Characterization of selenoprotein P as a selenium supply proteinYoshiro Saito* and Kazuhiko Takahashi Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkai
Trang 1Characterization of selenoprotein P as a selenium supply protein
Yoshiro Saito* and Kazuhiko Takahashi
Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
Selenium (Se) is well known to be essential for cell culture
when using a serum-free medium, but not when a medium
containing serum is used This finding suggests that serum
contains some usable form of Se To identify the Se-supplier,
T-lymphoma (Jurkat) cells were cultured for 3 days in the
presence of human serum immunodepleted of Se-containing
serum protein, selenoprotein Por extracellular glutathione
peroxidase The Se-dependent enzyme activities (glutathione
peroxidases and thioredoxin reductase) and Se content
within the cells markedly decreased only when cultured with
selenoprotein P-depleted serum Compared with other Se-containing proteins, the addition of purified selenopro-tein Pto the selenoproselenopro-tein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status) These results suggest that selenoprotein Pfunctions as a Se-supply protein, delivering Se to the cells
Keywords: selenium; selenoprotein; glutathione peroxidase
Se is an essential micronutrient, and is incorporated into
proteins in the form of selenocysteine (Sec) and
selenomethi-onine The term selenoprotein is restricted to Sec-containing
proteins [1], and is to be distinguished from proteins that
nonspecifically incorporate selenomethionine Sec is encoded
by a UGA codon, formerly known only as a stop codon, in
the open reading frame of selenoprotein mRNA that is
accompanied by a Sec insertion sequence element in the
3¢-untranslated region in eukaryotes [2] More than 15
selenoproteins have been found in animals, and some of them
have been shown to exert biological functions [3] Four types
of glutathione peroxidase (GPx) [4–7], three types of thyroid
hormone deiodinase [8–10], three types of thioredoxin
reductase (TR) [11–13], selenophosphate synthetase [14]
and selenoprotein P(SeP) [15] were identified as enzymes
Other selenoproteins with as yet unidentified functions, such
as selenoprotein W [16] and a 15-kDa selenoprotein [17], have
also been reported Recently, selenoprotein W was reported
as a glutathione-dependent antioxidant in vivo [18]
Some pathological conditions of Se deficiency, such as
cancer, coronary heart disease and liver necrosis, are
thought to be due to a decrease in selenoprotein levels [19] It is also well known that Se is essential for cell culture when a serum-free medium is used, but not when a medium containing serum is used [20] This finding suggests that serum contains some usable form of Se Identification of the
Se supplier is critical to understanding the distribution of Se
in the body There are three serum Se-containing proteins that are regarded as candidates for the Se-supply protein: extracellular GPx (eGPx), SeP and albumin eGPx, a tetramer containing one Sec per subunit, reduces both hydrogen peroxide and phospholipid hydroperoxide in the presence of glutathione (GSH) and thioredoxin [5,21–23] SePis a Se-rich extracellular glycoprotein [24–26] The sequence of the cDNA predicts that human SePcontains 10 selenocysteines encoded by UGA stop codons in the open reading frame of its mRNA [27] Several lines of evidence
in vivosuggest that SePis a free radical scavenger [28,29] Recently, we demonstrated that SePreduces phospholipid hydroperoxide in the presence of GSH [15] Albumin may contain Se in the form of selenomethionine, and does not contain the element in stoichiometric amounts [30]
In the present study, we describe the first identification of SePas a major source of Se for the cells cultured in human serum We also demonstrate that SePis more effective as a
Se supplier than are any other Se-containing proteins and compounds so far tested We propose that SePfunctions not only as an antioxidative enzyme but also as a Se supplier
E X P E R I M E N T A L P R O C E D U R E S
Chemicals Diisopropyl fluorophosphate was obtained from Kishida Chemical Co., Osaka, Japan; tertiary butyl hydroperoxide and hydrogen peroxide from Nacalai, Kyoto, Japan; GSH, GSH reductase, RPMI-1640 medium, selenocystine, sele-nomethionine, DMEM and Hepes from Sigma-Aldrich Co., St Louis, MO, USA; recombinant human insulin and human transferrin from Wako, Osaka, Japan; and nickel-nitrilotriacetic acid agarose from Qiagen Inc., Chatsworth,
CA, USA Recombinant human thioredoxin was kindly
Correspondence to K Takahashi, Department of Hygienic Chemistry,
Graduate School of Pharmaceutical Sciences, Hokkaido University,
Kita 12 Nishi 6, Kita-ku, Sapporo, 060-0812, Japan.
Fax: + 81 11 706 4990, Tel.: + 81 11 706 3244,
E-mail: kazu@pharm.hokudai.ac.jp
Abbreviations: GPx, glutathione peroxidase; cGPx, cellular
glutathi-one peroxidase; eGPx, extracellular glutathiglutathi-one peroxidase; GSH,
glutathione; PH-GPx, phospholipid hydroperoxide glutathione
peroxidase; Sec, selenocysteine; SeP, selenoprotein P;
TR, thioredoxin reductase.
Enzymes: glutathione peroxidase (EC 1.11.1.9); phospholipid
hydro-peroxide glutathione peroxidase (EC 1.11.1.12); glutathione reductase
(EC 1.8.1.7); thioredoxin reductase (EC 1.8.1.9).
*Present address: Human Stress Signal Research Center,
National Institute of Advanced Industrial Science and Technology,
1-8-31 Midorigaoka, Ikeda, Osaka 563–8577, Japan.
(Received 10 June 2002, revised 5 September 2002,
accepted 8 October 2002)
Trang 2provided by Ajinomoto, Co Inc., Kawasaki, Japan.
Human serum albumin and human outdated frozen plasma
was kindly donated by the Hokkaido Red Cross Blood
Center Ebselen was kindly provided by Daiichi
Pharma-ceutical Co., Ltd, Tokyo, Japan Other chemicals were of
the highest quality commercially available
Preparation of SeP- and eGPx-depleted human serum
Six monoclonal antibodies (BD1, BD3, BF2, AE2, AH5
and AA3) against human SePwere immobilized as
described previously [31] After the incubation of
anti-human SePmAbs-Sepharose 4B with anti-human serum for 1 h
at 4C, the obtained supernatant was used as a
SeP-depleted human serum The depletion of SePwas confirmed
by a sandwich enzyme-linked immunosorbent assay for
human SeP, as described previously [31] SeP was
undetect-able (< 2%) after this treatment eGPx-depleted human
serum was prepared using anti-human eGPx polyclonal Ig–
conjugated Sepharose 4B The depletion of eGPx was
confirmed by coupled enzymatic assay with GSH reductase,
as described previously [5] The enzyme activity of eGPx was
undetectable (< 2%) after this treatment Anti-human TR
mAb (KB12)–conjugated Sepharose 4B was used for the
control experiment [32]
Se assay
Levels of Se in each serum and in the cell were determined
according to the fluorometric method of Bayfield and
Romalis [33]
Cell culture and cytosol preparation
Jurkat E6-1 cells, human T-leukemia (American Tissue
Type Collection, Rockville, MD, USA), were maintained in
RPMI-1640 medium containing 10% (v/v) fetal bovine
serum at 37C under an atmosphere of 95% (v/v) air and
5% (v/v) CO2 For studies on the effects of
selenoprotein-depletion from human serum, the cells were cultured with
RPMI-1640 medium containing 5% (v/v) of each human
serum Serum-free medium (RPMI-1640 containing
5 lgÆmL)1 human insulin, 5 lgÆmL)1 human transferrin,
5 mgÆmL)1human serum albumin and 2 lMa-tocopherol)
was also used After culturing for the specified periods, the
cells were collected by centrifugation and resuspended in an
appropriate volume of 50 mMTris/HCl, pH 7.4, containing
0.25Msucrose, 0.1 mMEDTA, 0.7 mM2-mercaptoethanol
and 2 mMdiisopropyl fluorophosphate The cell suspension
was sonicated with an Ultra Sonic homogenizer VP-5s
(Taitec, Tokyo, Japan), and centrifuged at 105 000 g for 1 h
at 4C to obtain a cytosolic fraction
GPx enzyme assay
GPx activities were examined by following the oxidation of
NADPH in the presence of GSH reductase, which catalyzes
the reduction of the oxidized GSH formed by GPx [5] To
measure cellular GPx (cGPx) activities, both the sample
and reference cuvettes contained 0.1M Tris/HCl, pH 8.0,
0.2 mMNADPH, 0.5 mMEDTA, 2 mMGSH and 1 U of
GSH reductase in a total volume of 1 mL An aliquot of
the cytosolic fraction was added to the sample cuvette only
The reaction mixture was preincubated at 37C for 2 min, after which the reaction was started by the addition of
70 nmol tertiary-butyl hydroperoxide to both cuvettes To measure phospholipid hydroperoxide GPx (PH-GPx) activity, the reaction mixture contained 0.1M Tris/HCl,
pH 8.0, 0.2 mM NADPH, 0.5 mM EDTA, 1 mM NaN3,
5 mM GSH and 1 U of GSH reductase and 60 nmol 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-3-PtdCho hydroperoxide was added [15] The oxidation of NADPH was followed at 340 nm at 37C and activity was expressed as micromoles of NADPH oxidized per minute
TR enzyme assay
TR activity was examined by spectrophotometric insulin reduction assay, as described previously with a slight modification [34] Both sample and reference cuvettes contained 50 mM phophate buffer, pH 7.0, 1 mMEDTA, 0.2 mMNADPH, and 0.8 lMhuman recombinant thiore-doxin in a total volume of 1 mL An aliquot of the cytosolic fraction was added to the sample cuvette only The reaction mixture was preincubated at 37C for 2 min, after which the reaction was started by the addition of 80 nmol insulin
to both cuvettes The oxidation of NADPH was followed at
340 nm at 37C and activity was expressed as micromoles
of NADPH oxidized per minute
GSH reductase enzyme assay GSH reductase activities were examined by following the NADPH oxidation in the presence of oxidized glutathione [35] The assay mixture contained 50 mMphosphate buffer,
pH 7.6, 1 mM EDTA, 0.1 mM NADPH, and 1 mM oxidized glutathione Activity was calculated as micromoles
of NADPH oxidized per minute
Purification of SeP and eGPx SePand eGPx were purified from human plasma using conventional chromatographic methods as described previ-ously [5,15]
R E S U L T S
Immunodepletion of SeP and eGPx from human serum
To identify a Se supply protein for the cells cultured in human serum, we first prepared SeP- and eGPx-depleted human serum with immobilized antibodies The addition of immobilized anti-human SePand anti-eGPx Igs reduced the
Se content to 47 and 81%, respectively (Fig 1) This suggests that 53 and 19% of serum Se content is derived from SePand eGPx, respectively No decrease was observed after treatment with the immobilized control antibody The residual 28% of Se may be derived mainly from albumin in the form of selenomethionine [30]
Se deficiency in cells cultured with SeP-depleted human serum
We then investigated the effect of selenoprotein depletion from serum on the Se-dependent enzymatic activities and Se
Trang 3content of cultured Jurkat cells (Fig 2) When cultured
solely in the presence of SeP-depleted serum and not
eGPx-depleted or the control serum, the activity of cellular GPx
(cGPx), a major Se-dependent enzyme, decreased to 17% that of the control (Fig 2A) The activity of two other Se-dependent enzymes, phospholipid hydroperoxide GPx (P H-GP x) and TR, also decreased to 16 and 38%, respectively However, almost no change in the activities
of other antioxidant enzymes, such as superoxide dismutase and glutathione reductase, was observed (data not shown) When cultured with SeP-depleted serum, the Se content of the whole cell and the cytosol also decreased to 19 and 35% that of control cells, respectively (Fig 2B)
Next, we studied the time course of the cGP x activity (as
an indication of Se status) of cells cultured with SeP-depleted serum (Fig 3) cGPx activity was almost unde-tectable after 4 days The addition of 270 ngÆmL)1purified SeP, which corresponded to the SeP concentration of 5% serum, resulted in the complete recovery of cGPx activity within 2 days
Effect of addition of SeP on the cGPx activity
of Jurkat cells
To compare SePwith other Se-containing proteins or compounds as a Se supplier, we studied the effect of seven reagents on the cGPx activity of the cells (Fig 4A) SeP was the most effective with a 50% effective dose (ED50) of 5 nM (Se equivalent), followed by eGPx, sodium selenite, seleno-cystine, selenomethionine and albumin The ED50 of the former three reagents was 25 nM, and that of the latter two were 300 and 500 nM, respectively Ebselen had no effect up
to 500 nM To eliminate the effect of serum proteins on the activity of SePas a Se supplier, a similar experiment was conducted using a serum-free medium Essentially identical results were obtained using the serum-free medium (Fig 4B), and SePwas again the most effective
D I S C U S S I O N
SePis an extracellular protein that has been postulated to have an oxidant defense function [28,29] We recently reported that SePreduces phospholipid hydroperoxide in the presence of GSH [15] SePis also reported to have
Fig 2 Effect of selenoprotein depletion on the selenoenzyme activities
and Se content in Jurkat cells The cells were cultured for 3 days in
RPMI-1640 medium containing 5% (v/v) of each human serum.
cGPx, PH-GPx, TR activity and Se content were determined, as
des-cribed in Experimental procedures.
Fig 3 Effect of SePon the cGPx activity of Jurkat cells The cells were cultured in RPMI-1640 medium containing 5% (v/v) of SeP-depleted human serum, and cGPx activity was measured After 6 days, purified SeP, corresponding to the SeP concentration of 5% serum, was added.
Fig 1 Se concentration in selenoprotein-depleted human serum and the
estimation of Se components in human serum Preparation of each
human serum and measurement of Se concentration were conducted as
described in Experimental procedures (A) Se concentrations in
selenoprotein-depleted human serum and in the control are shown.
These values are the means of six experiments with the error bar
in-dicating SD (B) Se concentrations and percentages in each component
were calculated from the data shown in (A).
Trang 4survival-promoting properties for cultured neurons [36].
Because of its high Se content and extracellular localization,
SePhas also been suggested to be a Se supply protein
It is known that serum-free culture media for immune cells
and neurons contain insulin (as a growth factor), transferrin
(as an iron source) and sodium selenite (as a Se source)
Without these compounds, the cells can neither survive nor
proliferate This suggests that some usable form of Se in
serum can provide Se to the cells instead of sodium selenite
Using an in vitro cell culture system, we investigated which
Se-containing proteins or compounds supplies Se to the cells
Several methods have been employed to determine the
proportion of total Se in human serum (or plasma)
accounted for by SeP Using heparin–agarose, SeP
accoun-ted for 40% of the Se applied to the column [37] The SePin
the plasma of Chinese men of varying Se status accounted
for 50–60% of the total Se in their plasma [38] In another
approach based on immunoassay, 40–44% of the total Se
was reported to be in the form of SePin healthy US subjects
[39] In our study, we first prepared SeP- and eGPx-depleted
human serum Se analysis shows that SePand eGPx
contains 53% and 19% of the total serum Se content of
healthy Japanese human subjects, respectively (Fig 1B) The proportion of total Se in human serum accounted for
by SePvaried between regions and can be expected to reflect differences in Se intake Thus, our results essentially confirmed the previous findings described above
Jurkat cells that had been cultured in the presence of SeP-depleted serum became Se-deficient in a time-dependent manner Under these culture conditions, the cells duplicate
at 18 h intervals We speculate that the divided cells contain one half of the Se content, and that the cells became Se-deficient 4 days later When human peripheral lympho-cytes were incubated with SeP-depleted serum, the cells did not become Se-deficient (Y Saito and K Takahashi, unpublished observation) As peripheral lymphocytes do not proliferate, perhaps only proliferating cells (such as hemopoietic cells, spermatocytes and neurons) become Se-deficient when cultured with SeP-depleted serum The fact that new neurons are continually added to the neocortex of adult macaque monkeys has profound implications for the understanding of the cellular mechanisms of higher cogni-tive functions [40] However, the addition of purified SePto Se-deficient cells resulted in the recovery of cellular GPx activity within 2 or 3 days
Next, we compared the Se-supply activity of SePwith two other Se-containing serum proteins, eGPx and albumin The 50% effective dose (ED50) of SeP, eGPx and albumin was 5, 25 and 500 nM(Se equivalent), respectively The Se concentration of SeP, eGPx and albumin in 5% human serum was 42, 15 and 22 nM(Se equivalent), respectively This suggests that SePmainly supplies Se to cells under physiological conditions
Essentially identical results were obtained in a serum-free medium in the presence of 2 lMa–tocopherol In a serum-free medium without a-tocopherol, the cells could not survive in a Se-deficient state This suggests that a–toco-pherol protects Se-deficient cells from cell death by oxidative stress Actually 5% serum contains 0.9 lM a-tocopherol Thus, the cells could survive even in a Se-deficient state in the presence of SeP-depleted serum This speculation is supported by reports that SePis a suvival-promoting factor for cultured central neurons [36] In the study in which SeP was identified as a survival-promoting factor, the culture medium contained transferrin, insulin, albumin but not sodium selenite
Our observations indicate that SePfunctions as a Se supplier for the proliferating cells Little is known about the function of a group of serum proteins concerned in the supply of substances of small molecular weight The most studied of these is transferrin, which is concerned in the distribution of iron from the intestinal absorption sites to the various tissues requiring iron [41] Albumin is believed to supply many low-molecular-mass substances such as metals, amino acids and fatty acids [42] Transferrin and albumin noncovalently bind iron and many low-molecular-mass substances, respectively As Se is covalently bound in SeP, the supply mechanism is presumed to require digestion of SePby proteases and peptidases, and the breakdown of selenocysteine for release of its Se, and must be proved in the future
A serum-free medium containing Se (sodium selenite) was used to culture a variety of cells, neurons and hemopoietic (especially immune) cells Without Se, the cells could neither survive nor proliferate SePis taken up in greater amounts
Fig 4 Effect of the addition of Se-containing compounds on the cGPx
activity of Jurkat cells in serum or serum-free medium In the presence of
variable amounts of Se-containing compounds, the cells were cultured
with SeP-depleted serum (A) or serum-free medium (B), as described in
Experimental procedures After 3 days, the cells were collected and
cGPx activity was measured Open circles, SeP (185 nmol SeÆmg)1of
protein); closed circles, eGPx; (53 nmol SeÆmg)1of protein); open
squares, sodium selenite; closed squares, selenocystine; open triangles,
selenomethionine; closed triangles, human serum albumin (8.3 pmol
SeÆmg)1of protein); open circles with broken line, ebselen.
Trang 5by the brain but not by other organs in Se-deficient animals
[43], suggesting a critical function of this selenoprotein in
this organ It has survival-promoting properties for cultured
neurons [36] and its mRNA is present in the brain [44]
Furthermore, it is reported that astrocytes and cerebellar
granule cells secrete SeP[45] A recent finding that new
neurons are continually added to the neocortex suggests
that SePsecreted from astrocytes and cerebellar granule
cells may supply Se to proliferating neurons Numerous
studies suggest that a deficiency of Se is accompanied by a
loss of immunocompetence, probably not unconnected with
the fact that Se is normally found in significant amounts in
immune tissues such as the spleen, and lymph nodes Both
cell-mediated immunity and B-cell function can be impaired
[46] Supplementation with Se has marked
immunostimu-lant effects, including an enhancement of proliferation of
activated T cells [47]
In the present study, we identify SePas a Se supply
protein to proliferating T lymphoma cells The essential
roles of Se in brain and immune functions described above
and our in vitro studies strongly suggest that SePsupplies Se
to the proliferating brain and hemopoietic cells Thus, we
propose that SePfunctions not only as an antioxidative
enzyme but also functions as a Se supplier
A C K N O W L E D G E M E N T S
This work was supported in a party by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology of Japan We thank Hokkaido Red Cross Blood Center for
providing human plasma Y Saito is the recipient of a research
fellowship from the Japan Society for the Promotion of Science for
Young Scientists.
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