Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Oct 1;89(19):9151–9155. doi: 10.1073/pnas.89.19.9151

Expression cloning of a human cDNA encoding folylpoly(gamma-glutamate) synthetase and determination of its primary structure.

T A Garrow 1, A Admon 1, B Shane 1
PMCID: PMC50083  PMID: 1409616

Abstract

A human cDNA for folypoly(gamma-glutamate) synthetase [FPGS; tetrahydrofolate:L-glutamate gamma-ligase (ADP forming), EC 6.3.2.17] has been cloned by functional complementation of an Escherichia coli folC mutant. The cDNA encodes a 545-residue protein of M(r) 60,128. The deduced sequence has regions that are highly homologous to peptide sequences obtained from purified pig liver FPGS and shows limited homology to the E. coli and Lactobacillus casei FPGSs. Expression of the cDNA in E. coli results in elevated expression of an enzyme with characteristics of mammalian FPGS. Expression of the cDNA in AUXB1, a mammalian cell lacking FPGS activity, overcomes the cell's requirement for thymidine and purines but does not overcome the cell's glycine auxotrophy, consistent with expression of the protein in the cytosol but not the mitochondria.

Full text

PDF
9155

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  2. Bognar A. L., Osborne C., Shane B. Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J Biol Chem. 1987 Sep 5;262(25):12337–12343. [PubMed] [Google Scholar]
  3. Bognar A. L., Osborne C., Shane B., Singer S. C., Ferone R. Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. J Biol Chem. 1985 May 10;260(9):5625–5630. [PubMed] [Google Scholar]
  4. Bognar A. L., Shane B. Purification and properties of Lactobacillus casei folylpoly-gamma-glutamate synthetase. J Biol Chem. 1983 Oct 25;258(20):12574–12581. [PubMed] [Google Scholar]
  5. Chabner B. A., Allegra C. J., Curt G. A., Clendeninn N. J., Baram J., Koizumi S., Drake J. C., Jolivet J. Polyglutamation of methotrexate. Is methotrexate a prodrug? J Clin Invest. 1985 Sep;76(3):907–912. doi: 10.1172/JCI112088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cichowicz D. J., Shane B. Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme. Biochemistry. 1987 Jan 27;26(2):504–512. doi: 10.1021/bi00376a024. [DOI] [PubMed] [Google Scholar]
  7. Cichowicz D. J., Shane B. Mammalian folylpoly-gamma-glutamate synthetase. 2. Substrate specificity and kinetic properties. Biochemistry. 1987 Jan 27;26(2):513–521. doi: 10.1021/bi00376a025. [DOI] [PubMed] [Google Scholar]
  8. Clarke L., Waxman D. J. Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists. Arch Biochem Biophys. 1987 Aug 1;256(2):585–596. doi: 10.1016/0003-9861(87)90616-3. [DOI] [PubMed] [Google Scholar]
  9. Elledge S. J., Mulligan J. T., Ramer S. W., Spottswood M., Davis R. W. Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1731–1735. doi: 10.1073/pnas.88.5.1731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Foo S. K., Shane B. Regulation of folylpoly-gamma-glutamate synthesis in mammalian cells. In vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Chinese hamster ovary cells. J Biol Chem. 1982 Nov 25;257(22):13587–13592. [PubMed] [Google Scholar]
  11. Fournier R. E., Moran R. G. Complementation mapping in microcell hybrids: localization of Fpgs and Ak-1 on Mus musculus chromosome 2. Somatic Cell Genet. 1983 Jan;9(1):69–84. doi: 10.1007/BF01544049. [DOI] [PubMed] [Google Scholar]
  12. George S., Cichowicz D. J., Shane B. Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues. Biochemistry. 1987 Jan 27;26(2):522–529. doi: 10.1021/bi00376a026. [DOI] [PubMed] [Google Scholar]
  13. Hagan R. L., Duch D. S., Smith G. K., Hanlon M. H., Shane B., Freisheim J. H., Hynes J. B. Studies on the mechanism of antitumor action of 2-desamino-2-methyl-5,8-dideazaisofolic acid. Biochem Pharmacol. 1991 Mar 1;41(5):781–787. doi: 10.1016/0006-2952(91)90081-f. [DOI] [PubMed] [Google Scholar]
  14. Hendrick J. P., Hodges P. E., Rosenberg L. E. Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4056–4060. doi: 10.1073/pnas.86.11.4056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jones C., Kao F. T., Taylor R. T. Chromosomal assignment of the gene for folylpolyglutamate synthetase to human chromosome 9. Cytogenet Cell Genet. 1980;28(3):181–194. doi: 10.1159/000131529. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. McBurney M. W., Whitmore G. F. Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 1974 Jul;2(3):173–182. doi: 10.1016/0092-8674(74)90091-9. [DOI] [PubMed] [Google Scholar]
  18. McCloskey D. E., McGuire J. J., Russell C. A., Rowan B. G., Bertino J. R., Pizzorno G., Mini E. Decreased folylpolyglutamate synthetase activity as a mechanism of methotrexate resistance in CCRF-CEM human leukemia sublines. J Biol Chem. 1991 Apr 5;266(10):6181–6187. [PubMed] [Google Scholar]
  19. McGuire J. J., Hsieh P., Coward J. K., Bertino J. R. Enzymatic synthesis of folylpolyglutamates. Characterization of the reaction and its products. J Biol Chem. 1980 Jun 25;255(12):5776–5788. [PubMed] [Google Scholar]
  20. Moran R. G., Colman P. D. Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme. Biochemistry. 1984 Sep 25;23(20):4580–4589. doi: 10.1021/bi00315a011. [DOI] [PubMed] [Google Scholar]
  21. Moran R. G., Colman P. D., Rosowsky A., Forsch R. A., Chan K. K. Structural features of 4-amino antifolates required for substrate activity with mammalian folylpolyglutamate synthetase. Mol Pharmacol. 1985 Jan;27(1):156–166. [PubMed] [Google Scholar]
  22. Pristupa Z. B., Vickers P. J., Sephton G. B., Scrimgeour K. G. Folypolyglutamate synthetase from beef liver: assay, stabilization, and characterization. Can J Biochem Cell Biol. 1984 Jun;62(6):495–506. doi: 10.1139/o84-067. [DOI] [PubMed] [Google Scholar]
  23. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  25. Shane B. Folylpolyglutamate synthesis and role in the regulation of one-carbon metabolism. Vitam Horm. 1989;45:263–335. doi: 10.1016/s0083-6729(08)60397-0. [DOI] [PubMed] [Google Scholar]
  26. Shane B. Pteroylpoly(gamma-glutamate) synthesis by Corynebacterium species. Purification and properties of folypoly(gamma-glutamate) synthetase. J Biol Chem. 1980 Jun 25;255(12):5655–5662. [PubMed] [Google Scholar]
  27. Slusher L. B., Gillman E. C., Martin N. C., Hopper A. K. mRNA leader length and initiation codon context determine alternative AUG selection for the yeast gene MOD5. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9789–9793. doi: 10.1073/pnas.88.21.9789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sussman D. J., Milman G., Shane B. Characterization of human folylpolyglutamate synthetase expressed in Chinese hamster ovary cells. Somat Cell Mol Genet. 1986 Nov;12(6):531–540. doi: 10.1007/BF01671939. [DOI] [PubMed] [Google Scholar]
  29. Taylor R. T., Hanna M. L. Folate-dependent enzymes in cultured Chinese hamster cells: folypolyglutamate synthetase and its absence in mutants auxotrophic for glycine + adenosine + thymidine. Arch Biochem Biophys. 1977 May;181(1):331–334. doi: 10.1016/0003-9861(77)90512-4. [DOI] [PubMed] [Google Scholar]
  30. Toy J., Bognar A. L. Cloning and expression of the gene encoding Lactobacillus casei folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure. J Biol Chem. 1990 Feb 15;265(5):2492–2499. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES