Sep 25, 2005 - molecular mass of 23 kDa and several faster moving minor bands (Fig. ..... Hyslop, P. A., Hinshaw, B. D., Halsey, W. A., Jr., Schraufstatter,.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264, No.27,Issue of September 25, pp. 15850-158$5,1989 Printed in U.S.A.
Plasma Selenium-dependent Glutathione Peroxidase CELL OF ORIGIN AND SECRETION* (Received for publication, August 10, 1988)
Nelly AvissarS,John C. Whitin, Peter Z. Allen, Denisa D. Wagner, Patricia Liegey, and Harvey J. Cohen From the Department of Pediatrics and Cancer Center and the Department of Microbwiogy, Immunology, and Medicine, University of Rochester School of Medicine and DentistQ, Rochester, New York 14642
Human plasma glutathione peroxidase (GSHPx) has cause tissue damage by oxidizing DNA, proteins, and lipids a glycosylated selenoprotein distinct (1-3). Intracellular defense mechanisms to detoxify these been shown to be enzymatically, structurally,and antigenically from reactive oxygen species include the enzymatic systems superknown cellular glutathione peroxidases. The extracel- oxide dismutase, catalase, glutathione-S-transferase, and lular location of the enzyme and the fact that it is GSHPx‘ andthe lipid-free radical scavengers vitamin C, glycosylated suggested that it is a secreted protein. vitamin E, and 8-carotene (1-3).However, 0; produced Utilizing mutually non-cross-reactive antibodies to hu- mainly by phagocytic cells and endothelial cells is also seman cellular and plasma GSHPx,we conducted a creted into the extracellular fluids and might be a source of search to determine the tissue of origin for plasma either 0;derived free radicals or HzOz (4-7). GSHPx. The cells screened were endothelial cells beLittle is known about the oxidant detoxification enzymes cause they are the main source of extracellular super- of plasma and the mechanism of detoxification in the extraoxide dismutase, HL-60 cells (myeloid cell line) be- cellular milieu. Recently, an extracellular superoxide dismucause they are the main source of extracellular HzOZ, and Hep 6 2 cells (hepatic cell line) because they are tase distinct from the cellular form was characterized. The the source of many plasma proteins. Human umbilical enzyme is glycosylated and is secreted mainly from endothevein endothelial cells were metabolically labeled with lial cells (8-10). All the peroxidase activity in human plasma either [36S]methionineor [‘‘Se]selenious acid, and HL- can be attributed to GSHPx (11). Because of the small 60 cells and Hep G2 cells were metabolically labeled amounts of the enzyme in plasma compared with its quantity with [“Se]selenious acid. Proteins wereimmunopuri- in cells, it was thought tobe a contaminant present inplasma fied from the labeled cells and theirmedia with either due to cell breakage or leakage. Indications that the plasma GSHPx might be distinct from cellular GSHPx came from anti-red blood cell (RBC) GSHPx IgG or with antiplasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, studies of kinetics of enzyme appearance foIIowing seIenium only the cellular form of the enzyme was precipitated repletion in selenium deficient humans and animals (12), and from all thecells tested but not from their media. When from immunological studies showing that antibodies against anti-plasma GSHPx IgG was applied to the cells and purified RBC GSHPx precipitated other cellular forms of only from GSHPxbut do not react with plasma GSHPx (13). The their media, a selenoprotein was precipitated the media of Hep 6 2 cells. When Hep G2 cells were enzyme has been purified and characterized and was found to incubated in the presence of the carboxylic ionophore be structurally, enzymatically, and antigenically different monensin, an intracellular selenoprotein could be de- from RBC GSHPx (14-16). Furthermore, plasma GSHPx was tected using anti-plasma GSHPx IgG. The precipita- found to bind concanavalin A, and thisbinding capacity was tion of the cellular form from all three cell types was removed by treating the enzyme with glycopeptidase F indipartially inhibited by preincubation of the anti-RBC cating that theprotein is glycosylated (14, 17,181.Antibodies GSHPx IgGwith purified RBC GSHPx while the pre- raised in rabbits against human plasma GSHPx were found cipitation of the selenoprotein from the medium of Hep to be specific, precipitating, and non-cross-reacting with RBC G2 cells by anti-plasma GSHPx IgGwas prevented by preincubation of the antibody with purified plasma GSHPx (17,IS). These findings indicate that plasma GSHPx GSHPx. W e suggest that plasma GSHPx is synthesized is a unique selenoenzyme. Whether the differences between by and secreted from hepatic cells. This is, to the best the two enzymes are a result of them being products of two of our knowledge, the only known selenoprotein with different genes or whether they are due to post-translational since human a defined function that has been shown to be synthe- modification is not as yetknown.However, cellular GSHPx does not have a N-glycosylation site (Asp-Xsized for secretion by mammalian cells. Ser/Thr) (19), while the human plasma GSHPx is probably N-glycosylated (14, 18), the first hypothesis appears to be more likely. Although other possibilities exist, the most rational explanation for the presence of the enzyme in the HzOz,O;, and other free radicals are toxic to cells and can plasma is that itis secreted. This study was undertaken to identify the cell of origin of * This work was supported in part by United State Public Health Service Grant DK33231. The costs of publication of this article were plasma GSHPx and establish whether this cell secretes the defrayed in part by the payment of page charges. This article must enzyme. Plasma proteins are made and secreted mainly by therefore be hereby marked “advertisement” in accordance with 18 hepatocytes (20), endothelial cells (21), and lymphocytes (22). U.S.C. Section 1734 solely to indicate this fact. 4 To whom correspondence should be addressed: Dept. of Pediatrics, BOX777, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
The abbreviations used are GSHPx, glutathione peroxidase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; RBC. red blood cell.
15850
Cell of Origin of Plasma Se-glutathione Peroxidase Myeloid cell granules are another possible source of plasma proteins (22). Myeloid and endothelial cells are important sources of extracellular llZOz(4-7) and therefore, they could possibly produce the enzyme responsible for its detoxification. Moreover, extracellular superoxide dismutase which is thought to participate with extracellular GSHPx in oxidant detoxification, is produc:ed by endothelial cells (10). Therefore, we initially focused on the following three possible cells of origin for secretion of plasma GSHPx: endothelial cells, myeloid cells, and hepatocytes. Human umbilical vein endothelial cells were the source for endothelial cells and HL-60 cells served as a model of myeloid cells (23). Hep G2, a human hepatoma cell line, was chosen as a model for the hepatic cell (20). Our results show that 1) all three cell types synthesize cellular GSHPx which is found only within the cells and 2) plasma GSHPx is synthesized and secreted only by the hepatic cell line. Thus, unlike the otherextracellular antioxidant enzyme (superoxide disnnutase) GSHPx is made and secreted by hepatic cells and not by endothelial cells. Plasma GSHPx is, to thebest of our knowledge, the only known selenoprotein with adefined function that hasbeen shown to be synthesized for secretion by mammalian cells. EXPERIMENTAL PROCEDURES
Materials [75Se]Seleniousacid and [3sS]methionine were purchased from DuPont-New England Nuclear. “C-Methylated proteins were from Amersham Corp. or from Bethesda Research Laboratories. Eagle’s minimum essential medium, RPMI 1640, sodium pyruvate, glutamine, penicillin, streptomycin, and fetal bovine serum were obtained from GIBCO and McCoy’s 5A medium from Flow Laboratories Inc. (McLean, VA). Reduced glutathione, glutathione reductase, NADPH, tert-butylhydroperoxide, deoxycholate, phenylmethylsulfonyl fluoride, iodoacetic acid, protein A-Sepharose, and Nonidet P-40 were purchased from Sigma. Electrophoresis reagents were obtained from Schwartz/Mann. The Hep G2 cell line and the HL-60 cell line were obtained from American Type Culture Collection (Rockville, MD). All other reagents were of analytical grade.
15851
x IO7 Hep G2 or HL-60 cells or the medium in which these cells grew)was incubated a t room temperature for 30 min with either rabbit immune or nonimmune IgG and then samples of either lysed cells or media were added and thenmixed gently for an additional 90 min. The immunoprecipitate, whichwas coupled to the gel,was exhaustively washed in buffer A, boiled in nonreducing electrophoresis sample buffer, centrifuged for 2 min a t 12,000 rpm and an aliquot was analyzed by SDS-PAGE (24, 25). Purification of RBC and Plasm GSHPx and Preparation of Antibodies-RBC GSHPx was purified according to Awasthi et al. (26). Purification of plasma GSHPx was achieved by our previously published procedure (14). Both enzymes appeared as a single band on 12.5% SDS-PAGE (14). Polyclonal antibodies were obtained in rabbits against the purified enzymes and theIgG fractions isolated using DEAE-cellulose chromatography as previously described (13, 17, 18). The antibodies were found to be specific for the corresponding purifiedproteins. Theywere noninhibitory and precipitated the enzymatic activity. 1 unit of purified RBC GSHPx was totally precipitated by 2 mgof anti-RBC GSHPx IgG and 1 unit of plasma GSHPx was completely precipitated by 3-5 mg ofanti-plasma GSHPxIgG (1 unit of GSHPx activity is defined as the oxidation of 1 pmol NADPH/ min). Analytical Methods-SDS-PAGE, on 12.5%polyacrylamide gel and 5% stacking gel under nonreducing and reducing (100 mM dithiothreitol) conditions, was performed according to Laemmli (27). As radioactive electrophoretic standards the following l4C-rnethylated proteins were used myosin, 200 kDa; phosphorylase B, 92.5 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; (3-lactoglobulin,18.4 kDa, and lysozyme,14.3 kDa. The gels were autoradiographed, with exposure times being between 3-4 weeks for 35S gels and 4-8 h for 75Segels, unless otherwise stated. Scanning of gels was achieved using Zeineh soft laser scanning densitometer model SLR-SD/lD. The percent inhibition of specific immunoprecipitation by purified GSHPx was calculated from the area of the densitometry bands from the gel autoradiographs. The area of the precipitated protein band in the absence of the purified enzyme was considered to be 100%. RESULTS
In order to conduct a valid survey for the cell of origin of plasma GSHPx, asensitive method, able to differentiate cellular from plasma GSHPx, was needed. We used immunoprecipitations by specific antibodies followed by analysis on SDS-PAGE. On SDS-PAGE, purified RBC GSHPx had Methods higher mobility than purified plasma GSHPx (14, 16). The Cell and Culture Condit.ions-Hep G2 cells (human liver hepatoblastoma, ATCC No. HB 8065)were cultured in plastic flasks in antibodies raised against purified RBC and against purified Eagle’s minimum essential medium with nonessential amino acids, plasma GSHPx were found to be specific and non-crossEarle’s basic salt solution, 1 mM sodium pyruvate, 2 mM glutamine, reactive, i.e. antibodies against plasma GSHPx did not imand 10% heat inactivated fetal bovine serum. The medium was munoprecipitate RBC GSHPxorother cellular GSHPx’s replaced with fresh medium every 3 days, and the cells were subcul- tested, and antibodies against RBC GSHPx did not immutured weekly at a ratio of 1:5. Endothelial cells were obtained from human umbilical vein by mild proteolytic digestion as described noprecipitate plasma GSHPx (13, 17, 18). To increase the previously (24, 25). The cells weregrown in McCoy’s5A medium sensitivity of detection, cells were metabolically labeled prior containing 20% fetal bovine serum. HL-60 cells (human promyelo- to immunoprecipitation by protein A-Sepharose-antibody cytic leukemia cell line, ATCC No. CCL 240) werecultured in RPMI conjugate. The immunoprecipitates were thoroughly washed 1640 supplemented with 10% heat-inactivated fetal bovine serum and using detergents, to eliminate nonspecific precipitation and split inhalf weekly. All cu1t.ure media contained 25 units/ml penicillin analyzed by SDS-PAGE, a procedure which prevented nonand 25 pg/ml streptomycin.. The cells were incubated in a humidified specific attachment of radiolabel to theproteins. 37 “C atmosphere consisting of 5% C02,95% air. Fig. 1 shows the immunoprecipitation of proteins from For continuous metabolic labeling, expanding cultures of endothelial, Hep G2 and HL-60 cells were grown for 3 days in the presence endothelial cells using the antibodies directed against RBC of either [3sS]methionine (25 &i/ml, specific radioactivity 1128 Ci/ and againstplasma GSHPx.The immunoprecipitation of mmol) or [75Se]seleniousacid (2.5 pCi/ml, specific radioactivity 7742 [35S]methioninemetabolically labeled proteins from endothemCi/mmol). lial cells by anti-RBC GSHPxIgG resulted in specific purifiZmmurwpurification of QSHPx from Cells and Media-Metabolically labeled cells were lysed using 0.2% SDS, 2 mM N-ethylmaleim- cation of a protein which is indicated by the arrow (Fig. 1, ide, 2 mM iodoacetic acid, 4 mM phenylmethylsulfonyl fluoride, and cells lune 4). Several other proteins were also precipitated by 4 mM EDTA in 0.1 M Tris, buffer at pH 8.3. An equal volume of 1% this antibody but these were likely to be due to nonspecific Nonidet P-40 and 1%deoxycholate in 0.1 M Tris HCl, pH 8.3, was interactions since nonimmune rabbit IgG precipitated proadded to bring the constituents to a final concentration of 0.1 M Tris, teins of apparently identical molecular weight (Fig. 1, cells 0.1% SDS, 1 mM N-ethylmaleimide, 1 mM iodoacetic acid, 2 mM lanes 1 and 2). No protein could be specifically precipitated phenylmethylsulfonyl fluoride, 2 mM EDTA, 0.5% Nonidet P-40, and 0.5% deoxycholate (buffer A). Buffer A was also used for immuno- from the cells by anti-plasma GSHPx IgG (Fig. 1, cells lanes precipitation reaction and for washing the immunoprecipitate (24, 3 ) .In addition, nospecific protein could be found using either of these antibodies in endothelial cells medium (Fig. 1, me25). Protein A-Sepharose CL4B (30 mg/5 X lo6 endothelial cells or 2 dium lanes 3 and 4).
Cell of Origin of Plasma Se-glutathione Peroxidase
15852 Cells
Medium
Cells 1 2 3 4
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FIG. 1. SDS-PAGE of immunopurified [S6S]methionineproteins from endothelial cells and medium. Human umbilical vein endothelial cells were metabolically labeled for 3 dayswith [35S] methionine. GSHPx was immunopurified from lysates of 5 X lo6cells 14.3. and from the culture medium using rabbit IgGs. Purified reduced proteins were electrophoresed by SDS-PAGE andtheautoradiographs are presented. Lane 1, no IgG, lane 2, 150 pg of nonimmune FIG.2. SDS-PAGE of immunopurified ["5Se]seleniousacidIgG; lane 3, 150 pg of anti-plasma GSHPx; lane 4, 200 pg of antilabeled proteins from endothelial cells and medium. Human RBC GSHPx. umbilical vein endothelial cells were metabolically labeled for 3 days with [75Se]seleniousacid. GSHPx was immunopurified from lysates Selenium is known to be incorporated as selenoalanine of 5 X IO6 cells and from the culture medium using rabbit IgGs. (selenocysteine) into GSHPx and into relatively few other Purified reduced proteins were electrophoresed by SDS-PAGE, and proteins (28). In order to verify that the specifically precipi- the autoradiographs are presented. Lune 1,150 pg of nonimmune IgG; lane 2,150 pg of anti-plasma GSHPxIgG, lane 3,200 pg of anti-RBC tated protein was a selenoprotein and in order to increase the GSHPx IgG; lane 4, 200 pg of anti-RBC GSHPx IgG preincubated sensitivity and specificity of the method, the endothelial cells with 3.5 pg of purified RBC GSHPx. "
were metabolically labeled with [75Se]seleniousacid. The immunopurification procedure using the two specific antibodies was applied again. The results are presented in Fig. 2. Only one protein was immunopurified from the cells, a protein antigenically related to RBC GSHPx having subunit molecular mass of 24-25 kDa (Fig. 2, cells lane 3 ) .Prior incubation of anti-RBC GSHPx IgG with purified RBC GSHPx inhibited, by 77% (using scanning densitometry),the ability of the antibody to precipitate the 24-25-kDa protein from endothelial cells (Fig. 2, cells lane 4 ) . This enabled us to identify the protein immunopurified from endothelial cells as cellular GSHPx. Although the sensitivity of the assay was increased using ?3e, we could notdetecta [75Se]selenoprotein with antigenic determinants similar to plasma GSHPx either in the cells (Fig. 2, cells lane 2 ) or their media (Fig. 2, medium lane 2). It can be concluded that endothelial cells have a cellular GSHPx antigenically related to RBC GSHPx, but they do not possess or secrete plasma GSHPx within the sensitivity of the method. The same approach and techniques were used on HL-60 and Hep G2 cells. However, only results from [75Se]selenious acid-labeled cultures are presented. The findings for HL-60 cells are illustrated in Fig. 3. The immunopurification of a 24-25-kDa protein could be obtained from the cells only by using anti-RBC GSHPx IgG (Fig. 3, cells lane 4 ) . The immunoprecipitation of this protein was inhibited by 30%by preincubation of the IgG with RBC GSHPx (Fig. 3, cells lane 5). One or two 75Se-labeledbands of lower molecular weight were also seen (Fig. 3, cells lane 4 ) . But, since their precipitation was also inhibited by RBC GSHPx (Fig. 3, cells lane 5 ) they might be proteolytic fragments of HL-60 cellular GSHPx. As for endothelial cells the medium of HL-60 cells was devoid of any GSHPx protein (Fig. 3, medium lanes 1-4). The data
shown in Fig. 3 indicate that HL-60 cells synthesize only cellular GSHPx. The results obtained for 75Semetabolically labeled Hep G2 cells are presented in Fig. 4. Only in the presence of antiRBC GSHPx IgG was a [75Se]selenoproteinprecipitated from the cells (Fig. 4, cells lane 5). As in the case of endothelial and HL-60 immunopurification, the precipitation of this 24-25kDa protein was partially inhibited (65%) by RBC GSHPx (Fig. 4, cells lane 6). There was no indication that Hep G2 cells contain detectable intracellular plasma GSHPx under these conditions. However, it was possible to immunoprecipitate a selenoprotein with anapparent molecular weight slightly lower than that of cellular GSHPx from the medium of Hep G2 cells, and this was achieved only by using antiplasma GSHPx IgG (Fig. 4,medium lane 3 ) .The precipitation of this protein could be almost completely abolished (99.8%) by prior incubation of anti-plasma GSHPx IgG with purified plasma GSHPx (Fig. 4, medium lane 4 ) . In five experiments, the apparent subunit molecular mass obtained for the band precipitated from Hep G2 cells was 24.9 & 0.8 kDa and for that precipitated from the medium was 24.4 f 0.8 ( p < 0.05, paired t test). We conclude that Hep G2 cells produce a selenoprotein antigenically similar to plasma GSHPx. Since the metabolically labeled protein produced by the cells was found only in themedium of Hep G2 cells, it appears to be a secreted protein. Cellular (RBC and other) andplasma GSHPxs are known to be homotetramerscomposed of four identical subunits (1416,26,29). Inorder to determine if these subunits are attached by disulfide bridges, we subjected both the purified enzymes and the immunoprecipitates to electrophoresis under nonre-
Cell of Origin of Plasma Se-glutathione Peroxidase
15853
B
A 1 2 3 4
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-
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-
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-
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-
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-
FIG.3. SDS-PAGEof immunopurified ['6Se]selenious acidlabeled proteins from HL-60 cells and medium. HL-60 cells were metabolically labeled with [76Se]seleniousacid for 3 days after which GSHPx was immunopurified from cell lysates (2 X lo7 cells) and medium using rabbit IgGs. The purified reduced proteins were subjected to electrophoresis by SDS-PAGE and the autoradiographs are shown. Lane 1, no IgG; lune 2,150 pg of nonimmune IgG; lune 3, 150pg of anti-plasma GSHPxIgG; lune 4,200 pg of anti-RBC GSHPx IgG; lane 5,200 pg of anti-RBC GSHPx IgG preincubated with 3.5 pg of purified RBC GSHPx.
FIG.5. SDS-PAGEof non-reduced GSHPxs. Purified plasma and RBC GSHPxs and "Se-labeled immunopurified cellular and secreted GSHPxs from Hep G2 cells and media (described in Fig. 4) were analyzed by SDS-PAGE undernonreducing conditions. A , silver stain. Lanes 1 and 3 purified RBC and plasma GSHPx respectively; lanes 2 and 4 empty slots. The high molecular weight bands present also in the empty lanes are nonspecific contaminants in the gel. 23, autoradiographs of [75Se]seleniousacid-labeled proteins from Hep G2 cells and medium immunopurified as described in Fig. 4. L a n e s 5 and 6 cells. lunes 7 and 8 medium. Lune 5,200 pg of anti-RBC GSHPx IgG; lane 6, 200 pg of anti-RBC GSHPx IgG preincubated with 3.5 pg of purified RBC GSHPx; lane 7, 150 pg of anti-plasma GSHPx IgG; lane 8,150 pg of anti-plasma GSHPx I g G preincubated with 3.5 pg of purified plasma GSHPx.
ducing conditions. In Fig. 5, the nonreduced silver-stained electrophoretograms of purified plasma and RBC GSHPx and the nonreduced autoradiographs of immunopurified Hep G2 cellular and. secreted GSHPxs are presented. Purified RBC GSHPx appeared as a diffuse band apparently composed of several bands: a main component having an apparent subunit molecular mass of 23 kDa and several faster moving minor bands (Fig. 5, lune I ) . The main plasma GSHPx band under nonreducing conditions had an apparent molecular mass of 20 kDa (Fig. 5, lune 3). Most of the cellular selenoprotein 46 from Hep G2 cells was in one diffuse band with an apparent molecular mass of24.6 f 0.8 kDa (four experiments, not significantly different from its apparent molecular massunder reducing conditions) with minor lower molecular weightpro30teins (Fig. 5, lune 5), while the plasma-like GSHPx in the 4 medium migrated as one sharp band with an apparent molecular mass of20 k 1.2 kDa (four experiments, significantly different from its apparent molecular mass under reducing conditions, p < 0.001, paired t test) (Fig. 5, lune 7). The immunoprecipitations of the 24.6-kDa band from the cells 14.3 and the 20-kDa band from the mediumwere inhibited by prior incubation of the specific antibody with purified RBC FIG.4. SDS-PAGE of immunopurified [76Se]seleniousacid- and plasma GSHPxs, respectively (Fig. 5, lunes 6 and 8).No labeled proteins from Hep G2 cells and medium. Hep G2 cells higher molecularweight bands indicative of polypeptides were metabolically labeled with [75Se]seleniousacid for 3 days. Using cross-linked by disulfide bonds could be detected in either rabbit IgGs, proteins were immunopurified from cell lysates (2 X lo7 instance. cells) and medium and were subjected to SDS-PAGE under reducing Under standard conditions, we were not able to purify a conditions. Autoradiographs of the gels are illustrated. Lune 1, no labeled protein antigenically related to plasma GSHPx from IgG; lune 2,150 pg of nonimmune IgG; lane 3,150 pg of anti-plasma the Hep G2 cell. Since we believe that thisprotein is secreted GSHPx IgG; lane 4,150 pg of anti-plasma GSHPx IgG preincubated with 3.5 pg of purified plasma GSHPx; lane 5 , 200 pg of anti-RBC by these cells, we used an inhibitor of secretion in an attempt GSHPx; lune 6, 200 pg of anti-RBC GSHPx IgG preincubated with to detect the plasma enzyme in the Hep G2 cell. The carboxylic ionophore, monensin, is known to affect glycosylation by 3.5 pg of purified RBC GSHPx.
.
Cell of Origin of Plasma Se-glutathione Peroxidase
15854 A kDa -
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92.5 69 200
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However, in the presence of monensin, a bandwas specifically immunopurified by anti-plasma GSHPx IgG from both the medium (Fig. 6A: M, lane I+) and from the cells (Fig. 6A: C, lune I+). This band had a slightly higher mobility (23 kDa) than themobility of the secreted enzyme found in the medium of either control or monensin-treated cells (24 kDa). The mobility of the secreted protein was increased under nonreducing conditions to give an apparent subunit molecular mass of 20 kDa (Fig. 5B;Fig. 6B:M , lane I-, I+). Under nonreducing conditions, the mobility of the band of the protein precipitated from the cells with anti-plasma GSHPx IgG was also increased (Fig. 6B: C, lane I + ) . DISCUSSION
Since all cells require GSHPx for their protection against peroxidative damage, it was logical to expect that the cell producing GSHPx for secretion would also have its own + + + + cellular GSHPx. In searching for the cell of origin for plasma GSHPx, we required a means of differentiating between the B cellular and plasma GSHPxs. Our previously described speC M C M cific, precipitating and mutually noncross-reacting antibodies 1 2 1 2 kDa 2 against purified plasma and RBC GSHPxs, could be used to 200 detect each of the enzymes in the presence of the other (17, 18).In order to identify the cell of origin of the plasma enzyme, 97.4 it was necessary to demonstrate that theprotein was made by 68a cell and that the cell released it into the extracellular milieu. Therefore, we chose to investigate cell cultures (eitherprimary 43or immortal cell lines) and to search for the proteins in the cells as well as in the media. Immunoprecipitation of'%e metabolically labeled cells by specific antibodies proved to be 29 specific and sensitive enough to point to theliver cells as the cells producing extracellular GSHPx for secretion. A prominent [75Se]selenoproteinband, antigenically related to plasma GSHPx, could be immunoprecipitated from the medium of 10.4 the hepatic cell line (Hep G2). Similar results were obtained with [35S]methioninelabeling, but the specificity and sensitivity were less (data notshown). - + + + + In anassay based on immunoprecipitation of GSHPx activFIG. 6. Reduced and nonreduced SDS-PAGE of immuno- ity measured using HzOzas thesubstrate, we have previously purified [75Se]seleniousacid-labeled proteins from monensin- shown, that all the human liver cytosol selenium-dependent cellular form. The fact that treated Hep G2 cells and medium. Hep G2 cells were metabolically GSHPx could be attributed to the labeled for 3 days with [75Se]seleniousacid in the presence (+) and no protein corresponding to plasma GSHPx could be immuabsence (-) of 1 p~ monensin. GSHPx was immunopurified from noprecipitated from 75Se-labeledwhole lysates of 2 x lo7Hep using rabbit IgGs. Immu- G2 cells by the currently applied method indicates that either cell lysates (C) and from the medium ( M ) nopurified either reduced (A) or nonreduced ( B )proteins were electrophoresed by SDS-PAGE and the autoradiographs (24 h exposure) the extracellular enzyme is secreted rapidly after it is proare presented. Lane 1, 150 pg of anti-plasma GSHPx IgG, lane 2,200 duced, and is not stored in any cell compartment, or that antigenic determinants specific for the plasma enzyme are pg of anti-RBC GSHPx IgG. placed on the enzyme during the secretory process. The demonstration of the plasma enzyme in Hep G2 cell disrupting Golgi elements, resulting in incomplete glycosylation, and in many cases, failure of the glycoprotein to reach lysates only under conditions which partially inhibited its the plasma membrane (30, 31). Hep G2 cells were metaboli- secretion (in the presence of monensin), indicated that norcally labeled for 3 days with [75Se]seleniousacid in the pres- mally the protein is rapidly exported from the cell and the ence or absence of 1PM monensin. Cell growth was partially residual amounts in thecell are too low to be detected by our inhibited in the presence of monensin and thecells appeared standard method even after over-exposure of the gels. Monenmore rounded than the control nontreated cells (data not sin is known to inhibit protein processing in the Golgi comshown). GSHPxs were immunopurified from culture media plex, thereby affecting protein secretion (30, 31). The degree and cell lysates and were subjected to SDS-PAGE analysis. of processing of plasma GSHPx purified from the monensinThe gelswere exposed for 24 h in order to improve the treated cells is not known. However, monensin treatment sensitivity and the results are presented in Fig. 6 . Because of neither interfered with its ability to be recognized by antioverexposure, the cellular (a protein immunopurified by anti- plasma GSHPx IgG nor predisposed it to recognition by antiRBC GSHPx IgG (Fig. 6 ) . The antigenic components responRBC GSHPx IgG from the cells) (Fig. 6A: C, lane 2+ and 2-) and secreted (protein immunopurified by anti-plasma GSHPx sible for the immunological differences between the cellular IgG from the medium) (Fig. 6A: M , lane I+ and I-) GSHPxs and theplasma enzymes are eitherintrinsic to thepolypeptide are heavily stained. In addition, there are several other non- backbone or are introduced before the protein reaches the specific bands, which were shown to be precipitated by non- Golgi complex. It is not possible at this time to determine immune rabbit IgG in a parallel experiment (data notshown). whether the enzymes are products of two separate genes or 14.3 -
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Cell of Origin of Plasma Se-glutathione Peroxidase whether they are different due to post-translational modifications. The amountsof purified RBC and plasma GSHPx used to inhibit the precipitation of the enzymes by specific antibodies were chosen arbitrarily. The differences in degree of inhibition of the specific immuno:precipitation of the enzymes from the three cell types could be explained by the differences in the amounts of GSHPxs in thecultured cells and media and do not necessarily indicate antigenic variation. We could not immunopurify selenoproteins antigenically related to plasma GSHPx from media of endothelial and HL60 cells (Figs. 1-3) under conditions which enabled the purification of the enzyme from medium of Hep G2 cells (Fig. 4). We concluded that endothelial and HL-60 cells either do not synthesize and secrete plasma GSHPx or the amounts secreted are significantly lower than thosesecreted by Hep G2 cells and below the detection level of the method utilized. The apparent subunit molecular mass of cellular GSHPx from all the tested cell cultures of GSHPx secreted from Hep G2 cells and of purified plasma GSHPx was found to be between 24-25 kDa. There was a small significant difference in apparent subunit molecular weight when the cellular and extracellular GSHPxs arecompared within the same electrophoresis gel, the apparent subunit molecular mass of the extracellular enzyme being slightly lower. Purified RBC GSHPx, under the same conditions and within the same gel (data not shown), had lower subunit molecular mass of22 kDa (13, 14). Some of our RBC GSHPx preparations appear as doublets on SDS-PAGE (14) with a higher mobility band, all of which react withmti-RBC GSHPxIgG on immunoblots (data not shown). The same phenomenon was described for rat liver cellular GSHl?x (32). It is possible that during the multiple steps involved in purification of RBC GSHPx some proteolysis occurs leading to a slightly shorter polypeptide. Proteolysis was minimized in our current investigation by using protease inhibitors and the one step immunopurification procedure of GSHl?x from cultured cells and theirmedia. The human gene for cellular GSHPx isolated from kidney library was sequenced and the predicted subunit molecular mass was calculated to be 21.964 kDa (19). At this time, we have no explanation as to why the apparentsubunit molecular mass of the cellular GSHPxs, calculated from the mobility of their peptides on SDfS-PAGE, appears to be higher than predicted. It is possible that they are covalently linked to other material which gcts cleaved during purification. The change to highelr mobility of only the immunopurified plasma GSHPx under nonreducing conditions (Fig. 5) may reflect true differences between cellular and plasma GSHPx. This property was retained by the partially processed plasma GSHPx found within the monensin treated cells (Fig. 6B). This also indicates that the polypeptide backbones, rather than post-translational modifications, are responsible for the differences between the two enzymes. A change in themobility of a reduced versus a nonreduced protein on SDS-PAGE is due to thepresence of disulfide cross-bridges under nonreducing conditions. Higher mobility of a nonreduced protein indicates the possible formation of intrachain disulfide crossbridges. Under nonreducing conditions, the mobility of the immunopurified plasma GSHPx was increased, while that of the immunopurified cellular GSHPx was not (Figs. 5 and GB). Therefore, the plasma enzyme may be richer in intrachain disulfide cross-bridges. ‘Whetherthis is due to a higher content of SH groups or to their different topography within the polypeptide chain remains to be determined. The liver cell produces the vastmajority of plasma proteins
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and, Hep G2, ahuman hepatoma cell line, produces and secretes many of the proteins known to be secreted by liver cells (20). Although it can be argued that immortalized cell lines may not be true reflections of the normal cell, it seems reasonable to conclude that most of the extracellular GSHPx originates in theliver. Whether the liver cell is the only source of this enzyme remains to be determined. When selenium-deficient individuals are subjected to selenium repletion, plasma GSHPx activity reappears at a higher rate than blood cells GSHPx activity (33). Identification of the liver cell as thecell of origin of plasma GSHPx opens the possibility of investigating the rateof synthesis and secretion of the plasma enzyme versus the rate of synthesis of the cellular enzyme in selenium-deficient cells and during selenium repletion. 1. 2. 3. 4. 5. 6.
7. 8.
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