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-glutamate Synthetase: A Basis for Targeting Folate Antimetabolites1
Departments of Pharmacology and Toxicology [F. B. T., J. L. A., R. G. M.] and Microbiology and Immunology [S. M. T.] and the Massey Cancer Center [J. F., S. T., A. T., S. M. T., R. G. M.], Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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-glutamate synthetase (FPGS). Using 5'-rapid amplification of cDNA ends, RNase protection assays, transfection of cDNAs into FPGS-deficient cells, and kinetic analysis of recombinant enzymes expressed in insect cells, it was determined that the species of active FPGS in mouse liver and kidney was different from that in mouse tumor cells, bone marrow, and intestine. The NH2-terminal peptide of hepatic enzyme contained 18 amino acids not found in enzyme from dividing tissues, and the specificity of the two isoforms for antifolates also differed, suggesting different architecture of the active sites. In most tissues, the expression of one isozyme or the other was an all-or-nothing event. The exclusive use of one of two alternative sets of initial coding exons in different tissues underlies this phenomenon, suggesting the design of antifolates specific for activation by individual FPGS isoforms and hence tissue-selective targeting of antifolate therapy for cancer, arthritis, or psoriasis. |
Introduction |
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Transformation of cDNAs for FPGS Species into AUXB1 Cells.
The functional activity of FPGS species was tested by transforming cDNA constructs into FPGS-null function AUXB1 cells (16
, 17)
. Exon A1b linked to exons 2–8 or exons 1–8 were generated by reverse transcription-PCR using random-primed mouse liver or L1210 cDNA as a template, respectively. PCR was carried out using an editing mixture of Taq and Vent polymerases. Sense primers included each start ATG (bold) and a 5' HindIII site (italics): (a) mouse liver upstream ATG, 5'-CACGAAGCTTCAAGTGATGATGAAAAGCAGGA; (b) mouse liver downstream ATG, 5'-GAGTAAGCTTGCAGCCATGGAGTGGAAGGACCCATTCCCA; (c) L1210 upstream ATG, 5'-CTATAAGCTTAAGACTATGTCGTGGGCGCGGAGCCGAC; and (d) L1210 downstream ATG, 5'-CTCTAAGCTTCCGGGCATGGAGTATCAGGATGCTGTGC. The antisense primer (5'-ATGCCCCCTTTCTGCCATGC) was located in exon 8, downstream of a unique AccI restriction site. PCR products were cloned into pCRII and verified by sequence analysis. Inserts were released by digestion with HindIII and AccI and gel-purified. Exons 8 through the polyadenylation signal were isolated as a 1.5-kb AccI-EcoRI fragment of murine leukemia cell FPGS cDNA (18)
obtained from Dr. David Goldman (Albert Einstein Cancer Center, Bronx, NY). Each 5'-fragment was ligated to this 3'-fragment and HindIII/EcoRI-cut pcDNA3. Clones were verified by restriction enzyme analysis and limited sequencing to verify the 5' ends and start methionines.
The four cDNA expression plasmids were transfected by calcium phosphate precipitation into AUXB1 cells (5 x 104 cells/100-mm dish). Clones of transfectants were subsequently selected for integration of plasmid in modified 
+ medium [containing 1.2 mg/ml G418, 10 mg/liter thymidine, purines (ribo and deoxyribo adenine and guanine nucleosides, each at 10 mg/liter) and 50 mg/liter glycine] for complementation of cytosolic purine and thymidine auxotrophy, in - medium (containing G418 but lacking nucleosides) or for complementation of the mitochondrial glycine auxotrophy (+ medium without glycine but with G418). Selection was applied 48 h after transfection. Colonies were fixed and stained with Giemsa 10–12 days after transfection. Three to five plates were used for each condition in each of seven experiments. In some experiments, lower cell numbers (2 x 103 cells/100-mm dish) were transfected, and selection was only for the integration of plasmid with G418. One isolated colony was picked from each dish and mass-cultured, and the phenotype of cells expressing FPGS was tested by plating 120–150 cells/dish and then changing to selective media supplemented with glycine or purine and thymidine.
Preparation and Purification of Recombinant Enzymes in Insect Cells.
The coding region of each cDNA clone for mitochondrial and cytosolic FPGS species was cut from the pcDNA3 clones used in Fig. 2C
, inserted into pFastBac (Life Technologies, Inc.), and used to transform DH10-Bac cells. A recombinant bacmid was selected for each protein, and purified bacmid DNA was introduced into Sf9 insect cells growing as a monolayer using the Cell-fectin reagent (Life Technologies, Inc.). Virus produced by these cells was used to infect Sf9 cells at a multiplicity of infection of 1.3. The cells were harvested after 48 h, and enzymes were purified by 30% ammonium sulfate precipitation, followed by sequential Sephacryl S-100/HR (Pharmacia) and DEAE-Sephacel chromatography, a procedure previously used for purification of human CEM cell cytosolic FPGS expressed in insect cells (19)
. This sequence brought both mouse liver and L1210 cytosolic FPGS species to near electrophoretic homogeneity; these purified enzymes were used in kinetic experiments.
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was linked to exons 2–4 contained the cDNA found in mouse liver from a NcoI site at nt 69 in exon A1b to nt 335 in exon 4 [+1 was the A of the mitochondrial translational start codon (12)
]. The exon 1/2–4 probe contained the cDNA generated by 5'-RACE using mouse liver mRNA from a Tth111I site at nt 34 in exon 1 to nt 364 in exon 4. RNA transcripts were generated from linearized cDNAs using [-32P]UTP and either the SP6 or T7 RNA polymerase. RNase protection assay reactions contained deionized formamide, 2 x 105 cpm of probe, and 4 µg of poly(A)+ RNA for all tissues except bone marrow, for which 1.5 µg were used. Reactions were hybridized at 50°C overnight, and the samples were incubated with 100 µg/ml RNase A for 30 min (exon A1b/2–4 probe reactions at 37°C and exon 1/2–4 probe reactions at 16°C). The protected fragments were ethanol-precipitated and run on a 6% polyacrylamide urea gel. The sense complement of the probe sequence was transcribed in vitro and used as a RNA standard for each probe. This standard allowed the determination of the exact gel mobility of a specific protected RNA fragment representing a single molecular species. |
Results |
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. The substrates chosen for this study, aminopterin (•) and the 6-R (
) and 6-S diastereomers of DDATHF (
), are generally taken as metabolically inert in mammalian tissues, with polyglutamation being the only known biotransformation. The kinetics of addition of glutamic acid to the dihydrofolate reductase inhibitor aminopterin by FPGS from liver and leukemic cells was very similar, if not identical, but challenge of the FPGS from these tissues with the 6-R and 6-S diastereomers of DDATHF (20)
revealed some distinct differences. The enzyme from liver formed polyglutamate products from 6-R-DDATHF at a rate about the same as that seen with aminopterin, albeit characterized by a somewhat lower Michaelis constant. The 6-S diastereomer of DDATHF showed a distinct substrate inhibition with mouse liver enzyme but was otherwise as well accepted as 6-R-DDATHF. On the other hand, both 6-R- and 6-S-DDATHF had very different kinetics than aminopterin with FPGS extracted from mouse L1210 leukemic cells, characterized by a distinctly lower maximal velocity. Some time ago, Rumberger et al. (13)
also reported differences in the substrate specificities for methotrexate analogues of FPGS expressed by mouse small intestine, L1210 cells, and S-180 sarcoma cells. Although there could be several explanations for the results shown in Fig. 1
, a difference in the primary sequence of the FPGS expressed in mouse liver and leukemic cells seems to explain the observation. When mRNA from mouse liver and L1210 cells was reverse-transcribed from a primer complementary to the sequence in exon 4 (15)
of the mouse FPGS gene and the upstream sequence of FPGS transcripts in these tissues was determined by 5'-RACE, the sequence of the cDNA from liver and L1210 cells was identical from exons 2–4 but differed upstream of the exon 2/exon 1 border (Fig. 2A)
. Roy et al. (15)
have reported different species of cDNA isolated from libraries from mouse liver and leukemic cells that agree with these differences and have mapped the initial 5' cDNA sequence from mouse liver to two alternative exons (A1a and A1b) located approximately 10 kb upstream from the exon 1 used in mouse leukemic cells (Fig. 2B)
. Although the sequence downstream of the exon 1/exon 2 border in our RACE experiments was 100% identical between mouse liver and L1210 cells, the sequence upstream of this border showed only the level of homology expected of random matches (Fig. 2A)
. All (10)
of the L1210 cell 5'-RACE clones sequenced showed exon 1 spliced to exon 2; of 11 murine liver 5'-RACE clones, 10 contained exon A1b spliced to exon 2, and 1 had exon 1 spliced to exon 2.
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of this cDNA that we have previously proven to allow use as translational start codons for the production of mitochondrial and cytoplasmic species of FPGS in human CEM leukemic cells (12)
. In the mouse liver sequence, two sets of potential start codons were also found (Fig. 2A)
, although they were placed quite differently than those in the leukemic sequence. The fit to consensus for translational initiation of sequence surrounding the boxed ATG codons in Fig. 2A
was numerically compared by sequentially multiplying the frequency at which each of the nts at positions -1 to -6 and +4 occurs in the survey performed by Kozak (21)
. The fit to this consensus of each of the ATGs boxed in Fig. 2A
was similar to that of the upstream and downstream AUGs in human CEM cell FPGS, both of which are used to initiate translation (13)
. To directly test the function of these potentially translated open reading frames, cDNAs were constructed corresponding to full-length FPGS sequences initiated at each of the putative start codons noted in Fig. 2
. Thus, these cDNA constructs linked exon 1 found in mouse leukemic cells to mouse exons 2–15 and also linked exons A1a and A1b from mouse liver cDNA to exons 2–15. These cDNAs were transfected into AUXB1 cells, a subline of Chinese hamster ovary cells that does not express functional FPGS (17)
and, as a result, is auxotrophic for all of the end products of folate metabolism (16)
. As shown in Fig. 2C
, all four of these constructs (mouse liver and L1210 sequences from both upstream and downstream putative translational start codons boxed in Fig. 2A
) allowed synthesis of functional FPGS in AUXB1 cells, complementing either the thymidine and purine auxotrophy of the AUXB1 cells (an indication of restoration of the function of cytosolic FPGS in the transfectants) or the entire phenotype of auxotrophy for thymidine, purine, and glycine. Enzyme assays performed on cell-free extracts of cloned transfectant cell lines confirmed the expression of functional FPGS from all four cDNAs (data not shown). As seen previously with human CEM cells (12)
, cDNA initiating at the upstream ATG in the L1210 cell sequence complemented the glycine auxotrophy of the AUXB1 cells in addition to the thymidine and purine auxotrophies, indicating that the FPGS species made from the transcript initiating at the upstream ATG was capable of trafficking to and into the mitochondria. The cDNA from leukemic cells using the downstream start methionine complemented only the thymidine and purine auxotrophies, and the transfected cells remained glycine auxotrophs. Likewise, the cDNA constructed from the downstream ATG in the mouse liver sequence complemented only the thymidine and purine synthesis deficiencies typical of cytosolic folate metabolism in the AUXB1 cells, whereas the cDNA initiating at the upstream ATG also complemented the glycine metabolism known to require mitochondrial folate metabolism (12
, 22)
. This pattern was confirmed in experiments in which AUXB1 cells were transfected with the same set of cDNAs, and multiple independent cloned transfectants were selected solely on the basis of expression of G418 resistance; these clones were mass-cultured, and clones that expressed FPGS were phenotyped for requirement of purine and thymidine and/or glycine. The phenotypes of these transfectants were clear: cDNA constructs corresponding to FPGS initiating at the upstream ATG in either exon 1 or exon A1b (Fig. 2, A and B)
complemented both cytosolic and mitochondrial folate metabolism; whereas cDNAs initiating translation at the downstream ATGs in either exon 1 or exon A1b complemented only cytosolic folate metabolism (Table 1)
. The additional 42 amino acids between the two start codons in mouse L1210 cells and the equivalent 23 amino acids in the mouse liver protein both act as mitochondrial leader peptides. Hence, FPGS sequences isolated from mouse liver and those isolated from L1210 cells translate to enzymatically active FPGS in vivo, and the tissues direct enzyme to both mitochondrial and cytosolic compartments.
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are the result of the sequence heterogeneity shown in Fig. 2A
. To directly address this question, the cDNAs constructed for the experiments summarized in Fig. 2C
were recloned into a baculoviral transfer vector, and recombinant viruses were used to express these enzymes in insect cells. These single enzyme species were then purified to near homogeneity, and the experiment of Fig. 1
was repeated on the recombinant FPGS isoforms. With these pure recombinant proteins, the kinetic pattern seen with FPGS extracted from mouse liver and L1210 cells was recapitulated (Fig. 3)
. Thus, both diastereomers of DDATHF were used by recombinant mouse liver enzyme more efficiently than was aminopterin (Fig. 3A)
, but recombinant L1210 enzyme displayed a distinctly lower kcat when using 6-R- and 6-S-DDATHF than when using aminopterin (Fig. 3B)
. Data for cytosolic enzyme were used in these experiments, but enzyme produced from recombinant viruses containing the mitochondrial leader sequences of mouse liver and L1210 FPGS showed the same patterns (data not shown). Overall, we drew the conclusions that the differences in the substrate specificity seen in mouse L1210 cells compared to those seen with enzyme from normal mouse liver (Fig. 1)
were indeed due to differences in the primary structure of the FPGS made by these tissues and that the NH2-terminal sequence differences shown in Fig. 2A
were sufficient to account for the differences seen with endogenous enzyme expressed in mouse liver and leukemic cells (Fig. 1)
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due to the potential bias introduced by the reverse transcriptase step in either procedure. Hence, we used RNase protection assays to measure the degree to which this isomorphism is a qualitative or quantitative difference. A very clear but somewhat surprising pattern emerged (Fig. 4)
: in mouse leukemic cells, only the isoform encoded by the downstream initial exon (exon 1) was present; in mouse liver, the FPGS isoform found was almost exclusively that transcribed from the upstream initial exons (A1a and A1b; Fig. 4B)
. A small amount (approximately 3–5%) of the transcripts for FPGS in mouse liver appeared to include exon 1. The possibility that these minor transcripts were due to blood elements remaining in the perfused livers was ruled out by the observation that FPGS transcripts could not be detected in mRNA from unfractionated packed blood cells. Examination of the FPGS mRNA expressed in several mouse tumors indicated that the isoform defined in leukemic cells was again exclusively expressed in the HEPA-1 mouse hepatoma and Lewis lung carcinoma (Fig. 4C)
and also in the Neuro-2a neuroblastoma, S-180 sarcoma, and P19 teratocarcinoma cells (data not shown). Bone marrow and small intestine, the two mouse organs usually limiting to cytotoxic cancer chemotherapy, also expressed the same transcript for the isozyme found in these tumors, as does the spleen, but mouse kidney expressed both the form of enzyme typical of liver and that typical of tumor cells. We conclude that the pattern of differential utilization of the upstream (exons A1a and A1b) and downstream initial exons (exon 1) reflects a mechanism that controls expression of the liver isoform of FPGS in a highly tissue-specific pattern in differentiated cells, but that the form found in leukemic cells is found in many, if not all, dividing cell populations.
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Discussion |
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The definition of isozymes in human tissues is lagging behind the results we show for the mouse. All of the cultured mouse tumor cells we have examined to date express exclusively the FPGS isoform found in normal mouse stem cells. However, a species of FPGS analogous to that we find in mouse liver may be expressed in some human tumors. Should a primary tumor type be found that expresses the FPGS isoform which differs from that in normal human stem cells, it would represent an unprecedented opportunity for rationally designing cancer chemotherapy against that tumor based entirely on differences in the active sites of these FPGS isoforms. This hypothesis remains to be adequately tested.
Using crude preparations of FPGS from mouse tissues, both we (Fig. 1)
and Rumberger et al. (13)
observed differences in the substrate specificity of enzyme expressed in normal mouse tissues and tumors. In our studies, the differences in kinetics seen between FPGS extracted from mouse liver and FPGS extracted from L1210 cells were shown to be caused by isozyme-specific NH2-terminal peptides (Fig. 2)
by cloning, expressing, and purifying enzymes containing these differences and performing kinetic experiments on these molecularly defined, single enzyme species (Fig. 3)
. Using crude extracts, Rumberger et al. (13)
had previously published that crude FPGS from mouse intestine poorly accepted some substrates compared with extracts from L1210 and S-180 cells; aminopterin was used equally well by all of these extracts. Hence, we had originally thought that mouse intestine also expressed a different species of FPGS than mouse tumors, leading to the exciting possibility of selective chemotherapy targeting a tumor isozyme but not the isozyme in drug-limiting intestinal stem cells. This hypothesis was not supported by definitive RNase protection assays, which demonstrated that the same species of FPGS was expressed in mouse tumors, intestine, and bone marrow cells (Fig. 4)
. This raises the question of why the data of Rumberger et al. (13)
suggested differences in FPGS based on differences in polyglutamation in experiments with crude enzymes. Two possibilities suggest themselves: (a) either the mouse intestinal enzyme differs from the L1210 FPGS due to sequence differences downstream of exon 4; or (b) the results from kinetic experiments performed on unpurified enzymes were due to other enzymes in these tissue extracts. For instance, it has been shown that the level of conjugase, which hydrolyzes folylpolyglutamates to their respective monoglutamates, is quite high in the proliferating intestine but is low to undetectable in L1210 cells (24
, 25)
. Hence, kinetic experiments performed on crude extracts (13
, 14)
might reflect substrate specificity differences of the tissue conjugase rather than FPGS, and any such kinetic differences observed in tissue extracts need to be confirmed by studies on single protein species purified to homogeneity from recombinant systems.
The differences between these two isozymes of FPGS are all at the extreme NH2 terminus of the protein, in a domain homologous to an -helix that contacts the critical 
loop of the Lactobacillus casei FPGS (26)
. The extent to which those structural differences alter the substrate binding site in the mouse liver and dividing cell enzymes is under investigation. As can be seen from Figs. 1
and 3
, the kcat for DDATHF is substantially higher for the isozyme expressed in liver than for that expressed in dividing stem cells, although it is clearly not zero in the latter. We would therefore take DDATHF as a lead compound for the development of an antifolate that is a substrate for liver FPGS but not for the enzyme expressed in dividing cells.
An alternative major opportunity for rational drug design is suggested by the specificity of the expression pattern for FPGS isoforms shown herein. Although methotrexate was originally developed for use in cancer chemotherapy (1
, 2)
, it is widely used today for the treatment of rheumatoid arthritis and psoriasis (5
, 6)
. For both of these indications, low doses of methotrexate are given on a chronic schedule. It is commonly the case that hepatic toxicity forces discontinuation of methotrexate therapy, despite the continued activity of the drug. The frequency with which chronic methotrexate initiates severe hepatotoxicity has been estimated to be as high as 23% in recent large-scale trials in psoriatic patients (27)
. The differences in substrate specificity demonstrated in Figs. 1
and 3
suggest the design of folate antimetabolites that are activated only by the FPGS isoform expressed in actively growing tissues and not by that produced in hepatic tissue. Although the detailed biochemical mechanism of hepatotoxicity of methotrexate is only vaguely understood at the moment, it might be avoided by the selection or design of folate analogues that will not bind to hepatic FPGS. This approach may therefore prevent the onset of the hepatic toxicity seen (27
, 28)
with long-term low-dose administration of methotrexate in patients with rheumatoid arthritis or psoriasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant CA-39687 from the National Institutes of Health, DHHS. 
2 To whom requests for reprints should be addressed, at Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, 401 College Street, P. O. Box 980037, Richmond, VA 23298-0230. E-mail: rmoran{at}hsc.vcu.edu
3 The abbreviations used were: FPGS, folylpoly--glutamate synthetase (EC 6.3.2.17); 5'-RACE, 5'-rapid amplification of cDNA ends; DDATHF, 5,10-dideazatetrahydrofolate; poly(A)+ RNA, polyadenylated RNA; nt, nucleotide.
Received 8/23/99. Accepted 10/28/99.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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-glutamate synthetase gene. J. Biol. Chem., 270: 9579-9584, 1995.-glutamate synthetase. Biochim. Biophys. Acta, 1305: 11-14, 1996.[Medline]
-glutamate synthetase in insect cells. Protein Expr. Purif., in press, 2000.
-polyglutamates by folylpolyglutamyl hydrolase derived from various tumors and normal tissues of the mouse. Cancer Res., 46: 2230-2235, 1980.This article has been cited by other articles:
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  A. C. Racanelli, F. B. Turner, L.-Y. Xie, S. M. Taylor, and R. G. Moran A Mouse Gene That Coordinates Epigenetic Controls and Transcriptional Interference To Achieve Tissue-Specific Expression Mol. Cell. Biol., January 15, 2008; 28(2): 836 - 848. [Abstract] [Full Text] [PDF]
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 R. M. Flatley, S. G. Payton, J. W. Taub, and L. H. Matherly Primary Acute Lymphoblastic Leukemia Cells Use a Novel Promoter and 5'Noncoding Exon for the Human Reduced Folate Carrier That Encodes a Modified Carrier Translated from an Upstream Translational Start Clin. Cancer Res., August 1, 2004; 10(15): 5111 - 5122. [Abstract] [Full Text] [PDF]
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 I. Ifergan, A. Shafran, G. Jansen, J. H. Hooijberg, G. L. Scheffer, and Y. G. Assaraf Folate Deprivation Results in the Loss of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression: A ROLE FOR BCRP IN CELLULAR FOLATE HOMEOSTASIS J. Biol. Chem., June 11, 2004; 279(24): 25527 - 25534. [Abstract] [Full Text] [PDF]
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J. R. Whetstine, T. L. Witt, and L. H. Matherly The Human Reduced Folate Carrier Gene Is Regulated by the AP2 and Sp1 Transcription Factor Families and a Functional 61-Base Pair Polymorphism J. Biol. Chem., November 8, 2002; 277(46): 43873 - 43880. [Abstract] [Full Text] [PDF]
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G. J. Leclerc and J. C. Barredo Folylpoly-{{gamma}}-glutamate Synthetase Gene mRNA Splice Variants and Protein Expression in Primary Human Leukemia Cells, Cell Lines, and Normal Human Tissues Clin. Cancer Res., April 1, 2001; 7(4): 942 - 951. [Abstract] [Full Text]
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S. A. Titus and R. G. Moran Retrovirally Mediated Complementation of the glyB Phenotype. CLONING OF A HUMAN GENE ENCODING THE CARRIER FOR ENTRY OF FOLATES INTO MITOCHONDRIA J. Biol. Chem., November 17, 2000; 275(47): 36811 - 36817. [Abstract] [Full Text] [PDF]
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F. B. Turner, S. M. Taylor, and R. G. Moran Expression Patterns of the Multiple Transcripts from the Folylpolyglutamate Synthetase Gene in Human Leukemias and Normal Differentiated Tissues J. Biol. Chem., November 10, 2000; 275(46): 35960 - 35968. [Abstract] [Full Text] [PDF]
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90–95% purity as described in the text and used for kinetic experiments similar to those shown in Fig. 1
X174/HinfI DNA markers are designated in the far right lane. C, isoform expression patterns in differentiated and dividing tissues and tumors. Riboprobes were generated by in vitro transcription with the linearized cDNA templates cloned in pCRII using the SP6 or T7 RNA polymerase binding sites to generate antisense transcripts. The probes were gel-purified and then incubated with 4 µg of poly(A)+ RNA (1.5 µg for bone marrow). A single-stranded RNA standard complementary to the transcribed region of each probe was generated so that the protected product resulting from the standard RNA/probe hybrid would correspond exactly to the RNA segment being examined, as shown in the lanes labeled Exon 1 Std. and Exon A1b Std. In both panels, lanes for L1210, Hepa1, Lewis lung, liver, and kidney samples were exposed to film for 68 h; the other lanes were exposed to film for 190 h.