippocampal ASCT1 cDNA, and 0 System XAG , encoded by rat hippocampal EAAC1 cDNA, after expression in either the vaccinia/HeLa cell or EBNA-1/293c18 cell model. The results document that (i) the EBNA-1/293c18 expression system results in a larger increase over background of System ASCT1 transporter than does the vaccinia/HeLa expression system; (ii) the level of cDNA-induced EAAC1 activity over endogenous System X0AG activity in the EBNA-1/ 293c18 model exceeds the relatively high level of expression observed in the vaccinia/HeLa model; (iii) transfection of only the pDR2 vector does not affect the endogenous transport capacities of Systems ASC, 0 , or A; and (iv) exogenous cDNA-mediated induction XAG 0 of either System ASC or XAG activity in 293c18 cells does not adversely affect the endogenous transport capacity of the other system. We conclude that the EBNA1/293c18 cell expression model represents a useful transient expression regimen for the characterization of mammalian amino acid transport proteins, especially for those that exhibit relatively low activity in transient expression systems that lack a selection mechanism for expressing cells. MATERIALS AND METHODS
Preparation of plasmid constructs. A full-length ASCT1 cDNA was isolated and cloned from a human hippocampal cDNA library (4, 9) and a full-length EAAC1 cDNA was isolated from a rat brain hippocam-
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pal library by degenerate PCR followed by cDNA library screening (10). The ASCT1 or EAAC1 cDNAs were subcloned, as previously described (4), into pBluescript KS II (pBKS II) at the XhoI/EcoRI sites of the multiple cloning region for expression driven by either the T7 RNA polymerase or cytomegalovirus (CMV) polymerase promoter sequences. The ASCT1 or EAAC1 cDNAs also were subcloned between the SalI and BamHI sites of the pDR2 plasmid vector (Clonetech Laboratories, Palo Alto, CA). Expression of ASCT1 and EAAC1 transporters in HeLa cells. HeLa cells were purchased from American Tissue Culture Collection (Rockville, MD) and were maintained as described (4). Transient transporter expression in HeLa cells was performed as described (10, 11). Briefly, HeLa cells were plated at a density of 150,000 cells per well on Costar 24-well cluster trays and infected with recombinant vaccinia virus (vTF7-3, 15 pfu/cell) expressing T7 RNA polymerase (1). The cells were infected for 45 min at 377C. The virus-containing medium was then removed and replaced with minimal essential medium (OptiMEM I, GibcoBRL, Gaithersburg, MD) containing 12 ml/ml lipofectamine (GibcoBRL) and 4 mg/ml ASCT1 or EAAC1 cDNA in pBII KS. The cells were incubated for 8 h before assaying for amino acid transport activity. Background transport activity was assayed in cells transfected with the pBII KS vector that lacked a cDNA insert. Expression of ASCT1 or EAAC1 transporter in 293c18 cells. The 293c18 cell line was maintained according to the instructions of the supplier (Invitrogen, San Diego, CA). These cells have been stably transfected with the EBV transformation antigen EBNA-1 to permit the episomal maintenance of the pDR2 plasmid, which contains an EBV origin of replication. The 293c18 cells in passage numbers 2–5 were cultured until 80% confluent at 377C in 5% CO2 and Dulbecco’s minimum essential medium that contained streptomycin sulfate (100,000 mg/l), amphotericin B (250 mg/l), penicillin G sodium (100,000 U/l), and G418 sulfate (250 mg/l) and 10% fetal bovine serum (DMEM/FBS), where indicated. The cells were then transfected in OptiMEM by incubation with 4 mg/ml EAAC1–pDR2, or pDR2 vector lacking a cDNA insert, and 12 ml/ml lipofectamine. After 7 h, the transfection solution was removed and the cells were cultured in DMEM/FBS for 24 h and then for 10 days in DMEM/FBS that contained hygromycin B (400 U/ml) to select for pDR2-transfected cells. During the selection period, the medium was changed every 3 days. After 10 days, the cells were washed three times with PBS (pH 7.4), trypsinized, resuspended in DMEM/FBS that contained 10% DMSO, slow-frozen at 0807C for 1 day, and then stored in liquid N2 . After thawing, the cells were cultured in hygromycin B-containing DMEM/FBS until 80% con-
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fluent for use in whole-cell transport assays or determination of mRNA and protein content. Whole-cell transport assay. Amino acid uptake by HeLa or 293c18 cells was measured by the 24-well cluster tray method of Gazzola et al. (12) with modifications by our laboratory (13). To normalize the intracellular pools of amino acids before the transport assays, cells were incubated at 377C for 30 min in Na/ –Kreb’s– Ringer’s phosphate (NaKRP; 119 mM NaCl, 5.9 mM KCl, 1.2 mM MgSO4 , 1.2 mM KHCO3 , 5.6 mM glucose, 0.5 mM CaCl2 , 25 mM Na2HPO4 , pH 7.4). The amount of Na/-dependent uptake was calculated as the difference between uptake in Na/-containing KRP and Na/-free (choline-KRP) buffers. System 0 XAG /EAAC1 activity was calculated as the amount of 1 mM [3H]glutamate Na/-dependent uptake that was inhibitable by unlabeled 500 mM D-aspartate (14). System A activity was taken as the Na/-dependent uptake of 50 mM [3H]2-(methylamino) isobutyric acid (MeAIB) (15, 16). System ASC/ASCT1 activity was calculated as the amount of Na/-dependent 5 mM [3H]serine uptake that remained in the presence of unlabeled 5 mM MeAIB and 5 mM arginine to eliminate serine uptake by transport Systems A and y/L, respectively (5). Northern analysis. Total RNA was isolated (17) from cultured cells grown to 80% confluence. The RNA was size-separated in a 1% agarose gel by electrophoresis in the presence of 0.02 M formaldehyde. Densitometric analysis of ethidium bromide-stained 28S and 18S ribosomal RNA bands was used to confirm that the lanes were equally loaded with RNA. The RNA was transferred by downward capillary action (18) to 0.45mm nylon membranes, covalently cross-linked by UV light, and hybridized for 15 to 18 h at 677C with individual 32P-labeled cDNA probes in solutions that contained 1% BSA, 7% SDS, 0.5 M Na2HPO4 (pH 7.2), and 1 mM EDTA. The radiolabeled cDNA probes were prepared from full-length rat EAAC1 (2.2 kb) (10) and full-length human ASCT1 (4) by random priming extension using [32P]dCTP (Amersham Life Sciences Inc., Arlington Heights, IL) and a kit from GibcoBRL. After hybridization, the blots were washed 4 1 15 min at 677C in 40 mM Na2HPO4 (pH 7.2), 0.1% SDS, and 1 mM EDTA. Immunoblot analysis. Immunoblot analysis for EAAC1 was performed as described (19). Briefly, a plasma membrane-enriched fraction of both transfected cell types was generated by differential centrifugation (20). The membranes were resuspended in 0.25 mM sucrose, 10 mM Hepes–KOH (pH 7.5), 1 mM EGTA, and 2 mg/ml each of N-tosyl-L-phenylalanine chloromethyl-ketone, N-a-p-tosyl-L-lysine ketone, leupeptin hemisulfate, aproptinin, and pepstatin A to prevent proteolysis. Membrane proteins were separated by 7.5% SDS–PAGE (21) and then electrotransferred
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TABLE 1
Serine Uptake by System ASC by HeLa or HEK 293c18 Cells Expressing ASCT1 Vector Cells HeLa 293c18
ASCT1
(pmolrmg01proteinrmin01) 119 { 1.7 697 { 32
305 { 6.9* 4447 { 303*
Note. Each value is the mean { SE for Na/-dependent uptake of 5 mM L-[3H]serine (in the presence of 5 mM MeAIB). HeLa (n Å 3) or HEK 293c18 cells (293c18, n Å 4) were transfected with vector only (Vector) or ASCT1–vector (ASCT1), using pBKS II or pDR2 vectors, respectively. Uptake was measured for 1 min at 377C as described in the text. These values are representative of multiple experiments. * P õ 0.005 versus vector-only transfected cells.
to a 0.45-mm nitrocellulose membrane (Schleicher & Schuell, Keene, NH). For the detection of EAAC1 protein, blots were probed with 43 ng IgG/ml of the EAAC1 polyclonal antibody (19) or preimmune sera in blocking solution (1% nonfat dry milk and 2% casein hydrolysate in 10 mM Tris–Cl, pH 7.5, 300 mM NaCl) for 1 h at room temperature with agitation. Horseradish peroxidase-conjugated protein A (Amersham) was used to detect immunoreactive bands by visualization with a chemiluminescence kit (Amersham). Statistical analysis. Transport assays were performed in quadruplicate within each experiment and most experiments were repeated using independent batches of transfected cells. Differences between mean uptake values were determined by Student’s two-tailed t tests. RESULTS
Expression of human ASCT1 mRNA and activity in HeLa or 293c18 cells. The expression of the human ASCT1 transport protein in HeLa cells transiently transfected with pBSKII–ASCT1 plasmid or in 293c18 cells transfected with stably maintained pDR2–ASCT1 was evaluated. Consistent with previous reports (4, 5, 9), the activity of ASCT1 in HeLa cells transfected with the System ASCT1 cDNA was about 2.6 times that in cells transfected with the pBSKII plasmid only (Table 1). Previous Northern blot analysis by this laboratory has demonstrated that the steady-state ASCT1 mRNA content is increased to a much greater degree than the transport activity in HeLa cells that have been transfected with the pBSKII–ASCT1 construct (5). To determine if the expression of ASCT1 activity could be increased by employing the EBNA-1/293c18 cell expression system, 293c18 cells were transfected with the human ASCT1 cDNA sequence in pDR2. The ASCT1mediated Na/-dependent uptake of 5 mM [3H]serine by
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Activities of Amino Acid Transport Systems X0 AG , A, and ASC in pDR2-Transfected or Nontransfected HEK 293c18 Cells Nontransfected System X0 AG A ASC
FIG. 1. Steady-state expression of ASCT1 mRNA in HEK 293c18 cells transfected with ASCT1 transporter cDNA. Northern blot analysis of total cellular RNA (30 mg per lane) was performed as described under Materials and Methods. Replicate blots were hybridized with a 32P-labeled cDNA probe specific for human ASCT1 mRNA. Densitometry of a photographic negative of the ethidium bromide-stained gel was used to document that the amounts of 28S and 18S ribosomal RNA bands were equal between lanes.
the ASCT1-transfected cells was increased 6.4 times over control cells transfected with pDR2 vector only (Table 1). Consistent with the increase in System ASCT1 activity, ASCT1 mRNA was detected readily in ASCT1-transfected 293c18 cells (Fig. 1), whereas ASCT1 mRNA was not detectable in equal amounts of RNA isolated from pDR2-only-transfected cells. Therefore, the endogenous System ASC transport activity is likely to result from the expression of another member of the ASC gene family (22, 23). The expression of ASCT1 protein levels was not investigated in either expression system because antibodies are not available, but expression of activity is taken to represent functional protein residing in the plasma membrane. To determine whether the pDR2 expression system alters the endogenous amino acid transport capacity of 293c18 cells, the transport velocities of Systems ASC, 0 were compared between nontransfected A, and XAG 293c18 cells and pDR2-transfected 293c18 cells subjected to hygromycin B selection. The activity of these endogenous transport systems was not affected by pDR2 transfection/selection (Table 2), documenting that these procedures do not increase or decrease endogenous membrane transport. Expression of rat EAAC1 mRNA and activity in HeLa or 293c18 cells. To further assess the utility of the EBNA-1/293c18 expression model, EAAC1-mediated Na/-dependent anionic amino acid transport activity, mRNA, and protein content were measured in either 293c18 cells transfected with pDR2–EAAC1 vector or HeLa cells transiently transfected with pBKS II–
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pDR2-transfected
(pmolrmg01proteinrmin01) 34.7 { 4.9 149 { 12 847 { 10
27.8 { 5.7 127 { 10 795 { 32
Note. Each value is the mean { SE uptake (pmolrmg01proteinr min01; n Å 5 to 8 observations) by the indicated transport activity in HEK 293c18 cells transfected with the pDR2 plasmid lacking insert and selected in hygromycin B as described in the text. The Na/dependent uptake of 1 mM L-glutamate in the presence of 500 mM 0 D-aspartate (System XAG), 50 mM MeAIB (System A), or 5 mM L[3H]serine in the presence of 5 mM MeAIB and 5 mM L-arginine (System ASC) by cells transfected with vector only (pDR2) or not transfected (293c18) was measured for 1 min at 377C as described in the text. Uptake values between nontransfected and pDR2-transfected cells did not differ when compared with Student’s two-tailed t test (a Å 0.05 level).
EAAC1 cDNA (Fig. 2, Table 3). Two predominant species of mRNA (4.2 and 2.3 kb) were expressed in HeLa cells transfected with the pBKS II–EAAC1 plasmid, whereas the EAAC1 mRNA was below detectable levels in pBKS II-only-transfected cells (Fig. 2A). Immunoblot analysis revealed a similar increase in EAAC1 protein
FIG. 2. Steady-state expression of EAAC1 mRNA and protein following transfection of either HeLa or HEK 293c18 cells. Northern and immunoblot blot analyses were performed as described in the text for HeLa (A) or 293c18 (B) cells transfected with vector only (Vector) or EAAC1 cDNA (EAAC1), using pBKS II (HeLa cells) or pDR2 (293c18 cells) vectors, respectively. Total cellular RNA (7.5 mg per lane, HeLa; or 25 mg/lane, 293c18) was hybridized with a 32Plabeled cDNA probe specific for EAAC1 mRNA. Densitometry of a photographic negative of the ethidium bromide-stained gel was used to document that the amounts of 28S and 18S ribosomal RNA bands were equal between lanes. Plasma membrane proteins (35 mg) were subjected to 7.5% SDS–PAGE and immunoblotted with polyclonal antibodies to EAAC1, as described under Materials and Methods.
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Na/-Dependent Uptake of Aspartate by HeLa or HEK 293c18 Cells Expressing EAAC1 Vector
EAAC1
(pmolrmg01proteinrmin01)
Cells HeLa 293c18
50.2 { 0.2 61.1 { 4.6
1106 { 76* 2396 { 209*
Note. Each value is the mean { SE uptake of 50 mM L-[3H]aspartate (pmolrmg01proteinrmin01) by HeLa cells (n Å 3) or by HEK 293c18 cells (293c18, n Å 4) transfected with vector only (Vector) or EAAC1vector (EAAC1), using pBKS II (HeLa) or pDR2 (293c18 cells), respectively. Uptake was measured for 1 min at 377C as described in the text. These values are representative of multiple experiments. * P õ 0.005 versus vector-only transfected cells.
content in HeLa cells transfected with EAAC1–pBKS II (Fig. 2A). In both transfection treatments, a prominent band of about 60 kDa and a weakly expressed band of about 57 kDa were detected. For the pDR2– EAAC1-transfected 293c18 cells, a single EAAC1 mRNA species of 3.4 kb was detected by Northern analysis of total RNA (Fig. 2B), which is consistent with that detected in human kidney tissues (24). As with HeLa cells, no endogenous EAAC1 mRNA was detectable in 293c18 cells transfected with plasmid only. Expression of the transfected EAAC1 cDNA resulted in a single EAAC1 protein band of about 68 kDa (Fig. 2B). The appearance of multiple protein bands for EAAC1 has been reported in native tissue (19, 25), as well as after cRNA injection into oocytes (25) and cDNA transfection into cultured cells (26, 27). The difference in the apparent molecular size of EAAC1 protein when EAAC1 cDNA is expressed in HeLa versus 293c18 cells also is consistent with the observations of others who have documented protein sizes ranging from 61 to 78 kDa (19, 25–27). It is thought that these differences arise from cell-specific patterns of glycosylation because treatment with the appropriate glycosidase results in generation of a single band corresponding to the predicted core molecular size of 57 kDa (24, 26). The existence of different mRNA species following expression of the 2.2-kb EAAC1 cDNA probably arises from incomplete termination of RNA polymerase, incorrect processing of the nascent transcript, or differences in polyadenylation. The Na/-dependent uptake of 50 mM [3H]aspartate in EAAC1-transfected HeLa cells was about 22 times greater than that in vector-only-transfected controls (Table 3), a result consistent with a previously published report (10). The Na/-dependent uptake of 50 mM [3H]aspartate was also higher (approximately 39 times) in 293c18 cells transfected with the pDR2–EAAC1 plasmid than in vector-only-transfected controls (Table 3). The aspartate uptake was inhibited by either 5 mM
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L-glutamate (96%) or 5 mM D-aspartate (92%) (data not shown), consistent with published reports of EAAC1 0 transporter substrate specificity (28) and System XAG activity. For both expression systems, the increase in EAAC1-mediated Na/-dependent glutamate transport activity paralleled the increase in EAAC1 mRNA and protein content (Fig. 2). Given the lack of detectable endogenous EAAC1 mRNA, the endogenous System 0 XAG activity may be the result of expression of one or more of the other glutamate/aspartate transporter genes (29–33). To determine if the transfected cDNA altered the activity of other endogenous transporters, the endogenous System ASC activity was measured in pDR2EAAC1-transfected 293c18 cells and the endogenous 0 System XAG activity was measured in pDR2–ASCT1transfected 293c18 cells (Table 4). Consistent with the hypothesis that these cDNA sequences encode specific transporter proteins, the expression of either human ASCT1 or rat EAAC1 in 293c18 cells did not alter the activity of alternate endogenous amino acid transport systems within the cell. Furthermore, these observations, along with those in Table 1, indicate that endogenous transport in 293c18 cells is not adversely affected by the transfection process, the subsequent hygromycin B selection, or the expression of exogenous transport proteins.
DISCUSSION
This study demonstrates that the 293c18/pDR2 expression model for transporter expression in mammalian cells is a viable alternative to the often used vaccinia/HeLa cell system. Indeed, the data show that relative to the endogenous rate, the measurable activ-
TABLE 4
Consequence of EAAC1 or ASCT1 cDNA Expression on the Endogenous Transport Activities of Systems ASC and X0 AG in HEK 293c18 Cells Vector only Transfected cDNA
Endogenous system tested
ASCT1 ASCT1 EAAC1 EAAC1
ASC X0 AG ASC X0 AG
ASCT1 or EAAC1
(pmolrmg01proteinrmin01) 1338 32.5 427 13.4
{ 105 { 0.7 { 117 { 0.7
5007 32.6 499 342
{ 170* { 1.6 { 30 { 31*
Note. Each value is the mean { SE for System ASC-mediated uptake (pmolrmg01proteinrmin01) of 5 mM L-[3H]serine or System 3 X0 AG-mediated uptake of 1 mM L-[ H]glutamate by HEK 293c18 cells (n Å 6 to 8) after transfection with pDR2 plasmid-only (vector), ASCT1–pDR2 (ASCT1), or EAAC1–pDR2 (EAAC1). Uptake was measured for 1 min at 377C as described in the text. * P õ 0.05 versus vector-only transfected cells.
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ity of two different amino acid transporters, ASCT1 and EAAC1, is greater when their cDNAs are expressed in selectable 293c18/pDR2 cells than in HeLa/vaccinia cells. Compared to the 293c18 cell/ASCT1–pDR2 transfection protocol, the increase in System ASCT1 transport activity in ASCT1–pBKS II-transfected HeLa cells was relatively small, despite the fact that steady-state ASCT1 mRNA levels are much greater in ASCT1-transfected cells than in plasmid-only-transfected cells (5). These observations suggest that some transporter proteins, when expressed in vaccinia-infected HeLa cells, are produced in significant quantities, but that functional transport capacity may not be realized, possibly compromised by inadequate trafficking or the presence of other required regulatory proteins. As shown for a variety of transport proteins expressed with the vaccinia/HeLa system, the results from the present study show that EAAC1 mRNA, protein content, and activity are increased significantly in vaccinia virus-infected HeLa cells. A number of parameters were altered in an attempt to optimize the conditions for functional ASCT1 expression in vaccinia-infected cells. The transfection of several independent cell lines (each shown to be susceptible to vaccinia infection) with either ASCT1–pBKS II or ASCT1–pcDNA3 plasmid was evaluated, but each of the possible combinations gave similar low levels of increased System ASCT1 activity to that observed in several previous studies (4, 5). Likewise, several combinations of cDNA and lipofectamine concentrations were evaluated, as well as cell density and culture media composition. None of these parameters significantly enhanced the functional expression of System ASCT1 activity. Depending on the experimental goals, an additional limitation of the vaccinia-based transfection system can be the transient nature (usually 8 to 12 h) of the expression, imposed because of virus-induced deterioration of the cells with time of culture. Consequently, the sensitivity and reproducibility of transporter expression are compromised by transfection efficiency and viral infectivity, with little or no opportunity to normalize independently transfected culture dishes or wells. In contrast, one of the strengths of the 293c18/ pDR2 system is the ability to measure cells shortly after transfection or to select only those cells that have been transfected with pDR2 plasmid. Thus, after a selection period of the investigator’s choosing, cells from several large culture flasks can be combined to generate a common pool and then plated onto 24-well cluster trays. In this way, the transfection efficiency across all transport assays will be constant, even if the hygromycin B selection period was shortened and, thus, less than 100%. The maintenance of 10 to 20 copies of pDR2 plasmid as a replicating episome permits cell division without cDNA dilution. In this sense, the 293c18/pDR2
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system has similarities to both transient and stable transfections. The observation that the relative increase in transport activity paralleled the increase in mRNA and protein levels in cells transfected with transporter cDNA suggests that the 293c18/pDR2 expression system will be an effective model to use for the study of (i) amino acid transport protein function/structure, (ii) regulators of transporter expression and function, and (iii) cDNA library screening in mammalian cells. With regard to the later point, the fact that the pDR2 vector does not to integrate into cellular chromosomes, but instead is maintained as an episome, would allow use of the pDR2/293c18 system to screen cDNA expression libraries with subsequent recovery of the cDNA. There are several potential limitations of the 293c18/ pDR2 expression system that should be considered by prospective users of this expression model. First, if one wishes to select for 100% of expressing cells, this process takes up to 10 days. Second, the inability of EBV to replicate in rodent cells (7) eliminates its use in cells from important laboratory animals. Third, our experience indicates that the maximal level of increased transporter activity achieved after the hygromycin B selection period can only be maintained for 12 to 16 subsequent cell divisions (unpublished data). After this number of cell divisions, the activity significantly decreases. Despite these restrictions, as demonstrated by the present research, the expression of mammalian cDNAs with the pDR2 vector into 293c18 cells expressing EBNA-1 has the potential to be a flexible and useful model for the study of amino acid transport proteins in mammalian cells. ACKNOWLEDGMENTS This research was supported by the National Institutes of Health, the National Institute of Child Health and Human Resources (HD29934 to D.A.N.), the Institute for Diabetes, Digestive, and Kidney Diseases (DK-28374 to M.S.K.), and an Institutional National Research Service Award (DK07667 to J.C.M.).
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