October
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Vol. 172, No. 2, 1990
Pages
30, 1990
669-675
GLUTATHIONE S-TRANSFERASE IN YEAST: INDUCTION OF MRNA, CDNA CLONING AND EXPRESSION IN ESCHERICHIA m Hisanori Department
TAMAKI,'Hidehiko
of Food Science
Received
August
27,
KUMAGAI and Tatsurokuro
and Technology,
Kyoto
TOCHIKURA
University,
Kyoto
606,
Japan
1990
SUMMARY: Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast lssatchenkia orientalis approximately 37-fold by cultivation with oA cDNA library complementary to poly (A)+RNA of I. orientalis dinitrobenzene. grown with o-dinitrobenzene was screened by colony hybridization. Twenty positive clones were obtained from 6,000 clones and seven of twenty positive rl,lnrs expressed glutathione S-transferasc activity in --E. coli. One of the ILssing clones harboring plasmid pHT108 had 28 times more glutathione Sferase activity induced by Isopropyl-S-D-thio-galactopyranoside than a (-ain harboring plasmid pUC118. Expressed glutathione S-transferase Y-2 -1rotein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electruphoresis as detected by immunoblot analysis. i 1990 Academic press, rnc.
Glutathione
S-transferase(EC
glutathione
to
xenobiotics
which
conjugation.
a large
many
(1,2). herbicides
Transcriptional were
reported
S-transferase
glutathione
related
reported
mechanism the
dinitrobenzene(DNB) cell
growth
conjugation
have in
by toxic
detoxification of
been of
glutathione in yeast
distributed
isolated
and
glutathione
xenobiotics
about
from
in
to
detail
by drugs
and
The induction the
it
is
and
of
a
important
to
Recently
we
metabolism
DNB, which
was detuxifi~ed
of
existence very
regulation.
orientalis(5). 24 h,
bacteria
characterized
indicates
detoxification
lssatchenkia
glutathione
by
plants(3,4).
Therefore
of
Electrophilic
S-transferase
S-transferase
related
conjugation
detoxified
is widely
system.
of --L I orientalis for and further metabolized.
are
mammals and
glutathione
the
substrates.
and carcinogens
induction
also
catalyzes
electrophilic
S-transferase
isoenzymes
glutathione the
of
mutagens
Glutathione and
study
variety
are
mammals
2.5.1.18)
of
-o-
suppressed
by glutathione
' Fellow of the Japan Society for the Promotion of Science for Japanese Junior Scientists. Abbreviations used: SDS, Sodium dodecyl sulfate; DNB, g-Dinitrobenzene; PVDF, Polyvinylidene difluoride; TBS-T, Tris-buffered saline t Tween; Ig, lmmuno globulin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; lPTG, Isopropyl-fiD-thio-galactoppranoside.
Vol.
172,
No.
2, 1990
Keccntly, have
genetic
resulted
in
many
and
the
BIOCHEMICAL
AND
engineering
approaches
cDNA clones
isolated
been
reported(2).
Expression
also
been
in mammals and plants(6,7,8).
Although
much
transferase
in
concerning glutathione about
is
the
yeast
cells
is
isoenzymes
from
structural
and
One way
to
transferase report orientalis, and its
and
the
there in
of
reported
isolated
achieve
this
subunit
which
isolation expression
tar
made
plants,
in
S-transfcrasos these
have
cDNA in --E. coli
studies
of
been
little
has
subunits
mainly
from
and
glutathione
has
glutathione
S-
information
microorganisms.
glutathione the
and
is
to
distribution
Especially,
yeast,
but
information
S-transferase
of glutathione
characterized
-1. orientatis(l0). functional studies,
induction
sequences
S-tralrsfer;lse
in
lacking.
previously
yeasts(9)
been
S-transferase rote
also
glutathione
S-transferase
and is manufactured
physiological
We have in
abundant
has
and
glutathione
acid
COMMUNICATIONS
glutathionc
for
of glutathione
progress
mammals
amino
RESEARCH
to
coding
being
reported
deduced
BIOPHYSICAL
For
further
a large isolate
expresses
of glutathione
two
coding
quantity
of
quantities for
S-transferase
investigations
S-transferase
of cDNA clones
glutathione
such
enzyme
a cDNA encoding large
S-transferasc
the of
necessary.
glutathione
enzyme.
Y-L mKNA by glutathione
is
as enzyme
Here,
DNB in S-transferasr
yeast
SWC 21 Y-2
in 'II.-. coli MATERIALS
AND METHODS
Materials. Kestriction enzymes, other DNA modifying enzymes and p tasmid Oligo (dT)-cellulose pUC118 were purchased from TAKAKA Shuzo(Kyoto, Japan). kit, was from Collaborative Kes.(U%$). Wheat q m extract, 53 NA synthesis (aP)dC’I’P, (y- -P)A'i‘P and ( S)methionine were blotting detection kit, purchased from Amersham(UK). Eco RI /Not 1 adaptor was from f'harmacia Lysyl endopeptidasc was from Wako pure chemicals(Japan). (Sweden). T'he cultivation condition of 1. orientalis was Yeast and Bacterial culture. plasmid p-118 derivatives described before( Transformants harboring were cultured in LR broth with 50ng/ml ampicillin. Purified glutathione S-transferase Y-Z(lmg) was Protein sequence analysis. reacted with 3.4 pg of lysyl endopeptidase in 4 M urea, 0.01 M tris-HCl(pH Y) at 30 "C for 2 h. The reaction mixture was subsequently elutcd by Hl'LC in a O-60% CH.,CN gradjent containrng equipped with a Cosmosil 5C18-p column 0.1% trifluoroacetic acid and each peptide fragment wa& collected. The amino IJ g samples by a gas phase protein acid sequence was determined in 100 sequencer, Model 47714 (Applied Uiosystems, USA). Poly (A)+KNA was isolated from cells at early log Isolation of poly (A)+RNA. extraction and LiCl precipitation after Zymolyase phase by phenol treatment(l1) and oligo dT-cellulose chromatography. Poly (A)'KNA obtained from I. Translation of mRNA in a cell-free system. orientalis was translated in a wheat germ extract system for 1 h at 25 o C( 12JT extract, 20 1.11 1 M The reaction mixture (145 !~l) contained 75 1-11 wheat germ 13.9 MBq assium acetate, 10~1 1mM amino acids (without methionine), pYf Jncorporation of ( S)methionine (1000mCi/mmol) and 5iig of poly (A)+RNA. (35S)methionine was determined by spotting 3~1 reaction mixture on Whatman 3MM filter papers, which were subsequently soaked in 10 % trichloroacetic acid for After rinsing twice 10 min and boiled in 5 % trichloroaceLic acid for 3 min. the paper were dried and the radioactivity was counted in a liquid in ethanol, scintillation c(Junter , For analysis of total traIlslated protein, 20 u 1 670
Vol.
172,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1:lO diluted reaction mixtures were subjected directly to SDS-poly acrylamide by fluorography of dried gels to gel electrophoresis (SDS-PAGE) followed visualize the total protein. Glutathione S-transferase Y-2 was recovered from the Immunoprecipitation. by immunoprecipitation with anti-glutathione Stranslation mixtures Calbiochem(USA). transferase Y-2 immunoglobulin(lg) G and Pansorbin cells, Translation mixtures were diluted 1:lO with immuno buffer containing 50 IIIM 0.1 % Triton X-100 and 5 mM EDTA, and tris-HCl(pH 7.5), 0.15M NaCl, Pre-immune rabbit IgC(5ul) was added to centrifuged at 10,000 xg for 5 min. the supernatant and incubated for 1 h at room temperature followed by the After addition of 13Onl Pansorbin cells and another 1 h incubation. centrifugation, 5~1 of rabbit anti-glutathione S-transferase Y-2 IgG was added to the supernatant followed by addition of 130~1 Pansorbin cells and incubated as described above. The immunocomplex was pelleted by centrifugation and washed five times with immuno buffer. Antigen was eluted from immunocomplexes After centrifugation, supernatants by boiling in SDS-PAGE sample buffer. Radiolabeled glutathione containing antigen were subjected to SDS-PAGE(13). S-transferase Y-2 was identified by fluorography(l4) using Amplify(Amersham) and quantified by densitometric analysis of th+e fluorogram. Construction of a cDNA library. Poly (A) RNA isolated from I. orientalis cells cultured in the presence of DNB was fractionated on a 5-x % sucrose gradient. Each fraction was used for --in vitro translation of wheat germ extract followed by immunopr-eciprtation, and after SDS-PAGE enriched glutathione S-transferase Y-2 mRNA fractions were identified by fluorography. Complementary DNA (cDNA) to --L 1 orientalis mRNA was synthesized by AMV reverse transcriptase and the second strand DNA was synthesized by DNA polymerase I(15). Both 3'- and 5'- ends of cDNA were blunted by 'T4-DNA polymerase and Eco Rl/Not I Adaptors (Pharmacia) were Ligated to both sites of the doublestranded cDNA. After ligation, double-stranded cDNAs were ligated to the Eco RI site of pUC118 and transformed in E. coli DHSo(16). Enzyme assay for glutathione S-transferase. Glutathione S-transferase activity was assayed spectrophotometrically with 1-chloro-2,4-dinitrobenzene as the substrate according to Habig et a1.(17). One enzyme unit was defined as the amount of enzyme which produced l~mole of S-(2,4-dinitrophenyl) glutathione per min. Immunoblot analysis. or purified yeast glutathione S--E. co11 lysate transferase Y-Z were applied to SDS-PAGE then electroblotted onto polyvinylidene difluoride(PVDF) membranes (18). The PVDF membranes were blocked with 'I'BS-T(tris-buffered saline, pH 7.6, and 0.1 % Tween 20) containing 5 % dried milk, for 1 h at room temperature then incubated with a 1:400 dilution of rabbit antiserum to glutathione S-transferase Y-2(10) for 1 h at room temperature. After washing membranes once more with TBS-'I', they were incubated with a I:500 dilution of anti-rabbit immunoglobulin biotinylated species-specific whole antibody (from donkey) for 20 min at room temperature, washed and incubated with a 1:3000 dilution of streptavidinalkaline phosphatase conjugate and washed again. Glutathione S-transferase Y-2 was detected by adding nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate in diethanolamine-HCl buffer(pH9.5), after which, the membranes were washed with water and air dried.
RESULTS AND DISCUSSION translation --In vitro We reported previously presence
of
this lag glutathione
phase, cells S-transferase
mechanism,
and
200 M DNB,
poly
(A)+RNA
quantitation
that
when
cell-growth
of was
began to increase activity(g). was isolated
glutathione
Issatchenkia
S-transferase
orientalis
suppressed exponentially
for
about with
In order to investigate from I. orientalis cells -+ 671
Y-2
was cultured 24 h and a it-fold the cultured
mRNA. in
the
after higher
induction in
the
Vol.
172,
No.
presence
2, 1990
or
absence
germ celi-free ing
amino
of
fluorography. cultured
with
cultured this
total
as determined
translational
from
poly
band
with
cells
(
weight
Y-2
A-l,B-1).
These
orientalis
was
Electrophiles
IgG,
of
results
as DNB are and
l/3
that
used
to
chemical
were
IMMUNO e*
by
lane
transcription. the
cell
nucleophilic of might
chemical decrease
suppression
glutathione DNB or
S-
Y-2 mRNA in L of
Because
may cause
S-
glutathione
modify
organisms.
whether
in --L I S-transferase the
induction
PPT we*
MW - 94K - 67K -43 K -3OK -- 20.1 K -
A
B A-l
A-2B-1
- 14.4K
B-2
of total and immunoprecipitated acryl+amide gel Translation was (A) RNA fr+om L orientalis. of poly(A) RNA and an equal volume of total carried out with equal amount translation products or immune precipitated products were layered on to 12.5 % Total translation products(20 ~1 of 1:lO dilutron) SDS-poly acrylamide gel. without and with DNB are shown in of poly (A) RNA from I. orientalis grown Lanes>-1 and A-2 represent immunoprecipitated translation lanes A and B. grown without DNB using rabbit products of poly (A)+RNA from 1. orientalis S-transferase Y-2 (lane A-2) TgG. pre-immune (lane A-l) and anti-autathione Lanes B-l and A-2 represent those with DNB using pro-immune (lane B-1) and anti-glutathione S-transferase Y-2 (lane B-2) igC. Fig.
1. Fluorogram
translation
products
of
SDS-poly
using
poly
672
of
products
observed(Fig.1,
level
as to whether modification
TOTAL
of
in mKNA by DNB, translation which
spite
cells(Fig.1,
S-transferase the
chemically of
cells
no information
Y-2 mRNA is resistant
to
acids
normal
products DNB at
normally
approximately
cultured
place
cells
glutathione
incorporated
in
glutathione
groups
of
We have
orientalis.
was
thought
nucleic
of nucleophilic than
normally
by
as one polypeptide
purified
band
by
from In
detected
from
that
induced
those
from
translation
the
label-
followed
(A)+RNA
of
This
IgG
suggest
poly
that
no immunuprecipitable
proteins
modification
B-2). than
strongly
such
to
wheat
immunoprecipitable
incorporation.
DNB were
identical
pre-immune
The
immunoprecipitated with
as the
to SDS-PAGE
l/3
S)methionine
cultured
S)methionine
When
transferase
to less
(
than
the
to immunoprecipitation
IgG. from
less
the
Y-2 ;;bunit(Fig.l,lane more
A-2,).
groups
of
a molecular
37 times lane
by
difference,
(A)+RNA
transferase
B) ;;re
in
S)methionine
subjected
products
COMMUNICATIONS
translated
was subjected were
translation lane
(
Y-2
products
DNB(Fig.1,
cells
mixture
RESEARCH
RNA ;;s
using
S-transferase
translated
The
(A)
system
Each translation
and total
BIOPHYSICAL
poly
synthesizing
anti-glutathione
products
AND
DNB and each
protein
acid.
utilizing
BIOCHEMICAL
Vol.
172,
level
No.
of
BIOCHEMICAL
2, 1990
glutathione
chemical
S-transferase
modification
Partial
BIOPHYSICAL
Y-2
mRNA is
amino acid sequence of glutathione Purified
probes.
endopeptidation
glutathione
and 26 amino kinds
acids of
from
to
glutathione
each
Z),
using
and
2 (17
the
sequences
were
the
level
of
and
Model
1, amino
S-transferase
Y-2
of
48
Y-Z
64
a lysyl and
15
respectively(Fig.
probe
2)
to
amino
acids
were
different
2).
synthesized
acids
5-10
in
6-10 the
in
lysyl
respectively(Fig.
Biosystems).
and
and
sequenced
were
fragment(2),
381A(Applied
mixtures
Y-2 and synthesized
determined,
corresponding
Y-Z(l)
DNA synthesizer were
fragment
probes(probe
glutathione mer)
to
S-transferase
Y-2
N-terminus
S-transferase
endopeptidation
COMMUNICATIONS
superior
S-transferase
glutathione
S-transferase
oligonucleotide
complementary
RESEARCH
of mRNA.
oligonucleotide
Two
AND
Probe
1 (14 mer)
oligonucleotides,
respectively.
CDNAcloning described
and expression in --E. coli.
in
MATERIALS
hybridization
(19)
nucleotide both of
probes.
probes 20
about
positive
Antiserum
to
precipitin
two
Twenty
from
6,000
clones
expressed
glutathione
was
identical
(
clones
was
P)labeled
were
screened. Y-2 to
Cell-free
10
15
10
11
screened that
of
with
these
oligo-
hybridized
extracts
purified
as
by colony with
from
S-transferase
reacted that
constructed
synthesized
obtained
glutathione
was
seven
activity. and
formed
a
glutathione
S-
N-Thr-Phe-Ala-Thr-Val-Tyr-Ile-Lys-Pro-His-Thr-Prcl-Arg-61y-Asp 5’-UAU-AUU-AAA4DJXA-3’ C C G A Probe
1
C A G
3’-ATA-T&A-TTT-WeGT-5’ G G C G T T C 5
(2)
of
S-transferase
that
5 (1)
kinds
positive
clones
line
The :;brary
AND METHODS.
using
library
A cDNA
20
N-Gln-Phe-Gly-Val-Asp-Phe-Thr-His-Tyr-Pro-Asn-Val~lu-Arg-Phe-Thr~ly~lu-Val-Ser~ln-His-Pro-Ile-Ile-Lys 5’4AU-UUU-ACU-CALI-UAU-CC-3’ c c c c
Probe
2
c
3’-CTA-AAA-TGA-GTA-ATAGG-5’ G G G G G E
Fig.
2. N-Terminal amino acid sequence of glutathione S-transferase Y-2 and the lysyl endopeptidation glutathione S-transferase Y-2 fragment, and synthetic oligonucleotides used as probes. N-Terminal amino acid sequences were determined as described in MATERIALS AND METHODS. Oligonucleotide probes were synthesized complementary to all the possible DNA sequences correkponding to amino acids 6-10 in glutathione S-transferase Y-2 (probe 1) and 5-10 in the lysyl endopeptidation glutathione S-transferase Y-2 fragment(probe 2). 673
25
Vol.
172, No. 2, 1990
BIOCHEMICAL
1. Enzyme activity
Table
AND BIOPHYSICAL
of cloned
RESEARCH COMMUNICATIONS
glutathione
Specific
S-transferasc
activity
(mU/mg)
DH5nIpHT108
DHSdpUCl18
78.7 20.1
IPTG+
IPTG-
2.8 2.9
plasmid pHT108 (DH5~/pHT108) --E. coli strain DH5cr harboring and plasmid plJC118 (DH5aIpUC118) were grown in the presence (IPTG+) and absence (IPTG-) of 1mM IPTG for 13 h at 37°C. Glutathione S-transferase activity was assayed with I-chloro2,4-dinitrobenzene as the substrate.
transferase coli
Y-2
strain
in 28
DH5 a containing transferase had
also
3A).
DH5a/pHT108 weight
of
a
run
new band Y-2
examined
Y-Z(Fig.
pUCl18
which
comigrated
of
yeast
with 2).
The
I).
(A)
glutathione
by western purified
hand,
pHT108
glutathione
samples
were
S-transferase
was detected Y-2
and
DH5a/pHT108
yeast
to glutathione protein
S-
blotting,
3-B).
same diluted
other
Plasmid
1).
expressed
IPTG induction('fable
S-transferase
On the
in
2
3
1
2
cl molecular
no band
was detected
was cleaved
by Eco RI and
3 MW -94K -67K -43
K
-3OK
-2O.lK - 14.4K
Fig. 3. Western blot analysis of cell lysate of E. culi strain DB5a and (A) SDS-polyacrylamide gel was purified yeast glutathione S-transferasc Y-2. transferred to a PVDF membrane and detected by immune blot analysis with rabbit antiserum to glutathione S-transferase Y-2 . Lane 1.; Cell lysate of Cell lysate of E. coli plasmid pUC118. Lane 2.; --E. coli DH5a harboring DH5u harboring plasmid pHT108. Lane 3.; Purified yeast glutathsne Stransferase Y-Z. (B) SDS-polyacrylamide gel was transferred to PVDF membrane and detected by coomasie diluted samples of coomasie
brilliant blue
blue staining
674
staining. were used.
Fur
both
with
(6) 1
E. than
purified
the
antiserum
glutathione
/pHTI08)
staining(E'ig.
immunoreactive
2,3).
llB(Fig.?-A,lane
and
clones,
activity
% SDS-PAGE followed
using
23,500(Fig.3-A,lane
DH5ol/pUC
after
blue
positive
(DH5a
(DH5n/pUCllS)
%B,lane
band
the
S-transferase
by Coomassie
by immunoblotting and purified
pHl'108
DHSu/pUC118
subunit(Fig.
A single
One of
glutathione
on 12.5
was detected
test.
plasmid more
DHSa/pHT108,
Y-2 were
S-transferase
in
of
protein
lysates
times
plasmid
lysates
total
immunodiffusion
DH5a containing
approximately Cell
the
immunoblnttlng
l/40
Vol.
172,
No.
analyzed
by
southern
blot
and
about
650
is
suggested
electrophoresis From
this
it
result,
cDNA encoding We are transferase
BIOCHEMICAL
2, 1990
glutathione in
the
process
AND
BIOPHYSICAL
hybridization bp
with
two probes hybridized
cDNA insert that
S-transferase
RESEARCH
plasmid
pH'l'lO8
COMMUNICATIONS
after with contains
agarose gel both probes. full
length
Y-2.
of DNA sequencing
tht
cDNA encoding
glutathione
S-
Y-2.
ACKNOWLEDCMlWTS We thank Dr. R. Sasakj, Dr. M. Yoshikawa and Dr. K. Ikura for protein We also thank sequencing analysis and for synthesizing oligonucleotides. Dr.H. Matsui, Dr. R.B. Wickner, Dr. K. Tanaka and Mr. M. Wakaura for helpful advice about cDNA cloning. We thank Dr. Y. Sasaki, Dr. I(. Hitomi, Mr. Y. Nagano and Mr. N. Sutoh for their helpful advice. 02453129 and 017904j% This work was supported by Research Grants-in-Aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES MANNERVIK,B.(1985) Adv. Enzymol.,57, 357-417. MANNEKVIK,B. and DANIELSON, U. H.(1988) CRC Crit. Rev. Biochem., 23, 283337. Annu. Kev. Biochem. 58, 743-764. 3) PlCKETl', C.B. and LU, A.Y.H.(lY89) HARDING, E.L., DIAZ-COLLIER, J., 4) WIEGAND, R.C., SHAH, D.M., MOZER, T.J., SAUNDERS, C., JAWORSKI, E.G. and 'TlEMEIER, D.C.(1986) Plant Mol. Biol. 7, 235-243. TAMAKI, H., KUMAGAI, H., SHIMADA, Y., KASHIMA, T., OBATA, H., KIM, C.-S., UENO, T. and TOCHIKURA, 'T., Agric. Biol. Chem. Submitted. Biochem. J., 248, 937-941. 61 BOARD, P.G. and PIERCE, K.(1987) Arch. Blochem. Biophys., 7) WANG, K.W., PICKETT, C.B. and LU, A.Y.H.(1989) 269, 5X6-543. WJEGAND, R.C. 8) MOORE, R.E., DAVlES, M.S., O'CONNELL, K.M., HARDING, E.I., and TIEMEIER, D.C.(1986) Nucl. Acids Res., 14, 7227-7235. 9) KUMAGAI, H., TAMAKI, H., KOSHINO, Y., SUZUKI, H. and TOCHIKUKA, T. (1988) Agric. Biol. Chem., 52, 1377-1382. TAMAKJ, H. KLJMAGAI, H. and 'I'OCHIKURA, T.(1989) J. Bacterial., 171, 117310) 1177. C.E. and WARNEK, J.K.(1978) Methods Cell Biol. , 20, 61-81. 11) SRIPATI, 121 MARCU, K. and DUDOCK, B. (lY74) Nucl. Acids Res., 1, 1385-1397. LAEMMLI, U.K. (1970) Nature, 227, 680-685. 13) BONNEK, W.M. and L,ASKEY, R.A. (lY74) Eur. J. Biochem., 46, 83-88. 14) 15) GUBLER, U. and HOFFMAN, B.J. (1983) Gene, 25, 263-269. vol. 1, ~~109-135, IRL Press, Oxford, IJK. 161 HANAHAN, D. (1985) DNA cloning, HABIG, W.H., PABST, M.J. and JAKOBY, W.B.(1974) J. Biol. Chem., 249, 17) 7130-7139. J. Biol. Chem., 262, 10035-10038. 18) MATSUDAIRA, P. (1987) and MANIATIS, T. (1989) Molecular Cloning 19) SAMBROOK, J., FRITSCH, E.F., 2nd. ed. Cold Spring Harbor Lab., Cold Spring Harbor, NY.
1) 2)
675
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BIOCHEMICAL
Vol. 172, No. 2, 1990
Pages
30, 1990
669-675
GLUTATHIONE S-TRANSFERASE IN YEAST: INDUCTION OF MRNA, CDNA CLONING AND EXPRESSION IN ESCHERICHIA m Hisanori Department
TAMAKI,'Hidehiko
of Food Science
Received
August
27,
KUMAGAI and Tatsurokuro
and Technology,
Kyoto
TOCHIKURA
University,
Kyoto
606,
Japan
1990
SUMMARY: Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast lssatchenkia orientalis approximately 37-fold by cultivation with oA cDNA library complementary to poly (A)+RNA of I. orientalis dinitrobenzene. grown with o-dinitrobenzene was screened by colony hybridization. Twenty positive clones were obtained from 6,000 clones and seven of twenty positive rl,lnrs expressed glutathione S-transferasc activity in --E. coli. One of the ILssing clones harboring plasmid pHT108 had 28 times more glutathione Sferase activity induced by Isopropyl-S-D-thio-galactopyranoside than a (-ain harboring plasmid pUC118. Expressed glutathione S-transferase Y-2 -1rotein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electruphoresis as detected by immunoblot analysis. i 1990 Academic press, rnc.
Glutathione
S-transferase(EC
glutathione
to
xenobiotics
which
conjugation.
a large
many
(1,2). herbicides
Transcriptional were
reported
S-transferase
glutathione
related
reported
mechanism the
dinitrobenzene(DNB) cell
growth
conjugation
have in
by toxic
detoxification of
been of
glutathione in yeast
distributed
isolated
and
glutathione
xenobiotics
about
from
in
to
detail
by drugs
and
The induction the
it
is
and
of
a
important
to
Recently
we
metabolism
DNB, which
was detuxifi~ed
of
existence very
regulation.
orientalis(5). 24 h,
bacteria
characterized
indicates
detoxification
lssatchenkia
glutathione
by
plants(3,4).
Therefore
of
Electrophilic
S-transferase
S-transferase
related
conjugation
detoxified
is widely
system.
of --L I orientalis for and further metabolized.
are
mammals and
glutathione
the
substrates.
and carcinogens
induction
also
catalyzes
electrophilic
S-transferase
isoenzymes
glutathione the
of
mutagens
Glutathione and
study
variety
are
mammals
2.5.1.18)
of
-o-
suppressed
by glutathione
' Fellow of the Japan Society for the Promotion of Science for Japanese Junior Scientists. Abbreviations used: SDS, Sodium dodecyl sulfate; DNB, g-Dinitrobenzene; PVDF, Polyvinylidene difluoride; TBS-T, Tris-buffered saline t Tween; Ig, lmmuno globulin; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; lPTG, Isopropyl-fiD-thio-galactoppranoside.
Vol.
172,
No.
2, 1990
Keccntly, have
genetic
resulted
in
many
and
the
BIOCHEMICAL
AND
engineering
approaches
cDNA clones
isolated
been
reported(2).
Expression
also
been
in mammals and plants(6,7,8).
Although
much
transferase
in
concerning glutathione about
is
the
yeast
cells
is
isoenzymes
from
structural
and
One way
to
transferase report orientalis, and its
and
the
there in
of
reported
isolated
achieve
this
subunit
which
isolation expression
tar
made
plants,
in
S-transfcrasos these
have
cDNA in --E. coli
studies
of
been
little
has
subunits
mainly
from
and
glutathione
has
glutathione
S-
information
microorganisms.
glutathione the
and
is
to
distribution
Especially,
yeast,
but
information
S-transferase
of glutathione
characterized
-1. orientatis(l0). functional studies,
induction
sequences
S-tralrsfer;lse
in
lacking.
previously
yeasts(9)
been
S-transferase rote
also
glutathione
S-transferase
and is manufactured
physiological
We have in
abundant
has
and
glutathione
acid
COMMUNICATIONS
glutathionc
for
of glutathione
progress
mammals
amino
RESEARCH
to
coding
being
reported
deduced
BIOPHYSICAL
For
further
a large isolate
expresses
of glutathione
two
coding
quantity
of
quantities for
S-transferase
investigations
S-transferase
of cDNA clones
glutathione
such
enzyme
a cDNA encoding large
S-transferasc
the of
necessary.
glutathione
enzyme.
Y-L mKNA by glutathione
is
as enzyme
Here,
DNB in S-transferasr
yeast
SWC 21 Y-2
in 'II.-. coli MATERIALS
AND METHODS
Materials. Kestriction enzymes, other DNA modifying enzymes and p tasmid Oligo (dT)-cellulose pUC118 were purchased from TAKAKA Shuzo(Kyoto, Japan). kit, was from Collaborative Kes.(U%$). Wheat q m extract, 53 NA synthesis (aP)dC’I’P, (y- -P)A'i‘P and ( S)methionine were blotting detection kit, purchased from Amersham(UK). Eco RI /Not 1 adaptor was from f'harmacia Lysyl endopeptidasc was from Wako pure chemicals(Japan). (Sweden). T'he cultivation condition of 1. orientalis was Yeast and Bacterial culture. plasmid p-118 derivatives described before( Transformants harboring were cultured in LR broth with 50ng/ml ampicillin. Purified glutathione S-transferase Y-Z(lmg) was Protein sequence analysis. reacted with 3.4 pg of lysyl endopeptidase in 4 M urea, 0.01 M tris-HCl(pH Y) at 30 "C for 2 h. The reaction mixture was subsequently elutcd by Hl'LC in a O-60% CH.,CN gradjent containrng equipped with a Cosmosil 5C18-p column 0.1% trifluoroacetic acid and each peptide fragment wa& collected. The amino IJ g samples by a gas phase protein acid sequence was determined in 100 sequencer, Model 47714 (Applied Uiosystems, USA). Poly (A)+KNA was isolated from cells at early log Isolation of poly (A)+RNA. extraction and LiCl precipitation after Zymolyase phase by phenol treatment(l1) and oligo dT-cellulose chromatography. Poly (A)'KNA obtained from I. Translation of mRNA in a cell-free system. orientalis was translated in a wheat germ extract system for 1 h at 25 o C( 12JT extract, 20 1.11 1 M The reaction mixture (145 !~l) contained 75 1-11 wheat germ 13.9 MBq assium acetate, 10~1 1mM amino acids (without methionine), pYf Jncorporation of ( S)methionine (1000mCi/mmol) and 5iig of poly (A)+RNA. (35S)methionine was determined by spotting 3~1 reaction mixture on Whatman 3MM filter papers, which were subsequently soaked in 10 % trichloroacetic acid for After rinsing twice 10 min and boiled in 5 % trichloroaceLic acid for 3 min. the paper were dried and the radioactivity was counted in a liquid in ethanol, scintillation c(Junter , For analysis of total traIlslated protein, 20 u 1 670
Vol.
172,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1:lO diluted reaction mixtures were subjected directly to SDS-poly acrylamide by fluorography of dried gels to gel electrophoresis (SDS-PAGE) followed visualize the total protein. Glutathione S-transferase Y-2 was recovered from the Immunoprecipitation. by immunoprecipitation with anti-glutathione Stranslation mixtures Calbiochem(USA). transferase Y-2 immunoglobulin(lg) G and Pansorbin cells, Translation mixtures were diluted 1:lO with immuno buffer containing 50 IIIM 0.1 % Triton X-100 and 5 mM EDTA, and tris-HCl(pH 7.5), 0.15M NaCl, Pre-immune rabbit IgC(5ul) was added to centrifuged at 10,000 xg for 5 min. the supernatant and incubated for 1 h at room temperature followed by the After addition of 13Onl Pansorbin cells and another 1 h incubation. centrifugation, 5~1 of rabbit anti-glutathione S-transferase Y-2 IgG was added to the supernatant followed by addition of 130~1 Pansorbin cells and incubated as described above. The immunocomplex was pelleted by centrifugation and washed five times with immuno buffer. Antigen was eluted from immunocomplexes After centrifugation, supernatants by boiling in SDS-PAGE sample buffer. Radiolabeled glutathione containing antigen were subjected to SDS-PAGE(13). S-transferase Y-2 was identified by fluorography(l4) using Amplify(Amersham) and quantified by densitometric analysis of th+e fluorogram. Construction of a cDNA library. Poly (A) RNA isolated from I. orientalis cells cultured in the presence of DNB was fractionated on a 5-x % sucrose gradient. Each fraction was used for --in vitro translation of wheat germ extract followed by immunopr-eciprtation, and after SDS-PAGE enriched glutathione S-transferase Y-2 mRNA fractions were identified by fluorography. Complementary DNA (cDNA) to --L 1 orientalis mRNA was synthesized by AMV reverse transcriptase and the second strand DNA was synthesized by DNA polymerase I(15). Both 3'- and 5'- ends of cDNA were blunted by 'T4-DNA polymerase and Eco Rl/Not I Adaptors (Pharmacia) were Ligated to both sites of the doublestranded cDNA. After ligation, double-stranded cDNAs were ligated to the Eco RI site of pUC118 and transformed in E. coli DHSo(16). Enzyme assay for glutathione S-transferase. Glutathione S-transferase activity was assayed spectrophotometrically with 1-chloro-2,4-dinitrobenzene as the substrate according to Habig et a1.(17). One enzyme unit was defined as the amount of enzyme which produced l~mole of S-(2,4-dinitrophenyl) glutathione per min. Immunoblot analysis. or purified yeast glutathione S--E. co11 lysate transferase Y-Z were applied to SDS-PAGE then electroblotted onto polyvinylidene difluoride(PVDF) membranes (18). The PVDF membranes were blocked with 'I'BS-T(tris-buffered saline, pH 7.6, and 0.1 % Tween 20) containing 5 % dried milk, for 1 h at room temperature then incubated with a 1:400 dilution of rabbit antiserum to glutathione S-transferase Y-2(10) for 1 h at room temperature. After washing membranes once more with TBS-'I', they were incubated with a I:500 dilution of anti-rabbit immunoglobulin biotinylated species-specific whole antibody (from donkey) for 20 min at room temperature, washed and incubated with a 1:3000 dilution of streptavidinalkaline phosphatase conjugate and washed again. Glutathione S-transferase Y-2 was detected by adding nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate in diethanolamine-HCl buffer(pH9.5), after which, the membranes were washed with water and air dried.
RESULTS AND DISCUSSION translation --In vitro We reported previously presence
of
this lag glutathione
phase, cells S-transferase
mechanism,
and
200 M DNB,
poly
(A)+RNA
quantitation
that
when
cell-growth
of was
began to increase activity(g). was isolated
glutathione
Issatchenkia
S-transferase
orientalis
suppressed exponentially
for
about with
In order to investigate from I. orientalis cells -+ 671
Y-2
was cultured 24 h and a it-fold the cultured
mRNA. in
the
after higher
induction in
the
Vol.
172,
No.
presence
2, 1990
or
absence
germ celi-free ing
amino
of
fluorography. cultured
with
cultured this
total
as determined
translational
from
poly
band
with
cells
(
weight
Y-2
A-l,B-1).
These
orientalis
was
Electrophiles
IgG,
of
results
as DNB are and
l/3
that
used
to
chemical
were
IMMUNO e*
by
lane
transcription. the
cell
nucleophilic of might
chemical decrease
suppression
glutathione DNB or
S-
Y-2 mRNA in L of
Because
may cause
S-
glutathione
modify
organisms.
whether
in --L I S-transferase the
induction
PPT we*
MW - 94K - 67K -43 K -3OK -- 20.1 K -
A
B A-l
A-2B-1
- 14.4K
B-2
of total and immunoprecipitated acryl+amide gel Translation was (A) RNA fr+om L orientalis. of poly(A) RNA and an equal volume of total carried out with equal amount translation products or immune precipitated products were layered on to 12.5 % Total translation products(20 ~1 of 1:lO dilutron) SDS-poly acrylamide gel. without and with DNB are shown in of poly (A) RNA from I. orientalis grown Lanes>-1 and A-2 represent immunoprecipitated translation lanes A and B. grown without DNB using rabbit products of poly (A)+RNA from 1. orientalis S-transferase Y-2 (lane A-2) TgG. pre-immune (lane A-l) and anti-autathione Lanes B-l and A-2 represent those with DNB using pro-immune (lane B-1) and anti-glutathione S-transferase Y-2 (lane B-2) igC. Fig.
1. Fluorogram
translation
products
of
SDS-poly
using
poly
672
of
products
observed(Fig.1,
level
as to whether modification
TOTAL
of
in mKNA by DNB, translation which
spite
cells(Fig.1,
S-transferase the
chemically of
cells
no information
Y-2 mRNA is resistant
to
acids
normal
products DNB at
normally
approximately
cultured
place
cells
glutathione
incorporated
in
glutathione
groups
of
We have
orientalis.
was
thought
nucleic
of nucleophilic than
normally
by
as one polypeptide
purified
band
by
from In
detected
from
that
induced
those
from
translation
the
label-
followed
(A)+RNA
of
This
IgG
suggest
poly
that
no immunuprecipitable
proteins
modification
B-2). than
strongly
such
to
wheat
immunoprecipitable
incorporation.
DNB were
identical
pre-immune
The
immunoprecipitated with
as the
to SDS-PAGE
l/3
S)methionine
cultured
S)methionine
When
transferase
to less
(
than
the
to immunoprecipitation
IgG. from
less
the
Y-2 ;;bunit(Fig.l,lane more
A-2,).
groups
of
a molecular
37 times lane
by
difference,
(A)+RNA
transferase
B) ;;re
in
S)methionine
subjected
products
COMMUNICATIONS
translated
was subjected were
translation lane
(
Y-2
products
DNB(Fig.1,
cells
mixture
RESEARCH
RNA ;;s
using
S-transferase
translated
The
(A)
system
Each translation
and total
BIOPHYSICAL
poly
synthesizing
anti-glutathione
products
AND
DNB and each
protein
acid.
utilizing
BIOCHEMICAL
Vol.
172,
level
No.
of
BIOCHEMICAL
2, 1990
glutathione
chemical
S-transferase
modification
Partial
BIOPHYSICAL
Y-2
mRNA is
amino acid sequence of glutathione Purified
probes.
endopeptidation
glutathione
and 26 amino kinds
acids of
from
to
glutathione
each
Z),
using
and
2 (17
the
sequences
were
the
level
of
and
Model
1, amino
S-transferase
Y-2
of
48
Y-Z
64
a lysyl and
15
respectively(Fig.
probe
2)
to
amino
acids
were
different
2).
synthesized
acids
5-10
in
6-10 the
in
lysyl
respectively(Fig.
Biosystems).
and
and
sequenced
were
fragment(2),
381A(Applied
mixtures
Y-2 and synthesized
determined,
corresponding
Y-Z(l)
DNA synthesizer were
fragment
probes(probe
glutathione mer)
to
S-transferase
Y-2
N-terminus
S-transferase
endopeptidation
COMMUNICATIONS
superior
S-transferase
glutathione
S-transferase
oligonucleotide
complementary
RESEARCH
of mRNA.
oligonucleotide
Two
AND
Probe
1 (14 mer)
oligonucleotides,
respectively.
CDNAcloning described
and expression in --E. coli.
in
MATERIALS
hybridization
(19)
nucleotide both of
probes.
probes 20
about
positive
Antiserum
to
precipitin
two
Twenty
from
6,000
clones
expressed
glutathione
was
identical
(
clones
was
P)labeled
were
screened. Y-2 to
Cell-free
10
15
10
11
screened that
of
with
these
oligo-
hybridized
extracts
purified
as
by colony with
from
S-transferase
reacted that
constructed
synthesized
obtained
glutathione
was
seven
activity. and
formed
a
glutathione
S-
N-Thr-Phe-Ala-Thr-Val-Tyr-Ile-Lys-Pro-His-Thr-Prcl-Arg-61y-Asp 5’-UAU-AUU-AAA4DJXA-3’ C C G A Probe
1
C A G
3’-ATA-T&A-TTT-WeGT-5’ G G C G T T C 5
(2)
of
S-transferase
that
5 (1)
kinds
positive
clones
line
The :;brary
AND METHODS.
using
library
A cDNA
20
N-Gln-Phe-Gly-Val-Asp-Phe-Thr-His-Tyr-Pro-Asn-Val~lu-Arg-Phe-Thr~ly~lu-Val-Ser~ln-His-Pro-Ile-Ile-Lys 5’4AU-UUU-ACU-CALI-UAU-CC-3’ c c c c
Probe
2
c
3’-CTA-AAA-TGA-GTA-ATAGG-5’ G G G G G E
Fig.
2. N-Terminal amino acid sequence of glutathione S-transferase Y-2 and the lysyl endopeptidation glutathione S-transferase Y-2 fragment, and synthetic oligonucleotides used as probes. N-Terminal amino acid sequences were determined as described in MATERIALS AND METHODS. Oligonucleotide probes were synthesized complementary to all the possible DNA sequences correkponding to amino acids 6-10 in glutathione S-transferase Y-2 (probe 1) and 5-10 in the lysyl endopeptidation glutathione S-transferase Y-2 fragment(probe 2). 673
25
Vol.
172, No. 2, 1990
BIOCHEMICAL
1. Enzyme activity
Table
AND BIOPHYSICAL
of cloned
RESEARCH COMMUNICATIONS
glutathione
Specific
S-transferasc
activity
(mU/mg)
DH5nIpHT108
DHSdpUCl18
78.7 20.1
IPTG+
IPTG-
2.8 2.9
plasmid pHT108 (DH5~/pHT108) --E. coli strain DH5cr harboring and plasmid plJC118 (DH5aIpUC118) were grown in the presence (IPTG+) and absence (IPTG-) of 1mM IPTG for 13 h at 37°C. Glutathione S-transferase activity was assayed with I-chloro2,4-dinitrobenzene as the substrate.
transferase coli
Y-2
strain
in 28
DH5 a containing transferase had
also
3A).
DH5a/pHT108 weight
of
a
run
new band Y-2
examined
Y-Z(Fig.
pUCl18
which
comigrated
of
yeast
with 2).
The
I).
(A)
glutathione
by western purified
hand,
pHT108
glutathione
samples
were
S-transferase
was detected Y-2
and
DH5a/pHT108
yeast
to glutathione protein
S-
blotting,
3-B).
same diluted
other
Plasmid
1).
expressed
IPTG induction('fable
S-transferase
On the
in
2
3
1
2
cl molecular
no band
was detected
was cleaved
by Eco RI and
3 MW -94K -67K -43
K
-3OK
-2O.lK - 14.4K
Fig. 3. Western blot analysis of cell lysate of E. culi strain DB5a and (A) SDS-polyacrylamide gel was purified yeast glutathione S-transferasc Y-2. transferred to a PVDF membrane and detected by immune blot analysis with rabbit antiserum to glutathione S-transferase Y-2 . Lane 1.; Cell lysate of Cell lysate of E. coli plasmid pUC118. Lane 2.; --E. coli DH5a harboring DH5u harboring plasmid pHT108. Lane 3.; Purified yeast glutathsne Stransferase Y-Z. (B) SDS-polyacrylamide gel was transferred to PVDF membrane and detected by coomasie diluted samples of coomasie
brilliant blue
blue staining
674
staining. were used.
Fur
both
with
(6) 1
E. than
purified
the
antiserum
glutathione
/pHTI08)
staining(E'ig.
immunoreactive
2,3).
llB(Fig.?-A,lane
and
clones,
activity
% SDS-PAGE followed
using
23,500(Fig.3-A,lane
DH5ol/pUC
after
blue
positive
(DH5a
(DH5n/pUCllS)
%B,lane
band
the
S-transferase
by Coomassie
by immunoblotting and purified
pHl'108
DHSu/pUC118
subunit(Fig.
A single
One of
glutathione
on 12.5
was detected
test.
plasmid more
DHSa/pHT108,
Y-2 were
S-transferase
in
of
protein
lysates
times
plasmid
lysates
total
immunodiffusion
DH5a containing
approximately Cell
the
immunoblnttlng
l/40
Vol.
172,
No.
analyzed
by
southern
blot
and
about
650
is
suggested
electrophoresis From
this
it
result,
cDNA encoding We are transferase
BIOCHEMICAL
2, 1990
glutathione in
the
process
AND
BIOPHYSICAL
hybridization bp
with
two probes hybridized
cDNA insert that
S-transferase
RESEARCH
plasmid
pH'l'lO8
COMMUNICATIONS
after with contains
agarose gel both probes. full
length
Y-2.
of DNA sequencing
tht
cDNA encoding
glutathione
S-
Y-2.
ACKNOWLEDCMlWTS We thank Dr. R. Sasakj, Dr. M. Yoshikawa and Dr. K. Ikura for protein We also thank sequencing analysis and for synthesizing oligonucleotides. Dr.H. Matsui, Dr. R.B. Wickner, Dr. K. Tanaka and Mr. M. Wakaura for helpful advice about cDNA cloning. We thank Dr. Y. Sasaki, Dr. I(. Hitomi, Mr. Y. Nagano and Mr. N. Sutoh for their helpful advice. 02453129 and 017904j% This work was supported by Research Grants-in-Aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES MANNERVIK,B.(1985) Adv. Enzymol.,57, 357-417. MANNEKVIK,B. and DANIELSON, U. H.(1988) CRC Crit. Rev. Biochem., 23, 283337. Annu. Kev. Biochem. 58, 743-764. 3) PlCKETl', C.B. and LU, A.Y.H.(lY89) HARDING, E.L., DIAZ-COLLIER, J., 4) WIEGAND, R.C., SHAH, D.M., MOZER, T.J., SAUNDERS, C., JAWORSKI, E.G. and 'TlEMEIER, D.C.(1986) Plant Mol. Biol. 7, 235-243. TAMAKI, H., KUMAGAI, H., SHIMADA, Y., KASHIMA, T., OBATA, H., KIM, C.-S., UENO, T. and TOCHIKURA, 'T., Agric. Biol. Chem. Submitted. Biochem. J., 248, 937-941. 61 BOARD, P.G. and PIERCE, K.(1987) Arch. Blochem. Biophys., 7) WANG, K.W., PICKETT, C.B. and LU, A.Y.H.(1989) 269, 5X6-543. WJEGAND, R.C. 8) MOORE, R.E., DAVlES, M.S., O'CONNELL, K.M., HARDING, E.I., and TIEMEIER, D.C.(1986) Nucl. Acids Res., 14, 7227-7235. 9) KUMAGAI, H., TAMAKI, H., KOSHINO, Y., SUZUKI, H. and TOCHIKUKA, T. (1988) Agric. Biol. Chem., 52, 1377-1382. TAMAKJ, H. KLJMAGAI, H. and 'I'OCHIKURA, T.(1989) J. Bacterial., 171, 117310) 1177. C.E. and WARNEK, J.K.(1978) Methods Cell Biol. , 20, 61-81. 11) SRIPATI, 121 MARCU, K. and DUDOCK, B. (lY74) Nucl. Acids Res., 1, 1385-1397. LAEMMLI, U.K. (1970) Nature, 227, 680-685. 13) BONNEK, W.M. and L,ASKEY, R.A. (lY74) Eur. J. Biochem., 46, 83-88. 14) 15) GUBLER, U. and HOFFMAN, B.J. (1983) Gene, 25, 263-269. vol. 1, ~~109-135, IRL Press, Oxford, IJK. 161 HANAHAN, D. (1985) DNA cloning, HABIG, W.H., PABST, M.J. and JAKOBY, W.B.(1974) J. Biol. Chem., 249, 17) 7130-7139. J. Biol. Chem., 262, 10035-10038. 18) MATSUDAIRA, P. (1987) and MANIATIS, T. (1989) Molecular Cloning 19) SAMBROOK, J., FRITSCH, E.F., 2nd. ed. Cold Spring Harbor Lab., Cold Spring Harbor, NY.
1) 2)
675
