Human Cytosolic and Mitochondrial Folylpolyglutamate Synthetase Are Electrophoretically Distinct

Abstract

Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic (≈67% of total) and mitochondrial (≈22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands (≈60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity. Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic (≈67% of total) and mitochondrial (≈22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands (≈60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity. folylpolyglutamate synthetase (EC 6.3.2.17) human cytosolic FPGS human mitochondrial FPGS head and neck squamous cell carcinoma Chinese hamster ovary cytochromec oxidase (EC 1.9.3.1) lactate dehydrogenase (EC1.1.1.27) methotrexate (2,4-diamino-10-methylpteroylglutamic acid) poly(γ-glutamyl) derivatives of MTX postnuclear supernatant polyacrylamide gel electrophoresis It has been known for years that mitochondria have their own folate-dependent enzymes and folylpolyglutamate pool, but it has not been established how that pool is acquired or maintained (reviewed in Refs. 1.Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (296) Google Scholar, 2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 3.Wagner C. J. Nutr. 1996; 126 (suppl.): 1228-1234Crossref Google Scholar). A central enzyme in establishing and maintaining folylpolyglutamate pools in whole cells is folylpolyglutamate synthetase (FPGS)1 (4.Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar, 5.McGuire J.J. Coward J.K. Blakley R.L. Benkovic S.J. Folates and Pterins: Chemistry and Biochemistry of Folates. 1st Ed. 1. John Wiley & Sons, Inc., New York1984: 135-190Google Scholar). Early studies indicated that FPGS activity was present in both cytosol and mitochondria (6.Gawthorne J.M. Smith R.M. Biochem. J. 1973; 136: 295-301Crossref PubMed Scopus (35) Google Scholar, 7.McGuire J.J. Kitamoto Y. Hsieh P. Coward J.K. Bertino J.R. Kisliuk R.L. Brown G.M. Chemistry and Biology of Pteridines. Elsevier Science Publishing Co., Inc., New York1979: 471-476Google Scholar), suggesting that folate monoglutamates were transport forms and that the essential (8.McBurney M.W. Whitmore G.F. Cell. 1974; 2: 173-182Abstract Full Text PDF PubMed Scopus (224) Google Scholar) folate polyglutamate forms were then synthesized independently in each compartment. Studies with isolated mitochondria have confirmed that folate monoglutamates are transported by a carrier-mediated facilitated-diffusion mechanism (9.Horne D.W. Holloway R.S. Said H.M. J. Nutr. 1992; 122: 2204-2209Crossref PubMed Scopus (44) Google Scholar); if folate polyglutamates pass the mitochondrial membrane, they do so slowly, since the cytosolic and mitochondrial folate pools are not in equilibrium (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 10.Horne D.W. Patterson D. Cook R.J. Arch. Biochem. Biophys. 1989; 270: 729-733Crossref PubMed Scopus (90) Google Scholar).More detailed studies later showed the presence of FPGS activity in Chinese hamster ovary (CHO) cell cytosol and mitochondria (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar). The function of FPGS within the two compartments has recently been studied. Shane and co-workers (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar, 12.Lowe K.E. Osborne C.B. Lin B.F. Kim J.S. Hsu J.C. Shane B. J. Biol. Chem. 1993; 268: 21665-21673Abstract Full Text PDF PubMed Google Scholar, 13.Osborne C.B. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21657-21664Abstract Full Text PDF PubMed Google Scholar) studied CHO AUXB1 cells (which lack expression of both cFPGS and mFPGS) transfected with Esherichia coli or human FPGS that was expressed in either or both compartments. In cells expressing cytosolic FPGS activity, mitochondrial folates were absent, and cells were auxotrophic for glycine and methionine (12.Lowe K.E. Osborne C.B. Lin B.F. Kim J.S. Hsu J.C. Shane B. J. Biol. Chem. 1993; 268: 21665-21673Abstract Full Text PDF PubMed Google Scholar). However, cells expressing activity only in mitochondria also contained cytosolic folates, although the levels were low, and as a consequence, folate metabolism was not optimal. The results indicate that both FPGS isoforms must be expressed to establish normal folate metabolism and optimal growth. They also suggest that cytosolic folylpolyglutamates cannot enter mitochondria, but the mitochondrial folylpolyglutamate pool may exit to the cytosol (11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar), at least slowly and to a limited extent.The effect of the subcellular localization of FPGS on the mechanism of action of the cancer chemotherapy drug methotrexate (MTX) was also studied (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar). Similar to our studies with acquired MTX resistance through decreased MTX polyglutamate (MTXGn) accumulation (15.Pizzorno G. Mini E. Caronnelo M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar, 16.McCloskey D.E. McGuire J.J. Russell C.A. Rowan B.G. Bertino J.R. Pizzorno G. Mini E. J. Biol. Chem. 1991; 266: 6181-6187Abstract Full Text PDF PubMed Google Scholar) and with the nonpolyglutamylatable analog γ-FMTX (17.Galivan J. Inglese J. McGuire J.J. Nimec Z. Coward J.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2598-2602Crossref PubMed Scopus (65) Google Scholar, 18.McGuire J.J. Graber M. Licato N. Vincenz C. Coward J.K. Nimec Z. Galivan J. Cancer Res. 1989; 49: 4517-4525PubMed Google Scholar), the authors (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar) conclude that MTXGn and, thus, FPGS are not required during continuous MTX exposure. Furthermore, the extent of metabolism to MTXGn is dependent on FPGS level; thus, in pulse exposure, where MTXGn allows intracellular retention in the absence of extracellular drug, MTX sensitivity is directly related to FPGS level. In addition, the authors (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar) show that MTX does not enter mitochondria and does not affect the pre-existing one-carbon pool in mitochondria, although its continued presence can limit the further accumulation of folates in mitochondria.Our work (15.Pizzorno G. Mini E. Caronnelo M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar, 16.McCloskey D.E. McGuire J.J. Russell C.A. Rowan B.G. Bertino J.R. Pizzorno G. Mini E. J. Biol. Chem. 1991; 266: 6181-6187Abstract Full Text PDF PubMed Google Scholar) shows that decreased expression of FPGS activity and protein (19.McGuire J.J. Russell C.A. Oncology Res. 1998; 10: 193-200PubMed Google Scholar) in whole cells can lead to very high levels of resistance to pulse MTX exposure, a regimen similar to that employed for clinical use of this drug. These findings and the observations that MTX is not accessible to mitochondrial FPGS suggest the hypothesis that selectively decreased expression of hcFPGS relative to hmFPGS could lead to high level resistance to pulse MTX; cytosolic folylpolyglutamate pools essential for growth would be supplied by slow leakage from the mitochondria (11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar). We have explored this hypothesis using activity assays and a recently developed 430Ab polyclonal antipeptide antibody to hFPGS (19.McGuire J.J. Russell C.A. Oncology Res. 1998; 10: 193-200PubMed Google Scholar). In the course of these studies we discovered that the two FPGS isoforms from whole cells exhibit different electrophoretic mobilities, indicating a physico-chemical difference between them that has not been reported previously. It has been known for years that mitochondria have their own folate-dependent enzymes and folylpolyglutamate pool, but it has not been established how that pool is acquired or maintained (reviewed in Refs. 1.Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (296) Google Scholar, 2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 3.Wagner C. J. Nutr. 1996; 126 (suppl.): 1228-1234Crossref Google Scholar). A central enzyme in establishing and maintaining folylpolyglutamate pools in whole cells is folylpolyglutamate synthetase (FPGS)1 (4.Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (289) Google Scholar, 5.McGuire J.J. Coward J.K. Blakley R.L. Benkovic S.J. Folates and Pterins: Chemistry and Biochemistry of Folates. 1st Ed. 1. John Wiley & Sons, Inc., New York1984: 135-190Google Scholar). Early studies indicated that FPGS activity was present in both cytosol and mitochondria (6.Gawthorne J.M. Smith R.M. Biochem. J. 1973; 136: 295-301Crossref PubMed Scopus (35) Google Scholar, 7.McGuire J.J. Kitamoto Y. Hsieh P. Coward J.K. Bertino J.R. Kisliuk R.L. Brown G.M. Chemistry and Biology of Pteridines. Elsevier Science Publishing Co., Inc., New York1979: 471-476Google Scholar), suggesting that folate monoglutamates were transport forms and that the essential (8.McBurney M.W. Whitmore G.F. Cell. 1974; 2: 173-182Abstract Full Text PDF PubMed Scopus (224) Google Scholar) folate polyglutamate forms were then synthesized independently in each compartment. Studies with isolated mitochondria have confirmed that folate monoglutamates are transported by a carrier-mediated facilitated-diffusion mechanism (9.Horne D.W. Holloway R.S. Said H.M. J. Nutr. 1992; 122: 2204-2209Crossref PubMed Scopus (44) Google Scholar); if folate polyglutamates pass the mitochondrial membrane, they do so slowly, since the cytosolic and mitochondrial folate pools are not in equilibrium (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 10.Horne D.W. Patterson D. Cook R.J. Arch. Biochem. Biophys. 1989; 270: 729-733Crossref PubMed Scopus (90) Google Scholar). More detailed studies later showed the presence of FPGS activity in Chinese hamster ovary (CHO) cell cytosol and mitochondria (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar). The function of FPGS within the two compartments has recently been studied. Shane and co-workers (2.Lin B.F. Huang R.F. Shane B. J. Biol. Chem. 1993; 268: 21674-21679Abstract Full Text PDF PubMed Google Scholar, 11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar, 12.Lowe K.E. Osborne C.B. Lin B.F. Kim J.S. Hsu J.C. Shane B. J. Biol. Chem. 1993; 268: 21665-21673Abstract Full Text PDF PubMed Google Scholar, 13.Osborne C.B. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21657-21664Abstract Full Text PDF PubMed Google Scholar) studied CHO AUXB1 cells (which lack expression of both cFPGS and mFPGS) transfected with Esherichia coli or human FPGS that was expressed in either or both compartments. In cells expressing cytosolic FPGS activity, mitochondrial folates were absent, and cells were auxotrophic for glycine and methionine (12.Lowe K.E. Osborne C.B. Lin B.F. Kim J.S. Hsu J.C. Shane B. J. Biol. Chem. 1993; 268: 21665-21673Abstract Full Text PDF PubMed Google Scholar). However, cells expressing activity only in mitochondria also contained cytosolic folates, although the levels were low, and as a consequence, folate metabolism was not optimal. The results indicate that both FPGS isoforms must be expressed to establish normal folate metabolism and optimal growth. They also suggest that cytosolic folylpolyglutamates cannot enter mitochondria, but the mitochondrial folylpolyglutamate pool may exit to the cytosol (11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar), at least slowly and to a limited extent. The effect of the subcellular localization of FPGS on the mechanism of action of the cancer chemotherapy drug methotrexate (MTX) was also studied (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar). Similar to our studies with acquired MTX resistance through decreased MTX polyglutamate (MTXGn) accumulation (15.Pizzorno G. Mini E. Caronnelo M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar, 16.McCloskey D.E. McGuire J.J. Russell C.A. Rowan B.G. Bertino J.R. Pizzorno G. Mini E. J. Biol. Chem. 1991; 266: 6181-6187Abstract Full Text PDF PubMed Google Scholar) and with the nonpolyglutamylatable analog γ-FMTX (17.Galivan J. Inglese J. McGuire J.J. Nimec Z. Coward J.K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2598-2602Crossref PubMed Scopus (65) Google Scholar, 18.McGuire J.J. Graber M. Licato N. Vincenz C. Coward J.K. Nimec Z. Galivan J. Cancer Res. 1989; 49: 4517-4525PubMed Google Scholar), the authors (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar) conclude that MTXGn and, thus, FPGS are not required during continuous MTX exposure. Furthermore, the extent of metabolism to MTXGn is dependent on FPGS level; thus, in pulse exposure, where MTXGn allows intracellular retention in the absence of extracellular drug, MTX sensitivity is directly related to FPGS level. In addition, the authors (14.Kim J.-S. Lowe K.E. Shane B. J. Biol. Chem. 1993; 268: 21680-21685Abstract Full Text PDF PubMed Google Scholar) show that MTX does not enter mitochondria and does not affect the pre-existing one-carbon pool in mitochondria, although its continued presence can limit the further accumulation of folates in mitochondria. Our work (15.Pizzorno G. Mini E. Caronnelo M. McGuire J.J. Moroson B.A. Cashmore A.R. Dreyer R.N. Lin J.T. Mazzei T. Periti P. Bertino J.R. Cancer Res. 1988; 48: 2149-2155PubMed Google Scholar, 16.McCloskey D.E. McGuire J.J. Russell C.A. Rowan B.G. Bertino J.R. Pizzorno G. Mini E. J. Biol. Chem. 1991; 266: 6181-6187Abstract Full Text PDF PubMed Google Scholar) shows that decreased expression of FPGS activity and protein (19.McGuire J.J. Russell C.A. Oncology Res. 1998; 10: 193-200PubMed Google Scholar) in whole cells can lead to very high levels of resistance to pulse MTX exposure, a regimen similar to that employed for clinical use of this drug. These findings and the observations that MTX is not accessible to mitochondrial FPGS suggest the hypothesis that selectively decreased expression of hcFPGS relative to hmFPGS could lead to high level resistance to pulse MTX; cytosolic folylpolyglutamate pools essential for growth would be supplied by slow leakage from the mitochondria (11.Lin B.F. Shane B. J. Biol. Chem. 1994; 269: 9705-9713Abstract Full Text PDF PubMed Google Scholar). We have explored this hypothesis using activity assays and a recently developed 430Ab polyclonal antipeptide antibody to hFPGS (19.McGuire J.J. Russell C.A. Oncology Res. 1998; 10: 193-200PubMed Google Scholar). In the course of these studies we discovered that the two FPGS isoforms from whole cells exhibit different electrophoretic mobilities, indicating a physico-chemical difference between them that has not been reported previously. We thank Dr. Alexander Maccubbin of this Center for providing the Lubrol PX detergent and Dr. Jennifer Black, Director of the Roswell Park Cancer Institute Cell Analysis Facility, for microscopic examination of the mitochondrial pellets.