Skip to main content
NCBI 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
. 1991 Aug 15;88(16):7361–7365. doi: 10.1073/pnas.88.16.7361

High-level expression of enzymatically active mature human gamma-glutamyltransferase in transgenic V79 Chinese hamster cells.

A Visvikis 1, C Thioudellet 1, T Oster 1, S Fournel-Gigleux 1, M Wellman 1, G Siest 1
PMCID: PMC52295  PMID: 1678521

Abstract

gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 cell line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 cells. We selected a cell line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this cell line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered cell line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

Full text

PDF
7361

Images in this article

7362Image
on p.7362
7363Image
on p.7363
7363Image
on p.7363
7363Image
on p.7363
7363Image
on p.7363
7364Image
on p.7364
7364Image
on p.7364

Selected References

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

  1. Altman R. A., Hughey R. P. The identification of two subcellular sites for cleavage of gamma-glutamyltranspeptidase propeptide. Biochem Int. 1986 Dec;13(6):1009–1017. [PubMed] [Google Scholar]
  2. Angele C., Wellman M., Thioudellet C., Guellaen G., Siest G. Expression of rat renal gamma-glutamyltransferase cDNA in Escherichia coli. Biochem Biophys Res Commun. 1989 May 15;160(3):1040–1046. doi: 10.1016/s0006-291x(89)80107-x. [DOI] [PubMed] [Google Scholar]
  3. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  4. Capraro M. A., Hughey R. P. Processing of the propeptide form of rat renal gamma-glutamyltranspeptidase. FEBS Lett. 1983 Jun 27;157(1):139–143. doi: 10.1016/0014-5793(83)81132-6. [DOI] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Curthoys N. P., Hughey R. P. Characterization and physiological function of rat renal gamma-glutamyltranspeptidase. Enzyme. 1979;24(6):383–403. doi: 10.1159/000458694. [DOI] [PubMed] [Google Scholar]
  7. Doehmer J., Dogra S., Friedberg T., Monier S., Adesnik M., Glatt H., Oesch F. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5769–5773. doi: 10.1073/pnas.85.16.5769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dogra S., Doehmer J., Glatt H., Mölders H., Siegert P., Friedberg T., Seidel A., Oesch F. Stable expression of rat cytochrome P-450IA1 cDNA in V79 Chinese hamster cells and their use in mutagenicity testing. Mol Pharmacol. 1990 May;37(5):608–613. [PubMed] [Google Scholar]
  9. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  10. Fournel-Gigleux S., Jackson M. R., Wooster R., Burchell B. Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture. FEBS Lett. 1989 Jan 30;243(2):119–122. doi: 10.1016/0014-5793(89)80111-5. [DOI] [PubMed] [Google Scholar]
  11. Fournel-Gigleux S., Sutherland L., Sabolovic N., Burchell B., Siest G. Stable expression of two human UDP-glucuronosyltransferase cDNAs in V79 cell cultures. Mol Pharmacol. 1991 Feb;39(2):177–183. [PubMed] [Google Scholar]
  12. Goldberg D. M. Structural, functional, and clinical aspects of gamma-glutamyltransferase. CRC Crit Rev Clin Lab Sci. 1980;12(1):1–58. doi: 10.3109/10408368009108725. [DOI] [PubMed] [Google Scholar]
  13. Goodspeed D. C., Dunn T. J., Miller C. D., Pitot H. C. Human gamma-glutamyl transpeptidase cDNA: comparison of hepatoma and kidney mRNA in the human and rat. Gene. 1989 Mar 15;76(1):1–9. doi: 10.1016/0378-1119(89)90002-4. [DOI] [PubMed] [Google Scholar]
  14. Hammarström S. Leukotrienes. Annu Rev Biochem. 1983;52:355–377. doi: 10.1146/annurev.bi.52.070183.002035. [DOI] [PubMed] [Google Scholar]
  15. Kaufman R. J., Wasley L. C., Dorner A. J. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem. 1988 May 5;263(13):6352–6362. [PubMed] [Google Scholar]
  16. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. 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]
  19. Laperche Y., Bulle F., Aissani T., Chobert M. N., Aggerbeck M., Hanoune J., Guellaën G. Molecular cloning and nucleotide sequence of rat kidney gamma-glutamyl transpeptidase cDNA. Proc Natl Acad Sci U S A. 1986 Feb;83(4):937–941. doi: 10.1073/pnas.83.4.937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin P. H., Selinfreund R., Wakshull E., Wharton W. Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding. Biochemistry. 1987 Feb 10;26(3):731–736. doi: 10.1021/bi00377a012. [DOI] [PubMed] [Google Scholar]
  21. Manson M. M., Legg R. F., Watson J. V., Green J. A., Neal G. E. An examination of the relative resistances to aflatoxin B1 and susceptibilities to gamma-glutamyl p-phenylene diamine mustard of gamma-glutamyl transferase negative and positive cell lines. Carcinogenesis. 1981;2(7):661–670. doi: 10.1093/carcin/2.7.661. [DOI] [PubMed] [Google Scholar]
  22. Matsuda Y., Tsuji A., Katunuma N. Studies on the structure of gamma-glutamyltranspeptidase. III. Evidence that the amino terminus of the heavy subunit is the membrane binding segment. J Biochem. 1983 May;93(5):1427–1433. doi: 10.1093/oxfordjournals.jbchem.a134278. [DOI] [PubMed] [Google Scholar]
  23. Orlowski M., Meister A. The gamma-glutamyl cycle: a possible transport system for amino acids. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1248–1255. doi: 10.1073/pnas.67.3.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Papandrikopoulou A., Frey A., Gassen H. G. Cloning and expression of gamma-glutamyl transpeptidase from isolated porcine brain capillaries. Eur J Biochem. 1989 Aug 15;183(3):693–698. doi: 10.1111/j.1432-1033.1989.tb21100.x. [DOI] [PubMed] [Google Scholar]
  25. Parekh R. B., Dwek R. A., Thomas J. R., Opdenakker G., Rademacher T. W., Wittwer A. J., Howard S. C., Nelson R., Siegel N. R., Jennings M. G. Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator. Biochemistry. 1989 Sep 19;28(19):7644–7662. doi: 10.1021/bi00445a021. [DOI] [PubMed] [Google Scholar]
  26. Pawlak A., Cohen E. H., Octave J. N., Schweickhardt R., Wu S. J., Bulle F., Chikhi N., Baik J. H., Siegrist S., Guellaën G. An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues. J Biol Chem. 1990 Feb 25;265(6):3256–3262. [PubMed] [Google Scholar]
  27. Pawlak A., Lahuna O., Bulle F., Suzuki A., Ferry N., Siegrist S., Chikhi N., Chobert M. N., Guellaen G., Laperche Y. gamma-Glutamyl transpeptidase: a single copy gene in the rat and a multigene family in the human genome. J Biol Chem. 1988 Jul 15;263(20):9913–9916. [PubMed] [Google Scholar]
  28. Pawlak A., Wu S. J., Bulle F., Suzuki A., Chikhi N., Ferry N., Baik J. H., Siegrist S., Guellaën G. Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney. Biochem Biophys Res Commun. 1989 Oct 31;164(2):912–918. doi: 10.1016/0006-291x(89)91545-3. [DOI] [PubMed] [Google Scholar]
  29. PetitClerc C., Shiele F., Bagrel D., Mahassen A., Siest G. Kinetic properties of gamma-glutamyltransferase from human liver. Clin Chem. 1980 Nov;26(12):1688–1693. [PubMed] [Google Scholar]
  30. Rajpert-De Meyts E., Heisterkamp N., Groffen J. Cloning and nucleotide sequence of human gamma-glutamyl transpeptidase. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8840–8844. doi: 10.1073/pnas.85.23.8840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sakamuro D., Yamazoe M., Matsuda Y., Kangawa K., Taniguchi N., Matsuo H., Yoshikawa H., Ogasawara N. The primary structure of human gamma-glutamyl transpeptidase. Gene. 1988 Dec 15;73(1):1–9. doi: 10.1016/0378-1119(88)90307-1. [DOI] [PubMed] [Google Scholar]
  32. Siest G., Strazielle N., L'Hôte H., Wellman M., Bagrel D., Batt A. M., Schiele F., Galteau M. M. Interprétation des variations de la gamma-glutamyltransférase plasmatique et tissulaire sous l'influence des médicaments. Therapie. 1989 Jan-Feb;44(1):19–28. [PubMed] [Google Scholar]
  33. Stenius U., Rubin K., Gullberg D., Högberg J. gamma-Glutamyltranspeptidase-positive rat hepatocytes are protected from GSH depletion, oxidative stress and reversible alterations of collagen receptors. Carcinogenesis. 1990 Jan;11(1):69–73. doi: 10.1093/carcin/11.1.69. [DOI] [PubMed] [Google Scholar]
  34. Tanaka T., Naganuma A., Imura N. Role of gamma-glutamyltranspeptidase in renal uptake and toxicity of inorganic mercury in mice. Toxicology. 1990 Mar 16;60(3):187–198. doi: 10.1016/0300-483x(90)90142-4. [DOI] [PubMed] [Google Scholar]
  35. Tate S. S., Galbraith R. A. In vitro translation and processing of human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase. Biochem Biophys Res Commun. 1988 Aug 15;154(3):1167–1173. doi: 10.1016/0006-291x(88)90263-x. [DOI] [PubMed] [Google Scholar]
  36. Tate S. S., Meister A. Affinity labeling of gamma-glutamyl transpeptidase and location of the gamma-glutamyl binding site on the light subunit. Proc Natl Acad Sci U S A. 1977 Mar;74(3):931–935. doi: 10.1073/pnas.74.3.931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tate S. S., Meister A. Subunit structure and isozymic forms of gamma-glutamyl transpeptidase. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2599–2603. doi: 10.1073/pnas.73.8.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tate S. S., Meister A. gamma-Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem. 1981 Sep 25;39:357–368. doi: 10.1007/BF00232585. [DOI] [PubMed] [Google Scholar]
  39. Tate S. S., Ross M. E. Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit. J Biol Chem. 1977 Sep 10;252(17):6042–6045. [PubMed] [Google Scholar]
  40. Visvikis A., Goergen J. L., Oster T., Bagrel D., Wellman M., Marc A., Engasser J. M., Siest G. Gamma-glutamyltransferase from human hepatoma cell lines: purification and cell culture of HepG2 on microcarriers. Clin Chim Acta. 1990 Nov 5;191(3):221–232. doi: 10.1016/0009-8981(90)90023-l. [DOI] [PubMed] [Google Scholar]
  41. Viña J. R., Blay P., Ramirez A., Castells A., Viña J. Inhibition of gamma-glutamyl transpeptidase decreases amino acid uptake in human keratinocytes in culture. FEBS Lett. 1990 Aug 20;269(1):86–88. doi: 10.1016/0014-5793(90)81125-8. [DOI] [PubMed] [Google Scholar]
  42. Wellman-Bednawska M., Artur Y., Siest G. Variations in sialic acid content of gamma-glutamyltransferase: a consequence for immunochemical determinations? Clin Chim Acta. 1985 May 15;148(1):21–30. doi: 10.1016/0009-8981(85)90296-7. [DOI] [PubMed] [Google Scholar]
  43. Yamashita K., Hitoi A., Tateishi N., Higashi T., Sakamoto Y., Kobata A. Organ-specific difference in the sugar chains of gamma-glutamyltranspeptidase. Arch Biochem Biophys. 1983 Sep;225(2):993–996. doi: 10.1016/0003-9861(83)90116-9. [DOI] [PubMed] [Google Scholar]
  44. Yamashita K., Tachibana Y., Hitoi A., Matsuda Y., Tsuji A., Katunuma N., Kobata A. Difference in the sugar chains of two subunits and of isozymic forms of rat kidney gamma-glutamyltranspeptidase. Arch Biochem Biophys. 1983 Nov;227(1):225–232. doi: 10.1016/0003-9861(83)90365-x. [DOI] [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