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Expression of Plasma Glutathione Peroxidase in Human Liver in Addition to
Kidney, Heart, Lung, and Breast in Humans and Rodents
By Fong-Fong Chu, Robert Steven Esworthy, James H. Doroshow, Khiem Doan, and Xian-Fang Liu
We analyzed the expression of plasma glutathione peroxidase (GSHPx-P) messenger RNA (mRNA) in mouse, rat, and
human tissues, using a human GSHPx-P cDNA clone as the
probe. Unlike the classical cellular glutathione peroxidase
(GSHPx-l), GSHPx-P expression appears t o be tissuespecific. In the mouse and rat, kidney expresses an mRNA at
a high level detected with the human probe. A signal is also
detected in mRNA isolated from mouse and rat heart, rat
cardiac myocytes, mouse lung, epididymis, and the mammary gland of midpregnant mice. No signal is detected in
mRNA isolated from mouse and rat liver, mouse brain,
uterus, and testis. In human tissues, an mRNA hybridizing t o
GSHPx-P cDNA is present in liver, as well as kidney, heart,
lung, breast, and placenta. We have shown that human
kidney expresses a GSHPx-P mRNA, and not a GSHPx-P-like
message, by isolating a cDNA clone from a human kidney
library in Agtll. From the 412-nucleotide partial sequence of
the kidney cDNA, which codes for the 40-170 amino acids of
GSHPx-P including the TGA codon for selenocysteine, we
found complete sequence identity of the kidney cDNA with
GSHPx-P isolated from placenta. The expression of GSHPx-P
mRNA in cell lines was also studied. There is some correlation of the expression of GSHPx-P in these cell lines with that
in normal tissues. Cell lines that expressed GSHPx-P mRNA
or protein included the human hepatocarcinoma HepG2,
Hep3B cells, human kidney carcinoma A498 cells, and the
human breast cancer SK-BR-3, T47D. MDA-MB-231, and
Adr‘MCF-7 cells. Cell lines that did not express GSHPx-P
included human choriocarcinoma BeWo cells, human breast
cancer MCF-7, ZR-75-1, and Hs578T cells, and mouse hepatoma Hepa-1 cells.
o 7992 by The American Society of Hematology.
M
We and others have previously reported that the human
hepatoma cell lines HepG2 and Hep3B secrete GSHPxP.4,10However, Takahashi et all1 failed to detect GSHPx-P
mRNA in human liver. Yoshimura et a1 studied GSHPx-P
protein expression by Western blotting in 13 rat tissues and
found signals in kidney and lung, but not in liver, heart,
spleen, thymus, bone marrow, erythrocytes, brain, pituitary
and adrenal glands, jejunum, duodenum, or skeletal muscle. These results raised the question of whether there is
any correlation between the expression of GSHPx-P in
normal tissues and that in cell lines derived from these
tissues. Therefore, we have analyzed several normal tissues
and cell lines of human, mouse, and rat origin for GSHPx-P
expression. We found that human liver, and not mouse or
rat liver, expresses GSHPx-P mRNA. Our results are
presented in this report.
ANY FORMS OF ACTIVE oxygen such as hydrogen peroxide, lipid hydroperoxides, superoxide, hydroperoxy and hydroxyl radicals, and singlet oxygen are
implicated in human disease. Evidence exists to support a
role for oxidant damage in the pathogenesis of rheumatoid
arthritis, reperfusion injury, cardiovascular disease, immune injury, and cancer.1,2Glutathione peroxidase (GSHPx,
glutathi0ne:hydrogen-peroxideoxidoreductase, EC 1.11.1.9)
has a selenocysteine residue at its active site that catalyzes
the reduction of hydrogen peroxide and organic hydroperoxides by oxidizing GSH. Four members of the GSHPx
family have been identified in human cells: (1) the classical
cellular enzyme, GSHPx-1; (2) the plasma GSHPx,
GSHPx-P; (3) the phospholipid hydroperoxide enzyme,
PHGPX; and (4) a GSHPx-1-like enzyme, GSHPx-2, expressed in human liver and hepatoma cell^.^-^ Additionally,
a mouse 24-Kd epididymal secretory protein regulated by
androgen has sequence homology with GSHPx, although it
is devoid of ~elenocysteine.~
Based on its epididymisspecific expression, it appears to be distinct from the other
members of GSHPx. Thus, it may be the fifth member in the
GSHPx family.*
Yoshimura et a19 detected a messenger RNA (mRNA)
that hybridized to a rat GSHPx-P cDNA, and a polypeptide
that reacts with a chicken antirat-GSHPx-P antisera on a
Western blot isolated from rat kidney. These results strongly
suggested that the kidney is the source of the plasma
GSHPx activity. However, there are multiple forms of
GSHPx that share significant sequence homology across
species: the nucleic acid sequence of GSHPx-P has 57%
homology with that of cellular glutathione peroxidase
(GSHPx-l), and has 67% homology with that of a 24-Kd
mouse epididymal secretory protein. It is possible that the
signal detected in various tissues is caused by crosshybridization with yet undetermined members of the GSHPx
family. To prove that kidney expresses GSHPx-P we used
direct sequencing of a GSHPx-P cDNA isolated from a
kidney library. In this study, we showed the sequence
identity of the cDNA isolated from a human kidney library
to be a GSHPx-P.
Blood, Vol79, No 12 (June 15). 1992:pp 3233-3238
MATERIALS AND METHODS
Materials. Human cDNA libraries in hgtll from placenta and
kidney were purchased from Clontech Lab (San Diego, CA);
GSHPx-P-specific oligonucleotides were synthesized by the Core
DNA Synthesis Lab of City of Hope Cancer Research Center
(Duarte, CA). Phage hgtll forward and reverse primers were
purchased from New England Biolabs, Inc (Beverly, MA).
Isolation of GSHfi-P cDNA clones. Human stretched placenta
and kidney cDNA libraries had 2.3 x 1Olo and 5.0 x 1O’O pfu/mL,
From the Department of Medical Oncology and Therapeutics
Research, City of Hope National Medical Center, Duarte, CA.
Submitted August 28,1991; accepted F e b u l y 12, 1992.
Supported by Grant-in-aid No. 901 GI-2 of American Heart
Association Greater Los Angeles Affiliate, Inc, and US Public Health
Services Grants No. CA31788 and CA33572.
Address reprint requests to Fong-Fong Chu, PhD, Dept. of Medical
Oncology, City of Hope National Medical Center, 1500 E Duarte Rd,
Duarte, CA 91010-0269.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-497119217912-0003$3.00/0
3233
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CHU ET AL
3234
respectively. Degenerate oligonucleotides were made according to
our previously described GSHPx-P peptide fragments." They were
used as probes to isolate GSHPx-P cDNA clone from a placenta
library. Specific oligonucleotides were made after we sequenced
the placenta GSHPx-P clone. These oligonucleotides were used to
screen for GSHPx-P clones in a human kidney library. Screening,
phage plating, and transfer to nitrocellulose filters (Schleicher &
Schuell Inc, Keene, NH) were performed in duplicate as described.I2 Secondary and tertiary screening were performed after
isolating the positive plaques to ensure that single clones were
obtained in each isolate.
DNA sequencing. We completely sequenced our placenta
GSHPx-P cDNA clone bidirectionally by subcloning the insert of
the Agtll clone into pBluescript (pBS; Stratagene, San Diego,
CA). For sequencing reactions, either Sequenase (US Biochemical, Cleveland, OH) or linear polymerase chain reaction (PCR)
sequencing13was used. Ig Suite from the Core DNA facility of the
Cancer Center, City of Hope (IntelliGenetics, Inc, Mountain View,
CA) was used to analyze and manage the sequencing data.
The kidney GSHPx-P cDNA clone was sequenced in A g t l l
directly. The insert was amplified with Agtll forward and reverse
primers by PCR. The amplified fragment was isolated from the
unincorporated primers by precipitation with 20% polyethylene
glycol in 2 N NaC1.14 It was then sequenced directly by the linear
PCR sequencing method.13
Northern analysis. Total RNAs were isolated from human and
animal tissues or cell lines. Tissue sources and numbers of
successful isolation are summarized as follows.
(1) Mouse tissues: liver: 1X from a 0 BaZb\c, 1X from each of a
6 and a 0 Swiss Webster, and 1X from a 0 BM-1 strains; kidney:
3X from 0 Balb\c, 1X from each of a 6 and a P Swiss Webster,
and 1X from a 6 Swiss CD-1 strains; heart: 1X from each of a 6
and a 0 Swiss Webster, and 4X from 6 Swiss CD-1 strains; lung:
1X from a 6 Swiss CD-1 strain; mammary gland: 2X from 14 day
pregnant P Swiss Webster strain; epididymis: 1X from a 6 BM-1,
and 1X from a 6 Swiss Webster strain; uterus: 1X from a 0 BaZb\c
strain; brain and testis: 1X from a 6 Swiss Webster strain.
(2) Rat tissues were all isolated from 6 Sprague-Dawley rats
(Charles River Lab, Wilmington, MA), these including: liver: 2 X
kidney cortex and medulla: 2X each; heart: lX, cardiac myocytes:
2x.
(3) Human tissues: liver: 3X normal tissue; kidney: 3X normal
tissue; heart: 1X normal tissue; lung: 3X normal and 1X tumor
tissue; breast: 4X normal tissue.
Isolation of cardiac myocytes from 6 Sprague-Dawley rats was
performed following a previously described procedure with minor
modification^.'^ After the heart was perfused on a LangendorB
apparatus with Ca*+-free Tyrode's solution, myocytes were obtained by perfusing the heart with 50 mL of recirculating Tyrode's
solution containing 50 mg of collagenase B (Boehringer-Mannheim
Biochemicals, Indianapolis, IN), 5 mg protease (type XIV, Sigma)
for 45 minutes at 37°C.
Total RNA was isolated using the acid guanidinium thiocyanatephenol-chloroform extraction method.I6 Ten micrograms of RNA
was resolved by formaldehyde-containing gels. Random primed
human 32P-GSHPx-P and chicken 32P-p-actin cDNA were used as
probes. The hybridizing and washing conditions were described
previously."
CeZZ lines and '5Se-ZabeZing. Human cell lines used included
hepatoma HepG2 and Hep3B cells, kidney carcinoma A498 cells,
breast carcinoma MCF-7, SK-BR-3, T47D, MDA-MB-231, ZR75-1, Hs578T cells, and choriocarcinoma BeWo cells (American
Type Culture Collection, Rockville, MD). An adriamycin-resistant
variant of MCF-7 cells, AdrIMCF-7, was kindly provided by
Kenneth Cowan (National Cancer Institute, Bethesda, MD).ls
Mouse hepatoma Hepa-1 cells were kindly provided by Oliver
Hankinson (University of California, Los Angeles, CA). A GSHPx-1
transfectant of MCF-7 cells, MCF-7H6, was established as described.17 At least two independent isolations of mRNA were
performed on each of these cell lines.
BeWo cells were cultured in Kaighn's nutrient mixture F-12
(Irvine Scientific, Santa Ana, CA) supplemented with 15% fetal
bovine serum. The other cells were grown in either Dulbecco's
modified Eagle's medium (DMEM)\F12 or Richter's improved
MEM zinc option medium (GIBCO Laboratories, Grand Island,
NY)supplemented with 10% fetal bovine serum. Selenoproteins
were labeled by incubating cells in growth media supplemented
with 100 nmol/L 75Se-selenious acid for 4 to 6 days before
harvesting.
Protein analysis and enzyme assays. Immunoprecipitation of
75Se-GSHPx-1 and 75Se-GSHPx-P was performed with rabbit
antihuman-RBC-GSHPx-1 antisera and antihuman-GSHPx-P antisera, as previously described." The immunoprecipitates were
resolved by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE).
RESULTS
We screened 60,000 plaques and isolated eight GSHPx-P
clones from a human placenta cDNA library. We found a
cDNA clone that includes the full coding sequence, and
extends to the polyadenylation tail. The coding sequence
that we have obtained is identical to the sequence published
by Takahashi et al," although the 3' noncoding sequence
contains a few discrepancies (EMBL accession number:
X58295). We have aligned the deduced amino acid sequences of five glutathione peroxidase-like cDNA clones as
shown in Fig 1. PxP is human GSHPx-P; Px4 is the porcine
monomeric phospholipid hydroperoxide glutathione peroxidase (PHGPX)I9; Px2 is a tetrameric and catalytically
active GSHPx expressed only in human liver and hepatoma
cells6 (manuscript in preparation); Pxl is the classical
human cellular GSHPx-1; and Epi is a 24-Kd mouse
epididymal secretory protein regulated by t e s t o ~ t e r o n e . ~ , ~
We have recently determined the nucleotide sequence of
315 bp of the coding region of a human PHGPX cDNA.
The deduced amino acid sequence of this portion has 95%
homology with porcine PHGPX (unpublished data).
First, we studied the expression of GSHPx-P mRNA in
mouse tissues and mouse hepatoma Hepa-1 cells using a
human GSHPx-P cDNA as the probe. As shown in lanes 1
to 8 of Fig 2A, and lane 9 of Fig 2C, kidney expresses a
hybridizing mRNA at a high level, and it is detected in
lesser abundance in epididymis, heart, and mammary gland
from midpregnant mice. Other mouse tissues, including
liver, testis, uterus, and brain, do not have a detectable
signal. Similar to mouse liver, Hepa-1, a mouse hepatoma
cell line, does not express any mRNA that hybridized with
the human GSHPx-P probe. Figure 2B shows the mRNA
signals hybridized with the chicken @-actinprobe that are
used as a control for the quality and quantity of the mRNA
preparation. The weak signal at higher molecular weight is
at the same location as the abundant 28s rRNA that often
hybridizes with the probes. The 18s rRNA has a molecular
weight similar to p-actin. Because actins are composed of a
number of isotypes highly conserved in evolution, the
p-actin probe appears to hybridize with a-skeletal and
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PLASMA GLUTATHIONE PEROXIDASE (GSHPX-P)
PXP
3235
1
&ascllslllaafvsasr&qeksk
Px4
Fig 1. Alignment of amino
acid sequences from potential
members of the glutathione peroxidase family. PxP is human
plasma glutathione peroxidase;
Pxl is the classic human erythrocyte glutathione peroxidaseza;
PxZ is expressed in human liver
or hepatoma cellso; Px4 is porcine phospholipid hydroperoxide glutathioneperoxidase'o; and
Epi is a 24-Kd, mouse epididymal
secretary protein regulated by
androgen.' Because the published Px4 and Epi sequences are
not full-length, we could not assign numbers for the amino acid
positions. The double underlined
PxP sequence contains a stretch
of hydrophobic amino acids representing the signal sequence for
membrane insertion. The boldface letters are the conserved
sequences in all five members
from three species.
SmhEFSAkdID GhmVnLdkYRGyVcivtNVASqCSeGkTevnYTQLvdLhaRyAecG
I
II
I l l
II
I
I
I l l
I1
I l l
II
I I I I I I
I
Ill
I
Px2
1
mAfIAkSfYdlSAisLD GEkVdFntfRGraVLIENVISLCSeGTTtRDfTQLNELQcRf PRr
Pxl
1 mcaarlaAaaAGSvYaFSArpLaGGEpVsLgs1RGKV1LIENVASLCSeGTTvRDYTQmNEIQrRLgPRG
PxP
30
I
I
I
I I I I I I l l I l l II II I I I I I
II
I I
I1111II1
I
II
I l l
I l l
mdchggIsGtiYEygAltIDGeEyIPFKQYaGKyVLFVNVASYCSBCLTg QYLELNALQEeLAPFG
I I IIIIII
I IIIIIII I
II IIIIIII I Ill
Ill
sLnGkEhIPFKQYRpKhVLFVNVAtYc GLTi QYpELNAIQEdLkPFG
Ep i
LrILaFPCNQFGrQEPGsdaEIkef
Px4
I
I IIIIIII I I
II
aaGYnvkFdmFsKicVNGddAHPLwkwmK
II
I
I
II
Ill
I
Px2
61 LWLGFPCNQFGHQENcqNEEILNS~~RPGGGYqPtFtLvqKCEVNGqNEHPvFAy~dkLPyPyD
Pxl
69 LWLGFPCNQFGHQENa~EEIqNSLK~RPGGGFePNFmLFEKCEVNGagAHPLFAFLreaLPaPSD
PxP
94 LVILGFPCNQFGKQEPGeNsEILPtLKWRPGGGhrPNFQLFEKGDVNGEkEQKf TFLKnSCPptSE
IIIIIIIIIIIIIIII IIII IIIIIlllIII I I I IIIIII
Epi
I I IIIIIIIII I I
I II
I I II I
I I I I I I I I I I I l l IIII I l l
I1
I I I I I I I I I I I I I I I I I I IIII I I I I I I I I I I I I I I I I I I I I I I I l l
II I I
I
IIII Ill
I
II
LVILGFPCNQFGKQEPG~lEILPgLKYVRPGkGFlPNFQLFaKGDVNGENEQKiFTF~rSCPhPSE
Px4
VqPKgrgml
gNaIkWNFtKFLIdknGcvV kRYgpmeepqVIEkDlpcYL.
II
I l l iiii
I
I
II
II I
I
IIIII
I1
Px2 129 DufsLMTDPKLIiWSPVrRsDVAWNFEKFLIGPeGePf RRYSRTFuTmnIEPDIkrLLkvai.
I L I I I I I I I I IIII I IIIIIIIIII II I I
IIIII 1 - 1
Pxl 137 DataLMTDPKLItWSPVcRNDVAWNFEKFLVGPDGVPl RRYSRrFqTidIEPDIeALLSqRpsca.
I
I I
I
IIIIIIIIIIIII I
II
I
PxP 165
LlgtSdrlfWEPmKVHDIRWNFEKFLVGPDGiPiMRWhHRTtVSnVKm DIlsYmrrqaalgvkrk.
Ep i
tvVmSKhtsWEPiKVHDIRWNFEKFLVGPDGVPVMRWfHqapVStVKs DImAYLShfkyi.
I
I l l IIII111111IIIIIIII I Ill I
a-cardiac actin mRNAs that are coexpressed at high levels
in striated muscles, in addition to two a-smooth muscle
actin mRNAs present in sarcomeric muscles.20-22This
results in double bands in some of the lanes in the p-actin
hybridized blots. To confirm the results obtained with
rodent tissues, we also analyzed several human tissues for
GSHPx-P expression. Multiple human samples were shown
in lanes 5 through 8 of Fig 2C and Fig 3A. With the
exception of one human heart mRNA preparation, which
was run twice in lane 5 of Fig 2C and lane 5 of Fig 3A, and
the normal and tumor lung mRNA in lanes 7 and 8 of Fig
3A, which were isolated from the same individual, all
human RNAs of different tissues are isolated from different
individuals. These data show that human liver, as well as
kidney, heart, lung, and breast, express GSHPx-P mRNA.
Because Yoshimura et a19 did not detect any signal from
rat heart by Western analysis, we wanted to know if the
signal that we detected in mouse heart was specific to mice.
As shown in lanes 1 through 4 of Fig 2C, we have detected
an mRNA isolated from rat heart and cardiac myocytes, in
addition to rat kidney cortex and medulla, that hybridizes
with the human GSHPx-P probe.
The kidney expresses a message that hybridizes strongly
with the human GSHPx-P probe; however, so does epididymis, which expresses a GSHPx-P-like mRNA. To establish
that the kidney mRNA codes for GSHPx-P, it was necessary
to sequence a kidney cDNA clone that hybridized to
GSHPx-P. Thus, we screened 35,000 plaques of a human
kidney cDNA library, and found two clones that hybridized
to a GSHPx-P-specific oligonucleotide. Despite the fact
I1 II
II
I
that both clones are truncated at their 5' ends, we obtained
a 412-nucleotide sequence coding for the 40-176 amino
acids, including the TGA codon for selenocysteine from
one of the kidney clones. It is identical to the GSHPx-P
sequence.
We have analyzed human cell lines to study the correlation of GSHPx-P expression in vitro versus in vivo. As
shown in Fig 2C, lanes 10 through 14, and lanes 3 and 5 of
Fig 2E, four of seven human breast cell lines express an
mRNA hybridized to GSHPx-P cDNA. SK-BR-3, T47D,
MDA-MB-231, and Adr'MCF-7 have a detectable message,
whereas ZR-75-1, Hs578T, and MCF-7 do not. Other
human cell lines analyzed for GSHPx-P mRNA expression
are shown in Fig 2E. These include hepatoma HepG2 (lane
l), kidney carcinoma, A498 (lane 2), and choriocarcinoma,
BeWo (lane 5), cell lines. HepG2 and A498 cells express
GSHPx-P mRNA, whereas BeWo cells do not have a
detectable mRNA.
We have examined the protein secreted by several human
cell lines to determine whether the mRNA expressed by
these cells, which hybridized with the GSHPx-P probe,
codes for GSHPx-P. As shown in Fig 4, A498 (lanes 1
through 3), Adr'MCF-7 (lanes 4 through 6), MCF-7H6
(lanes 7 through 9 and 13 through 15; a GSHPx-1 transfectant of MCF-7 cells), and HepG2 cells (lanes 10 through 12)
were labeled metabolically in media containing 100 nmol/L
75Se-selenious acid for 4 to 6 days and the 75Se-labeled
proteins were analyzed by SDS-PAGE. Figure 4 shows that
A498, Adr'MCF-7, and HepG2, but not MCF-7H6, secrete
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CHU ET AL
3236
A 1 2 3 4 5 6 7 8
‘1
2 3 4 5 6 7 8
w--
- 28s rRNA
- B-Actin
- a - Actin
--m=
GSHPX-P -
1‘
2 3 4 5 6 7 8 9 1011 121314
Dl2 3 4 5 6 7 8 9 1011 121314
-28s rRNA
-&Actin
a-Actin
GSHPX-P-
E l2 3 4 5
5 , 3 4 5
- 2 8 s rRNA
,a -@Actin
GSHPX-P -
Fig 2. Northern analysis of GSHh-P mRNA in human and animal tissues, and human cell lines. (A, C and E) show a 1.6-kb mRNA hybridized t o
a human GSHPx-P cDNA probe; and (B, D, and F) show a 1.9-kb mRNA hybridized t o an pactin probe. (A and B) show the same set of mouse RNAs
isolated from Hepa-1 (lane 1). kidney (lane 2). liver (lane 3). testis (lane 4). epididymis (lane 5). uterus (lane 6). heart (lane 7). and brain (lane 8). (C
and D) are the same set of RNAs isolated from rat kidney cortex (lane 1).rat kidney medulla (lane 2). rat heart (lane 3). rat cardiac myocytes (lane
4). human heart (lane 5). human lung (lane 6), human breast (lanes 7 and 8). the mammary glands of a pregnant mouse (lane 9). and five human
breast cancer lines (lanes 10 through 14). The breast cell lines are SK-BR-3 (lane 10). T47D (lane 11). MDA-MB-231(lane 12). ZR-75-1 (lane 13). and
Hs578T (lane 14). (E and F) are the same set of RNAs isolated from other human cell lines, including HepG2 (lane 1).A498 (lane 2), AdrMCF-7 (lane
3). BeWo (lane 4). and MCF-7 (lane 5).
2 3 4 5 6 7 8
‘1
2 3 4 5 6 7 0
4-28s rRNA
GSHPx-P-
+@-Actin
Fig 3. Northern analysis of
mRNA isolated from human tissue. (A) mRNA hybridized t o
GSHPx-P cDNA. (B) mRNA hybridized t o pactin. Duplicate
samples were from two individuals. Lanes 1 and 2 are from normal kidney, lanes 3 and 4 are
from normal liver, lane 5 is from
normal heart, lanes 6 and 7 are
from normal lung, and lane 8 is
from tumor lung of the same
individual as lane 7.
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3237
PLASMA GLUTATHIONE PEROXIDASE (GSHPX-P)
Media
1 Cytosol
HepG2
MCF-7H6
1011 12 13 14 15
Fig 4. 75Se-labeled proteins obtained from four
human cell lines resolved by SDS-PAGE. Lanes 1
through 9,11, and 12 are selenoprotein immunoprecipitatesrecovered in the conditioned media, whereas
lanes 13 through 15 are cytosolic selenoproteins that
are controls to show the specificity of the antisera.
Lanes 1, 4, 7, and 13 are immunoprecipitateswith
preimmune sera; lanes 2,5,8.11, and 14 are immunoprecipitateswith anti-RBC-GSHPx-1 antisera; lanes
3, 6, 9, 12, and 15 are immunoprecipitates with
anti-GSHPx-P antisera; and lane 10 is the pattern of
total cytosolic selenoproteins in HepG2 cells.
22.5 kDa b
a 22.5-Kd selenoprotein that is immunoprecipitated with
antihuman GSHPx-P antisera.
DISCUSSION
Yoshimura et a1,9 using immunoblotting and Northern
hybridization techniques, identified that rat kidney and
lung, but not rat liver or heart, expressed GSHPx-P in 13
tissues analyzed. These data suggest that kidney is the
source of plasma glutathione peroxidase. However, as
shown in Fig 1, there are apparently five isozymes in the
glutathione peroxidase family. To prove that the signal
detected in kidney by GSHPx-P probes is that of GSHPx-P
per se, a nucleic acid or an amino acid sequence analysis of
a kidney GSHPx-P cDNA or protein is necessary. Our
partial sequence of a cDNA clone isolated from a human
kidney cDNA library has shown its identity.
When the deduced amino acid sequence of five potential
isozymes in the glutathione peroxidase family are compared, there is higher homology between human GSHPx-P
and mouse epididymal 24-Kd protein than between human
GSHPx-P and human cellular GSHPx-1. When the nucleic
acid sequences at the coding region are compared, human
GSHPx-P has 67% homology with mouse epididymal 24-Kd
protein and 57% homology with human GSHPx-1. However, the 24-Kd epididymal secretory protein is not likely to
be the mouse GSHPx-P, based on the facts that (1) the
cDNA of the 24-Kd epididymal protein did not hybridize
with any mRNA isolated from kidney and other nonepididymal tissue: (2) the amino acid homology of human and rat
GSHPx-P is 90%, whereas that of human GSHPx-P and
mouse 24-Kd epididymal protein is 71%,7a9 and (3) the
cDNA coding for the 24-Kd protein does not appear to
contain a TGA codon.'
In addition to the mouse kidney, positive signals were
also detected in mRNA isolated from mouse epididymis,
heart, lung, and mammary gland. It is likely that the signal
detected in epididymis is caused by cross-hybridization of
the mRNA of the 24-Kd epididymal secretory protein with
a GSHPx-P cDNA probe. However, based on the specificity
of the probe, which was shown in the isolation of the human
kidney GSHPx-P cDNA, it is more likely that these other
tissues are expressing GSHPx-P mRNA. Additionally, the
HepG2, A498, and Adr'MCF-7 cell lines that expressed an
mRNA that hybridized to GSHPx-P cDNA also secrete a
selenoprotein that can be immunoprecipitated by antihuman GSHPx-P antisera. This evidence supports the conclusion that these tissues express a GSHPx-P mRNA.
We have detected GSHPx-P mRNA in human liver,
kidney, heart, lung, and breast. We are confident in these
results because, with the exception of human heart, multiple samples were analyzed. Takahashi et all1 have reported
that human liver does not express GSHPx-P mRNA, but no
data was shown, and they isolated GSHPx-P clones from a
human fetal liver cDNA library.ll However, because
GSHPx-P is not expressed in mouse and rat liver, there is a
definite species difference in its expression in liver. Additionally, unlike rodent kidncy, human kidney does not have
overwhelming levels of GSHPx-P mRNA. Considering the
mass of liver, the total GSHPx-P expressed in human liver
may be equivalent to that expressed in kidney.
We have also detected a signal in mRNA isolated from
rat heart, rat cardiac myocytes, mouse heart, and human
heart; mouse lung and human lung; mouse breast and
human breast. Our result indicates that there may be no
species difference in GSHPx-P expression in heart, lung,
and breast. However, GSHPx-P is expressed at a very low
level, if any, in one human tumorous lung sample. More
samples need to be analyzed to determine whether there is
a general pattern of reduced GSHPx-P mRNA level in
tumorous human tissues.
We have also studied the expression of GSHPx-P mRNA
or protein in 12 cell lines, and found a fair correlation with
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3238
CHU ET AL
the tissues from which they were derived. HepG2 and
Hep3B, human hepatoma cells, are similar to human liver
and express GSHPx-P; Hepa-1, a mouse hepatoma cell line
similar to mouse liver, does not. A498, a human kidney
carcinoma cell line, expresses a GSHPx-P mRNA and
protein as does human kidney; human breast cancer cells
SK-BR-3, T47D, MDA-MD-231, and Adr'MCF-7 are similar to human breast, and express GSHPx-P. However, other
breast cancer cells, ZR-75-1, Hs578T, and MCF-7, and
BeWo, a human choriocarcinoma cell line, are unlike their
tissues of origin, and do not express GSHPx-P.
Although the physiologic role of GSHPx-P has not been
established, its expression in heart, lung, and breast tissues,
in addition to kidney and human liver, suggest that the
antioxidant activity may be important in providing local
extracellular protection, in addition to providing a potential
oxidant scavenger role in the plasma. This local protection
maybe particularly important in humans, because human
plasma has several-fold lower levels of GSHPx activity
compared with rodent plasma (unpublished data). The
differential expression of GSHPx-P in breast cancer cell
lines is interesting. Because the normal mouse breast
tissues were obtained from pregnant females, and the
normal human breast were most likely obtained from
postpregnant females, its expression in this tissue may be
developmentally regulated.
ACKNOWLEDGMENT
We thank J.N.A. van Balgooy for his preparations of rat cardiac
myocytes.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1992 79: 3233-3238
Expression of plasma glutathione peroxidase in human liver in
addition to kidney, heart, lung, and breast in humans and rodents
FF Chu, RS Esworthy, JH Doroshow, K Doan and XF Liu
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