Selenoprotein P Expression, Purification, and Immunochemical Characterization
2000; Elsevier BV; Volume: 275; Issue: 9 Linguagem: Inglês
10.1074/jbc.275.9.6288
ISSN1083-351X
AutoresRosa M. Tujebajeva, John W. Harney, Marla J. Berry,
Tópico(s)Iron Metabolism and Disorders
ResumoMost selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10–12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein. Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10–12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein. polymerase chain reaction Chinese hamster ovary fetal bovine serum Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis human embryonic kidney wild type Selenocysteine, the 21st amino acid, is cotranslationally incorporated into selenoproteins at UGA codons, which typically function as stop codons. This rare amino acid is found in the active site of all selenoenzymes characterized to date. Most selenoprotein mRNAs contain a single UGA codon encoding a single selenocysteine residue per polypeptide chain and a single specific RNA secondary structure, termed a SECIS element, directing incorporation of this amino acid. Selenoprotein P is unique in that its mRNAs in different mammalian species encode 10–12 selenocysteine residues and contain two SECIS elements in the 3′-untranslated region (1.Hill K.E. Lloyd R. Yang J.G. Read R. Burk R.F. J. Biol. Chem. 1991; 266: 10050-10053Abstract Full Text PDF PubMed Google Scholar, 2.Hill K.E. Lloyd R.S. Burk R.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 537-541Crossref PubMed Scopus (161) Google Scholar, 3.Berry M.J. Banu L. Harney J.W. Larsen P.R. EMBO J. 1993; 12: 3315-3322Crossref PubMed Scopus (349) Google Scholar, 4.Saijoh K. Saito N. Lee M.J. Fujii M. Kobayashi T. Sumino K. Mol. Brain Res. 1995; 30: 301-311Crossref PubMed Scopus (50) Google Scholar). Selenoprotein P is a glycoprotein that carries up to 50% of the selenium in plasma, but its function is unknown. Although originally identified in plasma and shown to be expressed in liver, more recent studies have identified selenoprotein P mRNA in a wide variety of tissues. An abundance of selenoprotein P message is present in specific regions of the brain, in kidney, testis, lung, and heart (4.Saijoh K. Saito N. Lee M.J. Fujii M. Kobayashi T. Sumino K. Mol. Brain Res. 1995; 30: 301-311Crossref PubMed Scopus (50) Google Scholar, 5.Dreher I. Schmutzler C. Jakob F. Kohrle J. J. Trace Elem. Med. Biol. 1997; 11: 83-91Crossref PubMed Scopus (61) Google Scholar, 6.Burk R.F. Hill K.E. J. Nutr. 1994; 124: 1891-1897Crossref PubMed Scopus (157) Google Scholar). Selenoprotein P cDNA sequences in the GenBankTM data bases indicate expression in human thyroid, prostate, a T cell lymphoma, and an endometrial tumor, as well as in mouse myotubes and mammary gland. Response elements for hepatic nuclear factor, HNF3, and a brain-specific transcription factor, BRN-2, are present in the murine gene promoter (7.Steinert P. Bachner D. Flohe L. Biol. Chem. 1998; 379: 683-691Crossref PubMed Scopus (42) Google Scholar), consistent with expression of the mRNA in these tissues. Expression of selenoprotein P has been shown to be repressed by cytokines in HepG2 cells (8.Dreher I. Jakobs T.C. Kohrle J. J. Biol. Chem. 1997; 272: 29364-29371Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) and induced by dexamethasone treatment in normal rat kidney cells (9.Fujii M. Saijoh K. Sumino K. Kobe J. Med. Sci. 1997; 43: 13-23PubMed Google Scholar), suggesting possible roles in inflammatory responses and repression during the acute phase reaction. Immunohistochemical localization of selenoprotein P revealed its presence in the liver and brain bound to capillary endothelial cell walls. In kidney, the protein is found associated with the linings of the glomerulus but not in the tubules (10.Burk R.F. Hill K.E. Boeglin M.E. Ebner F.F. Chitum H.S. Histochem. Cell Biol. 1997; 108: 11-15Crossref PubMed Scopus (75) Google Scholar). In vivo, selenoprotein P is thought to serve as an antioxidant in protection from diquat-induced liver necrosis (11.Burk R.F. Hill K.E. Awad J.A. Morrow J.D. Kato T. Cockell K.A. Lyons P.R. Hepatology. 1995; 21: 561-569PubMed Google Scholar, 12.Burk R.F. Lawrence R.A. Lane J.M. J. Clin. Invest. 1980; 65: 1024-1031Crossref PubMed Scopus (220) Google Scholar). Observed in vitro functions include exhibiting a low level extracellular phospholipid hydroperoxide glutathione peroxidase-like activity (13.Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), promoting survival of neurons in primary culture (14.Yan J. Barrett J.N. J. Neurosci. 1998; 18: 8682-8691Crossref PubMed Google Scholar), and binding to complexes consisting of equimolar ratios of heavy metals and selenium,i.e. Hg-Se, Cd-Se, and Ag-Se (14.Yan J. Barrett J.N. J. Neurosci. 1998; 18: 8682-8691Crossref PubMed Google Scholar, 15.Yoneda S. Suzuki K.T. Biochem. Biophys. Res. Commun. 1997; 231: 7-11Crossref PubMed Scopus (160) Google Scholar, 16.Mostert V. Lombeck I. Abel J. Arch. Biochem. Biophys. 1998; 357: 326-330Crossref PubMed Scopus (57) Google Scholar). The protein has also been proposed to function in selenium transport or storage (6.Burk R.F. Hill K.E. J. Nutr. 1994; 124: 1891-1897Crossref PubMed Scopus (157) Google Scholar). The localization of selenoprotein P to the capillary endothelial cells may correlate with protection from products of oxidative stress or as a barrier to heavy metal uptake by cells. In addition to the presence of 10–12 selenocysteines, the protein has a high content of cysteine and histidine residues. The rat and human selenoprotein P sequences each encode two histidine-rich regions; the first region consists of 8 (rat) or 9 (human) histidines out of 14 residues, and the second a stretch of 7 (rat) or 4 (human) consecutive histidines. These regions, in conjunction with the cysteine and selenocysteine content, are likely responsible for the observed coordination to heavy metals (17.Suzuki K.T. Sasakura C. Yoneda S. Biochim. Biophys. Acta. 1998; 1429: 102-112Crossref PubMed Scopus (109) Google Scholar). This property has been utilized in designing affinity purification strategies, employing either zinc-Sepharose (14.Yan J. Barrett J.N. J. Neurosci. 1998; 18: 8682-8691Crossref PubMed Google Scholar) or nickel-agarose (16.Mostert V. Lombeck I. Abel J. Arch. Biochem. Biophys. 1998; 357: 326-330Crossref PubMed Scopus (57) Google Scholar). All studies to date reporting characterization or functional studies of selenoprotein P have relied on protein expressed endogenously by various cell lines or purified from plasma or serum. Progress has been made in defining the biochemical properties, such as heavy metal binding, and correlation with physiological effects, e.g.antioxidant activity and extension of neuronal survival. However, the function(s) and particularly the mode of action of selenoprotein P remain elusive, in part due to the lack of a transgenic or knockout model and the reported inability to express the recombinant protein in mammalian cells. Similarly, little is known about the efficiency of selenocysteine incorporation into selenoprotein P, either in cell lines or in the intact animal. Studies in transfected mammalian cells (18.Berry M.J. Harney J.W. Ohama T. Hatfield D.L. Nucleic Acids Res. 1994; 22: 3753-3759Crossref PubMed Scopus (87) Google Scholar) and in bacteria (19.Suppmann S. Persson B.C. Bock A. EMBO J. 1999; 18: 2284-2293Crossref PubMed Scopus (70) Google Scholar) suggest that selenocysteine incorporation at a single site per polypeptide may be inefficient. If so, this raises the question of how the translation machinery produces full-length selenoprotein P, outcompeting termination at 10–12 UGA codons. Herein, we describe expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line. We show that the expressed rat protein is glycosylated to a similar extent as human and hamster selenoprotein P expressed endogenously in cell lines from these two species. Taking advantage of the histidine-rich nature of the proteins, we utilized nickel-agarose affinity chromatography to purify the expressed recombinant protein. We show that commercially available antibodies prepared against 4 or 5 consecutive histidines detect the expressed protein in both Western blotting analysis and immunoprecipitation assays. The reactivity of these antibodies with unlabeled selenoprotein P in Western blots thus circumvents the need to use radiolabeled selenium to study the protein. These results provide an excellent foundation for investigation of the functions of selenoprotein P, as well as the mechanism and efficiency of selenocysteine incorporation into this protein. The rat selenoprotein P cDNA was a generous gift of Kristina Hill (Vanderbilt University, Nashville, TN). Two upstream PCR1 primers (Life Technologies Inc.), GGAATTCCGCAATGT GGAGAAGCCTAGGGCTTG and GGAATTCCACCATGG GGAGAAGCCTAGGGCTTG, were designed corresponding to the region of the initiation codon (italics). One amplifies the wild-type sequence, in which the ATG codon is found in a context predicted to be unfavorable for initiation (GCA ATG T). A second primer was designed to create a favorable Kozak context (ACC ATG G). These primers introduce an EcoRI site (underlined) just upstream of the coding region. A downstream PCR primer complementary to nucleotides 2550–2532 in the 3′-untranslated region and encoding a NotI site was used in conjunction with the upstream primers to amplify the selenoprotein P coding and 3′-untranslated region sequences. The ∼2.5-kilobase pair PCR products were subcloned into pUHD10-3 vector (20.Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar) via the unique EcoRI site and a NotI site introduced adjacent to the XbaI site. Constructs were verified by dideoxy sequencing to ensure that no mutations were introduced by PCR. Transient transfections in human embryonic kidney (HEK) 293 cells were carried out using the calcium phosphate method of transfection as described previously (21.Berry M.J. Kieffer J.D. Harney J.W. Larsen P.R. J. Biol. Chem. 1991; 266: 14155-14158Abstract Full Text PDF PubMed Google Scholar). Three days prior to transfection, cells were plated onto 60-mm culture dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were transfected with 10 μg of the pUHD10-3 based expression plasmids and 4 μg of the pUHD15 plasmid (20.Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar), which encodes a protein necessary for transcriptional activation of the pUHD10-3 promoter. To monitor transfection efficiencies, cells were cotransfected with 3 μg of an expression vector containing the human growth hormone cDNA under control of the herpes simplex virus-thymidine kinase promoter. On the day of transfection, media were changed to DMEM supplemented with 1% FBS. The day following transfection, media were changed to DMEM without serum to reduce the amount of albumin in the preparations. For labeling studies,75Se (3 μCi/ml) was added to the media at this time. Two days after transfection, media and cells were harvested. Cells were washed in phosphate-buffered saline and resuspended in 0.1m potassium phosphate, pH 6.9, 1 mm EDTA containing 0.25 m sucrose. For studies of the effects of selenium supplementation, graded amounts of sodium selenite were added to media the day before transfection. HepG2 human hepatoma cells were maintained in DMEM supplemented with 10% FBS. Chinese hamster ovary (CHO) cells were maintained in a 1:1 mixture of DMEM and Ham's F-12, supplemented with 10% FBS. Transfection with DEAE-dextran was performed according to the procedure described (22.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1992: 9.1.2Google Scholar). DEAE-dextran was prepared in phosphate-buffered saline supplemented with 0.1 mm chloroquine. Media were changed to DMEM without serum at the time of addition of 75Se as above, and labeling was continued for 24 h. Polysome preparation from transiently transfected HEK 293 cells was carried out according to a modification of the protocol described by White et al. (23.White M.W. Kameji T. Pegg A. Morris D.R. Eur. J. Biochem. 1987; 170: 87-92Crossref PubMed Scopus (58) Google Scholar). Cells were harvested as above, except that cycloheximide was added to 100 μg/ml 20 min before harvest. Cells combined from three 100-mm dishes were washed in PBS buffer and resuspended in low salt buffer (LSB, 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 3 mm MgCl2) containing 1 mm RNasin, 1 mm dithiothreitol. Cells were homogenized in a Dounce homogenizer in LSB containing 1.2% Triton X-100 and 0.25 m sucrose. Postmitochondrial supernatants were prepared by centrifugation in a benchtop microcentrifuge at 12,000 × g for 5 min. NaCl and MgCl2concentration were adjusted to 150 and 10 mm, respectively. Linear sucrose gradients (15–50% w/w in LSB) were prepared using a Searle Densi-flow IIC gradient maker with a sublayer of 1 ml of Fluorinert (ISCO, Lincoln, NE). Cytoplasmic extracts (1 ml) were layered onto 10.6-ml gradients and centrifuged at 150,000 ×g for 2 h. Fractions (0.75 ml) were collected using a gradient fractionator (Brandel) equipped with an ISCO UA-6 UV detector. Polysome profiles were detected by absorbance at 254 nm. RNA was isolated from 250 μl of each gradient fraction using TRI REAGENT LS (Molecular Research Center, Inc., Cincinnati) according to the manufacturer's specifications. RNAs were electrophoresed on denaturing agarose gels and vacuum-blotted to Nitran membrane (Schleicher & Schuell). A DNA fragment from the selenoprotein P cDNA (NspI fragment, 783 base pairs) was labeled by random priming (Random Primer Labeling Kit, Stratagene). Hybridization was carried out at 65 °C followed by high stringency washing at 65 °C. Protease or proteasome inhibitors were added to media at the time of 75Se labeling. CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals) containing inhibitors of chymotrypsin, thermolysin, papain, Pronase, and trypsin was used according to the manufacturer's specifications.N-Acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN, Sigma), a calpain I inhibitor (24.Sasaki T. Kishi M. Saito M. Tanaka T. Higuchi N. Kominami E. Katunuma N. Murachi T. J. Enzyme Inhibit. 1990; 3: 195-201Crossref Scopus (194) Google Scholar), was added to media at a final concentration of 50 μm. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), a proteasome uptake inhibitor (25.Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (87) Google Scholar), was added at a final concentration of 10 μm. Deglycosylation was performed using theN-Glycosidase F Deglycosylation Kit from Roche Molecular Biochemical according to the manufacturer's specifications. Nickel-agarose chromatography was performed using nickel-nitrilotriacetic acid spin columns (Qiagen, Valencia, CA) or Bio-Rad poly-prep columns (2-ml bed volume) filled with nickel-nitrilotriacetic acid. Columns were equilibrated with protein binding (PB) buffer, consisting of 20 mm Tris-HCl, pH 8.0, 0.3 m NaCl; 10 mm β-mercaptoethanol, 10% glycerol. Media from transfected cells (up to 3 column volumes) were allowed to percolate into the columns by gravity. Column resin was washed at least 3 times with PB buffer containing 0.5 m NaCl. Specifically bound proteins were eluted by washing columns with PB buffer containing 0.5m NaCl and 100–400 mm imidazole. If necessary, proteins were concentrated in Viva-spin columns (VivaScience, Lincoln, UK). Tetra-histidine- and penta-histidine-specific antibodies were obtained from Qiagen. Primary antibodies were diluted according to the manufacturer's specifications. Typically 5 μl were incubated with 300 μl of selenium-labeled media from cells. Immunoprecipitation was carried out according to Ref. 41.Qiagen Inc QIAexpress Detection and Assay Handbook. Qiagen, Valencia, CA1998Google Scholar except that Pansorbin (Calbiochem) was used as a source of protein A. Samples were boiled for 5 min in SDS sample buffer containing 0.5 m β-mercaptoethanol, and electrophoresed on 10% acrylamide gels (acrylamide:bisacrylamide, 29:1) or 12% ReadyGels (Bio-Rad). Samples were subjected to SDS-PAGE analysis on 10 or 12% polyacrylamide gels (acrylamide:bisacrylamide, 29:1), followed by electrotransfer to Immobilon membranes (Millipore, Bedford, MA) in 20% methanol, 25 mm Tris-HCl, pH 8.3, 192 mm glycine. Membranes were blocked with 3% (w/v) bovine serum albumin in TBS (10 mm Tris-HCl, pH 7.5, 150 mm NaCl), incubated with histidine-specific antibodies (Qiagen) at 1:1500 dilution in 3% (w/v) bovine serum albumin in TBS and then washed in TBS/Triton/Tween according to the manufacturer's instructions. This was followed by incubation with goat anti-mouse horseradish peroxidase-conjugated secondary antibody in 10% (w/v) nonfat milk in TBS (1:6000 dilution, Roche Molecular Biochemicals). Reaction products were visualized by enhanced chemiluminescence (Roche Molecular Biochemicals) and exposure to X-Omat film (Eastman Kodak Co., Rochester, NY). After extensive washing of the membrane and decay of the chemiluminescent signal the membrane was subjected to autoradiography. We hypothesized that one possible reason for the reported inability to express selenoprotein P in transfected cells was due to the unfavorable context, GCA ATG T, of the initiation codon in the wild-type (w.t.) rat selenoprotein P sequence. To test this hypothesis, the w.t. sequence was mutated to the Kozak consensus sequence (26.Kozak M. EMBO J. 1997; 16: 2482-2492Crossref PubMed Scopus (414) Google Scholar), ACC ATG G. W.t. and mutant selenoprotein P expression constructs were then transiently transfected into HEK 293 cells using the calcium phosphate precipitation method. We chose to visualize the expression of selenoprotein P first by polysome analysis. Determination of the number of ribosomes associated with an RNA reflects its translational activity. Cytoplasmic extracts were prepared and loaded onto sucrose gradients for polysome isolation and fractionation. The A 260 profile of a representative gradient is shown in Fig.1. RNA was prepared from each gradient fraction and analyzed by Northern hybridization to localize selenoprotein P mRNA in the gradient. Fig. 1 B shows that selenoprotein P mRNA expressed from the w.t. mRNA is found in fractions ranging from monosomes up to at least 7–8 ribosomes, with the peak of message in the four-ribosome region. This indicates that ribosomes are efficiently loaded onto the mRNA. No differences were detected in polysome profiles from the w.t. versus the Kozak consensus construct (not shown). Thus, problems with initiation of protein biosynthesis seem not to be the explanation for the reported inability to express selenoprotein P. Since selenoprotein P mRNA appeared to be translated efficiently, we analyzed media from cells transiently transfected with the selenoprotein P expression plasmid and labeled with 75Se. Forty-eight hours after transfection and after a 24-h labeling period,75Se-labeled selenoprotein P was present in the media from transfected cells (Fig. 2, lane 2). The recombinant protein migrated with the observed molecular mass of full-length or near full-length selenoprotein P, ∼57 kDa. No endogenously expressed selenoprotein P was detectable in the media from HEK cells following transfection with vector alone (Fig. 2, lane 1). We also analyzed the labeled selenoprotein profile in cell lysates. We were unable to detect any differences in the cell lysate labeling pattern between the selenoprotein P and vector-transfected cells. Thus, no new selenoprotein bands corresponding to the sizes of prematurely terminated or non-glycosylated selenoprotein P species were seen in cell lysates following transfection. However, the high background of endogenous selenoproteins in the size range of the full-length protein, including thioredoxin reductases and selenophosphate synthetase, might easily mask full-length or near full-length glycosylated selenoprotein P associated with cells (see below). Endogenous selenoproteins in the size range corresponding to cytoplasmic and phospholipid hydroperoxide glutathione peroxidase were also present in these cells. We next investigated the abilities of other cell lines to express the recombinant rat protein. Following 75Se labeling of CHO cells, endogenously expressed, secreted selenoprotein P was easily detected in the media (Fig. 2, lane 3). However, the expression of recombinant selenoprotein P was negligible, as there was no increase in the amount of selenoprotein P observed after transfection of the plasmid encoding the rat protein (not shown). The human hepatoma cell line, HepG2, is known to express several selenoproteins, including type 1 deiodinase 2R. M. Tujebajeva, J. W. Harney, and M. J. Berry, unpublished observations. and selenoprotein P (5.Dreher I. Schmutzler C. Jakob F. Kohrle J. J. Trace Elem. Med. Biol. 1997; 11: 83-91Crossref PubMed Scopus (61) Google Scholar). Following 75Se labeling, HepG2 cells also produced endogenous selenoprotein P (Fig. 2, lane 4) but no additional selenoprotein P upon transfection of the plasmid (not shown). Strikingly, the patterns of labeled proteins differed from these three cell lines. Transfection of the rat cDNA into HEK 293 cells produced a single prominent selenoprotein migrating as ∼57 kDa, whereas the human protein produced endogenously by HepG2 cells migrated slightly more slowly, in the 60-kDa size range. As the deduced molecular masses only differ by ∼170 Da, this may reflect differences in extent of glycosylation. In CHO cells, a faster migrating selenoprotein of ∼45 kDa was also observed, in addition to the ∼57-kDa protein. This may correspond to a prematurely terminated form of the protein (see "Discussion"). One potential difficulty in determining the extent of premature termination of endogenous or recombinant expressed selenoprotein P from these cell lines is that these products may be rapidly degraded within the cell, underestimating the level of termination. This has been shown to be the case for the UGA-terminated product produced upon transfection of type 1 deiodinase cDNA in HEK cells (see below). To address this question, we examined the effects of adding protease or proteasome inhibitors to cells during in vivo 75Se labeling. We tested the calpain I-specific protease inhibitor, ALLN (Sigma), which stabilizes the type 1 deiodinase UGA-termination product. 3G. Warner, personal communication. Addition of either ALLN or a protease inhibitor mixture, CompleteTM (Roche Molecular Biochemicals), had no detectable effect on the patterns of selenoproteins either in the media or in cell lysates (not shown) from any of the three cell lines. Similarly, the proteasome uptake inhibitor, MG132, did not affect the selenoprotein patterns. As selenium may be limiting for expression of selenoprotein P, we examined the effects of supplementation of media with unlabeled selenium. Sodium selenite was added to a final concentration of 0, 10, 30, or 100 nm the day before transfection. The selenium concentration in the "0 added" condition is estimated at less than 1 nm. Thus, the increase in unlabeled selenium to 100 nm results in a >100-fold decrease in specific activity. Supplementation with 10, 30, or 100 mm selenium led to 1.2-, 3.6-, and 7.2-fold increases, respectively, in labeling intensityversus the unsupplemented lane (data not shown). Examination of the labeling intensity over this range revealed a progressive increase in incorporation of label into selenoprotein P despite the large decrease in specific activity, indicating a dramatic increase in selenoprotein P production. No apparent changes in the ratios of individual labeled bands were detected with the increase in selenium concentration. In addition, no detectable differences in cell growth or morphology were observed during the time course of the experiment. Previous studies have shown that rat selenoprotein P undergoes N-linked glycosylation in at least three of the five potential sites (27.Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To determine if some of the observed differences in migration patterns could be accounted for by differences in glycosylation, selenoprotein P preparations were treated with endoglycosidase F. A decrease in the apparent molecular weights of the selenium-labeled proteins from all three cell lines was observed, indicating that the protein is glycosylated in all three lines (Fig.3). The mobility of the HepG2 human protein decreased from an apparent molecular mass of ∼60 to ∼45 kDa and the recombinant rat protein from ∼57 to ∼45 kDa. The larger and smaller selenoprotein species from CHO cells (∼57 and ∼45 kDa) decreased to ∼38 and ∼26 kDa following endoglycosidase treatment, representing changes of ∼19 kDa. This latter change indicates that the two forms produced by CHO cells are probably glycosylated to a similar extent (Fig. 3, lane 3 versus 4). Although the majority of selenoprotein P was expected to be in the media, and no premature termination products or deglycosylated forms were detected in the cell lysate, some glycosylated selenoprotein P might be present in the cell lysate fraction, due to either association with the outer cell membrane or perhaps in intracellular compartments in the process of being secreted. Glycosylated selenoprotein P would be predicted to comigrate with other endogenous selenoproteins, making it difficult to detect. We might be able to reveal its presence by deglycosylation, thus increasing its mobility relative to the other endogenous selenoproteins. Lysates from HEK cells transfected with the selenoprotein P plasmid or empty vector were treated with endoglycosidase F and analyzed by SDS-PAGE in parallel with untreated lysates and treated and untreated media. Upon overexposure of the autoradiogram, a very faint band, corresponding in size to the major selenoprotein P band in endoglycosidase-treated media, was detected in the endoglycosidase-treated selenoprotein P-transfected lysate (data not shown). This band was not seen in the endoglycosidase-treated vector-transfected lysate. Thus, the only selenoprotein P-specific band detectable in cell lysates corresponds to full-length or near full-length protein. As previous studies have utilized nickel-agarose or zinc-Sepharose chromatography to purify selenoprotein P from serum, we wanted to determine if the recombinant selenoprotein exhibited similar chromatographic behavior. Media were harvested from HEK 293 cells following transfection of the rat selenoprotein P plasmid and75Se labeling. Media (Fig. 4,lane 1) were percolated through a bed of nickel-agarose resin, the flow-through fraction collected (lane 2), and the resin washed with buffer containing 0.5 m NaCl to elute nonspecifically bound proteins (Fig. 4, lanes 3–5). This was followed by washes in the same buffer containing 100, 200, or 400 mm imidazole to elute specifically bound protein (lanes 6–8). The majority of the labeled protein eluted in the 100 mm imid
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