Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies

Studies with 75Se have shown the existence of a rat plasma selenoprotein in addition to glutathione peroxidase. Because the function of the protein is not known, it has been referred to as selenoprotein P. A partially purified preparation was used to produce a monoclonal antibody to selenoprotein P. The antibody did not bind glutathione peroxidase as evidenced by its failure to remove glutathione peroxidase activity from rat plasma by immunoprecipitation. An immunoaffinity column was prepared with the monoclonal antibody, and selenoprotein P was purified 1270-fold from rat plasma in a two-step procedure. The purified selenoprotein P migrated in a single band with an Mr of 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography demonstrated that this band contained 75Se when the protein was purified from rats which had received 75SeO2-(3). A competitive radioimmunoassay for selenoprotein P was developed. The selenoprotein P concentration in plasma of selenium-replete rats was determined with this assay to be 51 +/- 3.7 micrograms/ml. It was less than 5 micrograms/ml in plasma from selenium-deficient rats. Injection of 50 micrograms of selenium into selenium-deficient rats caused an increase in selenoprotein P from less than 10% of control to 52% of control in 6 h. Plasma glutathione peroxidase activity increased only from 2.2 to 3.1% of control. These experiments demonstrate that rat plasma contains a selenoprotein distinct from glutathione peroxidase. The concentration of this selenoprotein is depressed in selenium deficiency, as is glutathione peroxidase activity, but selenoprotein P increases more rapidly when selenium is supplied than does glutathione peroxidase activity.     

Selenoprotein W accumulates primarily in primate skeletal muscle,heart, brain and tongue

The human selenoprotein W coding region with the selenocysteine codon (TGA) changed to a cysteine codon (TGT) was fused to six histidine codons (at its 3 end), cloned into a prokaryotic expression vector (pTrc99a), and the corresponding mutated selenoprotein W was expressed in bacteria. The protein was purified by Ni-NTA agarose column and reverse phase HPLC. Polyclonal antibodies raised against this protein were used in Western blots to determine tissue distribution of selenoprotein W from rhesus monkeys fed a commercial chow. Selenoprotein W was found in several tissues with highest amounts in skeletal muscle and heart (muscle 6 fold greater than liver) and lowest levels in liver, but selenium concentrations were highest in kidneys (10 fold greater than muscle) and lowest in skeletal muscle. Northern blots using a human selenoprotein W cDNA probe indicated that mRNA levels were highest in monkey skeletal muscle and heart (2-2.5 fold greater than in liver), which is similar to the pattern found with a human multiple tissue Northern blot. However, as in the monkey, selenium concentrations were highest in human kidney and lowest in skeletal muscle and heart. Thus, selenoprotein W protein levels correlated with selenoprotein W mRNA levels but not with tissue selenium concentrations.     

Multiple selenocysteine content of selenoprotein P in rats

Partially purified selenoprotein P from rat plasma was digested with either trypsin, endoprotease Lys-C, or endoprotease Arg-C and analyzed by high pressure liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several 75Se-labeled peptides were detected. The moles of selenium in selenoprotein P were estimated based on the 75Se content of the 75Se-labeled peptide fragments. Using this method, selenoprotein P was shown to contain approximately 9 moles of selenium. This is the first report of a selenoprotein containing more than one selenium per polypeptide. These findings support the proposed function of this protein in selenium transport.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

High‐throughput quantification of selenium in individual serum proteins from a healthy human population using HPLC on‐line with isotope dilution inductively coupled plasma‐MS

In this study, a method, based on dual column affinity chromatography hyphenated to isotope dilution inductively coupled plasma–quadrupole MS, was developed for selenium determination in selenoprotein P, glutathione peroxidase, and selenoalbumin in human serum samples from a group of healthy volunteers (n=399). Method improvement was achieved using methanol‐enhanced isotope dilution which resulted in improved sensitivity and removal of isobaric interferences. Although no human serum reference materials are currently certified for their selenium species levels, method development was conducted using human serum reference material BCR 637 and 639 as their Se species content has been reported in the previous studies, and thus comparisons were possible. The mean selenium concentrations determined for the 399 healthy volunteer serum samples were 23±10 ng Se mL^?1 for glutathione peroxidase, 49±15 ng Se mL^?1 for selenoprotein P and 11±4 ng Se mL^?1 for selenoalbumin. These values are found to be in close agreement with published values for a limited number of healthy volunteer samples, and to establish baseline Se levels in serum proteins for an apparently healthy group of individuals, thus allowing for subsequent comparisons with respective values determined for groups of individuals with selenium related health issues, as well as assist in the discovery of potential selenium biomarkers. Also, the relationship between Se serum protein levels and some anthropometric characteristics of the volunteer population were investigated. Additionally, further development of the analytical method used in this study was achieved by adding a size exclusion chromatography column after the two affinity columns via a switching valve. This allowed for the separation of small selenium‐containing molecules from glutathione peroxidase and thus enhanced the overall confidence in its identification.     

Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum

Selenoprotein P is the second plasma selenoprotein to be purified. It is a glycoprotein and has been shown to be distinct from plasma glutathione peroxidase. This study characterizes selenoprotein P further. Deglycosylation of the protein shifts its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from Mr 57,000 to Mr 43,000, indicating it has a substantial carbohydrate component. Measurement of selenium indicates a selenium content of 7.5 +/- 1.0 atoms/molecule based on a polypeptide weight of 43,000. Amino acid analysis accounts for all the selenium as selenocysteine. The protein is also rich in cysteine (17 residues) and histidine (23 residues). Fragmentation of selenoprotein P by trypsin and by cyanogen bromide produces peptides with varying selenium content. This indicates that selenium-rich regions of the protein exist. The concentration of selenoprotein P determined by radioimmunoassay in serum from control rats is 26.3 +/- 4.5 micrograms/ml and in serum from selenium-deficient rats it is 2.7 +/- 0.8 micrograms/ml. Depletion of selenoprotein P from control serum using an immunoaffinity column indicates that over 60% of serum selenium in the rat is contained in this protein. These results demonstrate that selenoprotein P is the major form of selenium in rat serum. It is the first selenoprotein described which has more than one selenium atom/polypeptide chain.     

The cDNA for rat selenoprotein P contains 10 TGA codons in the open reading frame

Selenoprotein P is a plasma protein recently purified and characterized as containing 7.5 +/- 1.0 selenium atoms/molecule as selenocysteine. In rats maintained on a defined diet containing nutritionally adequate amounts of selenate as the sole selenium source, over half the selenium in plasma is accounted for by selenoprotein P. Its cDNA has been cloned from a rat liver library and sequenced. The sequence is highly unusual, containing 10 TGA codons in its open reading frame prior to the TAA termination codon. TGA designates selenocysteine in other selenoproteins, and limited peptide sequencing that included the amino acids encoded by two of the TGA codons verified that they correspond to selenocysteine. The deduced 366-amino acid sequence is histidine- and cysteine-rich and contains 9 of its selenocysteines in the terminal 122 amino acids. Comparison of the deduced amino acid sequence of selenoprotein P with those of other selenoprotein reveals no significant similarities. Selenoprotein P represents a new class of selenoproteins and is the first protein described with more than 1 selenocysteine in a single polypeptide chain. The primary structure of selenoprotein P suggests that it might be responsible for some of the antioxidant properties of selenium.     

Size isomers of testosterone-estradiol-binding globulin exist in the plasma of individual men and women

We isolated testosterone-estradiol-binding globulin TeBG rapidly and in high yield from pooled pregnancy plasma. It showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Both bands stained with three different monoclonal antibodies to TeBG, thus demonstrating their immunological similarity. Freshly drawn, individual sera, from men, women, and pregnant patients were submitted to microaffinity chromatography, a procedure which partially purifies TeBG in approximately 4 hr. The partially purified plasma was submitted to SDS PAGE, followed by immunoblotting. The blotted TeBG exhibited the same two bands seen in the isolated, purified protein. The size heterogeneity observed in TeBG purified to: proteolysis occurring during isolation; a peculiarity of pregnancy plasma; or heterogeneity attendant upon the use of pooled plasma for isolation.     

Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

Attenuation of hepatic expression and secretion of selenoprotein P by metformin

High serum selenium levels have been associated epidemiologically with increased incidence of type 2 diabetes. The major fraction of total selenium in serum is represented by liver-derived selenoprotein P (SeP). This study was undertaken to test for a hypothesized effect of hyperglycemia and the antihyperglycemic drug metformin on hepatic selenoprotein P biosynthesis. Cultivation of rat hepatocytes in the presence of high glucose concentrations (25 mmol/l) resulted in increased selenoprotein P mRNA expression and secretion. Treatment with metformin dose-dependently downregulated SeP mRNA expression and secretion, and suppressed glucocorticoid-stimulated production of SeP. Moreover, metformin strongly decreased mRNA levels of selenophosphate synthetase 2 (SPS-2), an enzyme essential for selenoprotein biosynthesis. Taken together, these results indicate an influence of metformin on selenium metabolism in hepatocytes. As selenoprotein P is the major transport form of selenium, metformin treatment may thereby diminish selenium supply to extrahepatic tissues.     

Serum selenium and selenoprotein P status in adult Danes – 8-year followup

Selenium is an essential micronutrient important to human health. The main objective of this study is to describe serum selenium and selenoprotein P status in two samples of the Danish population. In addition, the influence of various factors potentially associated with selenium status was investigated.Blood samples from a total of 817 randomly selected subjects from two cities in Denmark were analyzed. Half of the samples were collected in 1997–1998 and the other half in 2004–2005. Samples from women aged 18–22, 40–45 and 60–65 years, and men aged 60–65 years were selected for this study. All subjects had filled in a food frequency questionnaire (FFQ) and a questionnaire with information about smoking habits, alcohol consumption and exercise habits.Mean serum selenium level was 98.7±19.8 μg/L and median selenoprotein P level was 2.72 (2.18–3.49) mg/L. Serum selenium and selenoprotein P increased with age, and selenoprotein P was higher in men than in women. Serum selenium levels decreased by 5% on average from 1997–98 to 2004–05 (P<0.001), whereas selenoprotein P level increased (P<0.001). The intake of fish correlated weakly with serum selenium level (r=0.14, P<0.001) but not with selenoprotein P level. Smoking status, alcohol intake, exercise habits, BMI and medicine use did not influence selenium status.It is concluded that selenium status in this Danish population is at an acceptable level. No major groups with regard to age, sex or lifestyle factors could be identified as being in risk for selenium deficiency.     

Antithrombin Glasgow, 393 Arg to His: a P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41-year-old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin-Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C-terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.     

Rat plasma selenoprotein P properties and purification

A selenoprotein in rat plasma, selenoprotein P, was fractionated and characterized. Plasma collected from rats 3 h post injection of 75SeO3(2-) contained one 75Se-labeled protein, selenoprotein P. Selenoprotein P was fractionated using salt precipitation, Affi-Gel Blue, and DEAE chromatography. The 75Se-containing subunit of selenoprotein P was purified to 90% homogeneity using SDS-polyacrylamide gel electrophoresis followed by electroelution. This isolation resulted in an 850-fold purification of the 75Se-containing subunit of selenoprotein P with a 15% yield of 75Se radioactivity. The molecular weight of selenoprotein P in plasma was 98,000. The 75Se-containing subunit of selenoprotein P had a molecular mass of 57 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing under nondenaturing conditions resulted in a band of 75Se radioactivity at pH 5.4. A comparison of Coomassie Blue- and silver-staining properties of selenoprotein P in SDS-polyacrylamide gels was made. Reverse-phase HPLC and Sephadex G-50 chromatography of tryptic peptides of the 57 kDa subunit of selenoprotein P yielded several peaks of 75Se radioactivity. These results indicate that 75Se is present in several locations within the 57 kDa subunit of selenoprotein P.     

A large-scale purification of beta-glucuronidase from human liver by immunoaffinity chromatography.

We intend to purify beta-glucuronidase from human liver in a large quantity in order to facilitate the study of its biochemical structure and pathophysiologic roles in cholelithiasis and carcinogenesis. The initial purification procedure involved: (1) liver homogenization, (2) 25-45% saturated ammonium sulfate fractionation, (3) heat denaturation of protein at 56 degrees C, (4) gel filtration with Bio-Gel P-300 gel, (5) anion exchange chromatography with DEAE agarose, (6) cation exchange chromatography with CM agarose, and (7) hydroxyapatite chromatography (overall yield, 1%; overall purification, 169X). The final product was used to immunize rabbits and BALB/c mice for production of polyclonal and monoclonal antibodies, respectively. The antibodies, mainly IgG, were purified by using gamma-Protein A agarose column chromatography. The purified IgG, after periodate oxidation, was coupled to hydrazide gel by formation of a stable covalent hydrazone bond linkage. The new purification procedure involved the initial first three steps, followed by (4) polyclonal IgG immunoaffinity chromatography and (5) monoclonal IgG immunoaffinity chromatography (overall yield, 6.1%; overall purification, 3720X). Polyacrylamide gel electrophoresis indicated minor contaminants in the final product which could be further purified by electroelution. It is concluded that beta-glucuronidase constitutes 0.016 mg per gram of wet liver tissue and can be obtained on a large scale in a highly purified form within a 2-day cycle.     

A selenocysteine-containing selenium-transport protein in rat plasma

A selenocysteine-containing rat plasma protein (selenoprotein P) was examined for a possible role in the transport of selenium in the rat. A time-course study of the localization of injected 75Se from [75Se]selenite indicated that one-half of the selenium was sequestered by liver tissue 1 h after injection and that one-fourth of the 75Se in the plasma was attached to selenoprotein P 3 h after injections. By 25 h there was little 75Se in plasma, and much of the 75Se had accumulated in nonhepatic tissues. 75Se was incorporated into selenoprotein P by liver slices in a process that was sensitive to the protein synthesis inhibitor cycloheximide. The fate of 75Se from intracardially injected 75Se-labeled selenoprotein P was followed in rats maintained on selenium-deficient and selenium-sufficient diets. Substantially more of the injected 75Se was present per gram wet weight in the testes and kidneys than the livers of the selenium-deprived rats after 5 h. The results indicate that selenoprotein P is synthesized in rat liver and that it transfers selenium from the liver to extrahepatic tissues.     

Purification and Identification of the Fusicoccin Binding Protein from Oat Root Plasma Membrane

Fusicoccin (FC), a fungal phytotoxin, stimulates the H⁺-ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H⁺-efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on DEAE Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between ³H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.     

Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.     

Monoclonal antibodies against human acid α-glucosidase

Acid α-glucosidase purified from human placenta was used to immunize a mouse (strain Balb/cHeA) according to a procedure described earlier (Stähli, C., Staehlin, T., Miggiano, V., Schmidt, J. and Häring, P. (1980) J. Immunol. Methods 32, 297–304). After fusion of spleen cells with myelona cells, about 10% of the hybrid clones obtained produced antibodies against acid α-glucosidase. Finally, eight stable clones producing antibodies against the enzyme were obtained. When purified acid α-glucosidase is analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, two major major protein bands (mol. wt. 76 000 and 70 000), a minor band of mol. wt. 96 000 and several minor bands with a mol. wt. of 67 000 or lower are seen. Since all these component react with the monoclonal antibodies, they must have at least one antigenic determinant in common.     

Specific antibodies to bovine choline acetyltransferase raised in mice immunised with small amounts of partially purified enzyme

Choline acetyltransferase was purified approximately 18,000-fold from 300 g of bovine caudate nuclei to a specific activity of 21 μmol min mg protein. The overall procedure used was: extraction of the enzyme by high salt concentration, chromatography on carboxy-methyl-Sephadex, precipitation by ammonium sulphate, affinity chromatography on Blue-Sepharose and, finally absorption on hydroxylapatite. When the enzyme absorbed on hydroxylapatite was injected into mice, it provoked reproducibly a transient production of ‘inhibitory’ antibodies, followed by higher antibody titres mainly of ‘non-inhibitory’ type. These responses were elicited by injecting less than a total of 20 μg of immunogen. The highest antibody titre was obtained less than 2 months following the initial immunisation. Species cross reactivity was investigated. This procedure should prove to be of value in the production of monoclonal antibodies to choline acetyltransferase.     

Selenoprotein P in subjects exposed to mercury and other stress situations such as physical load or metal chelation treatment

In plasma, Se is found in plasma glutathione peroxidase (pGSH-Px), selenoprotein P (Sel-P), and albumins. The aim of the present study was to determine the influence of lifelong exposure to various levels of mercury vapor (Hg⁰) on plasma Se content and the fraction bound to Sel-P. Second, a pilot study was performed on the influence of short-term excessive physical stress and metal chelation (DIMAVAL) treatment. Samples of human plasma/serum obtained from a control group, Idrija residents living in a Hg-polluted environment because of the vicinity of the Idrija mercury mine (closed 1994), a few Idrija residents exposed to excessive physical stress, and two retired miners treated with the drug DIMAVAL were investigated. Selenoprotein P was isolated by affinity chromatography (heparin-Sepharose), and the concentrations of selenium were determined by radiochemical neutron activation analysis and hydride generation-atomic fluorescence spectrometry. Regardless of the group investigated, plasma Se average values were very similar (about 70–90 ngSe/g). A significant change of Sel P (7–24% decrease) was noted only in the group exposed to physical stress as compared to the same subjects before the test, to the control group, and to the Hg exposed group. The decrease of Se bound on Sel-P was accompanied by its increase in fraction of pGSH-Px with albumin.