John Galivan

Identification of single nucleotide polymorphisms in the human γ-glutamyl hydrolase gene and characterization of promoter polymorphisms

gamma-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Increased GGH activity has been found in rat hepatoma cells resistant to the cancer drug methotrexate (MTX). The aim of this study was to identify polymorphisms in the GGH gene that modulate GGH activity and that may affect methotrexate resistance. Exons of the human gamma-glutamyl hydrolase (hGGH) gene were amplified by polymerase chain reaction (PCR) from breast cancer tissue and leukemia cell lines. Single-stranded conformational polymorphism (SSCP) analysis was performed, and PCR products containing different patterns were cloned and sequenced. Six single nucleotide polymorphisms (SNPs) were identified, at bases -401C>T, -354G>T, -124T>G, +16T>C, +452C>T, and +1102A>G, relative to the A of the translation start codon being considered as +1. The SNP at +16, which changes codon -19 (relative to the start of the mature hGGH protein) in the endoplasmic reticulum targeting sequence of hGGH protein from cysteine to arginine, has previously been identified in this laboratory. The SNP at +452 changes the conserved hGGH protein codon 127 from threonine to isoleucine. The functions of SNPs in the promoter of the hGGH gene were studied by site-directed mutagenesis of a 516-bp region of the hGGH gene promoter in a luciferase reporter vector and transfection into HepG2 and MCF-7 cells. All of the promoter polymorphisms enhanced the production of luciferase compared to the wild-type hGGH gene promoter in HepG2 cells, and -401C>T and -124T>G enhanced luciferase expression in MCF-7 cells, suggesting that polymorphisms in the hGGH gene promoter may increase expression of hGGH protein.

Glutamyl hydrolase: pharmacological role and enzymatic characterization

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Abstract Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH 2 -10-CH 3 PteGlu 4 ). When the time-dependent synthesis of MTX polyglutamates (4-NH 2 -10-CH 3 PteGlu 2 and higher) at extracellular concentrations of 10 and 100 μ m methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μ m is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μ m intracellular methotrexate, respectively, after 24 h in the presence of 100 μ m extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μ m with both cell types which corresponded to 10 to 12 μ m intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu 4–5 ) are more avidly retained by the cells than the shorter ones (Glu 2–3 ).

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 microM extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 microM. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. The turnover of methotrexate polyglutamates in cells saturated with these derivatives occurred at approximately one-half the rate of net synthesis and was stimulated to nearly the same extent by folate depletion and insulin. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells (M. Balinska, J. Galivan, and J.K. Coward (1981) Cancer Res. 41,2751-2756) and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.

Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50–100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme’s contribution to the cell cycle may be more complex than believed previously.

IDENTIFICATION, CLONING, AND SEQUENCING OF A CDNA CODING FOR RAT GAMMA -GLUTAMYL HYDROLASE

Purified -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 -glutamyl hydrolase captured the enzyme activity and recognized the glycosylated and both deglycosylated forms of -glutamyl hydrolase. A complete cDNA sequence of -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 -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.

Glutamyl hydrolase and the multitargeted antifolate LY231514

Purpose: To examine the activity of glutamyl hydrolase (GH) on the poly-γ-glutamates of multitargeted antifolate (MTA) (LY231514) and the effect of enhanced GH on the pharmacological activity of MTA. Methods: Expressed and purified GH were used to study the enzymatic cleavage of MTA poly-γ-glutamates and wild-type and GH-enhanced H35 hepatoma cell lines to evaluate growth inhibition. Results: MTA tri- and penta-γ-glutamates were good substrates for human GH, having higher rates than MTX tri- and penta-γ-glutamates. Preferential hydrolysis with human enzyme occurred at the two γ-glutamyl bonds at the carboxyl end of the molecule, whereas the rat enzyme preferred the innermost γ-linkage. Incubation of rat H35 hepatoma cell lines with MTA resulted in the intracellular accumulation of primarily tetra-, penta-, and hexa-γ-glutamate. The formation of these were markedly reduced in H35D cells, which is a line resistant to antifolates chiefly through enhanced cellular levels of GH activity. Conclusions: MTA poly-γ-glutamates are effective substrates for GH and their pharmacological effectiveness bears an inverse relationship to cellular GH activity. This observation, along with enhanced resistance to MTA of thymidylate synthase-amplified cells, substantiates the importance of the poly-γ-glutamates of MTA inhibiting TS as the primary target. Further evidence for the inverse relationship of GH to classical antifolate pharmacological activity is established.

The properties of the secreted γ-glutamyl hydrolases from H35 hepatoma cells

γ-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-γ-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-γ-glutamate (PteGlu5), methotrexate penta-γ-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-γ-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-γ-glutamate, which is subsequently degraded to glutamic acid. When penta-γ-glutamate is the substrate, the cleavage of the γ-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-α-glutamate is not a substrate nor are pABA-γ-Glu5 or penta-γ-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-γ-glutamates are poor substrates for γ-glutamyl hydrolase from H35 hepatoma cells.

The Chromophore and Polypeptide Composition of Aplysia Ink

The composition of the ink of the sea hare, Aplysia, was studied in regard to its tetrapyrrole and polypeptide content. The ink was separated into three pigment components by both thin-layer and gel filtration chromatography. These three pigments have distinctive visible absorption spectra, and--by comparison with known tetrapyrroles--we have demonstrated that they are derived from the three tetrapyrrole chromophores (bilins) found on the biliproteins of certain red algae, which constitute a portion of the Aplysia diet. The red component is phycourobilin; the purple is phycoerythrobilin; and the blue is phycocyanobilin. Sodium dodecyl sulfate gel electrophoresis experiments were also performed. The results of these experiments showed several polypeptides, and major bands at 78,000 and 61,000 molecular weight were noted. Biliproteins, at most, would be minor components of the ink.

Protective effect of the pteroylpolyglutamates and phosphate on the proteolytic inactivation of thymidylate synthetase.

Abstract Thymidylate synthetase is readily inactivated by trypsin, chymotrypsin, and carboxypeptidase A when incubated in 10–20 m m potassium phosphate buffer (pH 7.0). The loss is activity produced by trypsin and chymotrypsin is accomplished by extensive protein degradation, while inactivation by carboxypeptidase A is accompanied by release of the carboxyl-terminal valine only (Aull et al. , 1974, J. Biol. Chem. , 249 , 1167–1172). In contrast, when the incubations are conducted in 100–200 m m potassium phosphate buffer (pH 7.0), the synthetase is not inactivated by any of the three enzymes and the results of amino acid analysis and sodium dodecyl sulfate disc gel electrophoresis demonstrate that proteolysis is prevented. The resistance of thymidylate synthetase to inactivation was shown not to be due to the inhibition of the proteolytic enzymes by the buffer. The inactivation is not prevented either by pteroyl mono glutamates or by 2′-deoxyuridine 5′-phosphate (dUMP) alone, but the presence of both is partially protective. The pteroyl poly glutamates, however, offer limited protection against carboxypeptidase A and chymotrypsin; in combination with dUMP, proteolytic inactivation of the snythetase by all three enzymes is prevented. Characterization of the properties of carboxypeptidase A-inactivated thymidylate synthetase reveals the following, (i) The binding of deoxynucleotides is unaltered, but the binding of a variety of pteroylpolyglutamate derivatives is reduced or abolished, (ii) Pteroylpolyglutamates are bound provided dUMP or an analog such as 5-fluorodUMP is present, (iii) Ternary complex formation between carboxypeptidase A-inactivated enzyme and (+)5,10-methylenetetrahydropteroyltetraglutamate plus 5-fluorodUMP occurs in the same molar binding ratio (1:2:2) at saturation as with the native enzyme, but differs from the native enzyme ternary complex in that the dissociation constant for 5-fluorodUMP is increased by approximately 10 5 . In addition, there is no evidence for the formation of covalent linkages between the ligands and enzyme, (iv) The treated enzyme cannot catalyze tritium release from [ 3 H 5 ]dUMP in the presence of either (+)5,10-methylenepteroylmonoglutamate or (+)5,10-methylenetetrahydropteroyltetraglutamate.