distribution of selenoproteins in mouse mammary epithelial ...reduction of organic hydroperoxides or...

[CANCER RESEARCH 46. 4582-4589, September 1986]

Distribution of Selenoproteins in Mouse Mammary Epithelial Cellsin Vitro and in Vivo1

Keith G. Danielson2 and Daniel Medina

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT

Resolution of selenium-containing proteins synthesized by mouse mammary gland cells was achieved using the technique of two-dimensionalgel electrophoresis. Radioactive selenium as i I "Si-th was incorporated

into relatively few proteins within mammary gland cells maintained invitro and cells of mammary gland tissue in vivo. The pattern of seleno-proteins obtained was identical qualitatively between a nontumorigenicdifferentiated cell line, COMMA-D, and a tumorigenic cell line, MOD.Eleven selenoproteins ranging in molecular weight from 12,000-78,000were detected and a total of 25 spots were visible indicating chargeheterogeneity of some of the proteins. A majorselenoprotein (M, 26,000)migrated identically with the subunit form of glutathione peroxidase, awell-characterized protein containing four selenocysteine residues. Othermajor selenoproteins had molecular weights of 58,000, 22,000, 18,000,and 14,000. Analysis of the total cellular protein extract and of each ofthe five major proteins indicated that selenium was incorporated asselenocysteine in the proteins. Incorporationof selenium as selenomethi-onine into cellular proteins was detected only when selenomethionine wasprovided in the culture medium. Cleavage of 75Se-labeled proteins with/V-chlorosuccinimide produced polypeptides of different molecularweights indicating that the M, 58,000, 26,000, and 22,000 selenoproteinswere dissimilar in the amino acid sequences containing the selenoaminoacid. The pattern of selenoproteins of mammary gland cells in vivo wassimilar to that obtained for cells in culture and most other tissues in vivo.These results provide evidence for the presence of multiple selenium-containing proteins in mammaryepithelial cells. The possible significanceof these proteins in selenium-mediated inhibition of cell growth awaitsfuture clarification.

INTRODUCTION

Selenium was first demonstrated to be an essential traceelement in animal nutrition by Schwarz and Foltz (1) in 1957.This observation was later confirmed by other investigators forboth birds and mammals (2, 3). More recently, the essentialityof selenium was extended to human diploid fibroblasts grownin culture (4). Additionally, experiments over the past 12 yearshave demonstrated the efficacy of selenium as an inhibitor ofcarcinogenesis in the mammary gland, liver, skin, colon, andstomach (for reviews, see Refs. 5-7). The demonstration of theimportance of selenium as an essential nutrient in low dosesand as a chemopreventive agent in high doses has generated aconsiderable effort devoted to the elucidation of the mechanisms of selenium action in biological systems. With the discovery in 1973 of the association of selenium with the enzymeglutathione peroxidase (8), the importance of selenium in mediation of a cellular antioxidant defense mechanism becameevident (9, 10). This enzyme contains selenium in the form ofselenocysteine and catalyzes the oxidation of glutathione andreduction of organic hydroperoxides or H2O2, thereby protecting membrane lipids and possibly other macromolecules fromoxidative damage. Whereas the antioxidant property of sele-

Received 1/6/86; revised 3/25/86, 6/3/86; accepted 6/3/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'This investigation was supported by USPHS Research Grants CA-11944,CA-32473, and CA-38650 from the National Cancer Institute.

1To whom requests for reprints should be addressed.

nium has been demonstrated as an important function at physiological levels of selenium, there is no evidence in the literatureto indicate that chemopreventive doses of selenium exert theireffects via such a mechanism (11-17).

Selenium has also been reported to be incorporated as selenocysteine in several bacterial redox enzymes such as glycinereducÃasecomplex, formate dehydrogenase, xanthine dehydro-genase, hydrogenase, and nicotinic acid hydroxylase. An additional bacterial enzyme, thiolase, contains selenium as theamino acid, selenomethionine. To date, 7 different bacterialselenoproteins have been described and have been the subjectof several review articles (6, 18-20). Evidence for the presenceof selenium in mammalian proteins other than glutathioneperoxidase has been reported by several investigators. A low-molecular-weight selenoprotein (M, 10,000) with characteristics similar to the cytochromes has been isolated from lambmuscle (21, 22). The absence of this protein has been implicatedin the cause of nutritional muscular dystrophy in selenium-deficient sheep. Several proteins (M, 57,000, 45,000, and15,000) containing selenium have been isolated from rat testiscytosol (23). The major testicular selenoprotein (M, 15,000)may be identical to a M, 17,000 protein found in tails of ratspermatozoa (24). A number of investigators have also reportedthe presence of a selenium-containing protein in rat and monkeyplasma (25-28). By gel filtration the relative molecular weightof this protein was estimated at 80,000 with a subunit size ofapproximately 45,000-49,000. The protein was shown to contain selenocysteine and play a role in selenium transport andstorage (27, 28). Finally, 6 selenocysteine-containing proteinsranging in molecular weight between 8,400 and greater than90,000 and distinguishable from glutathione peroxidase bycolumn chromatography have been detected in tissue extractsof rats equilibrated with radioactive selenium over a 5-monthperiod (29). The specific enzymatic or biochemical functions ofthese selenoproteins remain unknown.

To date, no study known to the authors has been done on theseparation and molecular weight determination of cellular selenoproteins using the powerful technique of two-dimensionalpolyacrylamide gel electrophoresis. The purpose of these experiments was to determine the number and molecular weightsof selenoproteins found in mammary cells. The long range goalof this experimental approach is to examine the significance ofselenoproteins as a mediator of the biological role of seleniumas a chemopreventive agent. The results presented herein showthat selenium is incorporated into specific polypeptides of 11different molecular weights which are resolvable as 25 distinctspots on a two-dimensional gel. Additional evidence of thedistribution of these selenoproteins in murine tissues andplasma is presented. For the purpose of this study, we are usingthe term "selenoprotein" to indicate proteins which bound

selenium over a rigorous extraction and separation procedure.

MATERIALS AND METHODS

Cell Lines. The COMMA-D cell line, derived from normal mousemammary tissue, has been characterized and was maintained as de-

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SELENOPROTEINS IN MAMMARY CELLS

scribed (30). The MOD cell line was derived from a mammary adeno-carcinoma arising spontaneously from the D2 preneoplastic mammaryoutgrowth line and is tumorigenic when injected s.c. into syngeneicBALB/c mice. The MOD cell line is routinely maintained in Dulbecco'smodified Eagle's medium supplemented with 10% fetal bovine serum,10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer, gen-tamycin sulfate (50 fig/ml), and insulin (5 ^g/ml). All cell cultures weremaintained in a humidified atmosphere (92.5% air-7.5% CO2) at 37"C.

Both cell lines exhibit an epithelial morphology in monolayer culture.Treatment of Cells with 75Se and Analysis of Selenoproteins. Cells

were seeded at a concentration of 1.5 x 10*cells/cm2 in 60-mm diameterdishes containing Dulbecco's modified Eagle's medium, 2% fetal bovine

serum, and the following growth factors: insulin (5 /in ml): epidermalgrowth factor (10 ng/ml); endothelial cell growth factor (10 ^g/ml);transferrin (10 //u ml): and fibronectin (1 numi)- This growth mediumcan be used to grow and maintain mammary epithelial cells in cultureand contains selenium, about 4 ng/ml. After 24 h the medium wasremoved and fresh medium added containing radioactive carrier-freeselenious acid (H275SeO3; specific activity, 356 mCi/mg) at a concentration of 2 fiCi/ml (5.6 ng "Se/ml). During the 4-day labeling period,

the medium was changed once after 2 days. For incorporation ofradioactive selenomethionine into cellular proteins, cell monolayerswere incubated for 7 h in methionine-free RPMI 1640 medium supplemented with insulin, epidermal growth factor, transferrin, and L-["Se]-

selenomethionine, 10 Â¿iCi/ml(specific activity, 179 mCi/mg).To prepare cell extracts the following steps were performed at or

below 4Â°C.The cell monolayers were rinsed 3 times with cold NaCI

solution composed of 8.0 g/liter NaCI, 0.2 g/liter KC1, 1.5 g/literanhydrous Na2HPO4, and 0.2 g/liter KH2PO4 and the cells were removed from the plastic substrate by scraping with a rubber policeman.The cells were placed in 15-ml conical glass centrifuge tubes andcentrifuged at 1000 rpm for 15 min. The supernatant was removed anddiscarded and the cell pellet was frozen in an ethanol/dry ice bath. Fiveml of acetone were added to each tube and the cell pellets were sonicatedbriefly. The sonicated cells were transferred to Eppendorf tubes and thecell protein was extracted 3 times with 5-ml volumes of cold acetonein the ethanol/dry ice bath (31). After each extraction the insolublepellets were collected by centrifugation for 5 min using a microcentri-fuge. Following the acetone extractions, approximately 0.5 ml of 40mM acetic acid was added to each pellet which was subsequently frozenand lyophilized. The lyophilized material was then dissolved in 0.1 -0.3ml of O'FarreH's lysis buffer A (32) prior to electrophoresis. Insoluble

material was removed by centrifugation.Two-dimensional polyacrylamide gel electrophoresis was carried out

using the procedure of O'Farrell et al. (33) with some modifications.

Nonequilibrium pH gradient electrophoresis of total cell proteins wasdone using glass tubes (6.5 mm long and 2.0 mm inside diameter) andampholytes of a pH gradient 3-10. Electrophoresis was for 2.5-3.0 husing an increasing voltage (50-400 V) for a total of 500-550 V-h.After the gels were extruded from the glass tubes, they were placedindividually into plastic Petri dishes containing 10 ml of equilibrationbuffer (32) and incubated on a rotary shaker for 1 h. Two gels werethen loaded on each 15% polyacrylamide slab gel with a 5% stackinggel for molecular weight determination of the proteins by SDS3 poly

acrylamide gel electrophoresis (34). After a constant current of 25 mA/gel was applied for about 6 h, the gels were soaked in a 50% ethanol/10% acetic acid solution overnight, stained by the silver method (35),dried, and exposed to Cronex 4 X-ray film (Dupont) for 12 days.

To prepare extracts of cells in culture for one-dimensional SDSpolyacrylamide gel electrophoresis, each cell monolayer in a 75-cm2

flask was rinsed 3 times with cold NaCI solution and dissolved directlyat 4Â°Cin 1 ml of sample buffer containing 4% SDS, 4% 2-mercapto-

ethanol, 5 mM Tris buffer (pH 6.8), 10% glycerol, bromphenol blue(0.05 mg/ml), and aprotinin (1000 kallikrein inactivating units/ml).The samples were then vortexed, placed in a boiling water bath for 5min, and passed through a 26-gauge needle prior to storage at â€”70Â°C.

Treatment of Experimental Animals with 75Se.Three-week-old femaleBALB/c mice were fed a selenium-deficient diet containing 0.03 ppmselenium by fluorometric analysis (36) for 1 month and then were given

3The abbreviation used is: SDS, sodium dodecyl sulfate.

injections i.p. of 1.0 mCi of H2"SeO3 (specific activity, 201 mCi/mg).

After 40 h the animals were anesthetized with ether and exsanguinatedby heart puncture. Heparin was added to each sample of blood for afinal concentration of 1 mg/ml. The plasma was separated from thecellular components by centrifugation and the resulting cell pellet waswashed several times in NaCI solution and recentrifuged. Aliquots ofplasma or washed blood cells were diluted 1:1 with double-strengthsample buffer and stored at -70Â°C.

Tissues were removed rapidly, washed briefly in ice-cold NaCI solution, and placed on a block of dry ice. The tissue fragments were placedin a mortar containing liquid nitrogen and ground into a fine powderwhich was weighed immediately and then stored at â€”70".To prepare

the tissue for one-dimensional gel electrophoresis, 1 ml of single-strength sample buffer was added to about 175-200 mg of tissue. Thesamples were then vortexed, placed in a boiling water bath for 5 min,and passed through a 26-gauge needle prior to storage at â€”70Â°C.

Insoluble material was removed by centrifugation in a microcentrifuge.Protein concentration in each sample was determined by the microassayof Bradford (37).

Selenocysteine Analysis. Confluent monolayers of COMMA-D cellsin 100-mm diameter dishes were incubated with 75Se as Na275SeO3

(specific activity, 100 mCi/mg; 2 ^Ci/ml) for 4 days. The medium waschanged once after 2 days. To harvest the cells, the cell monolayerswere washed 3 times with cold (4Â°C)NaCI solution, drained well, and

treated with lysis buffer (0.1 M Tris, 1 mM dithiothreitol, and 1% SDS,pH 8.0). The cells were scraped off the dish with a rubber policemanand the cell homogenate was placed in a boiling water bath for 15 min,sonicated for 1.5 min, and stored at -20Â°C.

Derivatization of selenocysteine (and also cysteine) residues to yieldcarboxymethylselenocysteine in the crude cell homogenate was done byalkylation with iodoacetic acid as described by Stadtman (38). The cellhomogenate in lysis buffer was placed under an argon barrier andpotassium borohydride was added to a final concentration of 5 mM.After 10 min, a neutralized solution of iodoacetic acid was added (10mM, final concentration). The reaction mixture was stored in the darkunder argon at room temperature for 5 h. The reaction was terminatedby addition of 2-mercaptoethanol (30 mM, final concentration). Toremove excess reactants, the cell homogenates were dialyzed against 4liters of 0.02% SDS and 10 mM 2-mercaptoethanol for 48 h. Thesamples were then lyophilized and dissolved in sample buffer for one-dimensional polyacrylamide gel electrophoresis. The cell homogenatefrom one 100-mm dish was loaded on each 1.5-mm thick gel (15%resolving gel). Following electrophoresis the gels were dried downdirectly without fixation and processed for autoradiography. The gelslices corresponding to 75Se-labeled proteins were removed and placed

in dialysis tubing. Electroelution of protein from the gel slices was doneat 30 mA using a flat-bed electrophoresis apparatus filled with 0.1%SDS and 10 mM ammonium bicarbonate. After 24 h, the current wasreversed for 5 min to release the protein from the wall of the dialysismembrane. The eluted protein samples were filtered through WhatmanNo. 1 filter paper and dialyzed exhaustively against water and twicelyophilized. For acid hydrolysis of the protein, the samples were dissolved in 100 n\ 6 M HC1 containing 10 ^1 0.1% aqueous phenol. Thehydrolysis tubes were evacuated of air, sealed under a nitrogen barrier,and heated at 110Â°Cfor 24 h. The HC1 was removed by drying down

the samples under a vacuum and the dried material was reconstitutedin 10 (/I of 0.01 M HC1 and processed for thin-layer chromatographyas described by Stadtman (38). The chromatograms were dried, processed for autoradiography, and the Rf values were calculated. Preparation of [MC]carboxymethylselenocysteine, which was used as the stand

ard for the derivatized selenocysteine, was done as described by Tappelet al. (39),

Cleavage of 75Se-labeled Proteins. Selective cleavage of tryptophanresidues by Â¿Y-chlorosuccinimideof 75Se-labeled proteins to yield partialmaps of the selenium-containing peptide sequence was done as described by Lischwe and Ochs (40). Briefly, gel slices from dried, unfixedgels corresponding to selenoproteins were cut out, incubated with orwithout 0.15 M W-chlorosuccinimide for 30 min, and loaded onto a 5%stacking gel above a 15% polyacrylamide resolving gel. Followingelectrophoresis, the gel was fixed in 50% methanol/10% acetic acid

4583




containing 0.05% Coomassie Brilliant Blue R250, destained, drieddown, and processed for autoradiography.

Materials. JV-chlorosuccinimide, jV.yV'-methylenebisacrylamide, so

dium dodecyl sulfate, glycine, and Tris base were obtained from SigmaChemical Company. A/,./V,./V',W-tetramethylethylenediamine, NP40,

and ammonium persulfate were obtained from LKB. Acrylamide andampholytes were obtained from Bio-Rad. Acetone and 2-mercaptoeth-anol were obtained from Fisher Scientific Company. Urea was ultrapuregrade from Schwartz-Mann. Selenious acid as H275SeO3was obtainedfrom New England Nuclear. Sodium selenite as Na275SeO3and iodoa-cetic acid as ICH214COOH were obtained from ICN. Selenomethionineas L-[75Se]selenomethionine was purchased from Amersham. Hydro

chloric acid (6 M) was purchased from Pierce Chemical Company.

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1NEPHGE

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RESULTS

Electrophoretic Analysis of Selenoproteins from Mouse Mammary Cells in Vitro and Tissues in Vivo. Figs. 1 and 2 illustratethe autoradiographic pattern of selenoproteins separated ontwo-dimensional nonequilibrium pH gradient gels for the twomouse mammary cell lines, COMMA-D and MOD. The patterns are very similar with approximately 25 spots visible ineach figure representing selenoproteins ranging in molecularweight from 12,000-78,000. The pattern of selenoproteinsproduced is not changed if the time that cell monolayers areexposed to 75Se is increased from 4-17 days. A number of

different isoelectric forms may exist for some of the selenoproteins, or polypeptides with different isoelectric points but of thesame molecular weight may represent unique selenoproteins.The presence of radioactive selenium is found predominantlyin the M, 12,000-26,000 class of proteins for both cell lines.Major selenoproteins in this class have molecular weights of14,000, 22,000, and 26,000. The M, 26,000 proteins are prob-

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Fig. I. Distribution of "Se-labeled proteins from COMMA-D cells as afunction of molecular weight and relative isoelectric point. Cells were labeled invitro with 11. 'Sc<), for 4 days. The sample applied to the gel contained 200,000

cpm of acetone-precipitable protein. The autoradiograph shown was exposed tothe gel for 12 days. In Figs. 1-4, the pH gradient (pH 4.S-8.8) is representedextending from right to left. NEPHGE, nonequilibrium pH gradient electropho-

- 14

- 12

Fig. 2. Distribution of 75Se-labeled proteins from MOD cells as a function of

molecular weight and relative isoelectric point. Cells were labeled as described inFig. 1. Note similarity with Fig. 1. The notable exception is the marked decreasein the M, 16,000 protein. Approximately 200,000 cpm was applied to the gel andthe autoradiograph was exposed to the gel for 12 days. NEPHGE, nonequilibriumpH gradient electrophoresis.

ably the subunit forms of glutathione peroxidase since purifiedglutathione peroxidase comigrates with these proteins (data notshown). The intensity of the spot on the autoradiogram representing a minor M, 16,000 selenoprotein is about 9-fold higheras measured by densitometry for the COMMA-D cell linecompared to the MOD cell line (Figs. 1 and 2). Other surrounding spots representing major selenoproteins, however, are equalin intensity between the two cell lines. The significance of thisdifference is not clear. Incorporation of selenium occurred alsoin proteins ranging in molecular weight from 50,000-78,000with the major selenoprotein having a relative molecular weightof 58,000. The identity and biological significance of theseselenoproteins remain unknown.

Fig. 3 illustrates the pattern of selenoproteins synthesizedintracellularly by the intact virgin mammary gland in vivo.Although fewer spots are present, the similarity of the autoradiograph to Figs. 1 and 2 is evident. The major discrepanciesare likely due to the significantly lesser amount of radioactivityrecovered from the mammary gland in vivo compared to cellsin culture.

Fig. 4 illustrates the pattern of selenoproteins contained byCOMMA-D cells if L-[75Se]selenomethionine is added in placeof methionine to the culture medium. During the 7-h labelingperiod, many proteins incorporate selenomethionine into theirpolypeptide chains. If COMMA-D cells are cultured for 4 daysin RPMI medium containing the normal concentration of methionine and 2 /Â¿Ci/mlof L-[7SSe]selenomethionine, the patternof selenoproteins produced by two-dimensional gel electrophoresis is identical to Fig. 4 (data not shown). These resultsconfirm earlier work (41,42) that selenomethionine can replacemethionine during protein synthesis. The dissimilarity of theselenoprotein profile between Figs. 4 and 1 suggests that 75Seincorporated as L-[7SSe]selenomethionine is not readily utilized

for production of the selenoproteins shown in Fig. 1 and that4584




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Fig. 3. Distribution of selenoproteins from the mammary gland labeled in vivowith H275SeO3. The sample applied to the gel contained 45.000 cpm and theautoradiograph was exposed to the gel for 21 days. Note similarity of the patternsof spots with Figs. 1 and 2. NEPHGE, nonequilibrium pH gradient electrophoresis.

mined by one-dimensional polyacrylamide gel electrophoresis(Figs. 5 and 6). Major selenoproteins of M, 58,000, 51,000,26,000, 22,000, 18,000, and 14,000 were found in most tissuesexamined including liver, kidney, pancreas, testis, mammarygland, ovary, and tumor tissue derived from s.c. injection ofMOD cells. In addition, testes exhibited major proteins of M,66,000 and 12,000. The COMMA-D cell line exhibited a similar profile of selenoproteins although a few additional minorselenoproteins present in tissues (M, 16,000 and 12,000) werevisible as major bands (Fig. 6, lane 1). Tissue derived from thebrain contained barely detectable levels of selenoproteins andno selenium-labeled protein was found in the lens. Sele-

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Fig. 5. Separation by one-dimensional polyacrylamide gel electrophoresis ofselenoproteins in various tissues labeled in vivo with HÂ¡"SeO3.One hundred fig

of protein were loaded in each lane. Lane I, liver, 27,900 cpm; Lane 2, kidney,63,700 cpm; Lane 3, pancreas, 12,700 cpm; Lane 4, stomach. 15,300 cpm; Lane5, testis, 48,200 cpm; Lane 6, mammary gland, 32,000 cpm; Lane 7, tumorderived from s.c. injection of MOD cells, 19,300 cpm; Lane S, plasma of a malemouse, 65,600 cpm; Lane 9, plasma of a female mouse, 53,500 cpm. Theautoradiograph was exposed to the gel for 14 days.

Fig. 4. Distribution of 75Se-labeled proteins from COMMA-D cells labeled invitro with i.-[15Se]selenomethionine for 7 h. The sample applied to the gel

contained 600.000 cpm and the autoradiograph was exposed to the gel for 7 days.Note that a very large number of selenium-labeled proteins are present whichcontrasts sharply with the few proteins labeled by selenium when the cells wereincubated with inorganic selenium (HÂ¡SeO3).NEPHGE, nonequilibrium pHgradient electrophoresis.

relatively few proteins contain selenium when 75Se is suppliedin the form of selenious acid (H275SeO3).

To detect possible heterogeneity of selenoprotein expressioni/i vivo, the distribution of selenoproteins in various tissues ofa mouse radiolabeled with selenium as H275SeO3 was deter-

Fig. 6. Separation by one-dimensional polyacrylamide gel electrophoresis of"Se-labeled proteins in COMMA-D cells and in the several tissues labeled in vivowith H/'SeOj. Lane I, COMMA-D cells, 152,700 cpm; Lane 2, ovary, 30,100

cpm; Lane 3, lens, 1,900 cpm; Lane 4, brain. 3,800 cpm; Lane 5, RBC of femalemouse, 1,800 cpm; Lane 6, RBC of male mouse, 1,200 cpm. One hundred jig ofprotein were loaded in each lane. The autoradiograph was exposed to the gel for14 days.

4585




noproteins in plasma washed free of cellular elements werefound only at a molecular weight of 58,000 and 26,000 (Fig. 5,lanes 8 and 9). RBC washed extensively with NaCl solutioncontained only the A/r 26,000 selenoprotein which comigratedwith glutathione peroxidase (Fig. 6, lanes 5 and 6).

Biochemical Analysis of Murine Selenoproteins. To determinethe biochemical form of selenium in the major selenium-containing proteins, protein hydrolysates containing 75Se-labeledproteins were subjected to 6 M HC1 at 110Â°Cfor 23 h and

analyzed by thin-layer chromatography (Fig. 7). Selenium wasfound to be incorporated predominantly as selenocysteinewhich migrated similarly to the [14C]selenocysteine standard(Fig. 7, lane 7). The presence of selenocysteine in 5 selenopro-teins ranging in molecular weight from 14,000-58,000 was alsoconfirmed (Fig. 7, lanes 2-6). Hydrolyzed samples of L-[75Se]-selenomethionine (lane 8) and Na27SSeO3 (lane 9) migrated

dissimilarly compared to selenocysteine.To determine if the polypeptide sequences containing the

selenoamino acids were unique or similar, the gel slices containing 75Se-labeled proteins of different molecular weights wereincubated with /V-chlorosuccinimide. This chemical cleavestryptophan residues selectively with high efficiency and hasbeen used to produce partial peptide maps from ng quantitiesof proteins (40). Figs. 8 and 9 present the gel electrophoreticpattern of the uncleaved and cleaved 75Se-labeled proteins as

revealed by autoradiography. Differences in the molecularweights of the selenium-containing polypeptide fragments generated by jV-chlorosuccinimide were evident among the M,

22

Fig. 8. Cleavage of Selenoproteins wilh (Y-chlorosuccinimide/urea. Gel slicescontaining "Se-labeled protein were incubated for 30 min in 0.15 M /V-chlorosuc-cinimide/urea. Peptide fragments were separated by one-dimensional SDS-poly-acrylamide gel electrophoresis on a 15% gel and processed for autoradiography.Lanes 1 and 2, Mr 58,000 selenoprotein, uncleaved and cleaved, respectively. Notepresence of a prominent cleavage peptide fragment of M, 13,500 (Lane 2). Lanes3 and 4, M, 26,000 selenoprotein, uncleaved and cleaved, respectively. Twocleaved polypeptides, M, 18,000 and 19,000 are present (Lane 4). Lanes 5 and 6,M, 22,000 selenoprotein, uncleaved and cleaved, respectively. A cleavage product(M, 16,500) is evident.

Fig. 7. Thin layer chromatography of selenoprotein hydrolysates on a cellulose-backed sheet. Total cell homogenate of "Se-labeled COMMA-D cells orselenoproteins electroeluted from one-dimensional polyacrylamide gels were Indrolyzed in 6 M HCI at 110'C and processed for thin-layer chromatography (see"Materials and Methods"). The solvent system used consisted of fert-butyl alco-

holimcthyl ethyl ketone:88% formic acid:water (40:30:15:15) and the runningtime was 2.5 h. Lane I. cell homogenate (CH), 5,200 cpm; Lane 2 M, 58.000selenoprotein (S8K), 5,300 cpm; Lane 3, M, 26,000 selenoprotein (26/0, 3,800cpm; Lane 4, M, 22,000 selenoprotein (22K), 5,100 cpm; Lane 5, M, I8.000selenoprotein (ISK). 500 cpm: Lane 6, M, 14,000 selenoprotein (14K). 5,400cpm; Lane 7, [-OOC-MCHjlcarboxymethyl selenocysteine (CM-Se-Cys), 52,400cpm; Lane 8, L-|7'Se]selenomethionine (Se-Met), 5,700 cpm; Lane 9, sodiumselenite as Na27*SeO3, 5,300 cpm. The autoradiograph was exposed to thecellulose-backed sheet for 14 days. K, thousands.

18-14-

Fig. 9. Cleavage of selenoproteins with A/-chlorosuccinimide/urea as describedin legend of Fig. 8. Lanes I and 2, M, 18.000 selenoprotein, uncleaved andcleaved, respectively; Lanes 3 and 4, M, 14,000 selenoprotein. uncleaved andcleaved, respectively. No cleaved polypeptides are detected.

58,000, 26,000, and 22,000 selenoproteins indicating that theseproteins were probably unique and not related (Fig. 8). Thepresence of one or two fragments produced per selenoproteinindicates few selenocysteine residues are present or alternativelyfew tryptophan cleavage sites. Cleaved selenopolypeptides werenot detected for the M, 18,000 and 14,000 selenoproteinsallowing no conclusion to be made regarding their similarity.

DISCUSSION

The results of this investigation provide evidence for theexistence of selenium-binding proteins in addition to glutathione peroxidase within mammary gland epithelial cells in vitroand murine tissues in vivo. Specifically, 11 subunit sizes weredetected ranging in molecular weight from 78,000-12,000. Themultiple spots detected by autoradiography (Figs. 1-3) for aspecific molecular weight size of selenoprotein could indicatethe presence of similar forms differing only in electrical charge,although selenoproteins of the same molecular weight but dissimilar isoelectric point could differ considerably in amino acidsequence. Major selenoproteins of M, 58,000, 26,000, 22,000,18,000, and 14,000 were found consistently in cells cultured invitro and tissue homogenates from 75Se-treated animals. These

proteins were shown to contain selenium stably associated withprotein in the form of selenocysteine.

Generally, our results are in agreement with the recent work4586




of Hawkes et al. (29) who separated 7 selenoproteins in rattissue homogenates by molecular exclusion column chromatog-raphy and detected 9 different Chromatographie forms by ionexchange chromatography. Interestingly, they proposed an upper limit of 22 unique selenoproteins which is remarkablysimilar in number to the 25 forms separated by two-dimensionalelectrophoresis as shown in Figs. 1 and 2. Similarly, they alsofound selenoproteins of M, 26,500, 20,100, and 14,700 andshowed that selenocysteine was the predominant form of selenium in the tissue homogenates; however, in contrast, theyreported the presence of several larger selenoproteins (M,36,300,46,400, and >89,200) that differed in molecular weightbut could be possibly related to the M, 51,000-78,000 selenoproteins described here. The reason for this discrepancy in themolecular weight of the larger selenoproteins is not clear butcould be related to differences in sample preparation or theseparation techniques used.

Also, in agreement with Hawkes et al. (29) we report a majorselenoprotein of M, 26,000 which comigrates with glutathioneperoxidase isolated from liver and probably represents thesubunit form of this enzyme. This selenoenzyme has beenpurified from a number of different tissues and consists of 4similar and possibly identical selenium-containing subunits ofabout M, 20,000-26,000 each (43-49). The multiple spotspresent at a molecular weight of 26,000 (Figs. 1 and 2) apparently reflects the charge heterogeneity of the subunits, an observation noted by other investigators (47, 48). Interestingly,the M, 26,000 selenoprotein found in plasma is not thought tobe glutathione peroxidase by Hawkes et al. (29) since theydetect little glutathione peroxidase activity in rat plasma.

Surprisingly, we found little 75Se to be associated with intra-

cellular proteins of molecular weight between 26,000 and50,000. Minor bands representing molecular weights of 33,000and 45,000 were detected by one-dimensional gel electrophoresis in testis and COMMA-D cells while a minor selenoproteinof molecular weight 30,000 was found in liver tissue. In plasma,a small amount of selenium was associated with a Mr 42,000protein; however, several selenium-containing proteins largerthan M, 50,000 were detected by gel electrophoresis with themajor spot at M, 58,000. The M, 78,000 protein revealed by 2-

dimensional gel electrophoresis may be similar to a M, 75,000selenoprotein isolated from rat kidney (50) and a Mr 57,000selenoprotein has been detected in rat testis cytosol (23).Clearly, the results of this investigation and others in recentyears show that a significant number of selenoproteins exist inthe mammalian cell in addition to glutathione peroxidase.

The presence of a M, 58,000 selenoprotein in almost alltissues and cells examined (except lens and RBC) is a significantnew finding of this study. The identity of this protein remainsunknown but it may be related to a selenocysteine-containingprotein (M, 45,000) detected by Motsenbocker and Tappel (27)in rat and monkey plasma. A similar selenium-binding plasmaprotein (M, 49,000) has also been described by Herrman (25).The apparent large amount of this protein in plasma mayindicate a transport and/or storage function of selenium forthis protein; indeed, Motsenbocker and Tappel (28) postulatedthat the protein is synthesized in the liver and transported toother tissues when a selenium deficiency exists in the animal.

The chemical form of selenium incorporated into 5 differentmolecular weight selenoproteins (M, 58,000, 26,000, 22,000,18,000, and 14,000) was shown specifically to be selenocysteineby thin-layer chromatography. The Rr for the carboxymeth-ylated selenocysteine standard was 0.41 which correlated wellwith the reported value of 0.47 (38). The presence of a second

spot running near the solvent front may represent an oxidizedform of carboxymethylselenocysteine (51); however, probablyonly partial oxidation has occurred and the presence of a spotat the correct Rf strongly suggests the presence of carboxymethylselenocysteine. We have also run [carboxymethyl-^C]-selenocysteine in another solvent system (2-propanol: 88%formic acid: water; 60:3:15 ratio) on cellulose thin-layer chromatography and it migrated with a correct Rf of 0.35; furthermore, a sample of 75Se-labeled protein hydrolysate also pro

duced a spot at the correct Rf value of 0.35 in this solventsystem. Thus, overall, the results indicate that selenium supplied as selenious acid or sodium selenite is primarily incorporated as selenocysteine into a discrete number of proteins;however, a more definitive determination of the selenium-labeled moiety in carboxymethylated 75Se-labeled selenopro

teins such as amino acid analysis is needed to substantiate thisfinding. To date, no mammalian protein is known to contain aselenium moiety in the form of selenomethionine although sucha selenoprotein has been described in bacteria (52). Recently,however, evidence for incorporation of selenium into an unusualacidic amino acid of the lens protein 7-crystallin was reported(53). This selenium-containing amino acid chromatographedseparately from selenocysteine and selenocystine and remainsunidentified; however, we were unable to detect by polyacryl-amide gel electrophoresis any selenium-containing proteins ina cell homogenate of the lens. Possibly, insufficient 75Se was

incorporated into protein of the lens to be detected by one-dimensional gel electrophoresis since glutathione peroxidasehas been isolated from bovine lens (54).

In the course of this work, we performed several experimentsto rule out the possibility that some of the selenoproteinsseparated by polyacrylamide gel electrophoresis may representthe formation of selenotrisulfides (protein-S-75Se-S-protein).Cell extracts containing 75Se-Iabeled proteins were dialyzed

extensively against a strong alkaline solution (ammonium hydroxide in water, pH 10) for several days to remove seleniumpresent as selenotrisulfide (55, 56). Cell extracts were alsotreated with 10 mM KCN for 22 h which has also been reportedto release selenium from selenotrisulfide (53, 57). Appropriatecontrols such as dialysis against water and omission of the KCNwere also done. The cell extracts were prepared for one-dimensional gel electrophoresis. Autoradiography of the dried gelrevealed an identical pattern of selenoproteins in all samplesloaded. No loss of selenoproteins was detected by either pre-treating the sample with KCN or exhaustive dialysis against anammonium hydroxide solution; thus, it is unlikely that theselenoproteins described in this paper represent selenotrisulfides. The selenium is stably incorporated into the protein asshown by thin-layer chromatography.

Recently, the existence of selenium-containing tRNAs inmammalian cells has been reported (58); however, since theselenium component in the tRNA is very alkali-labile and alsosensitive to boiling at neutral pH (58), it is unlikely that any75Se-labeled macromolecules detected by autoradiography of

polyacrylamide gels represent tRNAs.We have also found, as reported by Schwarz and Sweeney

(59) in 1964, that 75Se as selenious acid or sodium selenite

when added exogeneously to a nonradioactive cell extract willbind readily to protein macromolecules;4 however, no selenium-

containing proteins were detected in these cell extracts by gelelectrophoresis and subsequent autoradiography. Presumably,the selenium is loosely bound to protein and then is released

' Unpublished data.

4587




when the samples are prepared in sample buffer and heated to100Â°Cfor 5 min. Seleni Â»risiiIlick's also would not be expected

to be stable under conditions which denature proteins such ashigh concentrations of urea, detergent, or 2-mercaptoethanol(60).

In summary, these experiments support the concept of multiple selenoproteins in mammalian cells. At the present time,the subcellular localization and functions of these proteinsremain unknown; however, the purification and subsequentanalysis of these proteins using current immunological andmolecular biology techniques should provide a fund of newinformation on the significance of these proteins in seleniumbiology and chemoprevention.

ACKNOWLEDGMENTS

The authors wish to thank Drs. Egon Durban, Fred R. Harmon, andJanice E. Knepper, Baylor College of Medicine, for valuable advice anddiscussions and Dr. C. Channa Reddy, Pennsylvania State University,for a generous gift of purified glutathione peroxidase. Thanks are alsodue Brenda Cipriano for expert typing of the manuscript.

REFERENCES

1. Schwarz, K., and Foltz, C. M. Selenium as an integral part of factor 3 againstdietary necrotic liver degeneration. J. Am. Chem. Soc., 79:3292-3293,1957.

2. Thompson, J. N., and Scott, M. L. Role of selenium in the nutrition of thechick. J. Nutr., 97: 335-342, 1969.

3. McCoy, K. E. M., and Weswig, P. H. Some selenium responses in the ratnot related to vitamin E. J. Nutr., 98: 383-389, 1969.

4. McKeehan, W. L., Hamilton, W. (... and Ham, R. G. Selenium is an essentialtrace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Nati.Acad. Sci. USA, 73: 2023-2027, 1976.

5. Griffin, A. C. Role of selenium in the chemoprevention of cancer. Adv.Cancer Res., 29:419-442, 1979.

6. Shamberger, R. J. Biochemistry of Selenium, pp. 207-271. New York:Plenum Press, 1983.

7. Medina, D. Selenium and murine mammary tumorigenesis. In: B. Reddy andL. Cohen (eds.), Diet, Nutrition and Cancer: A Critical Evaluation. Vol. 2,pp. 23-44. Boca Raton, FL: CRC Press, 1986.

8. Rotruck. J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D.G.. and Hoekstra, W. G. Selenium: biochemical role as a component ofglutathione peroxidase. Science (Wash. DC), 179: 588-590, 1973.

9. Sunde, R. A., and Hoekstra, W. G. Structure, synthesis and function ofglutathione peroxidase. Nutr. Rev., 38: 265-273, 1980.

10. Rotruck, J. T. Discovery of the role of selenium in glutathione peroxidase./;/. J. E. Spaltholz, J. L. Martin, and H. E. Ganther (eds.), Selenium inBiology and Medicine, pp. 10-16. Westport, CT.: AVI Publishing Co., Inc.,1981.

11. Lane, H. W., and Medina, D. Selenium concentration and glutathioneperoxidase activity in normal and neoplastic development of the mousemammary gland. Cancer Res., 43: 1558-1561, 1983.

12. Medina, D., Lane, H. W., and Tracey, C. M. Selenium and mouse mammarytumorigenesis: an investigation of possible mechanisms. Cancer Res., 43:2460-2464, 1983.

13. Jacobs, M. M., Forst, C. F., and Beams, F. A. Biochemical and clinicaleffects of selenium on dimethylhydrazine-induced colon cancer in rats. Cancer Res., 41: 4458-4465, 1981.

14. Birt, D. F., Lawson, T. A., Julius, A. D., Runice, C. E., and Salmasi, S.Inhibition by dietary selenium of colon cancer induced in the rat by bis(2-oxopropyl)nitrosamine. Cancer Res., 42:4455-4459, 1982.

15. Ip, C., and Siulia. D. Anticarcinogenic effect of selenium in rats treated withdimethylbenz[a]anthracene and fed different levels and types of fat. Carci-nogenesis (Lond.), 2:435-438, 1981.

16. Lane, H. W., and Medina. D. Mode of action of selenium inhibition of 7,12-dimethylbenz[a]anthracene-induced mouse mammary tumorigenesis. J. Nati.Cancer Inst., 75:675-679, 1985.

17. Horvath, P. M., and Ip, C. Synergistic effect of vitamin E and selenium inthe chemoprevention of mammary carcinogenesis in rats. Cancer Res., 43:5335-5341,1983.

18. Stadtman, T. C. Selenium-dependent enzymes. Annu. Rev. Biochem., 49:93-110, 1980.

19. Stadtman, T. C. New biologic functionsâ€”selenium-dependent nucleic acidsand proteins. Fund. Appi. Toxicol., 3: 420-423, 1983.

20. Sunde, R. A. The biochemistry of selenoproteins. J. Am. Oil Chem. Soc., 61:1891-1900, 1984.

21. Pedersen, N. D.. Whanger, P. D., Weswig, P. H., and Muth, O. H. Seleniumbinding proteins in tissues of normal and selenium responsive myopathielambs. Bioinorg. Chem., 2: 33-45, 1972.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

Whanger, P. D., Pedersen, N. D., and Weswig, P. H. Selenium proteins inovine tissues. II. Spectral properties of a 10,000 molecular weight seleniumprotein. Biochem. Biophys. Res. Commun., S3: 1031-1035, 1973.McConnell, K. P., Burton, R. M., Kute, T., and Higgins, P. J. Selenoproteinsfrom rat testis cytosol. Biochim. Biophys. Acta, 588: 113-119, 1979.Calvin, H. I. Selective incorporation of selenium-75 into a polypeptide of therat sperm tail. J. Exp. Zool., 204:445-452, 1978.Herrman, J. L. The properties of a rat serum protein labelled by the injectionof sodium selenite. Biochim. Biophys. Acta, 500: 61-70, 1977.Burk, R. F., and Gregory, P. E. Some characteristics of "Se-P, a selenopro-tein found in rat liver and plasma, and comparison of it with selenogluta-thione peroxidase. Arch. Biochem. Biophys., 213:73-80, 1982.Motsenbocker, M. A., and Tappel, A. L. Selenocysteine-containing proteinsfrom rat and monkey plasma. Biochim. Biophys. Acta, 704: 253-260, 1982.Motsenbocker, M. A., and Tappel, A. L. A selenocysteine-containing selenium-transport protein in rat plasma. Biochim. Biophys. Acta, 719: 147-153, 1982.Hawkes, W. C., Wilhelmsen, E. C., and Tappel, A. L. Abundance and tissuedistribution of selenocysteine-containing proteins in the rat. J InorgBiochem., 23: 77-92, 1985.Danielson, K. G., Oborn, C. J., Durban, E. M., Butel, J. S., and Medina, D.Epithelial mouse mammary cell line exhibiting normal morphogenesis invivo and functional differentiation in vitro. Proc. Nati. Acad. Sci. USA, 81:3756-3760,1984.Leavitt, J., Goldman, D., Merril, C., and Kakunaga, T. Actin mutations in ahuman fibroblast model for carcinogenesis. Clin. Chem., 28:850-860, 1982.O'Farrell, P. H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem., 250: 4007-4021, 1975.O'Farrell, P. Z., Goodman, H. M., and O'Farrell, P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell, /_':1133-1142,1977.Lanford, R. E., and Butel, J. S. Antigenic relationship of SV-40 early proteinsto purified large T polypeptide. Virology, 97: 295-306, 1979.Sammons, D. W., Adams, C. D., and Nishizawa, E. E. Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis,2:135-141,1981.Lane, H. W., Tracey. C. K., and Medina, D. Growth, reproduction rates andmammary gland selenium concentration and glutathione peroxidase activityof BALB/c female mice fed two dietary levels of selenium. J. Nutr., 114:323-331, 1984.Bradford, M. M. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem., 72: 248-254, 1976.Stadtman, T. C. Occurrence and characterization of selenocysteine in proteins. Methods. Enzymol., /07: 576-581, 1984.Tappel, A. L., Hawkes, W. C., Wilhelmsen, E. C., and Motsenbocker, M. A.Selenocysteine-containing proteins and glutathione peroxidase. Methods Enzymol.,/07:602-619, 1984.Lischwe, M. A., and Ochs, D. A new method for partial peptide mappingusing ,V-chlorosuccinimide/urea and peptide silver staining in sodium dodecylsulfate-polyacrylamide gels. Anal. Biochem., 127: 453-457, 1982.McConnell, K. P., and Hoffman, J. L. Methionine-selenomethionine parallels in rat liver polypeptide chain synthesis. FEBS Lett., 24:60-62, 1972.Lecocq, R. E., Hepburn, A., and Lamy, F. The use of L-["S]methionine andL-[75Se]selenomethionine for double-label autoradiography of complex protein patterns on two-dimensional polyacrylamide gels: a drastic shorteningof the exposure time. Anal. Biochem., 127: 293-299, 1982.Oh, S. H.. Ganther, H. E., and Hoekstra, W. G. Selenium as a componentof glutathione peroxidase isolated from ovine erythrocytes. Biochemistry, 13:1825-1828, 1974.Nakamura, W., Hosoda, S., and Hayashi, K. Purification and properties ofrat liver glutathione peroxidase. Biochim. Biophys. Acta, 358: 251-261,1974.Forstrom, J. W., Zakowski, J. J., and Tappel, A. L. Identification of thecatalytic site of rat liver glutathione peroxidase as selenocysteine. Biochemistry, 17: 2639-2644, 1978.Tappel, A. L. Glutathione peroxidase and its selenocysteine active site. In: J.E. Spallimi/. J. L. Martin, and H. E. Ganther (eds.). Selenium in Biologyand Medicine, pp. 44-53. Westport, CT: AVI Publishing Co., Inc., 1981.Ladenstein, R., Epp, O., Huber, R., and Wendel, A. The structure of glutathione peroxidase from bovine red blood cells. In: J. E. Spaltholz, J. L.Martin, and H. E. Ganther (eds.), Selenium in Biology and Medicine, pp.33-43. Westport, CT: AVI Publishing Co., Inc., 1981.Lyons, D. E., Wilhelmsen, E. C., and Tappel, A. L. Rapid, high-yieldpurification of rat liver glutathione peroxidase by high performance liquidchromatography. J. Liq. Chromatogr. 4: 2063-2071, 1981.ChaudiÃ¨re, J., and Tappel, A. L. Purification and characterization of sele-nium-glutathione peroxidase from hamster liver. Arch. Biochem. Biophys.,220:448-457, 1983.Motsenbocker. M. A., and Tappel, A. L. Selenium and selenoproteins in therat kidney. Biochim. Biophys. Acta, 709: 160-165, 1982.Cone, J. E., Del Rio, R. M., Davis, J. N., and Stadtman, T. C. Chemicalcharacterization of the selenoprotein component of clostridial glycine reducÃase:identification of selenocysteine as the organoselenium moiety. Proc.Nati. Acad. Sci. USA, 73: 2659-2663. 1976.Hartmanis, M. G. N., and Stadtman. T. C. Isolation of a selenium-containingthiolase from Clostridum kluyveri: identification of the selenium moiety asselenomethionine. Proc. Nati. Acad. Sci. USA, 79:4912-4916, 1982.

4588




53. Shearer, T. R., Ridlinglon, J. W., Goeger, D. E., and Whanger, P. D. Uptake 57. Ganther, H. E., Prohaska, J. R., Oh, S. H., and Hoekslra, W. G. The reactionof "Se in cataractous rat lens proteins. Biol. Trace Elem. Res.. 6: 195-205, of cyanide with glutathione peroxidase. Fed. Proc., 36: 1094, 1977.

, ,1.9^4', ,,.,.,. , , . . , , 58. Ching,W.-M. Occurrence of selenium-containing t-RNAs in mouse leukemia54. Holmberg, N J. Purification and properties of glutathione peroxidase from ce,|s Proc Na(, Acad Â«^ UÂ§Ag] 30|0_3OI3 19g4

bovine lens. Exp. Eye Res., 7:570-580, 1968. Â«noi. v , r- t- â€¢â€¢L- j- .*55. Ganther, H. E Selenotrisulfides. Formation by the reaction of thiols with 59' ?chwarf: K'' and Sweeney- E- SelenlÂ« binding to sulfur ammo acids. Fed.

selenious acid. Biochemistry, 7: 2898-2905, 1968. Proc- 2S: 421> I96456. Ganther. H. E., and Corcoran, C. Selenotrisulfides. II. Cross-linking of 60. Jenkins, K.J. Evidence for the absence of selenocystine and selenomethionine

reduced pancreatic ribonuclease with selenium. Biochemistry. S: 2557-2563. Â¡nthe serum proteins of chicks administered selenite. Can. J. Biochem.. 46:1969. 1417-1425,1968.

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1986;46:4582-4589. Cancer Res Keith G. Danielson and Daniel Medina

in Vivo and in VitroCells Distribution of Selenoproteins in Mouse Mammary Epithelial

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