THE JOURNAL OF BIOLOC~CAL CHEMISTRY Vol. 265, No. 3, Issue of January 25, pp. 1588-1593,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Amino Acid Sequence of Rat Kidney y-Glutamylcysteine Synthetase”

(Received for publication, August 2, 1989)

Ning Yan$ and Alton Meister From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021

y-Glutamylcysteine synthetase catalyzes the first step in the synthesis of glutathione. The enzyme iso- lated from rat kidney has two subunits (heavy, M, 73,000; and light, M, 27,700) which may be disso- ciated by treatment with dithiothreitol. The heavy sub- unit exhibits all of the catalytic activity of the isolated enzyme and also feedback inhibition by glutathione. The light subunit has no known function and may not be an integral part of the enzyme. cDNA clones encod- ing rat kidney y-glutamylcysteine synthetase were iso- lated from a Xgt 11 cDNA library by immunoscreening with antibody against the isolated enzyme and further screening with oligonucleotide probes derived from several peptides whose sequences were determined by the Edman method. The nucleotide sequence of the mRNA for the heavy subunit was deduced from the sequences of the cDNA of three such clones. The se- quence, which codes for 637 residues (Mr 72,614), contains all four of the independently determined pep- tide sequences (-100 residues). This amino acid se- quence shows extremely low overall similarity to that of y-glutamylcysteine synthetase isolated from Esche- riehia co&.

Glutathione, the tripeptide thiol that provides cells with their reducing environment, has important functions in me- tabolism, transport, and the protection of cells against the toxic effects of oxygen and other compounds (1). GSH is synthesized within cells by the actions of y-glutamylcysteine synthetase (Reaction 1) and GSH synthetase (Reaction 2) (2, 3).

L-Glutamate + L-cysteine + ATP (1)

8 L-y-glutamyl-L-cysteine + ADP + P,

L-y-Glutamyl-L-cysteine + glycine + ATP (2)

i? GSH + ADP + P,

The activity of y-glutamylcysteine synthetase, which cata- lyzes the committed step in GSH synthesis, is controlled by feedback inhibition by GSH; GSH binds to the glutamate site and to another site on the enzyme (4, 5).

y-Glutamylcysteine synthetase has been purified from sev-

* This work was supported in part by National Institutes of Health Grant 2 R37 DK 12034 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) J05181.

2 Provided part of this work from a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Cornell University Graduate School of Medical Sciences.

era1 sources (2,3). Studies in this laboratory (6-10) led to the isolation of highly purified preparations of the enzyme from rat kidney. The enzyme (Mr 104,000) is homogeneous on gel electrophoresis and dissociates under denaturing conditions to yield two apparent subunits (Mr 73,000 and 27,700, respec- tively). The enzyme may be dissociated under nondenaturing conditions by treatment with dithiothreitol. The heavy sub- unit obtained in this way exhibits all of the catalytic activity of the isolated enzyme; it also exhibits feedback inhibition by GSH (11). The light subunit is not enzymatically active, and its presence does not alter the catalytic properties or stability of the heavy subunit. Previously, the possibility was consid- ered that the enzyme is synthesized as a polypeptide chain of M, 104,000 which is cleaved to form subunits which remain associated by disulfide linkage (11). This interpretation was consistent with studies (11, 12) in which the mRNA for the M, 104,000 enzyme was tentatively identified and with the finding (11) that lo-25% of the isolated enzyme was not dissociated into subunits by treatment with dithiothreitol. However, as discussed below, the light subunit may be unre- lated to the heavy subunit, and the apparent subunits may become linked by disulfide bond formation after synthesis or during the isolation procedure. A preparation of y-glutamyl- cysteine synthetase from rat erythrocytes (13) was found to have a subunit structure similar to that of the rat kidney enzyme; the subunit structures of other preparations of the enzyme from mammalian sources have apparently not been examined. y-Glutamylcysteine synthetase has also been pu- rified from bacterial sources (5, 12, 14, 15). y-Glutamylcys- teine synthetase purified from a strain of Escherichia coli enriched in its genes for GSH synthesis was found to have a M, of -58,000 (5,14). The amino acid sequence of this enzyme was deduced from the sequence of the corresponding genomic DNA (16). In contrast to the kidney enzyme, the E. coli enzyme does not have an active-site thiol (5).

This study was undertaken to elucidate the structure of rat kidney y-glutamylcysteine synthetase and to explore its struc- tural relationship to the y-glutamylcysteine synthetase of E. coli. The amino acid sequence of the heavy subunit of rat kidney y-glutamylcysteine synthetase was deduced from the nucleotide sequence of the corresponding cDNA clones and from the independently determined amino acid sequences of several peptides derived from the isolated enzyme by chemical and enzymatic cleavage.

EXPERIMENTAL PROCEDURES’

RESULTS

General Approach-cDNA clones that encode the enzyme were isolated from a Xgtll rat kidney library by screening

’ Portions of this paper (including “Experimental Procedures,” Figs. l-4 and 7, and Tables I and II) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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y-Glutamylcysteine Synthetase Sequence 1589

with antibody raised against the isolated enzyme. The clones were further screened against three chemically synthesized oligonucleotide probes whose sequences were predicted by the amino acid sequences of internal peptides isolated from the enzyme. The amino acid sequence of the enzyme was deduced from that of the cDNA of three overlapping clones. The amino acid sequence thus obtained was found to contain the four peptide sequences that were determined independently by the Edman method.

Isolation and Characterization of Peptides Obtained from Heavy Subunit-The enzyme was isolated from rat kidney (lo), and the subunits were separated (see “Experimental Procedures”). The reduced and carboxymethylated heavy sub- unit was subjected to cleavage with cyanogen bromide and, in separate studies, with Lys-C protease; the peptides formed were separated by high performance liquid chromatography. Four apparently homogeneous peptides were obtained, and their sequences were determined by the automated Edman method (Fig. 1).

Design of Oligonucleotide Probes-Three oligonucleotide probes were synthesized (Fig. 2). Probe II is a unique sequence deduced from peptide II (Fig. 1) and is based on optimum codon usage of 11 rat mRNA sequences (GenBanke). This probe generated weak hybridization signals. Probe III is a mixed sequence of 64 different 18-mer oligonucleotides in- cluding all possible codons derived from the sequence of peptide II. In the synthesis of probe IV, deoxyinosine was substituted at the wobble positions in seven of the codons (27); this 27-mer oligonucleotide probe proved to be highly specific.

Isolation of y-Glutamylcysteine Synthetase cDNA Clones- A rat kidney cDNA expression library was screened with antiserum against isolated y-glutamylcysteine synthetase. This antiserum, whose specificity is shown in Fig. 3, recog- nizes both heavy and light subunits of the enzyme. From a primary screen of -5 x lo5 phages, 17 positive clones were found. cDNA from 12 of these hybridized with the three oligonucleotide probes; the insert sizes were 0.3-2 kilobases. The specific antibodies that were selected from the original antiserum by the fusion proteins expressed by the 17 positive cDNA clones were used to probe the protein blots (34). The results indicated that these 12 cDNA clones might encode the heavy subunit of the enzyme since the specific antibody bound only to the heavy subunit bands’ on Western blots (Fig. 4) of the purified enzyme and of kidney homogenate; this indicates that the fusion proteins were antigenically similar or identical to the purified heavy subunit. (Two positive clones (GCS (where GCS represents y-glutamylcysteine synthetase) 202 and GCS 222) were found to express fusion proteins that appear to be antigenically similar to the light subunit.) After subcloning the inserts from the heavy subunit cDNA clones into pUC19, restriction mapping was performed, and the group of 12 clones was found to exhibit a set of related bands. Two clones (GCS 245 and GCS 258) were chosen for DNA sequence analysis because of their longer insert size; GCS 243 was also sequenced because it contained the 5’-end of the sequence.

Nucleotide Sequence of y-Glutamylcysteine Synthetase mRNA (Heavy Subunit&The sequence was derived from those of the cDNA inserts of the three recombinant clones as outlined in Fig. 5. The (+)-strand was sequenced at least twice from different overlapping sets using either deletion subclones

‘The heavy subunit gives two bands under these conditions be- cause it breaks down on storage to form an M, 63,000 species which is enzymatically inactive, but which reacts with the antibody; this is discussed below.

- - An3

GCS 243

GCS 245 ----- TAG

FIG. 5. Sequencing strategy used for recombinant clones GCS 243, GCS 245, and GCS 258. The cDNA inserts are verti- cally aligned to show the extent of overlap. GCS 243 starts at position 1 and ends at position 1506 of the composite sequence, GCS 245 starts at position 99, and GCS 258 starts at position 132. Horizontal arrows indicate the direction and extent of DNA sequence obtained from individual mp18 and pUC19 subclones, deletion derivatives, and internal primer-initiated sequencing, ATG, putative initiator codon; TAG, stop codon of protein-coding region; AATAAC, potential poly(A) addition signal.

or internal primers. The entire nucleotide sequence was con- firmed by sequencing the complementary strand. No differ- ences were found in overlap regions of the nucleotide se- quences derived from these three cDNA clones.

The composite cDNA sequence corresponding to y-glutam- ylcysteine synthetase mRNA is presented in Fig. 6. The sequence contains 2168 nucleotides and has a continuous reading frame of 1911 nucleotides. The mRNA, minimally, has a 5’-nontranslated region of 39 nucleotides and a 3’- nontranslated region of 232 nucleotides. The first ATG is presumed to be the initiator codon because (a) the nucleotide sequence surrounding this codon (. . .GCCATGG.. .) agrees with the consensus sequence for eukaryotic initiation sites described by Kozak (38) and (6) the open reading frame starting with this ATG encodes a protein of 637 residues with a calculated n/i, of 72,614, which agrees with the molecular weight (73,000) estimated by sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (10). The first methionine in the deduced sequence is followed by glycine; this may facilitate removal of the amino-terminal methionine (39) to form the mature enzyme. The hexanucleotide AATAAC located at nucleotides +2098 through 2103 represents a potential signal for polyadenylation (40, 41). The coding sequence ends with a termination codon (TAG) at position +1912, followed by a second termination codon (TGA) at position +1972, a third one (TAG) at position +2017, and a fourth one (TGA) at position +2077. The predicted protein sequence, which con- tains the four independently determined internal peptide sequences (Fig. l), was found to be unique when compared with the protein sequences given in the Protein Identification Resource and GENTRANS data bases.

Codon Usage-Codon usage in the heavy subunit of rat y- glutamylcysteine synthetase mRNA is summarized in Table I. All but one (CGT for arginine) of the possible 61 codons are used. Those that differ from the codon utilization data compiled from other rat protein-coding sequences include phenylalanine (TTT instead of TTC), glycine (GGA but not GGC), and threonine (ACA but not ACC). These unusual codon preferences accounted for part of the incorrectly pre- dicted sequence in probe II.

Properties of Predicted Sequence of y-Glutamylcysteine Syn- thetase-The deduced sequence has 39% nonpolar amino acids, 32% uncharged polar amino acids, and 29% charged amino acids. Hydropathy analysis (Fig. 7) (42) shows a dis- tribution typical of water-soluble proteins, with two short hydrophobic regions (residues 240-300 and 430-460) and

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y-Glutamylcysteine Synthetase Sequence

FIG. 6. cDNA sequence derived from GCS 243, GCS 245, and GCS 258. The sequence is that of the sense strand. The coding sequence of the mRNA begins at nucleotide +l and ends at nucleotide +1911. The deduced amino acid sequence is shown above the cDNA sequence. The residues are numbered above the sequence, and the nucleotides are numbered below. Independently determined internal peptide sequences (Fig. 1) are underlined. Asterisks indicate the stop codon. A potential signal for poly(A) addition is underlined.

three hydrophilic regions (residues 70-130,300-430, and 570- 590).

Tissue nRNA Expression-Tissue-specific mRNA expres- sion was estimated by Northern blot hybridization of rat total RNA and rat kidney poly(A)+ mRNA with a nick-translated probe obtained from the GCS 258 insert. Autoradiographs revealed strong hybridization in the studies on rat kidney. In the other tissues, hybridization bands that were less than 5% of the intensity found in kidney were detected.

DISCUSSION

The deduced amino acid sequence leads to a molecular weight (72,614) which is close to that (73,000) previously estimated for the heavy subunit. The sequence contains all four of the peptide sequences determined by the Edman method (-100 residues). The calculated amino acid composi- tion is in fair agreement with that determined by amino acid analysis (Table II). In some preparations, a minor band (M, -63,000) was found on gel electrophoresis to copurify with the heavy subunit. After storage for several months, the intensity of the M, 63,000 band increased significantly.3 West- ern blot analysis (Figs. 3 and 4) showed that antibody against isolated y-glutamylcysteine synthetase and the affinity-se- lected antibody (against the expressed fusion proteins encoded by isolated cDNA clones) recognized the M, 63,000 band, indicating that this species is antigenically related to the heavy subunit. The findings suggest that the isolated enzyme undergoes degradation to yield a species of somewhat lower molecular weight; the nature of this phenomenon requires further study.

As discussed above, the light subunit does not contribute to catalysis or to the feedback behavior of the enzyme. The possibility that the light subunit might be part of a proenzyme form of the enzyme was considered (ll), but the subsequent findings seem to mitigate against this. The cDNA clones obtained here code for a protein with the predicted properties of the heavy subunit. The cDNA sequence contains stop codons, indicating that the light subunit cDNA sequence is not located at the 3’-end of the mRNA sequence. There is no evidence for such a sequence at the 5’-end, although it cannot be unequivocally excluded. Thus, it seems unlikely that the enzyme subunits are formed by cleavage of a proenzyme encoded by a common mRNA, as has been found, for example, for y-glutamyltranspeptidase (43,44). The immunoscreening did not identify a cDNA clone whose fusion protein could affinity-select antibodies against both heavy and light sub- units in the Western blot, and the oligonucleotide probes derived from the heavy subunit did not recognize a cDNA containing both heavy and light subunit sequences. Recent studies in this laboratory3 have led to preparations that can be completely dissociated into subunits and which therefore do not contain a species similar to that previously postulated (11) to be a possible precursor protein. It has also been found that the amount of light subunit associated with heavy subunit varies with different enzyme preparations. It thus appears that the light subunit is not an integral part of the enzyme; it may be derived from another source and be trapped by disul- fide bond formation on the heavy subunit after synthesis or during isolation. Despite these considerations, the constant finding of the light subunit in preparations of the enzyme is of interest; additional study will be required to determine the origin of this protein.

Northern blot hybridization studies with nick-translated probe prepared from heavy subunit cDNA showed that the mRNA for the enzyme is well expressed in kidney, whereas the mRNA levels in other tissues (brain, heart, liver, lung, and muscle) gave bands that were less than 5% of those found with kidney. The findings are in general agreement with determinations of the activities of y-glutamylcysteine synthe- tase in these tissues; thus, rat liver homogenates exhibit about 4% of the enzymatic activity found in rat kidney homogenates (45).

Previous studies showed that rat kidney y-glutamylcysteine synthetase is inactivated by (a) L-2-amino-4-oxo-5-chloro-

3 C.-S. Huang, unpublished data.

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y-Glutamylcysteine Synthetase Sequence 1591

Rat GCS 264-285

E.a GCS 196-218

Rat GCS 309-421

E.a GCS 281-392

Rat GCS 9-27

E.d GCS 8-26

YLYDQLATICPIvMALSAAS-PF : : : : : ::

YLFGASPAICSSFMGKPTSLPF

REERGLEPLKNNRFKISKSRYDSIOS-YLSKCGEKYNDIDLTID :: : :

KDCKRLQ-INS&IENELtAP~RPKR"TRSGESPSDALLRGG

TEIYEQLLEEGIDHLLAQHVAHLFIRDPLTLFEEKIHLDDANES : : : : : :: : :::

IE-YIEVRSLDINPFSPIGVDEQQVR-FLDLFMVWCALADAPEM

DHFE-NIQSTNWQTMRFKPPPPNSDIG :: : :

SSSELACTRVNWNRVILEGRKPGLTLG

PLSWEETQRHADHVRRHGI ::: : ::

ALAWLEKHPQALKGIQRGL

FIG. 8. Comparison of amino acid sequences of enzymes from rat kidney and E. coli; partial alignment of sequences (see text). The comparison was performed by use of a program (1FASTA) made available by The Rockefeller University Computer Service. GCS, y-glutamylcysteine synthetase.

pentanoate (46) (inactivation is accompanied by stoichiomet- ric binding of the inactivator to the heavy subunit), (b) cystamine (5, 47-49) (inactivation is reversed by treatment with dithiothreitol, indicating that cystamine forms a mixed disulfide between cysteamine and an enzyme thiol), (c) the L- and D-isomers of 3-amino-l-chloro-2-pentanone (49) (inacti- vation by these compounds as well as by cystamine and L-2- amino-4-oxo-5chloropentanoate is prevented by addition of L-glutamate), (d) y-methylene D-glutamate (50) (which forms a covalent linkage with the active-site thiol through a Mi- chael-type addition reaction), and (e) S-sulfocysteine and S- sulfohomocysteine (51) (these compounds bind tightly, but not covalently to the active site). In contrast to the rat kidney enzyme, the E. coli enzyme is not inactivated by compounds listed above (b, d, e) (51, indicating that the bacterial enzyme does not have a thiol at its active center.

It was previously suggested (49) that a thiol group may be involved in the mechanism of the reaction catalyzed by the kidney enzyme. Alternatively, the thiol group in the kidney enzyme may not play a role in catalysis, but may be suffi- ciently close to the glutamate-binding site to react with cer- tain glutamate analogs. It is also possible that a thiol moiety plays a role in the reactions catalyzed by both enzymes and that it is hindered from reaction with glutamate analogs in the active site of the E. coli enzyme (5). The rat kidney enzyme and the E. coli enzyme catalyze the same overall reaction and exhibit similar turnover numbers, specificity, and apparent K, values; both enzymes are feedback-inhibited to the same extent by GSH (5), and both are inhibited by sulfoximine analogs (52-54) of y-glutamyl phosphate. How- ever, despite these close similarities of function, it is possible that the active-site structures of these enzymes are different. Since the amino acid sequences of both enzymes are now known, it is of interest to compare them. Computer-aided comparison revealed an extremely low level of overall similar- ity. However, three regions exhibit some possibly significant similarities (Fig. 8); in these, a total of 39 identical residues was found, 16 of which occur in pairs. Although the level of similarity is higher (about 25%) in these regions than found in the overall comparison (about 8%), one cannot, without further study, draw definite conclusions about the sequence- function relationships of these enzymes. The residues that constitute the active sites are probably located at different regions of the respective sequences. The enzymes may there- fore have evolved independently to achieve functionally sim- ilar active centers.

Acknowledgments-We thank Drs. C. J. Lusty and W. A. Abbott for their help in preliminary experiments. We thank Drs. R. P.

Simondsen, A. P. Seddon, M. E. Anderson, M. Sudol, M. Teintze, and M. V. Chao for very helpful discussions and Dr. D. Wellner for performing part of the peptide sequencing work. We thank Chin- Shiou Huang for providing the antibody used in this work. We are grateful to Drs. A. Sehgal and H. D. Robertson for their expert advice and for constructive criticisms of this manuscript.

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N Yan and A MeisterAmino acid sequence of rat kidney gamma-glutamylcysteine synthetase.

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