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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Glutathione is a tripeptide thiol (L-gamma-glutamyl-L- cysteinylglycine) present in animal tissues, plants, and microorganisms. It is found intracellularly in high (0.1 to 10 mM) millimolar concentrations and is thus the most prevalent cellular thiol and the most abundant low molecular weight peptide typically found in mammals.
• the two characteristic structural features of glutathione - the gamma-glutamyl linkage and a sulfhydryl group - promote its intracellular stability and are intimately associated with its multiple biochemical functions.
• Glutathione protects cells from the toxic effects of reactive oxygen compounds and is an important component of the system that uses reduced pyridine nucleotide to provide the cell with its reducing properties which promote, for example, intracellular formation of cysteine and the thiol forms of proteins; glutathione functions in catalysis, metabolism, and transport; it participates in reactions involving the synthesis of proteins and nucleic acids and in those that detoxify free radicals and peroxides; it forms conjugates with a variety of compounds of endogenous and exogenous origin and is a cofactor for various enzymes.
• Glutathione functions as a co-enzyme for formaldehyde dehydrogenase, maleylacetoacetate isomerase, glyoxalase, prostaglandin endoperoxidase isomerases, and dichlorodiphenyltrichloroethane dehydrochlorinase and similar enzymes.
• the hemimercaptal formed nonenzymatically by reaction of methylglyoxal and glutathione (GSH) is converted by glyoxalase I to S-lactyl-Glutathione, which is split by glyoxalase II to D-lactate and Glutathione.
• GSH is synthesized by the actions of gamma- glutamylcysteine synthetase and Glutathione synthetase as shown in the following reactions.
• Gamma-glutamylcysteine catalyzes the first of these reactions and is the rate limiting reaction in Glutathione synthesis.
• L-glutamate + L-cysteine + ATP L-gamma-glutamyl-L-cysleine + ADP + Pi
• the activity of the holoenzyme is feedback inhibited by GSH [see J. Biol. Chem. 250:1422 (1975)].
• GSH feedback inhibited by GSH
• Such inhibition which provides a mechanism that regulates the GSH level in various tissues, is accompanied by reduction of the enzyme and by competitive inhibition by GSH with respect to glutamate [see J.
• the recombinant heavy subunit obtained by expression of the cDNA in E. coli exhibits a much higher K m value for glutamate and a greater sensitivity to feedback inhibition by GSH than the holoenzyme.
• Another aspect of the present invention is to describe the amino acid sequence of the light subunit of ⁇ - glutamylcysteine synthetase.
• Figure 1 represents the light subunit expression plasmid, pRGCSL, according to the present invention
• Figure 3 represents the sequencing strategy for Clones 71 and 62 according to the present invention
• Figure 4 represents a tracing showing 2 peaks of the separated light and heavy subunits according to the present invention.
• oligonucleotide probe was designed and synthesized corresponding to the sequence deduced from peptide I.
• the probe is a mixture of 32 different 20-mer oligonucleotides corresponding to all codons combination derived from peptide I, above.
• the letter I in the sequence below represents deoxyinosine, and has been substituted at the wobble positions in two of the codons.
• EXAMPLE IV Isolation of the cDNA clones for the light subunit of rat kidney ⁇ GCS was carried out as follows: A rat kidney cDNA expression library (Clontech), having an average insert size of 1.1 kb (range from 0.6 to 3.8 kb) and 1.2 x 1 ⁇ 6 independent clones in vector ⁇ gtll, was immunoscreened as described by Sambrook [see J. Sambrook, E.f. Fritsch and T. Maniatis, "Molecular Cloning, a Laboratory Manual” Cold Spring Harbor Laboratory Press (1989)] using antibody, prepared in accordance with Example I, to the light subunit. An overnight culture of E.
• coli Y1090r- (Clontech) grown in LB medium containing 0.2% maltose and 10 mM MgS04, was divided into ten 0.1 ml portions. Each tube containing a portion was infected with 5 x 10 4 plaque formation units (pfu) of the bacteriophage ⁇ gtll expression library (Clontech) at 37° C for 15 min. After mixing with 7 ml of top agarose (LB medium containing 0.75% agarose), the infected bacteria were poured onto 10 LB agar plates (150 x 35 mm) containing ampicillin (100 ⁇ g/ml) and incubated at 42° C for 3.5 hr.
• IPTG isopropylthiogalactoside
• the two sets of nitrocellulose filters were treated with blocking buffer (TNT containing 5% nonfat dry milk) for 1 hr followed by the same buffer containing diluted (1 :500) antibody to the light subunit for an additional 4 hrs. After washing with blocking buffer (TNT containing 5% nonfat dry milk) for 1 hr followed by the same buffer containing diluted (1 :500) antibody to the light subunit for an additional 4 hrs. After washing with blocking buffer (TNT containing 5% nonfat dry milk) for 1 hr followed by the same buffer containing diluted (1 :500) antibody to the light subunit for an additional 4 hrs. After washing with
• a rat kidney cDNA ⁇ gtll expression library containing the cDNAs in the phage EcoRI site, was screened with antibody to the light subunit. From about 5 x 10 5 phages, 38 positive clones were obtained. The expressed fusion protein from 21 of those clones reacted with the antibody when tested by western blot analysis. The phage DNA from those clones were isolated, and the two clones (numbered 62 and 71 ) with the largest inserts were chosen for further analysis. When digested with EcoRI followed by agarose gel electrophoresis, two DNA bands that represent the insert cDNAs were obtained from each of the two clones.
• clone 62 was found to contain a 1 kb and a 0.4 kb band, while a 0.7 and a 0.4 kb bands were found contained in clone 71. These results indicate that the cDNAs in these two clones most likely contains similar DNA sequences. Southern blot analysis showed that the 1 kb band from clone 62 and the 0.7 kb band from clone 71 hybridized with the oligonucleotide probe described above. It was the #1.0, 0.7, and 0.4 kb DNAs that were subcloned into the ECoRI site of phagmid pBluescript KS-(+) for sequence analysis.
• Recombinant ⁇ phage particles (1 x 10 6 pfu), obtained from the positive clones, were separately incubated with E. coli Y1090r- (1 x 10 8 cells) at 37° C for 20 min.
• the infected cells were inoculated in 50 ml of prewarmed LB medium until the cells lysed (about 6-8 hrs). After removal of cellular debris by centrifugation (3000 x g for 5 min), the ⁇ phage was precipitated by adding NaCI (2.9 g) and polyethylene glycol (MW 8000; 5 g) to the medium. After standing on ice for 2 hrs, the precipitated phage particles were recovered by centrifugation (5000 x g for 10 min).
• TM buffer 50 mM Tris-HCI (pH 7.5) and 10 mM MgS ⁇ 4); and the excess PEG8000 was removed by extracting the solution with 4 ml of chloroform.
• the aqueous layer was passed through a DE52 column (4 ml) pre- equilibrated with TM buffer. The column was washed with 3 ml of TM and the effluent (7 ml) was collected. Isopropanol (7 ml) and NaCI (400 #1 ; 4 M) were added to the effluent and the solution was placed on ice for 1 hr.
• the phage was precipitated by centrifugation (8000 x g for 10 min) and resuspended with 500 ⁇ l TE buffer (10 mM Tris-HCI; pH 8.0, and 1 mM EDTA).
• the phage solution was extracted with 500 ⁇ l phenol (saturated with Tris- HCI buffer, pH 8.0), followed by extraction with the same volume of phenol/chloroform (1 :1 ). This extraction by phenol and phenol/chloroform was repeated several times until no precipitate appeared at the interface of the extraction.
• the solution was then extracted with chloroform and the DNA was precipitated with 40 ⁇ l 0.3 M sodium acetate and 1 ml of ethanol.
• EXAMPLE VI Southern blot analysis of the recombinant DNA was conducted as follows:
• Recombinant ⁇ gtll DNA (3 g) from Example 5 was digested with EcoR I and the resulting fragments were separated by agarose (0.8%) gel electrophoresis.
• the digested DNA was transferred by capillary action to a Nitran membrane filter in 10x SSPE Buffer (20x SSPE: 3 M NaCI, 0.2 M NaH2P04, and 20 mM EDTA).
• the filter was incubated at 45° C for 3 hr in prehybridization buffer [6x SSPE, 5x Denhardt's solution (0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1 % bovine serum albumin, 0.5% SDS, and 100 ⁇ g/ml denatured and fragmented salmon sperm DNA) followed by incubation at 45° C overnight in the same buffer containing 32 P-labeled oligonucleotide probe (2x 10 5 cpm; 10 9 cpm/ ⁇ g).
• the probe was synthesized according to the sequence deduced from the peptide sequence obtained from tryptic digestion of the light subunit.
• the filter was washed with 2x SSPE containing 0.5% SDS for 5 min at room temperature followed by washing with Ix SSPE 30 min at 45° C. Autoradiography was performed at room temperature overnight.
• the inserts of the recombinant ⁇ gtll phage DNA were excised by treatment with EcoRI, and isolated from agarose gel and purified using a GenecleanTM kit in accordance with the manufacture's instructions.
• the cDNAs were subcloned into the EcoRI site of the phagemid pBluescript KS-(+) (Strategene).
• the nucleotide sequence was determined on either pBluescript single or double stranded DNA by dideoxynucleotide chain termination method [see Proc. Natl. Acad. Sci. USA 74:5463 (1977)] using Sequenase (U.S. Biochemicals) according to the manufacturer's instructions.
• T7, SK primers, as well as the primers corresponding to internal light subunit sequence were used. Sequence analysis was performed using PC/Gene software.
• ATG (position 61 ) is presumed to be the initiation codon because (a) the nucleotide sequence surrounding this codon (...GCCATGG%) agrees with the consensus sequence for eukaryotic initiation sites described by Kozak [see Nucleic Acid Research 12:857 (1984)] and (b) expression of the cDNA using this ATG as initiation codon produces a protein that co-migrates with the light subunit of isolated holoenzyme.
• the open reading frame sequence ends with a termination codon (TAA) at position 883, followed by 10 other termination codons.
• the predicted protein sequence which contains the two independently determined peptide sequence (total 41 residues; 139-156 and 219-241 ), was found to be unique when compare with the protein sequence given in the Genbank# data base.
• the expressed open reading frame for the light subunit of ⁇ -glutamylcysteine synthetase according to the present invention provides for the following peptide in which the two independently determined peptide sequence above are underlined is:
• amino acid composition of the light subunit of rat kidney ⁇ -glutamylcysteine synthetase is as follows: Amino acid Isolated subunit Deduced fr ⁇ n the cDNA. sequence
• the light subunit cDNA in plasmid pBluescript KS was digested with Ncol; the DNA was filled-in with four dNTPs using
• T4 DNA polymerase and subsequently treated with BamHI [see Sambrook, supra].
• the resulting DNA fragment (1 kb) was ligated [see Sambrook, supra ⁇ into expression vector pT7-7 which had previously been digested with Ndel (filled-in) and BamHI.
• the resulting plasmid (pRGCSL) (see Figure 1 ) contains the light subunit cDNA immediately downstream of a T7 promoter.
• This plasmid is on deposit at the Cornell University Medical College, 1300 York Avenue, New York, New York, and will be made available to anyone requesting the plasmid from the inventors hereof in accordance with the Budapest Treaty.
• the expression plasmid for the heavy subunit pRGCSH [see J. Biol. Chem (1993)] was digested with BstBI. The DNA was filled- in [see Sambrook, supra] using T4 DNA polymerase followed by digestion with Hind 111. The resulting 2-kb DNA fragment that contains a T7 promoter and the heavy subunit cDNA was ligated to plasmid pRGCSL (see Figure 1) which had been previously treated with Clal (filled-in) and Hindlll. The plasmid obtained (pRGCSHL) (see Figure 2) contains two T7 promoters in opposite directions immediately followed by the heavy and the light subunit respectively.
• Co-expression plasmid pRGCSHL (1 ng) was transformed into E. coli BL21 (DE3). This organism is on deposit at the Cornell
• the recombinant holoenzyme was expressed according to known methods as described in the literature [see J. Biol. Chem (1993)].
• the enzyme was purified in the manner similar to that used in purification of the recombinant heavy subunit using recognized techniques [see Sambrook, supra].
• the enzyme isolated from the ATP-agarose column was further purified on a ProteinPakTM 300 (Waters) HPLC gel filtration column previously equilibrated with imidazole buffer (10 mM; pH 7.4) containing 1 mM EDTA.
• the purified recombinant holoenzyme exhibited a specific activity of 1 ,250 when assayed in a assay solution that contains 10 mM glutamate as depicted in the following table. This specific activity is similar to that of the holoenzyme isolated from rat kidney but is much higher.
• the following table presents the Km values for the holoenzyme and heavy subunit of the GSH enzyme.
• the Km value is a reflection of the affinity of the substrate for the enzyme; a high value meaning a low affinity, and a low value means a high affinity.
• Recombinant holoenzyme (mixed) 2.8 1.2 0.2
• Recombinant heavy subunit 18.2 0.8 0.2
• this table illustrates that the two types of recombinant enzyme (one made by co-expressing cDNA for light and cDNA for heavy subunits - and the other by simply mixing the separately expressed subunits) have affinity for glutamate, cysteine (and alpha-aminobutyrate) that is about the same as the isolated holoenzyme.
• the recombinant heavy enzyme has a value of 18.2 mM for glutamate indicating that this enzyme has about a 10-fold lower affinity for glutamate.
• nucleotide sequences may be directly synthesized on an automated DNA synthesizer such as the Applied Biosystems Model 380A.
• a large number of base, ribose and phosphate modifications can also be incorporated by substitution of the appropriate reagents for normal phosphoramidite chemistry.
• Small oligonucleotides are spontaneously taken up from the surrounding medium by some cells, and this technique may be used to introduce the oligonucleotide according to the present invention into the appropriate cells to increase GSH levels within animal tissues.
• antisense sequences to the oligonucleotides according to the present invention and to introduce such antisense sequences into appropriate cells to inhibit GSH levels within animal tissues.
• Such uptake of both sense and antisense oligonucleotides may be facilitate by modification of the nucleic acid, as as derivatization with a hydrophobic moiety, substitution of methylphosphonates, phosphorothioates or dithioates for normally occurring phosphates.
• Liposome fusion provides another mode of delivering nucleic acid-based reagents to cells. Such techniques for manufacturing and delivery of oligonucleotides and peptides are well know in the art.
• TGTTGAGTTT AAGTACCTCC CTGGCGTCTG CAGCAGCGCA CTCACAGGAA 1082 GCATTGTATT CTCTTCATTA AACTCTTGGT TTCTAACTGA AATCGTCTAT 1132 AAAGAAAAAT ACTTGCAATA TATTTCCTTT ATTTTTATGA GTAATAGAAA 1182 TCAAGAAAAT TTGTTTTAAG ATATATTTTG GCTTAGGCAT CAGGGTGATG 1232 TATATACATA TTTTATTT CTAAAATTCA GTAACTGCTT CTTACTCTAT 1282

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• 1994
  • 1994-04-07 WO PCT/US1994/003856 patent/WO1994024276A1/en not_active Application Discontinuation
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