Role of cysteine residues in the activity of rat glutathione transferase P (7-7): Elucidation by oligonucleotide site-directed mutagenesis

Vol. 179, No. 2, 1991 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16, 1991

Role

Pages

of

Gysteine

Residues

in

the

Transferase

ELUCIDATION Katsuto

Tamai,*

Hongxie

Satoh,*

* Second

Department

Akira

Shen ,* Yasui,t

t the

Received

August

Tuberculosis

Pharmacology

of

SITE-DIRECTED

Shigeki

Tsuchida,*

Atsushi

Oikawa,t Hirosaki

Hirosaki

036,

Division,

and Cancer,

Rat

Glutathione

(7-7):

of Biochemistry, Medicine,

for

P

BY OLIGONUCLEOTIDE

Kimihiko

Institute

Activity

790-797

Tohoku

MUTAGENESIS' Ichiro

Hatayama,* Sato *,2

and Kiyomi University

School

of

Japan the Research University,

Sendai,

980, Japan

1, 1991

Sumnary: To clarify the role(s) of thiol (sulfhydryl) groups of cysteine (Cys) residues in the activity of the rat glutathione transferase P (7-7) pGP5, containing the entire coding sequence of form (GST-P), a cDNA clone, GST-P (Y. Sugioka et al., (1985) Nucleic Acids Res. 12, 6044-6057) was inserted into the expression vector pKK233-2 and the recombinant GST-P (rGST-P) expressed in E. coli JMl09. All four Cys residues in rGST-P were independently substitu-h alanine (Ala) by site-directed mutagenesis, the resultant mutants as well as the rGST-P being identical to GST-P purified from Liver preneoplastic nodules with regard to molecular weight and immunochemical staining. Since all mutants proved as enzymatically active towards I-chloro-2,4-dinitrobenzene as Liver GST-P, it was indicated that none of the four Cys residues is essential for GST-P activity. However, the mutant with Ala at the 47th position from the N-terminus (Ala47) became resistant to irreversible inactivation by 0.1 mM N-ethylmaleimide (NEM), whereas the other three mutants remained as sensitive as the nonmutant type (rGST-P). Ala47 was also resistant to inactivation by the physiological disulfides, cystamine or cystine, which cause mixed disulfide and/or intraor inter-subunit disulfide bond formation. These results suggest that the 47-Cys residue of GST-P may be located near the glutathione binding site, and modulation of this residue by thiol/disulfide exchange may play an important role in regulation of activity. 0 1991 Academic Press, Inc.

This work was supported in part by Grants-in-Aid for Cancer Research rom the Ministry of Education, Science and Culture of Japan. To whom correspondence should be addressed. Abbreviations used: GST, glutathione transferase; GST-P, rat GST 7-7; recombinant GST-P; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide rGST-P, NEM, N-ethylmaleimide; MCE, 2-mercaptoethanol; CDNB, gel electrophoresis; 1-chloro-2,4-dinitrobenzene; GSH, reduced glutathione; DTT, dithiothreitol; IPTG, isopropyl-H-D-thiogalactopyranoside; EDTA, kbp._ . kilo base pairs: ethylene diaminetetraacetic acid; PMSF, pheny lmethylsulfony 1 fluoride; &, dUTPase; ""g, uracil N-glucosylase; MCS, multi-cloning site. DDO6-291X/91 Copyright All rights

$1.50

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

790

Vol.

179,

No.

We have Class be

Pi;

namely

for

from

the

which

playing

has

to been

In the

other

or

cystine,

the

MATERIALS

roles

Cys

using

AND

(2).

in

residues

the

catalytic role

of

site-directed

the

class

thiol

N-ethyl-

47th

peroxide

position

be

responsible

laboratories

erythrocyte

GSTs

can

hydrogen

may

other

horse to

Pi

as

in

GST-MI1

e.g.

subunits

and

2.5.1.18)

mouse

such at

COMMUNICATIONS

, EC

modifiers;

from

had

GST

(5),

also

both

group-modifiers. have

thiol

groups

reaction.

played

GST-P

with

residue

sensitive the

irreversible

thiol

Cys

(4)

that

the

together

Reports

are

(GSTs

respective

GST

Pi,

study,

respectively

clarified

NEM

RESEARCH

metabolites

the

the

placenta

present three

of of

suggested

functional

with oxygen

alteration

Class

GST-7T

treatment

N-terminus

bovine

BIOPHYSICAL

transferases

human

active

with

belong it

Thus,

or

inactivation that

and

by

that the

AND

glutathione

GST-P

(1,2)

and

(31,

described

was

rat

(NEM)

(47-Cys)

that

inactivated

maleimide

of

reported

markedly

(H202)

BIOCHEMICAL

2, 1991

in and

by

thiol

groups

inactivation

by

reversible

thiol

of NEM

the and

47th

and

cystamine

group-modifiers,

mutagenesis.

METHODS

Materials. Plasmid pKK233-2 was purchased from Amersham. Plasmid pGP5, containing 734 bp of GST-P cDNA ligated to pUC8, was the generous gift of Dr. M. Muramatsu, University of Tokyo. E. coli JM109, CJ236 and MV1190 T4 DNA ligase, T4 kinase, and the Klenow were obtained from Bio-Rad. fragment of DNA polymerase I were from Takara Shuzo Co. (Kyoto). Cystine, cystamine and CDNB were from Wako Pure Chemical Industries (Tokyo). All other chemicals were of analytical grade. GST preparations and assay. Rat GST-P was purified from livers bearing preneoplastic hyperplastic nodules, the antibody being prepared as described previously (6). GST activity was assayed using CDNB as substrate as described by Habig et al. (7). Construction of expression vector of GST-P cDNA. The plasmid pKKS was constructed to contain an additional WI site in the multiple cloning site (MCS) of pKK233-2 as follows. The plasmid pKK233-2 was restricted with EcoRI and Sal1 to remove an internal SphI site located outside of the Y MCS, treated wiTthe Klenow fragment, and ligated with T4 DNA ligase. A 18 bp fragment containing an WI site was isolated from the plasmid pUC18 by digestion with PstI and HindIII, and inserted between PstI and Hind111 7 sites in the MCS of the pKK233-2, and the resultant plasmid was designated Two synthesized oligonucleotides (61 bases and the complepKKS (Fig. 1). mentary 53 bases) coding 20 amino acids from the N-terminus of GST-P were annealed and the double strand fragment was ligated at the NcoI and WI sites with the expression vector pKKS. This construct (pKKS-PI) was digested with HindIII, treated with the Klenow fragment of DNA polymerase I and digested with S+I. The resulting 3.8 kbp fragment was separated by electrophoresis on 0.75% agarose gel and extracted. The plasmid pGP5 containing the full length cDNA of rat GST-P (81, was cleaved at the SalI 7 site, treated with the Klenow fragment, and then cleaved at the -1 site. This 670 bp fragment containing the cDNA encoding amino acids from number 21 to the end of GST-P was inserted into. pKKS-Pl between the *I and Klenow fragment-treated HindIII sites. The resulting expression vector of GST-P was designated pKKGP5 (Fig. 1). E. coli JM109 cells transformed Expression and purification of rGST-P. with the plasmid pKKGP5 were grown overnw be confluent in 10 ml of LB medium supplemented with 50 ug/ml ampicillin at 37°C with vigorous shaking. The medium was mixed with 1 1 of fresh LB medium, and cultured

791

Vol.

BIOCHEMICAL

179, No. 2, 1991

partially

synthesized

GST-P

5’ CATG inserted of pKKS

N and

RESEARCH COMMUNICATIONS

NPSH

cDNA

CATG between

AND BIOPHYSICAL

3

S sites

S

F.&.-L Construction of the expression vector of GST-P cDNA. N, P, S, H, E, HI1 and Sal represent recognition sites of restriction enzymes -1, %I, WI, Hi&III, %I, H&II, and XI, respectively. Ptac, -tat resistance gene. The inserted GST-P cDNA is promoter; Amp , ampicillin shown to consist of two shadowed portions separately constructed.

for about 2 h. At log phase, IPTG was added at a final 1 mM concentration. After an additional 14 h culture, the cells were harvested by centrifugation, resuspended in 10 ml of the lysing buffer (20 mM Tris-HCl, pH 8.0, 1 mM 22 mM NH4C1, 0.1 mM PMSF), and disrupted DTT, 1 mM EDTA, 5% (v/v) glycerol, using a sonicator (Model 200M, Kubota, Tokyo). The cell free extract obtained by centrifugation at 105,OOOxg for 60 min at 0°C was subjected to affinity chromatography as described previously (6). A HincII fragment (Fig. Construction and expression of Ala mutant GST-P. 1) was isolated from the pKKGP5 and inserted into the phage M13mp19 to produce M13mp19-GP5. This derivative was used as a single strand DNA template for mutagenesis of the respective Cys-residues at the 14th, 47th, 1Olst and 169th positions. E. coli CJ236 (dut , 9 ) were grown overnight in 10 ml of YT medium containing3mp19-GP5 phages and 34 pg/ml chloramphenicol at 37°C with vigorous shaking. The medium was centrifuged for 10 min at 2OOOxg, and 1 ml of supernatant containing Ml3mp19-GP5 phages and 1 ml of the E. Coli CJ236 overnight culture were mixed together with 100 ml of YT mediumagain incubated overnight. The culture medium was centrifuged at 10,OOOxg for 10 min, and supernatant was used as the template DNA. One tenth pmol of the template DNA was annealed with 20 pmol of the synthetic oligonucleotides shown in Table 1 in lop1 of annealing buffer (20 mM TrisHCl, pH 7.4, 10 mM MgC12, 50 mM NaCl). After primer extension a?d liga+tion, the DNA was used to transform the competent E. coli MV 1190 (E , 3 >. The transformed cells were plated on 2% agarxning YT medium, and incubated overnight at 37°C. Ten clear plaques were picked up to prepare single stranded and replicative form DNAs of M13mp19-GP5.

792

Vol.

179,

No.

BIOCHEMICAL

2, 1991

Table

1.

Nucleotide

sequences

used

Position

of

AND

for

site

of

BIOPHYSICAL

the

RESEARCH

synthetic

directed

COMMUNICATIONS

oligonucleotides

mutagenesis

Sequence

cysteine

changed

14

TGT -.GCT

5'-CCAGTTCGAGGGCG&TGAGGCCACGCGCATG-3'

47

TGT +GCT

5'-TCGCTCAAGTCCACTgCTGTATGGGCAGCTC-3'

101

TGC + GCC

169

TGC -

5'-GTGGAGGACCTTCGAEAAATATGGTACCCTCA-3' 5'-CTGGCCCCTGCC%CTGGACAACTTCCCCCTG-3' -

*

position

of

Synthetic

GCC

changed

oligonucleotide

sequence

sequence.

To select single stranded M13mp19-GP5 inclyging the desired mutation, dot blot hybridization was carried out with 5'P-labeled synthetic oligonucleotides containing the respective mutations. DNAs that gave positive signals were sequenced to confirm the mutations (9). Replicative forms of the M13mpl9-GP5 were restricted with HincII and ligated at two HincII sites of the expression plasmid pKKS. The plasmid DNAs were used to transform E. coli JMl09, and the mutants of rGST-P (Ala14, Ala47, Ala101 and Ala1691 were expressed and purified using the same method as used for the wild type rGST-P.

RESULTS AND DISCUSSION GST-P

Expression Since

in

rat

GST-P

AGGA sequence initiation

in

for

medium

under free

protein infected (lane the

bands

than

that

sequence rGST-P sequence the

described

which

JM109

(15 units/mg) of the

subunit (8).

1).

first

of

rGST-P

This

from

GST-P acid

liver

with

60% of the

that

of GST-P

rGST-P

N-terminus. 793

ZA),

(lane protein

subunit

(lane

the an extra

2) and rGST-P migrated

to

41,

and all

of

GST-P

(Fig.

2B).

(Table

previously

(6).

the

N-terminus

deduced

from

contained

culture

of E. coli

band

to liver

from

with

500 ml of the

fraction

extra

described

the

pKKGP5 exhibited

the

units/mg

the

transformed

chromatography

rat

residues

in

with in

the antibody

was 11.3

of liver 20 amino

was identical However,

GST-P

from

On SDS-PAGE (Fig.

affinity

with

site,

Shine-Dalgarno

E. coli

protein

obvious

(lane

reacted

as the

transformed

was not

as authentic

activity

upstream

above.

by S-hexylglutathione

similarly

8 bases

of GST-P gene.

pKKS as a control

same position

binding

1 mg of rGST-P

of E. Coli 21,

a ribosomal

GST-P cDNA was used

expression

conditions

(lane

with

The specific

at

the

contain

pKKGP5 located

approximately

3) purified

these

not

plasmid

ATG of the

extract band

coli

cDNA did

efficient

pKKGP5 produced

cell

the

codon

sequence

Escherichia

the

21,

lower

The of the cDNA base

a methionine

Vol.

179, No. 2, 1991

BIOCHEMICAL

Ml

2

AND BIOPHYSICAL

3

1

4

RESEARCH COMMUNICATIONS

2

3

4

Fin. 2. SDS-PAGE and Western blotting of rGST-P expressed in E. coli. ALiquots of the cell free extract of E. coli JM109 transformed~lasmid pKKS or pKKGP5, purified rGST-P and 1-T-P were subjected to SDS-PAGE (A) according to the method of Laemmli (10) and Western blotting (B) as described by Towbin et al. (11). Lanes 1 and 2, extracts (200 pg each) of E. coli containing pKKS and pKKGP5, respectively. Lane 3, rGST-P (1.5 pg) purified from E. coli extract by S-hexylglutathione affinity chromatography; lane 4, purifiedliver GST-P (1.0 pg). Lane M, marker proteins; phosphorylase a (94 kDa), bovine serum albumin (67), ovalbumin (431, carbonic anhydrase (30), soybean trypsin inhibitor (20.1) and(Y-lactoalbumin (14.4). Proteins were stained with Coomassie Brilliant Blue. In Pig. B, immunostaining using anti-GST-P antibody was performed on the proteins separated in A.

Kinetic

Properties As

shown

specific for

either

of

and

mM, of

the

Table

2,

activities

values

0.30

in

of

GSH the

the

than and

Type

Ala

101

Ala14,

CDNB

two

three

Wild

were

and

substrates

at

1 mM.

than

those

type

(Table

2).

For

2.

Ala69

Ala47,

larger

Table

Mutants

Specific

Wild

activity

wild

type

11.3

the

-

0.11

and type

Ala

all

kinetic and

14

forms

higher

type the

GSH,

(0.10 CDNB,

wild

fixing

For

rGST-P

significantly

the

by

activities rGST-P

of had

and

determined

times

wild

Specific

and

(rGST-P).

Km

concentration Km value mM)

of

had

of of

the

Ala47 other

similar

properties

11.0

47

12.3

Ala

of

101

20.6

the

Ala

169

18.5

(units/mg) Km for

GSH (mM)*

0.11

0.10

0.30

0.11

0.11

Km for

CDNB

5.0

5.0

5.0

4.9

5.0

*

calculated

(mM)* from

Lineweaver-Burk

plot.

794

mutants

Km values.

mutants

Ala

was

Vol.

BIOCHEMICAL

179, No. 2, 1991

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Preincubation Fip. 3. Inactivation and cystamine (B). each) were incubated Ala47; n , AlalOl;

Inactivation

of

Ala101

and

shown

These

3A,

NEM

with

a similar

However,

Ala14

and

4A

nonreducing

After

treatment molecular

the

native

GST-P

48,

but be

not

in

produced

CyslOl

this

within

Ala47 in

more

and (lane

association a subunit

of extra

with DTT band

Ala169 3)

of

(lane and

of

a Mr (lane (21.5 5)

as (lane

disulfide

GST-P

the

kDa)

was

well

as 41,

(lane

with

the

in

wild

band

was

bond

cystamine

an

addition it of in

the

Ala14

type

(lane

considered

between formation),

extra to

but

detected

type.

buffer.

of

2),

the

formation

wild

restoration

also

with 3B).

by

in

kDa

inacti-

sample

detected

disulfide

795

than

the

is

inactivated (Fig.

kDa

23.5

the

GST-P

amount

along

5 min, activity.

residue

to

a small

of

for the

inactivated from

Ala14,

47-Cys

30 min,

3)

of

was

liver

removed

bond

(intra-subunit

the

inactivated

was

and

mM NEM 80%

Ala101 and

liver

Cystamine

resistant

while

was

21.5

Ala101

with

more

type

for

(Mr)

also

strongly

MCE

0.1

only

and type

about

that

wild

patterns

band

Because 2)

others,

the

i.e.

subunit addition

lane

the to

NEM

wild

with

report

1 mM cystamine

after

the

retained

was

were

weight

by

of

Ala47 than

SDS-PAGE

Mutants

still

previous

manner

with

with

(Fig.

our

Ala

preincubation

Ala47

conditions;

band

activity.

after

Ala169,

shows

under

disappeared

lost whereas

1 mM cystamine in

and activities

inactivation.

cystamine

Fig.

Type) initial

confirmed

in

vation

the

were

Fig.

results

involved

(Wild

90% of

Ala169 in

30

Time (min.)

of the rGST-P (wild type) and Ala mutants by NEM (A) Purified wild type and Ala mutants of rGST-P (0.1 unit A, Ala14; 0, with 0.1 mM NEM or 1 mM cystamine. A, Ala169; 0, wild type.

rGST-P

than

More

as

20

10

6

1) to

Cys47 causing

and

Vol.

179, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fie. SDS-PAGE of GST-P, the wild type and the Ala mutants under the nonreducing conditions. In Fig. A., 5 pg of protein was used for each lane. Lane 1, nontreated GST-P; lane 2, GST-P treated with 1 mM cystamine for 30 min; lane 3, GST-P reactivated with DTT. In Fig. B, the wild type and the Ala mutants were similarly treated with 1 mM cystamine for 30 min, as in Fig. 3B. 4 pg of protein was used for each lane. Lane 1, the wild lanes 2 to 5, Alal4, Ala47, Ala101 and Ala169, respectively. M, the type; same standard proteins as used in Fig. 2. Molecular weights (kDa) of extra GST-P bands are relative values estimated on the assumption of the native GST-P subunit being 23.5 EDa. Proteins were stained with Coomassie Brilliant Blue.

the

apparent

extra

band

(23.5

kDa),

reduction with

between

disulfide

bond

shown),

that

Ala14

wild

type

the

and and

extra

mutants

band than

but

of

the

reported

complete

GST-P

inactivated

Taken

together,

vicinity

weight

NEM

to

the

a low

explained

(Fig.

GSH,

by

thiol/disulfide

thought

21.5

kDa

the to

by that

the

47-Cys

The

fact than

larger

the

amounts

produced

with

inactivates

DTT

treatment

the

formation of

the

of

these

and

alkylation

an in

796

the

may

element cells

and

by

of

disulfide

addition

(3).

with the

in

the

group

of

the

low

molecular

binding

the

occurrence

(1.2)

which

facilitate

(2).

located

thiol

disturb

processes,

or

and

affinity be

exchange

important the

may of

GST-P

inactivation

column residue

carcinogenic

ratio

the

(results

cystamine

that were

at

range

formation.

mechanism(s)

metabolites

be

cystamine

formation

a basic

(H202)

thiol/disulfide

oxygen In

with

GST-P

S-hexylglutathione

that site,

of

4B).

the

its

binding

site.

of

suggesting

lost

active

evidence

achieved

in

(inter-subunit

by

the

bond

subunits

inactivated by

another subunit

disulfide

disulfide

bond

showed GST-P

with

toward

peroxide

is

NEM

is

disulfide

mixed

4)

native

produced

shift

strongly

modulation

active

with

type

by

GSH or

with pI

hydrogen

(lane the

different

from

that

of

inactivation

weight

involved

or

stress

be

speculate

disulfides

oxidative induce

the

by

the

more

be

we

of

residue

GSH

again

in

mostly

wild

with

may

residues

reactivation

thioltransferase

bond(s)

be

molecular

with

We have -r,

can

with

might

partly were

3B)

a dimer

evident

further

Ala101

that

Thus,

was

mutant

twice

associated

Ala169

(Fig.

The

about

47-Cys

be

as

(3).

kDa,

formation). to

residue,

not

Mr

37 that

the

considered

47-Cys

of

suggesting

formation

was

in

Mr

of of might

formation

Vol.

179,

of

No.

protein

which

are

control of

mixed

disulfides.

increased

in

at the

their

BIOCHEMICAL

2, 1991

roles

AND

The possibility

preneoplastic

thiol/disulfide in carcinogenesis

BIOPHYSICAL

RESEARCH

that

GST forms

and neoplastic

level,

is

and drug

of great

cells, interest

COMMUNICATIONS

in Class

might for

Pi,

be under elucidation

metabolism.

ACKNOWLEDGMENT Tokyo

The authors thank for the generous

Professor Masami Muramatsu gift of plasmid pGP5.

of

the University

of

REFERENCES 1.

2. 3. 4.

Sato, K. (1989) Adv. Cancer Res. 52, 205-255. Tamai, K., Satoh, K., Tsuchida, S., Hatayama, I., Maki, T., K. (1990) Biochem. Biophys. Res. Commun. 167, 331-338. Shen, H., Tamai, K., Satoh, K., Hatayama, I., Tsuchida, S., K. (1991) Arch. Biochem. Biophys. 286, 178-182. Schaffer, J., Gallay, O., and Ladenstein, R. (1988) J. Biol. 263,

5. 6. 7. 8. 9. 10. 11. 12.

and Sato, and Sato, Chem.

17405-17411.

Ricci, G., Del Boccio, G., Pennelli, A., Aceto, A., Whitehead, E. P., and Federici, G. (1989) J. Biol. Chem. 264, 5462-5467. Satoh, K., Kitahara, A., Soma, Y., Inaba, Y., Hatayama, I., and Sato, K. (1985) Proc. Natl. Acad. Sci. USA. 82, 3964-3968. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974) J. Biol. Chem. 249, 7130-7139. Sugioka, Y., Kano, T., Okuda, A., Sakai, M., Kitagawa, T., and Muramatsu, M. (1985) Nucleic Acids Res. 13, 6049-6057. Sanger, F., Nicklen, S., and Coulson, A, R. (1977) Proc. Natl. Acad. Sci. USA. 74, 5463-5467. Laemmli, U. K. (1970) Nature 227, 680-685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354. Cerutti, P. A. (1985) Science 227, 375-381.