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FASEB J. Jul 2011; 25(7): 2180–2187.
PMCID: PMC3114536

A GAG trinucleotide-repeat polymorphism in the gene for glutathione biosynthetic enzyme, GCLC, affects gene expression through translation

Abstract

A guanine-adenine-guanine (GAG) repeat polymorphism with 5 different alleles (4, 7, 8, 9, and 10 repeats) in the 5′ untranslated region (UTR) of GCLC has been associated with altered GCL activity and glutathione (GSH) levels. We investigated whether this polymorphism affects either transcription or translation using luciferase reporter constructs containing variant GCLC 5′ UTRs. Higher luciferase activity was observed in HepG2 and human embryonic kidney 293 (HEK293) cells transfected with constructs containing either 8 or 9 repeats than in constructs containing 4, 7, or 10 repeats (P<0.05). In cell-free lysates, GAG repeat number had no effect on luciferase mRNA yield. In vitro translation of mRNAs from luciferase constructs resulted in differences similar to those found in cell cultures (P<0.05). A similar association of GAG repeat with GCLC phenotype was observed in vivo in healthy adults, as individuals with GAG-7/7 genotype had lower GCL activity and GSH levels in lymphocytes compared to those with GAG-9/9 (P<0.05). Higher GCL activity and GSH levels observed in red blood cells (RBCs) from individuals with GAG-7/7 compared to GAG-9/9 are likely due to differences in GCL regulation in RBCs. Altogether, these results suggest that GAG polymorphism affects GCLC expression via translation, and thus may be associated with altered risk for GSH-related diseases and toxicities.—Nichenametla, S. N., Lazarus, P., Richie, J. P., Jr. A GAG trinucleotide-repeat polymorphism in the gene for glutathione biosynthetic enzyme, GCLC, affects gene expression through translation.

Keywords: γ-glutamylcysteine ligase, short tandem repeats, 5′ untranslated region

Glutathione (GSH), the most abundant intracellular antioxidant, has numerous functions in addition to protecting cells against oxidative stress, including the detoxification of toxins and carcinogens (13), maintenance of thiol groups in proteins, and post-translational regulation of protein function (4). Nearly all tissues in the body can synthesize GSH by sequential addition of the precursor amino acids, cysteine (Cys), glutamic acid, and glycine through enzymatic catalysis by 2 ATP-dependent enzymes, γ-glutamylcysteine ligase (GCL) and GSH synthetase (GS) (5). Because of the wide-ranging and essential physiological roles of GSH, the maintenance of optimal tissue levels of GSH is critical for maintaining health and preventing the development of numerous diseases. Even partial GSH depletion has been shown to decrease immune function (6) and increase susceptibility to a wide range of xenobiotics (7) and oxidative damage (8). For example, lower GSH levels have been associated with diseases such as cancer (9), cardiovascular diseases, arthritis, and diabetes (1011).

GSH levels can be regulated by enzymatic activity of GCL and GS, availability of the precursor amino acids Cys and methionine, and efflux of oxidized GSH (GSSG) from cells. However, activity of GCL, the rate-limiting enzyme for GSH biosynthesis, appears to be the major factor that determines GSH levels in healthy individuals. Genetic polymorphisms in the genes that code for the catalytic and modulator subunits of the heterodimer GCL (GCLC and GCLM, respectively) are associated with lower GSH levels in vitro and altered susceptibility to certain diseases (1214).

A guanine-adenine-guanine (GAG) trinucleotide short tandem repeat polymorphism has been identified in the 5′ untranslated region (UTR) of GCLC, positioned 10 bp upstream of the ATG start site in exon 1. The polymorphism consists of 5 different alleles, each with 4, 7, 8, 9, and 10 consecutive GAG repeats (represented as the GAG-4, GAG-7, GAG-8, GAG-9, and GAG-10 alleles, respectively). The most prevalent alleles are GAG-7 and GAG-9, followed by GAG-8, with GAG-4 and GAG-10 being rare and found primarily in African Americans (15). An association between the GAG repeat alleles and intracellular GSH levels was demonstrated in tumor cell lines, suggesting that it may be functional (15). The GAG repeat polymorphism is also associated with several disease conditions, such as schizophrenia (16), chronic beryllium disease (17), and diabetes (18). In a previous study, we demonstrated that healthy adult individuals with the GAG-9/9 genotype have 15% lower GSH levels and 21% lower GCL activity in blood than individuals with GAG-7/9 and GAG-7/7 genotypes, respectively (19). In addition, the GCLC protein levels were 50 and 46% lower in individuals with GAG-9/9 genotype than GAG-7/7 and GAG-7/9 genotypes, respectively (unpublished results).

While the GAG repeat polymorphism appears to have some functionality, the molecular events by which it may affect GCL activity are not known. Studies on GAG repeat polymorphisms in other genes, such as PAX7 and PHGDH, and secondary structure predictions of the GAG sequence suggest both transcriptional and translational regulatory roles for the repeat sequence (2021). In this study, it was hypothesized that the GAG repeat polymorphism alters GCL activity by affecting translation of the GCLC mRNA. To test this, we examined the effect of variant 5′ UTRs of GCLC with different number of GAG repeats on luciferase expression in vitro in cell culture and in cell-free lysate systems.

MATERIALS AND METHODS

Luciferase reporter constructs

Five different luciferase reporter constructs (denoted as GAG4, GAG7, GAG8, GAG9, and GAG10) were made by inserting GCLC 5′ UTRs with 4, 7, 8, 9, or 10 GAG repeats, respectively, into the PGL3 control vector. The resulting constructs had SV40 promoter 5′ to the GCLC UTR, and the 3′ end was followed by the luciferase gene, poly(A) signal, and SV40-enhancer (Fig. 1). In addition to these 5 constructs, a sixth construct (RAN9), with the same length (27 nt) as GAG9 but with the GAG repeat region substituted by a random sequence, was also made and used as a random control. The PGL3 vector without the GCLC 5′ UTR region inserted was also included in each analysis.

Figure 1.
Schematic representation of the PGL3 construct. GCLC 5′-UTR regions with different numbers of GAG repeats (4, 7, 8, 9, and 10) were inserted between 3′ end of the SV40 promoter and 5′ end of the luciferase gene. PGL3 vector without ...

Luciferase reporter constructs were made by 3 tandem PCRs. Genomic DNA from an individual with the GAG-7/7 genotype was used as a template for the first PCR. The product of the first PCR was used as the template for the second PCR, and the product of the second PCR was used as template for the third PCR. Forward and reverse primer sequences for all of the PCRs are outlined in Table 1. Primers for the first PCR amplified the entire 385 bp of the GCLC 5′ UTR. To change the number of GAG repeats in the GCLC 5′-UTR region, a second set of forward and reverse primers was used. 5′-UTR inserts with 4, 8, 9, and 10 GAG repeats were made by using a single forward primer and 4 different reverse primers. The reverse primers contained different numbers (4, 8, 9, and 10) of CTC (complimentary to GAG nucleotides) repeat nucleotides. A fifth reverse primer was used to generate a 5′-UTR insert with random nucleotides. This primer was made by substituting the 9 CTC repeats of reverse primer used for generating the 9 GAG repeat-5′-UTR insert with 27-nt random sequence. Finally, in the third PCR, restriction sites for HindIII and NcoI were added on either side of the 5′-UTR regions. Reaction conditions for all three of the PCRs were the same except for melting temperature (Table 1). The 100-μl reaction mixture consisted of 100 ng of DNA, 10 μl of 10× Pfu buffer, 0.8 μl each of 25 mM dNTPs, 2.5 μl of 20 mM forward and reverse primers, 2 μl of Pfu turbo polymerase (Stratagene, Wilmington, DE, USA), and 20 μl glycerol. Thermocycling parameters included initial denaturation at 98°C for 45 s, followed by 30 cycles of denaturation (98°C for 45 s), 45 s of melting (for primer specific melting temperatures, see Table 1), and 60 s of extension at 72°C, with a final extension for 10 min at 72°C.

Table 1.
Template and primer sequences used to synthesize GAG repeat inserts

Each of the 6 PCR-generated GCLC 5′ UTRs was inserted into the PGL3 vector after digestion of both the PCR-generated 5′ UTRs and the PGL3 vector with the restriction endonucleases Hind-III and NcoI to generate sticky and compatible ends. Each digestion reaction was carried out in a final volume of 25 μl consisting of 500 ng of either the PGL3 control vector or the GCLC 5′-UTR inserts, 2.5 μl of 10× buffer E (6 mM Tris-HCl, 6 mM MgCl2, 100 mM NaCl, and 1 mM DTT, pH 7.5), 0.2 μl of BSA, and 10 U each of HindIII and NcoI. The reaction mixtures were incubated at 37°C for 4 h, after which the enzymes were inactivated by incubating at 60°C for 30 min. Restriction-digested PGL3 vector and GCLC 5′-UTR inserts were run in 0.5% and 4% agarose gels, respectively, and purified using Qiaquick gel extraction kits (Qiagen, Valencia, CA, USA) following manufacturer's recommendations. The purified, digested GCLC 5′-UTR inserts and PGL3 vector were ligated using T4-DNA ligase (Promega, Madison, WI, USA). The ligation reactions consisted of a total of 90 ng of one of the five 5′-UTR inserts and 400 ng of the PGL3 vector (molar ratio 3:1), 2.5 μl of T4-DNA ligase, and 5 μl of 10× reaction buffer (300 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, and 10 mM ATP, pH 7.8) in a final volume of 50 μl. The reactions were incubated at 14°C overnight, run in 0.5% agarose gels, and purified using the Qiaquick gel extraction kit. An aliquot of the ligated DNA was sequenced to confirm the presence of the GCLC 5′ UTR and the expected number of GAG repeats. GCLC 5′-UTR-PGL3 vectors were transformed into competent DH5α Escherichia coli cells (New England Biolabs, Ipswich, MA, USA) using the manufacturer's protocol. Individual colonies were grown overnight at 37°C in ampicillin-resistant lactose-broth agar plates, single colonies were selected and grown overnight at 37°C, and plasmid DNA was extracted in minipreps using illustra bacteria genomic Prep Mini Spin Kit (GE Healthcare, Piscataway, NJ, USA). DNA from each construct was sequenced to verify the presence of GCLC 5′-UTR inserts and the expected number of GAG repeats. After a large-scale extraction of each GCLC 5′-UTR-PGL3 vector using the Qiagen Plasmid Maxi Prep following the manufacturer's protocol, constructs were again sequenced to confirm the presence of GCLC 5′-UTR-PGL3 vectors and GAG repeats.

Cell culture, transfection, and luciferase activity measurement

HepG2 and human embryonic kidney 293 (HEK293) cells were grown in Dulbecco's modified Eagle medium supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin (PS; Invitrogen). Each well of a 24-well plate was plated with 8 × 104 cells and incubated at 37°C, 5% CO2, and 95% humidity until 75% confluent. When cells reached 75% confluence, they were washed with PBS and conditioned for transfection with 400 μl of DMEM without FBS and PS for 2 h. All transfections were carried out using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. Cells in each well were transfected with 600 ng of each of the allelic constructs along with 120 ng of an internal control vector, pRL-tK (Renilla luciferase), to account for differences in transfection efficiency. After transfection for 6 h, medium was supplemented with 10% FBS. Luciferase activity was assayed using the dual luciferase assay kit (Promega, Madison, WI, USA), following the manufacturer's instructions. Cells were washed with PBS and lysed with 100 μl passive lysis buffer. Firefly and Renilla luciferase activities were determined in a plate reader (Synergy HT; Biotek, Winooski, VT, USA) by measuring the luminescence produced with the addition of 100 μl of LARII reagent and 100 μl of Stop and Glo reagent, respectively, to 20 μl of the lysate at room temperature.

Transcription in HeLa cell nuclear lysates

GCLC 5′-UTR-PGL3 constructs (2 μg) were incubated with HeLa cell nuclear lysates (Helascribe; Promega, Madison, WI, USA), for 2.5 h at 30°C. The 25-μl reaction mixture consisted of 0.4 mM each of the 4 rNTPS, 6 mM MgCl2, and 1× reaction buffer (20 mM HEPES, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% glycerol at pH 7.9 and 25°C). After incubation, the GCLC 5′-UTR-PGL3 constructs were digested with 2.5 μl DNase I (Invitrogen) at 25°C for 15 min. Immediately, DNase I was inactivated by the addition of 2.5 μl of 50 mM EDTA (65°C for 10 min), and 175 μl of HeLa stop solution was added and vortexed for 2 min. mRNAs were extracted using Tris-EDTA-saturated phenol:chloroform:isoamyl alcohol (PCI; 25:24:1; Ambion, Austin, TX, USA). The resulting pellet was washed in 100% ethanol and dissolved in RNase-free water. RNA concentrations were measured using a fluorescent intercalating dye, Ribogreen (Invitrogen), as recommended by the manufacturer.

Translation in rabbit reticulocyte lysates

Luciferase protein was synthesized using nuclease-treated rabbit reticulocyte lysate (Rabbit Reticulocyte Lysate System; Promega). Equal amounts of luciferase mRNAs, which were obtained by incubating each construct in the HeLa cell nuclear lysate (Promega), were incubated in a 50-μl reaction consisting of 70% lysates (35 μl) and 10 μmol amino acid mixture with fluorescently labeled lysine (FluoroTect GreenLys tRNA, Promega) for 2.5 h at 30°C. The translation reaction was stopped by incubating the reaction on ice for 10 min, and 50 μl of the reaction mixture was used for resolving proteins in 10% SDS-PAGE. Gels were scanned on a fluorescent bioimager (Typhoon 9300; Amersham Biosciences, Piscataway, NJ, USA), and the protein bands were quantified by densitometry (Visionworks LS; UVP, Upland, CA, USA).

Genotype/phenotype study

Fasting blood samples were obtained from a group of 23 healthy adults (23–35 yr of age), who were recruited as part of a previously described study (22). All study protocols were approved by the Penn State University College of Medicine Human Subjects Committee. Blood was collected by venipuncture into EDTA-containing tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using centrifugation on Ficoll-Hypaque gradient.

Total GSH was assayed enzymatically in metaphosphoric acid extracts of red blood cells (RBCs) or PBMCs, as described previously (23). GCL activity was determined in RBCs and PBMCs by measuring the product γ-glutamylcysteine, formed after incubating cell lysates with cysteine and glutamic acid, as described previously (19). Protein concentrations were measured by the bicinchoninic acid procedure (Pierce, Rockford, IL, USA). Hemoglobin was determined spectrophotometrically using Drabkin's reagent (24).

Determination of GAG repeat genotype

To isolate DNA, PBMCs were lysed in lysis buffer (10 mM Tris, 10 mM EDTA, 0.1 M NaCl, and 2% SDS) and incubated with proteinase K (0.1 mg/ml) at 58°C for 3 h. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol, as described previously (19). DNA containing the GAG trinucleotide repeat region was amplified by P32-labeled PCR amplification, as described previously (19, 25).

Statistics

Univariate ANOVA was used to determine whether the luciferase enzyme activities, mRNA expression, and protein quantities were different among groups. Post hoc analysis by Duncan's method was used to identify specific groups that were different from each other. Groups were considered to be significantly different at values of P ≤ 0.05. All statistical analyses were performed using SAS 9.1 (SAS Inc, Cary, NC, USA).

RESULTS

Effect of GAG repeat number on luciferase activity in transfected cell lines

The effect of GAG repeat number on luciferase activity was determined using a series of PGL3 constructs that contain the GCLC 5′ UTR with varying GAG repeat numbers that are naturally observed in humans (GAG4, GAG7, GAG8, GAG9, and GAG10). In cells transfected with luciferase vectors containing variant GCLC 5′-UTRs, luciferase activity was shown to vary significantly with different numbers of GAG repeats (Fig. 2). In HepG2 cells, luciferase activity was lowest for GAG4 and, with increasing numbers of GAG repeats, reached a maximum at GAG8 and GAG9, which were ~2-fold higher than GAG4 (Fig. 2). However, with a further increase in the number of repeats (GAG10), luciferase activity decreased. Luciferase activity in GAG4 was 27% lower than GAG7 (P=0.001), which, in turn, was 26% lower than GAG8 (P<0.001). Luciferase activity for GAG10 was 27 and 23% lower than GAG8 and GAG9, respectively (P<0.05). A similar pattern of activity was also found in HEK293 cells (Fig. 2), where luciferase activity with GAG4 was 28% lower than with GAG7 (P<0.001), whose activity was 18% lower than GAG8. Luciferase activity with GAG10 was 21 and 36% lower than with GAG8 and GAG9, respectively (P<0.001). In both HepG2 and HEK293 cells, RAN9 (construct with scrambled sequence) exhibited lower luciferase activity than its corresponding GCLC GAG repeat construct (GAG9). While the effect of GAG repeat number was similar in both cell lines, the effect of the addition of the UTR to the PGL-3 (regardless of GAG repeat number) on luciferase expression differed between HepG2 and HEK293 cells. In HepG2 cells, expression levels with the PGL-3 vector alone were increased compared to PGL-3 containing the UTR with the random repeat, while in HEK293 cells, expression was the same with the vector alone and with the UTR with the random repeat.

Figure 2.
Effect of GAG repeats on luciferase activity. Expression of PGL3 constructs, in HepG2 (A) and HEK293 (B) cells, with 4 (GAG4), 7 (GAG7), 8 (GAG8), 9 (GAG9), and 10 (GAG10) GAG repeats. PGL-3 vector had no GCLC 5′ UTR, while the random control ...

Effect of GAG repeat number on luciferase transcription in vitro

The effect of GAG repeat number on gene transcription was determined by transcribing the constructs described above in the Helascribe cell-free transcription system and quantifying mRNA levels by Ribogreen. All constructs with different numbers of GCLC GAG repeats or scrambled sequence yielded similar amounts of mRNA and were the same as the PGL-3 vector alone without 5′ UTR (Fig. 3).

Figure 3.
Effect of GAG repeats on in vitro transcription of luciferase gene. Transcriptional efficiency of different constructs was determined in HeLa cell nuclear lysates by quantifying mRNA obtained after incubating equal amounts of construct DNA containing ...

Effect of GAG repeat number on luciferase translation

To examine whether the GAG repeat number in the 5′ UTR of GCLC could affect translation, identical amounts of in vitro-synthesized GCLC 5′-UTR-luciferase mRNAs with different numbers of GAG repeats were translated in rabbit reticulocyte lysates. Differences were observed in luciferase protein levels obtained from mRNAs with different numbers of GAG repeats (Fig. 4). The effect of GAG repeat number on translation was very similar to the effect on luciferase expression in HepG2 and HEK293 cells. Protein levels obtained for GAG4 and GAG7 were significantly lower than those obtained for GAG8 and GAG9 (P<0.001). Even though GAG10 had higher activity than GAG4 and GAG7 and lower activity than GAG8 and GAG9, the differences were not significant. GAG4 yielded 21% lower protein level than GAG7, which had 35 and 32% lower protein than GAG8 and GAG9, respectively. GAG10 had 25 and 22% lower protein levels than GAG8 and GAG9, respectively. Relative expressions of GAG9 and its random control RAN9 were similar to luciferase expression in HepG2 and HEK293 cells; i.e., GAG9 expression was higher than that of RAN9.

Figure 4.
Effect of GAG repeats on in vitro translation of luciferase. Translational efficiency of mRNAs having different numbers of GAG repeats was determined in rabbit reticulocyte lysates by measuring the quantity of fluorescent protein obtained by incubating ...

Effect of GCLC genotype on PBMC and RBC GSH levels and GCL activity

The association of the GCLC genotype with GCL activity and GSH levels in RBCs and PBMCs was investigated in vivo in healthy adults. In PBMCs, GSH levels and GCL activity were higher by 40 and 69%, respectively, in individuals with GAG-9/9 genotype compared to those with GAG-7/7 genotype (P<0.05). In contrast, RBC GSH levels and GCL activity were lower by 18 to 20% in individuals with GAG-9/9 genotype compared to those with GAG-7/7 genotype (P<0.05, Table 2). To examine for possible allele-specific effects on GSH levels and GCLC activity, subjects were classified on the basis of the presence or absence of GAG-7, GAG-8, or GAG-9 (i.e., GAG-7 vs. non-GAG-7 and so on). GSH levels and GCL activity in PBMCs were lower by 23 and 34%, respectively, in individuals with ≥1 GAG-7 compared to those without GAG-7 (P<0.05). In contrast, GSH levels and GCL activity in RBCs were higher by 15 to 18% in individuals with ≥1 GAG-7 allele compared to those without GAG-7 (P<0.05). In individuals with ≥1 GAG-9, PBMC GSH levels and GCL activity were 34 to 36% higher and RBC GCL activity was 13% lower compared to those without GAG-9 (P<0.05). Since GAG-8 and GAG-9 gave similar results in our luciferase reporter studies, we also analyzed GAG-8 and GAG-9 alleles combined. GSH levels and GCL activity in PBMCs were lower by 23 to 30% in individuals without GAG-8 or GAG-9 allele compared with those with ≥1 GAG-8 or GAG-9 (P<0.05). In contrast, in RBCs, GSH levels were lower by 13% in individuals with ≥1 GAG-8 or GAG-9 allele compared to those without GAG-8 or GAG-9 (P<0.05).

Table 2.
Association of PBMC and RBC GSH levels and GCL activity with GCLC genotype

DISCUSSION

The results of this study demonstrate that the number of GAG repeats in the 5′ UTR of GCLC can affect the gene expression via a translational mechanism. Higher levels of luciferase activity in cell cultures and luciferase protein quantity in cell-free lysates were observed with GAG repeat constructs of 8 or 9 repeats compared to those with 4, 7, and 10 repeats. No differences were observed in in vitro transcription assays using luciferase constructs containing variant GCLC GAG repeats. These results suggest that cells exhibiting homozygous or heterozygous genotypes of the alleles GAG-8 or GAG-9 will have a higher capacity for GCLC protein expression than those with homozygous or heterozygous genotypes of the alleles GAG-4, GAG-7, or GAG-10, and thus have a higher capacity for GSH biosynthesis.

Although this is the first study to report a specific effect of the GCLC GAG-repeat polymorphism on translation, results are consistent with a previous study of a panel of cell lines, where higher GSH levels were associated with alleles GAG-8 and GAG-9 (15). However, no correlation was found between GAG genotype and GCLC mRNA levels in the same cell lines, suggesting that the effect was not due to changes in transcription, but rather due to translation (15, 26). Results from our study are consistent with these previous observations in that GAG8 and GAG9 had higher capacity for translation.

In a recent report, Butticaz et al. (27) have examined the effect of GAG repeat number on luciferase expression and found no effects on luciferase expression under basal conditions. However, in this study, a PGL3 vector was used that lacked an enhancer that we used in the present study (located on the 3′ end of the poly-A signal; Fig. 1). Because the activity of the PGL3 vector without enhancer is ~50- to 100-fold lower than with enhancer, it is likely that that the ability to detect differences caused by the addition of different numbers of GAG repeats would be substantially reduced (2829).

The effect of GAG repeat number on luciferase expression and protein levels was not linear. While a linear association with gene function is common among trinucleotide expansions of repeat lengths on the order of thousands, nonlinear relationships are often observed for shorter repeats (3036). In general, luciferase activity increased with increasing number of GAG repeats up to 8 or 9, and then decreased with more repeats, suggesting that the constructs with 8 and 9 GAG repeats are most favorable for luciferase translation. However, elimination of the entire GAG-repeat sequence by disrupting the GAG repeat sequence resulted in decrease of both luciferase activity and protein levels, implying that the presence of the GAG repeat sequence enhances translation.

Our in vitro findings using the luciferase expression system are consistent with a previous cell culture study (15), as well as with our in vivo findings in PBMCs that the GAG-7 allele was associated with decreased GCL activity and GSH levels. However, these results are in contrast to our previous in vivo study in RBCs, where the GAG-7 allele was associated with increased GCL activity and GSH levels (19). This in vivo relationship in RBCs has been confirmed in our present data, as in the same subjects, the opposite association of the GAG repeat genotype with GCL activity and GSH levels in PBMCs was observed. It is likely that these contrasting results are due to differences in the mechanism by which GCLC is regulated in RBCs, which lack the mechanisms for transcriptional and translational regulation, compared to other cell types. Consequently, up-regulation of GCL via post-translational mechanism in individuals with the GAG7/7 genotype may account for higher RBC activities compared to those with the GAG9/9. Such compensatory mechanisms are consistent with results from previous findings in mice, where food withdrawal resulted in GSH depletion in tissues such as liver, but increases in blood (37). Similarly, RBC GSH after severe exercise was elevated, despite decreases in other cells (38).

While the molecular mechanism linking the GAG repeats with translation is not known, previous studies suggest that secondary structures formed by GAG repeats and/or altered protein binding might be playing a role. Polypurine sequences of adenine and guanine, such as GAA and GGA repeats, are known to form secondary structures, such as intra- and intermolecular triplexes (39), hairpins (21), and quadruplexes (40). Such complex structures were shown to enhance translation by binding to ssRNA- and dsRNA-binding proteins (41) and impede the scanning of the 5′ UTR by 40S ribosomes (4244). The occurrence of GAG trinucleotide repeats is very rare among genes, and there are few studies on secondary structures of GAG repeats (45). However, secondary structure prediction of mRNA with GAG repeats suggested that they can form stable secondary structures under physiological pH and temperature (21). A GAG repeat polymorphism in the promoter region of PAX7 (with alleles GAG8, GAG10, and GAG11) was shown to affect transcription by altering the binding affinity for the transcription factor SP1 (GAG11>GAG8 or GAG10) (46). A similar effect could be possible with the GAG repeats in GCLC mRNA by recruitment of RNA binding proteins in the repeat region. Other studies have shown that TNRs can also affect mRNA stability, but most commonly when present in 3′ UTRs rather than in 5′ UTRs (47). However, an effect on mRNA stability by the 5′ UTR GAG repeat in the GCLC remains a possibility.

GCLC regulation is one of the major factors that determine GSH levels. Several studies have shown that GCLC is regulated transcriptionally and post-translationally by agents such as drugs, dietary antioxidants, etc. (48). However, most of these studies were on exogenous factors and did not address naturally occurring interindividual differences in GSH levels. Results from our study propose an additional level of GCLC regulation, during the translation of mRNA, which could be different among individuals with different GAG repeat genotypes. These differences could be enhanced when GCLC expression is induced under conditions such as oxidative stress, cancer, and toxic exposures. Thus, one possible consequence is that individuals with homozygous or heterozygous genotypes of GAG-4, GAG-7, or GAG-10 might be at greater risk for these diseases as compared to those with GAG-8 or GAG-9.

Acknowledgments

This study was supported in part by Public Health Service grants P01-CA68384 (P.L., principal investigator; J.P.R, project leader) and R01-DE13158 (P.L.) from the U. S. National Institutes of Health.

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