Publications

• Effects of gamma-glutamyl hydrolase on folyl and antifolylpolyglutamates in cultured H35 hepatoma cells.

  Yao R., Rhee M.S., Galivan J.

  A subline of H35 hepatoma cells (H35D cells) that have been made resistant to 5,10-dideazatetrahydrofolate exhibits an increase in gamma-glutamyl hydrolase (GH) activity. GH is a lysosomal enzyme in H35 and H35D cells on the basis of comparison of the distribution of enzyme activity with other kno ... >> More

  A subline of H35 hepatoma cells (H35D cells) that have been made resistant to 5,10-dideazatetrahydrofolate exhibits an increase in gamma-glutamyl hydrolase (GH) activity. GH is a lysosomal enzyme in H35 and H35D cells on the basis of comparison of the distribution of enzyme activity with other known lysosomal enzymes. The hydrolysis rate of methotrexate polyglutamate with isolated, intact lysosomes is 4-5-fold greater in H35D cells than in H35 cells. GH activity in isolated lysosomes is in part dependent on the presence of a reducing agent such as mercaptoethanol. Permeabilization of lysosomal preparations from both cell types by Triton X-100 causes a 10-fold enhancement in GH activity. The result of the enhanced activity of GH in H35D cells is a marked reduction in antifolylpolyglutamate concentration, with the parent antifolate being the predominant intracellular species found under all conditions tested. Unlike antifolates, the total intracellular folate concentration is nearly identical in both cells under standard culture conditions up to 10 microns folic acid. However, the chain length of folylpolyglutamates consists of predominantly triglutamates and tetraglutamates in H35D cells with increased GH, whereas it consists of pentaglutamates and hexaglutamates in the parental cells. At 50 and 100 microns folic acid, the folate accumulation in H35D cells is less than half that of H35 cells, and the predominant polyglutamate species in the H35D cells are the diglutamates through the tetraglutamates. The results demonstrate that the two H35 cell lines having equal folylpolyglutamate synthetase but that one with enhanced lysosomal GH activity exhibits a marked reduction in the amount and gamma-glutamyl chain length of folylpolyglutamates and antifolylpolyglutamates. << Less

  Mol Pharmacol 48:505-511(1995) [PubMed] [EuropePMC]

• The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells.

  Wang Y., Nimec Z., Ryan T.J., Dias J.A., Galivan J.

  gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column ... >> More

  gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells. << Less

  Biochim. Biophys. Acta 1164:227-235(1993) [PubMed] [EuropePMC]

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

  Yao R., Schneider E., Ryan T.J., Galivan J.

  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 hyd ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 93:10134-10138(1996) [PubMed] [EuropePMC]

• Plant gamma-glutamyl hydrolases and folate polyglutamates: characterization, compartmentation, and co-occurrence in vacuoles.

  Orsomando G., de la Garza R.D., Green B.J., Peng M., Rea P.A., Ryan T.J., Gregory J.F., Hanson A.D.

  gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal ... >> More

  gamma-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K(m) values (0.5-2 microm) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with approximately 50 and approximately 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16-60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack. << Less

  J Biol Chem 280:28877-28884(2005) [PubMed] [EuropePMC]

• Tomato gamma-glutamylhydrolases: expression, characterization, and evidence for heterodimer formation.

  Akhtar T.A., McQuinn R.P., Naponelli V., Gregory J.F., Giovannoni J.J., Hanson A.D.

  Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer an ... >> More

  Folates typically have gamma-linked polyglutamyl tails that make them better enzyme substrates and worse transport substrates than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme gamma-glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs, whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When coexpressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, whereas LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had one-half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed approximately 85% reduction in folate polyglutamates, but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously and to have catalytic characteristics midway between those of the corresponding homodimers. << Less

  Plant Physiol 148:775-785(2008) [PubMed] [EuropePMC]

• Identification, cloning, and sequencing of a cDNA coding for rat gamma-glutamyl hydrolase.

  Yao R., Nimec Z., Ryan T.J., Galivan J.

  Purified gamma-glutamyl hydrolase secreted from rat H35 hepatoma cells has been characterized as a diffuse band of 55 kDa on SDS-polyacrylamide gel electrophoresis that is converted to bands of 35 and 33 kDa after enzymatic removal of N-linked carbohydrate. Polyclonal antibodies against 55-kDa gam ... >> More

  Purified gamma-glutamyl hydrolase secreted from rat H35 hepatoma cells has been characterized as a diffuse band of 55 kDa on SDS-polyacrylamide gel electrophoresis that is converted to bands of 35 and 33 kDa after enzymatic removal of N-linked carbohydrate. Polyclonal antibodies against 55-kDa gamma-glutamyl hydrolase captured the enzyme activity and recognized the glycosylated and both deglycosylated forms of gamma-glutamyl hydrolase. A complete cDNA sequence of gamma-glutamyl hydrolase was obtained using degenerate oligonucleotides derived from peptide sequences, screening of a rat hepatoma cDNA library, and reverse transcription polymerase chain reaction. Based upon the deduced amino acid sequence the peptide component of gamma-glutamyl hydrolase had a molecular weight of 33,400. The results of amino acid analysis of the purified protein agreed with the deduced amino acid sequence in which there are seven potential asparagine-containing glycosylation sites. << Less

  J. Biol. Chem. 271:8525-8528(1996) [PubMed] [EuropePMC]