Stem Cells, Vol. 14, No. 1, 33-40,
January 1996
© 1996 AlphaMed Press
Advances in Cancer Treatment: The Chabner Symposium |
Biochemical and Molecular Studies of Human Methenyltetrahydrofolate Synthetase
Jacques Jolivet,
Alain Dayan,
Myriam Beauchemin,
Dounia Chahla,
Aline Mamo,
Richard Bertrand
Centre de Recherche, Hôtel-Dieu de Montréal, Montréal, Québec, Canada
Key Words. Leucovorin • Folate metabolism • 5-fluorouracil
Dr. Jacques Jolivet, Centre de Recherche, Hôtel-Dieu de Montréal, 3850 Saint-Urbain Street, Montréal QUE H2W 1T8, Canada.
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Abstract
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5-FormylH4folate is administered clinically under the name LeucovorinTM in association with the antineoplastic agent 5-fluorouracil (5-FU) to enhance the cytotoxic effects of 5-FU. The combination has been shown to be superior to 5-FU alone in the treatment of patients with metastatic colorectal carcinoma. Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the transformation of 5-formyltetrahydrofolate to methenylH4folate, which is the obligatory initial metabolic step prior to the intracellular conversion of 5-formylH4folate to other reduced folates and the increase in intracellular folate pools required for 5-FU potentiation. In the following paper, we will summarize results of biochemical and molecular studies of human MTHFS.
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Introduction
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Methenyltetrahydrofolate (methenylH4folate) synthetase (MTHFS) catalyzes the transformation of 5-formyltetrahydrofolate (5-formylH4folate) to methenylH4folate. 5-formylH4folate is administered clinically under the trade name Leucovorin TM (LV) either in association with the antineoplastic pyrimidine analog 5-fluorouracil (5-FU) to enhance its cytotoxic effects [1], or to rescue normal cells from the toxic effects of high-dose antifolate therapy [2]. MTHFS catalyzes the obligatory initial metabolic step of 5-formylH4folate prior to its intracellular conversion to other reduced folates. Thus, the level of expression of this enzyme in tumor cells might play a role in the folate's clinical efficacy. MTHFS may also be involved in the regulation of a number of the folate-dependent reactions of amino acid, purine and pyrimidine synthesis through modulation of intracellular 5-formylH4folate polyglutamates which are potent inhibitors of many folate-dependent enzymes [3].
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Folate Physiology
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Folates are synthesized in plants and certain bacteria but not in mammalian cells. Folic acid is thus a required vitamin in mammalian organisms which, upon reduction to the tetrahydro form H4folate, functions as a coenzyme for the transfer, oxidation and reduction of single carbon units used for the biosynthesis of thymidylate, purines, methionine, serine, glycine and many other compounds [4]. All folates are present in cells mostly in the form of polyglutamates, folates to which 
-linked glutamic acid residues have been added to the terminal glutamyl moitiety of H4folate by the enzyme folylpolyglutamate synthetase. These forms have greater affinity for the folate-dependent enzymes than their monoglutamyl counterparts and most of these enzymes are active in vivo only when the folate is in the polyglutamyl form [5]. There are six known one-carbon substituted derivatives of H4folate and each is associated with a particular metabolic cycle as shown in Figure 1
. 10-formylH4folate is involved in purine synthesis (cycle A), methyleneH4folate in thymidylate synthesis (cycle B) and 5-methylH4folate in methionine synthesis (cycle C). There are three additional H4folate derivatives which are not directly involved in biosynthetic pathways: 5-formimino-, methenyl- and 5-formylH4folate. The reversible conversion of serine and H4folate to glycine and methyleneH4folate, as catalyzed by serine hydroxymethyltransferase (SHMT), is the primary entry point of one-carbon units into one-carbon metabolism [6].
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Fig. 1. Metabolic pathways associated with cytosolic one-carbon metabolism. MTHFS represents methenyltetrahydrofolate synthetase; SHMT, serine hydroxymethyl transferase; 5-FdUMP, 5-fluorodeoxyuridylate monophosphate; cycle A, de novo purine synthesis; cycle B, thymidylate synthesis; cycle C, methionine synthesis; cycle D, MTHFS-SHMT futile cycle. Reduced folate metabolism in plasma following Leucovorin administration [24] is illustrated in the bottom of the panel. Closed cycles represent transmembrane transport.
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It has also been shown in recent years that mammalian cell mitochondria contain pteroylpolyglutamates and a number of folate-dependent enzymes, including SHMT and folylpolyglutamate synthetase, and that H4folate metabolism is highly compartmentalized between the cytosol and mitochondria [7]. While methionine, purine and pyrimidine syntheses occur mainly in the cytoplasm, the interconversion of serine and glycine occurs in both compartments. The mitochondrial SHMT activity appears more important than cytosolic activity in Chinese hamster ovary cells as auxotrophic mutants deficient in the mitochondrial activity are glycine-dependent, even though the cytoplasmic activity is retained [8].
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Combination Chemotherapy with 5-FU and LV
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5-FU's main site of action is the enzyme thymidylate synthase, a protein which catalyzes the transfer of the methylene group from methyleneH4folate to deoxyuridylate to form thymidylate, a step essential for DNA synthesis (Fig. 1
, cycle B). H2folate is produced by this reaction and is regenerated to H4folate via dihydrofolate reductase. The 5-FU metabolite 5-FdUMP inhibits thymidylate synthase activity as it forms a stable inactivating ternary complex with the enzyme and methyleneH4folate [9]. Low methyleneH4folate levels increase the ternary complex dissociation rate leading to incomplete thymidylate synthase inhibition and can be responsible for decreased fluoropyrimidine activity [10]. MethyleneH4folate polyglutamates containing 
3 glutamyl residues bind more tightly to thymidylate synthase than do monoglutamates or diglutamates [11], and are probably essential in increasing the stability of the ternary complex with 5-FdUMP and potentiating 5-FU activity [12].
Experimentally, 5-FU can also exert its cytotoxic effects through incorporation of 5-FU as 5-FUTP in RNA [9] or as 5-FdUTP into DNA [13]. The mechanisms of resistance to the drug have been directly examined in tumor specimens of patients treated with 5-FU [14–16]. The parameters affecting the inhibition of thymidylate synthase by 5-FU, such as the levels of 5-FdUMP, dUMP, methyleneH4folate and of the target enzyme thymidylate synthase, were found to be critical in predicting 5-FU's clinical efficacy. These studies confirm the central role played by thymidylate synthase inhibition in 5-FU's antitumor activity, and underline the fact that low intracellular methyleneH4folate levels are probably the only correctable parameter associated with clinically impaired 5-FU activity.
The cytotoxicity of 5-FU in several experimental models can effectively be enhanced by increasing intracellular folate concentrations through coadministration of fluoropyrimidines with LV [17–19]. This product contains a mixture of the natural and unnatural diastereomers of 5-formylH4folate with an S chirality at the 6 carbon of the tetrahydropterine ring [20] with the natural and unnatural isomers designated respectively (6S)- and (6R)-5-formylH4folate. The 5-FU/LV combination has also been extensively clinically tested. It has been shown to be superior to 5-FU alone in the treatment of patients with metastatic colorectal carcinoma, increasing the average response rate from 11% to 23% without, however, improving overall survival [21]. Following intravenous LV administrations, (6S)-5-formylH4folate disappears rapidly from plasma while (6R)-5-formylH4folate has a markedly slower clearance and accumulates to higher concentrations than the physiological stereoisomer for prolonged periods (Fig. 1) [22–25]. We and others have shown that the unnatural isomer has no measurable impact on the natural isomer's replenishment of intracellular folate pools and potentiation of 5-FU cytotoxicity in vitro [26–29]. 5-methylH4folate also rapidly appears in plasma following LV administration (Fig. 1). It can also potentiate 5-FU toxicity in vitro [18] but must first be metabolized by vitamin B12-dependent methionine synthase (Fig. 1, cycle C). In MCF-7 human breast cancer cells, methyleneH4folate was detected intracellularly following 5-formylH4folate but not 5-methylH4folate exposures, however [30], and 5-methylH4folate has been shown to be less effective than 5-formylH4folate potentiating 5-FU activity in certain cell lines [31, 32]. Recent in vitro studies have also shown that (6R)-5-formylH4folate is almost completely bound to albumin at physiological protein plasma concentrations with 5-methylH4folate 45% bound and (6S)-5-formylH4folate not bound at all [33]. It thus seems likely that (6S)-5-formylH4folate is the major contributor to intracellular folate replenishment after intravenous LV administration.
(6S)-5-formylH4folate requires metabolism through four sequential enzymatic steps prior to transformation to methyleneH4folate (Fig. 1). MTHFS is the initial metabolic step in this metabolism and, in one study, low MTHFS activity was associated with decreased intracellular folate interconversion to other folates in a rat tumor model [34]. Despite prolonged exposures to up to 1,000-fold elevations in extracellular 5-formylH4folate concentrations, intracellular folate levels increase only two to six-fold in many experimental systems [28, 31, 35–37]. In addition, as extracellular 5-formylH4folate concentrations are increased, reduced folates accumulate intracellular mostly as monoglutamate or short-length H4folate polyglutamates [38]. Since 5-formylH4folate is a poor substrate for folylpolyglutamate synthetase [39] and that unpolyglutamylated 5-formylH4folate will readily efflux cells, it is possible that MTHFS could be a rate-limiting step in intracellular folate accumulation following cellular exposures to 5-formylH4folate.
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MTHFS Biochemistry
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MTHFS (5-formylH4folate cyclodehydrase, EC 6.3.3.2) catalyzes the unidirectional transformation of 5-formylH4folate to methenylH4folate and requires ATP and Mg++ (Fig. 1, cycle D). The product is then interconverted into the other one-carbon substituted H4folates. MTHFS activity has been purified from sheep liver [40], Lactobacillus casei [41] and rabbit liver [42], and we have purified it from human liver as described below [43]. The primary amino acid structure and the 5-formylH4folate polyglutamate binding site of rabbit liver MTHFS have recently been determined [44].
Stover and Schirch have established that 5-formylH4folate polyglutamates, the endogenous substrates for MTHFS, are produced by a second catalytic activity of SHMT, the irreversible hydrolysis of methenylH4folate to 5-formylH4folate [45]. The 5-formylH4folate polyglutamates thus formed are tight-binding inhibitors of SHMT [46]. Since endogenously formed 5-formylH4folate polyglutamates are also known to be potent inhibitors of a number of other folate-dependent enzymes, MTHFS and SHMT thus seem to constitute together an apparent futile cycle that buffer the intracellular concentration of 5-formylH4folate (Fig. 1, cycle D) [3]. Thus, the relative activities of each enzyme could influence intracellular 5-formylH4folate polyglutamate levels and consequently a number of folate-dependent synthetic activities.
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Enzyme Kinetics and Physiological Function
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We purified to homogeneity enzyme from human liver obtained at necropsy and found it to have enhanced affinity for 5-formylH4folate pentaglutamate compared to the monoglutamate substrate [43]. Conversely, pentaglutamates of folates and methotrexate were much more potent enzyme inhibitors than monoglutamates. The Km value for the natural 6S stereoisomer of 5-formylH4folate was approximately half of that seen when using a mixture of both the natural (6S) and unnatural (6R) stereoisomers, confirming that the unnatural isomer was not a substrate for MTHFS.
Since the physiological function of MTHFS was unknown, we examined the consequences of the enzyme's inhibition in MCF-7 human breast cancer cells by a potent in vitro MTHFS inhibitor, the 5-formylH4folate analog 5-formyltetrahydrohomofolate [47]. The analog inhibited MCF-7 human breast cancer cell growth with an IC50 of 2.0 µM during 72 hour exposures and this effect could be fully reversed by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. Furthermore, a correlation was observed between increases in intracellular 5-formylH4folate polyglutamate levels, the accumulating substrate following MTHFS inhibition, and cell growth arrest. De novo purine synthesis was inhibited in the MCF-7 cells at the level of 5-aminoimioazole 4-carboxamide ribonucleotide (AICAR) formyltransferase (Fig. 1; cycle A), the second folate-dependent enzyme in this pathway, and 5-formylH4folate pentaglutamate was found to be a potent inhibitor of purified MCF-7 AICAR formyltransferase in vitro. Our results suggest that MTHFS is, like dihydrofolate reductase, a housekeeping enzyme needed to prevent de novo purine synthesis inhibition by the 5-formylH4folate polyglutamates formed in vivo.
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Mitochondrial Compartmentation
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In view of the importance of mitochondrial SHMT activity, and its potential inhibition by mitochondrial 5-formylH4folate [48], we recently examined mitochondria for the presence of MTHFS and identified activity in the matrix of mitochondria purified from human liver biopsies [49]. Mitochondrial and cytoplasmic MTHFS-specific activities were similar with 85% of the total cellular MTHFS activity in the cytoplasm and both native enzymes had similar molecular weights. Studies using purified mitochondrial MTHFS from CA-46 human Burkitt lymphoma cells revealed that mitochondrial MTHFS behaved kinetically like the cytoplasmic enzyme. Thus, MTHFS is compartmentalized between the cytosol and mitochondria in mammalian cells and possibly forms with mitochondrial SHMT a futile cycle to buffer local 5-formylH4folate concentrations.
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MTHFS Molecular Biology
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We recently isolated a family of human cDNA isoforms for MTHFS, one of which encodes for the active protein [50]. Degenerate oligonucleotides corresponding to a sequenced heptapeptide from purified human liver MTHFS were used to amplify a 389 bp cDNA using a 3' rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) protocol [51]. The PCR product was then subcloned into the Bluescript SK+ vector (Stratagene) for sequence analysis and found to contain nucleotides corresponding to the sequenced heptapeptide and to an open reading frame of 97 amino acids. This partial clone was first used as a probe to screen a 
DR2 human liver cDNA library (Clontech, Palo Alto, CA). When it became clear that multiple cDNA isoforms were being isolated, two other libraries were screened: a GT-10 cDNA library generated from LS180 human colorectal carcinoma cells [52], and a cDNA library that we constructed from CA-46 Burkitt lymphoma cells in the ZAP-cDNA synthesis system (Stratagene, La Jolla, CA) using a specific antisense primer to the first MTHFS isoform cloned in the human liver library. A total of seven clones with complete 5' coding areas were sequenced, five of which were different. These five clones contained 872, 832, 856, 867 and 1,219 bp and were named MTHFS-1 to –5, respectively (Fig. 2). MTHFS-1 was isolated twice from the human liver library along with one copy each of MTHFS-4 and –5. MTHFS-1 and –3 were obtained from the LS180 cell library while MTHFS-2 was isolated from the CA-46 cell library. The MTHFS isoforms have a common central 714 bp sequence with differences in the 5'-end noncoding and coding areas and the end of the 3'-noncoding area. MTHFS-1 to –4 have open reading frames corresponding to 203, 177, 165 and 164 amino acids with calculated molecular weights of 23,240, 20,336, 19,021 and 19,035, respectively. MTHFS-5 has an additional 146 bp segment inserted within the common 714 bp sequence. This insert, bordered by the consensus splice dinucleotides GT and AG, could be an unspliced intron. It contains stop codons and thus MTHFS-5 has a prematurely truncated open reading frame for an 80 amino acid protein of 9,230 Da. MTHFS-1 to -5 cDNAs could be detected by PCR in human liver and Burkitt lymphoma cell mRNA preparations. A Northern blot of poly(A)+ RNA from a variety of normal tissues was performed using the originally cloned 389 bp cDNA corresponding to the common 3' end for all isoforms. The MTHFS probe detected a transcript, approximately of 0.9 kb in size in liver with little expression in the heart, brain, placenta, lung, skeletal muscle, kidney and pancreas [50].
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Fig. 2. Cloned methenyltetrahydrofolate synthetase cDNAs. Comparisons between the five cloned MTHFS cDNAs are illustrated. Thick lines represent sequence similarities and narrow lines sequence differences. ATG represents ATG initiation, TAA or TGA protein termination and AAAA polyadenylation sites. The box at the right side of the panel contains the number of base pairs (bp), amino acids (aa) and molecular weight in kilodaltons (kDa) for each cloned cDNA.
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The molecular weight of MTHFS-1 predicted protein (23 kDa) is similar to that determined by gel filtration for purified human cytosolic [43] and mitochondrial MTHFS [49]. There is a 77% amino acid identity with the rabbit liver MTHFS amino acid sequence recently determined directly from purified protein [44]. Comparison with other folate-dependent enzyme sequences revealed an homology to a sequence identified as potentially binding 10-formyltetrahydrofolate. Cook et al. have identified the HPSLLP peptide and a Gly residue four residues downstream as being conserved in almost all enzymes using 10-formyltetrahydrofolate as a substrate [53]. The SLLP peptide followed by Trp four residues downstream is found in the human MTHFS transcripts suggesting a possible role in folate binding. Sequence identity analyses revealed only one match (22% amino acid identity) with a bacterial protein [54]. The sequence of this E. coli protein was deduced from a 540 nucleotide sequence open reading frame (ORF) placed immediately after a 6S RNA gene and encodes a 22 kDa protein of unknown function. There is a consensus ATP binding site, a potential Mg++ binding site and a number of consensus phosphorylation sites: four protein kinase C sites, three caseine kinase 2 sites, one AMPc protein kinase-dependent site, and one tyrosine kinase site.
We confirmed that the MTHFS-1 cDNA encoded a protein with enzymatic activity by inserting it in pET11-c plasmid, an efficient prokaryotic expression vector in which high mRNA and protein expression is driven by T7 RNA polymerase. MTHFS-specific activity in extracts of BL21(DE3) E. coli transfected with the pET11-c plasmid containing the MTHFS ORF increased from 2.9 x 10–4 to a plateau of 4 x 10–2 µmole/min/mg after a 2-hour induction with 1 mM isopropyl-B-D-thiogalacto pyranoside at 27°C [50]. E. coli transfected with pET11-c vector alone were used as a negative control and did not show increased MTHFS activity. Sodium docetyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the appearance of a 27 kDa protein in bacterial extracts following induction, the same molecular weight as previously observed for purified human liver MTHFS [43]. Differences observed between the calculated (23 kDa) and observed molecular weights on the SDS gel probably relate to conformational structure changes during electrophoresis. Indeed, a similar difference was seen between the rabbit MTHFS molecular weight calculated from the primary amino acid sequence and that determined by SDS gel electrophoresis, and the correct identity of the lower weight was confirmed by mass spectral analysis of the purified protein [44].
The absolute conservation in the shared portions of the ORF and most of the 3'-untranslated sequences between the different isoforms strongly suggests that there is only one MTHFS gene, and that alternative splicing of its primary transcript is the mechanism from which the 5 mRNA variants are derived [55]. A Southern blot of human genomic DNA digested with various restriction enzymes was hybridized with a restricted fragment containing nucleotides almost completely common to the five MTHFS cDNA splice variants. There was a single band with the HindIII (9 kb) and BamHI (25 kb) digests while two bands were seen following EcoR I (4.5 and 1.8 kb), PstI (4.4 and 2 kb) and BglII (5 and 1 kb) [50]. These results suggest the existence of a single MTHFS gene.
Ongoing studies will directly assess the pharmacological importance of MTHFS's level of expression in determining the extent of the potentiation of 5-FU cytotoxicity by LV and its physiological role in regulating folate-dependent syntheses. If they indicate that MTHFS mRNA or protein levels are important for the success of 5-FU/LV therapy, MTHFS quantitation in primary tumor specimens could become a useful prognostic test. If increased production of MTHFS in tumor cells is shown to enhance the potentiating effect of LV on 5-FU cytotoxic activity, gene therapy strategies to increase MTHFS production in tumor cells could be developed.
Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.
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Acknowledgments
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Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society.
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Received June 16, 1995;
accepted for publication June 16, 1995.
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Abstract
Introduction
