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
. 1996 Sep 17;93(19):10134–10138. doi: 10.1073/pnas.93.19.10134

Human gamma-glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro.

R Yao 1, E Schneider 1, T J Ryan 1, J Galivan 1
PMCID: PMC38349  PMID: 8816764

Abstract

A cDNA encoding human gamma-glutamyl hydrolase has been identified by searching an expressed sequence tag data base and using rat gamma-glutamyl hydrolase cDNA as the query sequence. The cDNA encodes a 318-amino acid protein of Mr 35,960. The deduced amino acid sequence of human gamma-glutamyl hydrolase shows 67% identity to that of rat gamma-glutamyl hydrolase. In both rat and human the 24 amino acids preceding the N terminus constitute a structural motif that is analogous to a leader or signal sequence. There are four consensus asparagine glycosylation sites in the human sequence, with three of them conserved in the rat enzyme. Expression of both the human and rat cDNA in Escherichia coli produced antigenically related proteins with enzyme activities characteristic of the native human and rat enzymes, respectively, when methotrexate di- or pentaglutamate were used as substrates. With the latter substrate the rat enzyme cleaved the innermost gamma-glutamyl linkage resulting in the sole production of methotrexate as the pteroyl containing product. The human enzyme differed in that it produced methotrexate tetraglutamate initially, followed by the triglutamate, and then the diglutamate and methotrexate. Hence the rat enzyme is an endopeptidase with methotrexate pentaglutamate as substrate, whereas the human enzyme exhibits exopeptidase activity. Another difference is that the expressed rat enzyme is equally active on methotrexate di- and pentaglutamate whereas the human enzyme has severalfold greater activity on methotrexate pentaglutamate compared with the diglutamate. These properties are consistent with the enzymes derived from human and rat sources.

Full text

PDF
10135

Images in this article

10136Fig. 3
on p.10136

Selected References

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

  1. Bhandari S. D., Gregory J. F., 3rd, Renuart D. R., Merritt A. M. Properties of pteroylpolyglutamate hydrolase in pancreatic juice of the pig. J Nutr. 1990 May;120(5):467–475. doi: 10.1093/jn/120.5.467. [DOI] [PubMed] [Google Scholar]
  2. Chandler C. J., Wang T. T., Halsted C. H. Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization. J Biol Chem. 1986 Jan 15;261(2):928–933. [PubMed] [Google Scholar]
  3. Elsenhans B., Ahmad O., Rosenberg I. H. Isolation and characterization of pteroylpolyglutamate hydrolase from rat intestinal mucosa. J Biol Chem. 1984 May 25;259(10):6364–6368. [PubMed] [Google Scholar]
  4. Galivan J., Rhee M. S. Insulin-dependent suppression in glutamyl hydrolase activity and elevated cellular methotrexate polyglutamates. Biochem Pharmacol. 1995 Nov 9;50(10):1659–1663. doi: 10.1016/0006-2952(95)02064-0. [DOI] [PubMed] [Google Scholar]
  5. Izard J. W., Doughty M. B., Kendall D. A. Physical and conformational properties of synthetic idealized signal sequences parallel their biological function. Biochemistry. 1995 Aug 8;34(31):9904–9912. doi: 10.1021/bi00031a012. [DOI] [PubMed] [Google Scholar]
  6. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Lennon G., Auffray C., Polymeropoulos M., Soares M. B. The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression. Genomics. 1996 Apr 1;33(1):151–152. doi: 10.1006/geno.1996.0177. [DOI] [PubMed] [Google Scholar]
  8. Lyko F., Martoglio B., Jungnickel B., Rapoport T. A., Dobberstein B. Signal sequence processing in rough microsomes. J Biol Chem. 1995 Aug 25;270(34):19873–19878. doi: 10.1074/jbc.270.34.19873. [DOI] [PubMed] [Google Scholar]
  9. O'Connor B. M., Rotundo R. F., Nimec Z., McGuire J. J., Galivan J. Secretion of gamma-glutamyl hydrolase in vitro. Cancer Res. 1991 Aug 1;51(15):3874–3881. [PubMed] [Google Scholar]
  10. Priest D. G., Veronee C. D., Mangum M., Bednarek J. M., Doig M. T. Comparison of folylpolyglutamate hydrolases of mouse liver, kidney, muscle and brain. Mol Cell Biochem. 1982 Mar 19;43(2):81–87. doi: 10.1007/BF00423095. [DOI] [PubMed] [Google Scholar]
  11. Rhee M. S., Wang Y., Nair M. G., Galivan J. Acquisition of resistance to antifolates caused by enhanced gamma-glutamyl hydrolase activity. Cancer Res. 1993 May 15;53(10 Suppl):2227–2230. [PubMed] [Google Scholar]
  12. Samuels L. L., Goutas L. J., Priest D. G., Piper J. R., Sirotnak F. M. Hydrolytic cleavage of methotrexate gamma-polyglutamates by folylpolyglutamyl hydrolase derived from various tumors and normal tissues of the mouse. Cancer Res. 1986 May;46(5):2230–2235. [PubMed] [Google Scholar]
  13. 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]
  14. Silink M., Reddel R., Bethel M., Rowe P. B. Gamma-glutamyl hydrolase conjugase). Purification and properties of the bovine hepatic enzyme. J Biol Chem. 1975 Aug 10;250(15):5982–5994. [PubMed] [Google Scholar]
  15. Wang T. T., Chandler C. J., Halsted C. H. Intracellular pteroylpolyglutamate hydrolase from human jejunal mucosa. Isolation and characterization. J Biol Chem. 1986 Oct 15;261(29):13551–13555. [PubMed] [Google Scholar]
  16. Wang Y., Nimec Z., Ryan T. J., Dias J. A., Galivan J. The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells. Biochim Biophys Acta. 1993 Aug 7;1164(3):227–235. doi: 10.1016/0167-4838(93)90253-n. [DOI] [PubMed] [Google Scholar]
  17. Yao R., Nimec Z., Ryan T. J., Galivan J. Identification, cloning, and sequencing of a cDNA coding for rat gamma-glutamyl hydrolase. J Biol Chem. 1996 Apr 12;271(15):8525–8528. doi: 10.1074/jbc.271.15.8525. [DOI] [PubMed] [Google Scholar]
  18. Yao R., Rhee M. S., Galivan J. Effects of gamma-glutamyl hydrolase on folyl and antifolylpolyglutamates in cultured H35 hepatoma cells. Mol Pharmacol. 1995 Sep;48(3):505–511. [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