https://orcid.org/0000-0003-1018-5232
Abstract
We previously used high pressure liquid chromatography coupled with Se-specific inductively coupled plasma mass spectrometry and molecule specific (ESI Orbitrap MS/MS) detection to study the increase in liver Se in turkeys and rats supplemented as selenite in high-Se (5 µg Se/g diet) and adequate-Se diets. We found that far more Se is present as selenosugar (seleno-N-acetyl galactosamine) than is present as selenocysteine (Sec) in true selenoproteins. In high-Se liver, the increase in liver Se was due to low molecular weight selenometabolites such as glutathione-, cysteine-, and methyl-conjugates of the selenosugar, but also as high molecular weight species as selenosugars decorating general proteins via mixed Se-S bonds. To demonstrate selenosugar binding to proteins, aqueous liver extracts from animals fed Se-adequate and high-Se were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Native-PAGE with and without pretreatment with β-mercaptoethanol (βME). The separated proteins were then electrophoretically transferred to membranes, and the membranes subsequently were subjected to laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) analysis of 78Se profiles. Without βME treatment, Se was widely distributed across the molecular weight profile for both SDS-PAGE and Native-PAGE, whereas βME pretreatment dramatically reduced Se binding, reducing the profile to true Sec-selenoproteins. This reduction was ∼50% for both high-Se rat and turkey extracts. The increased Se in non-βME treated samples was distributed across the full profile. The use of LA-ICP-MS indicates that selenosugar residues are bound to protein subunits of multiple sizes, and that targeted attachment of selenosugars to a single or limited number of protein subunits does not occur.
βME pretreatment releases selenosugar from selenosugar-decorated proteins resulting in just true selenoproteins in PAGE profiles
Introduction
Animals accumulate selenium (Se) in tissues when supplemented with high dietary Se, whereas selenoenzymes only increase marginally above Se-adequate levels when fed high dietary Se [1]. Our initial focus was on the turkey because they have higher dietary Se requirements (0.4 µg Se/g diet) than rodents (0.1 µg Se/g diet) for maximal glutathione peroxidase-1 (Gpx1) activity, and are more resistant to Se toxicity [2]. We previously used high pressure liquid chromatography (HPLC) coupled with Se-specific inductively coupled plasma mass spectrometry (ICP—MS) and molecule specific (ESI Orbitrap MS/MS) detection to study the six-fold increase in liver Se in turkeys fed 5 vs. 0.4 µg Se/g diet as selenite [3]. We found that far more Se is present as selenosugar (seleno-N-acetyl galactosamine, SeGalNac) in adequate-Se turkey liver than is present as selenocysteine (Sec) in true selenoproteins. In high-Se liver, the increase in liver Se was due to both low molecular weight (LMW) selenometabolites such as glutathione-, cysteine-, and methyl-conjugates of the selenosugar, but also as high molecular weight (HMW) species with selenosugar apparently decorating general proteins via mixed Se-S bonds, as dithiothreitol (DTT) treatment released the parent selenosugar. In high-Se turkey liver, these ‘selenosugar-decorated’ proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to the LMW selenometabolites [3]. Subsequently, we studied selenometabolites in rats fed graded levels (0–5 µg Se/g diet) as either selenite [4] or selenomethionine [5], and also found substantial apparent selenosugar-decorated proteins in rats fed high dietary Se.
In the turkey, DTT treatment of the aqueous HMW Se species released the majority of the Se which was shown by Orbitrap ESI-MS/MS to be the parent selenosugar [3]. Similarly in the rat, DTT reduction of the HMW liver fraction released the parent selenosugar as shown by ESI-MS/MS, although DTT treatment of the aqueous HMW Se species only removed a fraction of the Se [4]. In 1988 [6] using conventional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the labelling of selenoproteins in rats administered 75Se, we reported that pretreatment of samples with SDS plus β-mercaptoethanol (βME), followed by fixation, gel slicing, and 75Se counting; this resulted in electrophoretically removing Se from Se-binding proteins which otherwise obscured true selenoproteins in conventional gel filtration chromatography. Importantly in these studies, cycloheximide pretreatment to prevent protein synthesis completely blocked incorporation of 75Se into protein in rat liver, demonstrating that SDS-PAGE plus βME could be used to distinguish true selenoproteins from Se binding proteins [6]. Thus, our approach here was to develop PAGE without βME vs. plus βME reduction to further examine selenosugar binding to proteins.
The use of laser-ablation coupled to ICP-MS to analyse for Se species in biological samples was proposed as early as 2002; eight and five Se-containing protein spots from American avocet and largemouth bass were reported, respectively, after reduction with βME [7]. This and later studies were focused solely on selenoproteins, and exclusively used reductive pretreatments with βME [8–10] and/or DTT [9, 11–15] to release LMW Se species; thus just true Sec-containing selenoproteins and selenomethionine (SeMet) proteins (containing SeMet incorporated into the peptide backbone in place of methionine) were detected. Often alkylation with iodoacetate or iodoacetamide was also used to prevent oxidation and loss of Sec [12–16]. These laser-ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) studies detected Gpx in red blood cells [11, 16, 17], apparent Se-containing proteins in yeast [14, 16], formate dehydrase in E. coli [9], multiple selenoproteins in Desulfococcus multivorans [9], SeMet and Sec substitution for methionine and cysteine (Cys) residues in glutenin and gamma-gliadin proteins in Se-enriched wheat [13] and rice [15], only Sec-substitution for Cys in ten 32–91 kDa proteins in Lactobacillus reuteri with no SeMet substitution [10], multiple Se-containing proteins in African catfish [12], and even calmodulin with SeMet substitution for methionine expressed in E. coli [8]. A number of these LA-ICP-MS studies directly compared PAGE with more conventional protein separation methods, and further used tryptic digestion and subsequent electrospray ion MS to identify specific detected selenoproteins [17]. This focus on selenoproteins using reduction and acetylation [18], however, excluded Se-binding selenoproteins, including species linked via S-Se linkages. One group [10] further noted that ‘considerable amounts of the Se was not covalently bound to protein but rather nonspecifically complexed to HMW compounds,’ and thus was not detected due to use of denaturing conditions. They proposed that this system could be used to study covalent and noncovalent Se binding.
To further examine selenosugar binding to proteins, and to develop tools to more thoroughly characterize the nature of these proteins, aqueous liver extracts from animals fed Se-adequate and high dietary Se were subjected to SDS-PAGE and Native-PAGE with and without pretreatment with βME. The separated proteins were then electrophoretically transferred to membranes, and the membranes subsequently were subjected to LA-ICP-MS for analysis of 78Se profiles. We found that without βME treatment, Se was widely distributed across the protein molecular weight profile for both SDS-PAGE and Native-PAGE of turkey and rat liver extracts, whereas βME pretreatment dramatically reduced Se binding across the profile, especially for high-Se samples, stripping away released Se and revealing just specific Se labelling of true selenoproteins. While we had previously shown that DTT reduction released parent selenosugars from bulk HMW fractions, the paired −/+ βME treatments and LA-ICP-MS were used here to examine selenosugar binding to proteins of mixed sizes.
Methods
Diet and animals
This study was approved by the Research Animal Resources Committee at the University of Wisconsin–Madison (protocol no. A005368). The present study used liver from previous studies where day-old male turkey poults were supplemented with 0.4 µg Se/g (adequate-Se) or 5 µg Se/g diet (high-Se) as selenite for 28 days [3], and where 21–23 day old weanling male rats were supplemented with 0.08 µg Se/g (adequate-Se) or 5 µg Se/g diet (high-Se) as selenite for 28 days [4].
Aqueous extraction of Se species
As described previously [3], replicate (n = 3 per treatment) samples of –80°C liver were freeze-dried, and then homogenized. Portions (∼100 mg) of each homogenized sample were extracted in 1 ml water by incubation at 25°C for 1.5 h, with vigorous vortexing every 15 min. Supernatants were prepared by centrifugation (16 000 ×g × 30 min at 5°C), and aliquots were removed for protein determination by the Lowry protein assay [19]. Aqueous extracts were frozen at –20°C until PAGE.
PAGE
For SDS-PAGE [6, 20], samples were prepared to load 250 µg protein per lane in 30 µl onto BioRad (#5 671 093) 4%–20% TGX precast gels, by diluting extracts sufficiently with MilliQ water and combining with an equal amount of BioRad 2X Laemmli sample buffer, which contains SDS but not βME. For βME pretreatment, 2.2% βME replaced MilliQ water in the loaded sample. Samples were vortexed, then boiled for 7 min, and then remixed.
For Native-PAGE, samples were prepared to load 250 µg protein per lane in 30 µl onto BioRad (#5 671 043) 12% TGX precast gels, by diluting extracts with sufficient MilliQ water and combining with an equal amount of BioRad 2X Native sample buffer. For βME pretreatment, 2.2% βME replaced the MilliQ water in the loaded sample. Samples were vortexed, incubated for 30 min at 37°C, and remixed.
For both SDS- and Native-PAGE, samples pretreated with βME were analysed on separate gels from untreated samples to prevent βME effects on untreated samples. Both individual replicates (n = 3 per treatment) and treatment equal-protein mixes were also analysed. For each gel, 10 µl coloured MW standards (containing SDS and DTT) were loaded. Electrophoresis was conducted using a BioRad Criterion Vertical Electrophoresis Cell (#1 656 001) at 200 V until the dye front was close but not off the gel, following manufacturer's protocols.
Electrophoretic transfer
Gels were immediately removed and equilibrated in transfer buffer for 10 min. Membranes (BioRad PVDF #1 620177, 13 × 8.7 cm) were incubated (1–2 min) in 100% methanol. Using a BioRad Criterion Blotter (#1 704 070), the transfer sandwich was assembled, and gels were electrophoresed at 100 V for 40 min. After sandwich disassembly, the membrane was gently incubated in phosphate buffered saline (PBS, pH 7.4) for 10 min. Membranes were placed protein-side up on a paper towel, immediately photographed, and dried overnight.
For some blots, duplicate gel lanes were quickly separated from the blot, returned to the PBS and stained with Ponceau for ∼5 min, washed briefly with fresh PBS, photographed immediately, and then dried overnight.
LA-ICP-MS
As described previously [10, 21], a NewWave Research 213 laser (Freemont, CA) was coupled with Agilent 7700 ICP-MS (Agilent, Hachi-Oji, Japan) using a 60 cm Tygon tube (5.0 mm i.d.). The laser was operated in a focused spot mode at the repetition rate of 20 Hz with a scan speed of 100 µm/s and 250 µm spot size. Ablation was carried out with a He gas flow of 800 µl/min. The ablated aerosol was mixed in a T-connector with carrier gas of ICP MS delivered at 1.1 l min/min aerosol (with the Micromist nebulizer and a double pass Scott spray chamber removed from the configuration). Pulse energy was 30% and fluence was 1.45 J/cm2. 78Se was monitored in the collision cell mode.
Western blotting
In a preliminary experiment, frozen liver from adequate-Se rats was homogenized to prepare supernatant as described previously [6, 22]. The sample was 50 µg supernatant protein with an equal amount of BioRad 2X Laemmli sample buffer with 2% βME. SDS-PAGE and electrophoretic transfer were conducted for LA analysis, and conventional immunoblot analyses on mini-blots were performed as described [23]. Briefly, the membrane was first stained with Ponceau S solution (Sigma, P7170, 2 ml in 20 ml in PBS) to show relative total protein loading. Immunoblotting was initiated by blocking membranes for 1 hr in 5% blotting-grade blocker (Bio-Rad 1 706 404). The primary antibody was Sigma anti-Gpx1 (SAB 1302824 from rabbit) at 1:1000 dilution in blocking buffer applied at room temperature (RT) for 1 h with rocking. The membrane was then washed 3 × 5 min with wash buffer, and secondary antibody (Sigma A0545 anti-rabbit peroxidase, 1:100 000 in blocking buffer) was applied for 1 h at RT with rocking, followed by washing 2 × 10 min and then 2 × 5 min with wash buffer. The membrane was incubated in chemiluminescent reagent (ThermoFisher P134095) for 5 min with rocking, then transferred to a heat-sealed bag and exposed to X-ray film (Thermo Fisher 34 091) for 15 s.
Statistical analysis
Three individual liver samples for each treatment were subjected to SDS- and Native-PAGE as described to qualitatively verify 78Se profiles, and to determine the cumulative 78Se retained on the blots. Individual data points ≥1000 cpm greater than adjacent points on each side were replaced by the average of the two adjacent points. Profiles in a set of replicates were adjusted to align the Gpx peak, and then smoothed by replacing each point by the average (n = 3) with the average of the point and its two adjacent neighbours, to provide the profiles shown in Figs. 2 and 3. Cumulative 78Se cpm were compared using the t-test (n = 3, P <.05).
Results
For these studies, we shifted from our previous use of larger 3 mm acrylamide gels in 75Se metabolism studies, followed by slicing and 75Se counting [6, 20], Here, BioRad 1 mm acrylamide mini-gels were used for these experiments. In a preliminary experiment, adequate-Se rat liver extract was subjected to conventional SDS-PAGE with 2% βME, and separated proteins then transferred to membranes. Example protein staining using 50 µg protein per lane is shown in Fig. 1A. Western blotting with anti-Gpx1 antibodies is shown in Fig. 1B. This preliminary experiment demonstrates that this procedure readily separates proteins by MW with transfer to membranes for subsequent LA-ICP-MS. In our previous 75Se metabolism studies, we successfully used 1500 µg protein per lane with the larger 3 mm gels without loss of resolution for individual 75Se-labelled proteins [6, 20]. For these current LA-ICP-MS studies with 1 mm gels, we have increased loading to 250 µg protein per lane.
SDS-PAGE and western blotting of 50 µg adequate-Se rat liver supernatant for Gpx1. (A) coloured MW standards and ponceau protein staining of blot membrane; (B) western blot for Gpx1; (C) MW standard migration curve.
LA-ICP-MS of blots of aqueous liver extract from adequate-Se rats (0.08 µg Se/g diet) subjected to conventional SDS-PAGE with βME resulted in a single 78Se peak (Fig. 2A) corresponding to the Gpx1 20 kDa subunit observed in rat liver [6] and in liver from rats labelled with 75Se [20]. Minor ∼60 kDa 78Se peaks eluting at ∼3.5 cm were also detected, just as with 75Se labelling. In extracts from high-Se (5 µg Se/g) rat liver (Fig. 2C), the singular 78Se Gpx1 peak was also observed. There was good agreement of the equal-protein mix profiles with individual profiles (see Fig. S1 example for rats fed 5.0 g Se/g) upon alignment of the Gpx1 peaks. There was also little effect of βME on SDS-PAGE cumulative 78Se after LA-ICP-MS in adequate-Se extract. In contrast, βME treatment of high-Se extract reduced the cumulative 78Se detected by LA-ICP-MS by more than 50% (Table 1). The increased 78Se in non-βME treated samples was distributed across the full profile, indicating that these selenosugar residues are bound to protein subunits of multiple sizes in rat liver (Fig. 2C).
LA-ICP-MS analysis for 78Se in SDS-PAGE blots of aqueous liver extracts from rats (A, C) and turkeys (B, D) fed adequate-Se and high-Se diets without βME (red/grey in B&W) and with βME (black). (A) rats fed 0.08 µg Se/g; (B) turkeys fed 0.4 µg Se/g; (C) rats fed 5 µg Se/g; (D) turkeys fed 5 µg Se/g. Plotted are averages of three replicates per treatment. Dashed lines show cumulative profile 78Se. Approximate protein MW are: 1.8 cm, 250 kDa; 2.3, 150 kDa; 2.85, 100 kDa; 3.25, 75 kDa; 3.95, 50 kDa; 4.60, 37 kDa; 4.37, 25 kDa; 5.70, 20 kDa; 6.35, 15 kDa, 6.95, 10 kDa.
Cumulative profile 78Se cpm and βME relative reduction.a
| SDS-PAGE | non βME x103 | plus βME x103 | βME reduction | t-test |
|---|---|---|---|---|
| Rat 0.08 µg Se/g | 761 ± 140 | 873 ± 29 | −15% | 0.384 |
| Rat 5.0 µg Se/g | 3397 ± 400 | 1210 ± 39 | 64% | 0.028 |
| Turkey 0.4 µg Se/g | 726 ± 173 | 727 ± 31 | 0% | 0.765 |
| Turkey 5.0 µg Se/g | 2569 ± 475 | 938 ± 23 | 63% | 0.024 |
| Native-PAGE | nonβME x103 | plusβME x103 | βME reduction | t-test |
| Rat 0.08 µg Se/g | 783 ± 148 | 647 ± 416 | 17% | 0.945 |
| Rat 5.0 µg Se/g | 1870 ± 290 | 1049 ± 54 | 44% | 0.032 |
| Turkey 0.4 µg Se/g | 658 ± 20 | 775 ± 60 | −18% | 0.056 |
| Turkey 5.0 µg Se/g | 2089 ± 203 | 647 ± 95 | 69% | 0.013 |
| SDS-PAGE | non βME x103 | plus βME x103 | βME reduction | t-test |
|---|---|---|---|---|
| Rat 0.08 µg Se/g | 761 ± 140 | 873 ± 29 | −15% | 0.384 |
| Rat 5.0 µg Se/g | 3397 ± 400 | 1210 ± 39 | 64% | 0.028 |
| Turkey 0.4 µg Se/g | 726 ± 173 | 727 ± 31 | 0% | 0.765 |
| Turkey 5.0 µg Se/g | 2569 ± 475 | 938 ± 23 | 63% | 0.024 |
| Native-PAGE | nonβME x103 | plusβME x103 | βME reduction | t-test |
| Rat 0.08 µg Se/g | 783 ± 148 | 647 ± 416 | 17% | 0.945 |
| Rat 5.0 µg Se/g | 1870 ± 290 | 1049 ± 54 | 44% | 0.032 |
| Turkey 0.4 µg Se/g | 658 ± 20 | 775 ± 60 | −18% | 0.056 |
| Turkey 5.0 µg Se/g | 2089 ± 203 | 647 ± 95 | 69% | 0.013 |
Cumulative 78Se cpm recovery in LA-ICP-MS analysis of individual replicates (n = 3; Mean ± SEM). βME reduction (or 78Se lost) is % of total non-βME cpm that were not present in the plus-βME cpm; a negative βME reduction indicates that more 78Se was recovered in the plus-βME than in non-βME profiles.
Cumulative profile 78Se cpm and βME relative reduction.a
| SDS-PAGE | non βME x103 | plus βME x103 | βME reduction | t-test |
|---|---|---|---|---|
| Rat 0.08 µg Se/g | 761 ± 140 | 873 ± 29 | −15% | 0.384 |
| Rat 5.0 µg Se/g | 3397 ± 400 | 1210 ± 39 | 64% | 0.028 |
| Turkey 0.4 µg Se/g | 726 ± 173 | 727 ± 31 | 0% | 0.765 |
| Turkey 5.0 µg Se/g | 2569 ± 475 | 938 ± 23 | 63% | 0.024 |
| Native-PAGE | nonβME x103 | plusβME x103 | βME reduction | t-test |
| Rat 0.08 µg Se/g | 783 ± 148 | 647 ± 416 | 17% | 0.945 |
| Rat 5.0 µg Se/g | 1870 ± 290 | 1049 ± 54 | 44% | 0.032 |
| Turkey 0.4 µg Se/g | 658 ± 20 | 775 ± 60 | −18% | 0.056 |
| Turkey 5.0 µg Se/g | 2089 ± 203 | 647 ± 95 | 69% | 0.013 |
| SDS-PAGE | non βME x103 | plus βME x103 | βME reduction | t-test |
|---|---|---|---|---|
| Rat 0.08 µg Se/g | 761 ± 140 | 873 ± 29 | −15% | 0.384 |
| Rat 5.0 µg Se/g | 3397 ± 400 | 1210 ± 39 | 64% | 0.028 |
| Turkey 0.4 µg Se/g | 726 ± 173 | 727 ± 31 | 0% | 0.765 |
| Turkey 5.0 µg Se/g | 2569 ± 475 | 938 ± 23 | 63% | 0.024 |
| Native-PAGE | nonβME x103 | plusβME x103 | βME reduction | t-test |
| Rat 0.08 µg Se/g | 783 ± 148 | 647 ± 416 | 17% | 0.945 |
| Rat 5.0 µg Se/g | 1870 ± 290 | 1049 ± 54 | 44% | 0.032 |
| Turkey 0.4 µg Se/g | 658 ± 20 | 775 ± 60 | −18% | 0.056 |
| Turkey 5.0 µg Se/g | 2089 ± 203 | 647 ± 95 | 69% | 0.013 |
Cumulative 78Se cpm recovery in LA-ICP-MS analysis of individual replicates (n = 3; Mean ± SEM). βME reduction (or 78Se lost) is % of total non-βME cpm that were not present in the plus-βME cpm; a negative βME reduction indicates that more 78Se was recovered in the plus-βME than in non-βME profiles.
LA-ICP-MS of blots of aqueous liver extract from adequate-Se turkeys (0.4 µg Se/g diet) subjected to conventional SDS-PAGE with βME resulted in two major 78Se peaks (Fig. 2B) corresponding to the Gpx1 20 kDa and Gpx4 18 kDa peaks observed in turkeys labelled with 75Se [24]. This doublet is expected as adequate-Se turkeys have 6X the level of Gpx4 and 1/10th the level of Gpx1 as compared to rats [25]. There was only a modest effect of SDS-PAGE without βME on cumulative 78Se after LA-ICP-MS in adequate-Se turkey extract. Just as in rats, SDS-PAGE of high-Se turkey extract with βME reduced the cumulative 78Se detected by LA-ICP-MS by more than 50% (Table 1). The increased 78Se in non-βME treated turkey samples was also distributed across the profile indicating Se labelling of protein subunits of multiple-sizes in the turkey (Fig. 2D). Notably, there were no distinct peaks in the 78Se SDS-PAGE profiles in both rats and turkeys observed when extracts were denatured and boiled in SDS-containing Laemmli sample buffer without βME, other than the singular Gpx peaks, suggesting that targeted attachment of selenosugars to a single or limited number of protein subunits does not occur.
Incubation with βME at 37°C of high-Se samples for both rats and turkeys, followed by Native-PAGE, reduced 78Se recovery on the blots by ∼50%, just as with SDS-PAGE analysis (Fig. 3C, D, Table 1). Without βME pretreatment, there were no distinct singular detected 78Se peaks in adequate-Se rat or turkey extracts (Fig. 3A, B). Pretreatment with βME resolved the broader high MW 78Se peaks at ∼1.0 cm into more singular >250 kDa peaks in these extracts not subjected to SDS-denaturation. These 78Se peaks are almost certainly Gpx species, as liquid chromatography as well as SDS-PAGE analysis clearly shows that the Gpxs are the major liver true selenoproteins [26, 27]. This may imply that there is a redistribution of 78Se to protein(s) of this apparent size, but a more likely explanation is that βME reduction reduces protein-protein interactions and dissociation of HMW mixed-protein species. In addition, addition of βME to the adequate Se samples prior to incubation for 30 min at 37°C, resulted in an increased profile of 78Se, suggesting that this added βME may be inhibiting degradation of selenoproteins as well as other species during preincubations.
LA-ICP-MS analysis for 78Se in native-page blots of aqueous liver extracts from rats (A, C) and turkeys (B, D) fed adequate-Se and high-Se diets without βME (red/grey in B&W) and with βME (black). (A) rats fed 0.08 µg se/g; (B) turkeys fed 0.4 µg se/g; (C) rats fed 5 µg se/g; (D) turkeys fed 5 µg se/g. plotted are averages of three replicates per treatment. dashed lines show cumulative profile 78Se. approximate protein MW are: 1.35 cm, 250 kDa; 1.75, 150 kDa; 2.40, 100 kDa; 3.05, 75 kDa; 4.00, 50 kDa; 4.75, 37 kDa.
Discussion
LA-ICP-MS of blot membranes for 78Se readily detected Gpx subunits when rat and turkey βME-pretreated liver extracts were subjected to conventional SDS-PAGE and electroblotting. Both the major single Gpx1 peak in rat liver, and the major Gpx1 plus Gpx4 doublet in turkey liver were prominently observed, along with minor broad ∼60 kDa 78Se peaks; these profiles are similar to SDS-PAGE 75Se profiles observed with gel slicing/counting of 75Se-supplemented animals [6, 20]. The 78Se MW profiles for the Gpx proteins were similar in 5 µg Se/g and adequate-Se animals, as expected because Se supplementation above the requirements only modestly increases selenoprotein synthesis [1]. In preliminary trials (data not shown), LA-ICP-MS of blots of supernatants prepared from frozen liver by conventional homogenization [6, 20] in buffer or water also resulted in 78Se profiles that were nearly identical to profiles from the aqueous extraction used in this study.
It should be emphasized that these pretreatments, protein electrophoresis, and blotting procedures provide ample opportunity for LMW Se species to be lost before the LA-ICP-MS analysis. The lost LMW Se includes <2 kDa LMW selenosugar species and LMW selenometabolites. The resulting detected Se in these LA-ICP-MS profiles thus is firmly bound Se attached to protein. In high-Se extracts, βME treatment with or without SDS released ∼50% of this protein-bound Se, indicating that this Se is linked to protein via Se-S bonds.
We initiated these collaborative studies to better understand the nature of Se that accumulates in animals fed high inorganic Se. True Sec-containing selenoproteins plateau in Se-adequate animals and are used to set the dietary Se requirements, whereas liver Se increases dramatically with 5 ug Se/g dietary supplementation [1]. We first studied the turkey because their Se requirement is 4X that of rodents, and yet turkeys accumulate the same level of liver Se with high-Se feeding as rats without overt Se toxicity [1, 24, 25, 28, 29]. As expected, ICP-MS found that Sec in liver was not increased by feeding 5 vs. 0.4 ug Se/g diets to turkeys, confirming that total selenoproteins were not increased; importantly, no SeMet was detected by ESI-MS/MS in either water-soluble or water-insoluble fractions of liver in both selenite-fed turkeys [3] or rats [4]. What we did find was that liver LMW selenosugars increased dramatically, but also that HMW Se species (>10 kDa) increased dramatically and accounted for ∼50% of the Se upon reverse-phase-ICP-MS in the turkey (Fig. S2). Clearly, this HMW Se was attached to proteins of multiple sizes separated by HPLC. Further analysis of just the HMW aqueous fraction after DTT reduction, by reverse-phase-ICP-MS followed by ESI-MS/MS, found that 92% of the released Se was the parent SeGalNac species (remaining 8% was methyl-SeGalNac). The need for DTT reduction clearly indicated that these SeGalNac moieties are initially covalently attached via mixed selenodisulphide bonds to cysteine residues in these HMW proteins [3]. Similar analyses of the HMW fraction from rats fed of 5 ug Se/g as selenite [4] or SeMet [5] also showed that the major Se species detected by ESI-MS/MS after DTT was the parent SeGalNac. Collectively, these investigations showed that these HMW Se species can be separated by protein chromatography, and that SeGalNac is the major Se species released by thiol reduction of HMW fractions in bulk, leading to our designation of these HWM selenospecies as ‘selenosugar-decorated proteins’ [3]. Future direct analysis is yet needed to demonstrate that selenosugars are the species bound to proteins and released by βME treatment, including further identification of specific protein(s) labelled with selenosugar.
In the present studies, SDS-PAGE of βME-treated high-Se extracts (Fig. 2C, D) resulted a 50% reduction in detected Se vs. βME-pretreated extracts across the full profile of MW protein subunits, indicating that targeted attachment of selenosugars to a single or limited number of protein subunits does not occur. These increased levels correspond with increased HMW Se species observed by size-exclusion-ICP-MS of high-Se turkey [3] and rat [4] liver extracts; this Se after DTT reduction [3, 4] was further shown by HPLC-ICP-MS to be present as the parent selenosugar, SeGalNac.
βME pretreatment in Native-PAGE, lacking denaturing SDS in the sample buffer, still resolved the broad HMW Se peaks into more singular Se species in both adequate- and high-Se liver samples in both rats and turkeys. This undoubtedly contains Gpx1 and Gpx4, the predominant selenoproteins in liver, with established MWs of ∼80 kDa for Gpx1 and 18 kDa for Gpx4 [20, 30]. Eluting at ∼1.0 cm and >300 kDa, this shows that larger aggregates of Se-binding proteins are present in aqueous extracts in Native-PAGE buffer and remain even after βME treatment. Without βME pretreatment, Native-PAGE alone resulted in poorly resolved profiles showing a heterogeneous collection of both true liver selenoprotein plus additional Se-binding species. In previous size-exclusion chromatography studies of 75Se-labelled proteins, inclusion of 0.3 M NaCl in the elution buffer was needed to resolve Gpx1 into a single 80 kDa peak rather than the apparent larger species [26, 27]. Interestingly, in adequate-Se liver, there was little difference with or without βME pretreatment in cumulative Se release for both rat and turkeys, but SDS-PAGE with βME pretreatment of high-Se extract from rat liver (Fig. 2C) removed more Se as compared to turkey liver (Fig. 2D). This is likely because more than 50% of the Se species upon size-exclusion HPLC is present in turkey extracts as < 2 kDa LMW Se species [3], whereas only 15% were LMW Se species in rat extracts (Fig. S2).
This group at IPREM previously [8–10, 13–15, 17] used LA of 1D- and 2D-gels to identify Se-containing protein spots, followed by tryptic digestion of excised Se spots from parallel experiments which were subjected to HPLC and ESI-MS/MS peptide mapping. In wheat, seven Se-containing storage proteins were identified, with the Se present in both SeMet and Sec [13]. In Lactobacillus reuteri, 7 Se-containing proteins were identified, where the Se was found exclusively to be Sec [10]. Similar approaches have been reported for Se-containing proteins in catfish [12]. The use of βME/DTT and Laemmli sample buffer in all of these prior studies, however, excluded finding selenosugars or other Se species linked by S-Se bonds to proteins. Similar follow-up studies LA-PAGE-ICP-MS and ESI-MS/MS are planned to further demonstrate that selenosugar is the Se species released from these proteins, and to identify proteins that are decorated by selenosugars in high-Se animals.
The underlying objective of this research is to better understand how animals and humans adapt to high Se. Both turkeys and rats acquire the same concentrations of liver Se, with Gpx1 and Gpx4 the major selenoproteins in turkey liver and Gpx1 the sole major selenoprotein in rodents; in selenite-supplemented turkeys and rats, there is negligible accumulation of liver Se as selenomethionine [3–5]. Furthermore, turkeys have minimum dietary Se requirements for maximized Gpx expression that are 4X higher than rodent requirements; 5 µg Se/g diet is not toxic to growing turkey [31] but depresses growth in rats [29], suggesting that differences in Se metabolism may modulate the toxicity of Se in these animals. Our previous Se metallomics experiments found near equivalent levels of LMW vs. HMW selenosugars in aqueous liver extracts from turkeys fed 5 µg Se/g, whereas there was a far higher proportion of HMW vs. LMW selenosugars in rat liver, with additional nonselenosugar LMW Se species in rat liver extract accounting for the difference. These differences in rats vs. turkeys may contribute to increased Se toxicity in rats vs. turkeys.
Conclusions
These results show that studies comparing non-βME pretreatment vs. βME treatment in PAGE followed by LA-ICP-MS are feasible and can effectively follow Se bound to protein both in SDS-PAGE and Native-PAGE. These current studies further indicate that ‘selenosugar-decorated proteins’ are present in the HMW aqueous liver extracts from both turkeys and rats with selenosugar residues bound to protein subunits of multiple sizes. These differences in underlying Se metabolism, selenosugar synthesis, and selenosugar binding to protein are likely to modulate high Se status and Se toxicity. Lastly, selenosugar and protein-bound selenosugars have potential as biomarkers of excess dietary Se intake and high Se status.
Acknowledgments
None declared .
Author contributions
K.B. and R.S. carried out the experiments. K.B., J.S., R.L., and R.S. planned the work. K.B., J.S., and R.S. prepared the draft. All authors completed and approved the manuscript.
Conflicts of interest
None declared.
Funding
This research was supported in part by the National Institute of Food and Agriculture, United States Department of Agriculture (www.csrees.usda.gov), Hatch project 1004389 and Multi-State project NC1170, and by the Wisconsin Alumni Foundation (http://www.uwalumni.com) Selenium Nutrition Research Fund (no. 12046295). The funding of the FT MS platform by the EQUIPEX ANR-11-EQPX-0027 MARSS project is acknowledged.
Data availability
Data available on request.
