Uptake and Utilization of Selenium from Selenoprotein P

Abstract

Selenoprotein P (SELENOP) is a serum glycoprotein that is required for proper selenium distribution in mammals, particularly in supplying selenium to the brain and testes. As the sole mechanism for providing essential selenium to developing spermatozoa, SELENOP metabolism is central to male fertility in all mammals. In addition, this process is important for proper brain function, especially under conditions of limited dietary selenium. Several specific and nonspecific mechanisms for SELENOP uptake in target tissues have been described, but the utilization of SELENOP as a source of selenium for intracellular selenoprotein production has not been systematically characterized. In this report, we examine the process of SELENOP uptake using a robust selenium uptake assay that measures selenium utilization in cells fed ⁷⁵Se-SELENOP. Using a series of inhibitors and modulators we have identified specific regulators of the process and found that SELENOP must be in an oxidized state for uptake. This assay also demonstrates that SELENOP uptake is not highly sequence specific as the zebrafish protein is recognized and processed by mammalian cells.

Keywords: Oxidation; Selenium; Selenocysteine; Selenoprotein P.

Figure 1. ⁷⁵Se-SELENOP can be used as a selenium source for intracellular selenoprotein production

(A) Conditioned medium from HepG2 cells grown in the presence of ⁷⁵Se-selenite was concentrated ~50-fold and a sample was treated with PNGaseF. The samples were resolved by SDS-PAGE and SELENOP was detected by immunoblot (left) and phosphorimage analysis (right). (B) HeLa cells were incubated with ~2 pmol of ⁷⁵Se-SELENOP or 100 nM ⁷⁵Se selenite for the time points indicated then switched to normal growth medium and allowed to grow for a total of 24 hours. Endogenous selenoproteins that utilized the ⁷⁵Se were resolved by SDS-PAGE and detected by PhosphorImage analysis. (C) HeLa cells were labeled with ⁷⁵Se-SELENOP as described in (A) and lysates were incubated with either an anti-thioredoxin reductase or anti-SELENOP antibody. Immunoprecipitated ⁷⁵Se-labeled proteins were resolved by SDS-PAGE and detected by PhosphorImage analysis. (D) ⁷⁵Se-selenite was used to label the endogenous selenoproteins in the cell types indicated: mouse Leydig cells [TM3], mouse Sertoli cells [TM4], human glioblastoma [U87], human hepatoma [HepG2], rat hepatoma [McArdle 7777], human epithelial [HeLa]. After 24 hours of labeling, 4% of the media (left) and 10% of the lysate (right) was resolved by SDS-PAGE and detected by PhosphorImage analysis.

Figure 2. DIDS is a specific inhibitor of SELENOP uptake

(A) HeLa cells were labeled with ⁷⁵Se selenite or ⁷⁵Se-SELENOP in the presence of a range of 4,4-Diisothiocyanatostilbene-2,2-Disulfonic Acid (DIDS) as indicated. ⁷⁵Se labeled proteins were resolved by SDS-PAGE and detected by PhosphorImage analysis. (B) SELENOP was pre-treated with DIDS. Excess DIDS was removed by gel filtration and varying amounts of the DIDS-treated or untreated control protein was used to label endogenous HeLa selenoproteins as above.

Figure 3. Reduced SELENOP cannot deliver selenium to cells

(A) ⁷⁵Se-SELENOP was incubated with 10 mM DTT for 1h at 37 °C. DTT was removed by gel filtration and the protein was then used to label endogenous HeLa selenoproteins as described in Figure 1A. (B) Reduced SELENOP was incubated with 25 mM iodoacetamide (IA). Excess IA was removed by gel filtration and the protein was used to label endogenous HeLa selenoproteins as above.

Figure 4. Inhibitors reveal mechanistic details about SELENOP uptake and processing

(A) HeLa cells were treated with the indicated concentrations of Auranofin for 1 hour prior to the addition of ⁷⁵Se-selenite or ⁷⁵Se-SELENOP. After 24 hours, the cells were lysed and ⁷⁵Se labeled proteins were resolved by SDS-PAGE and detected by PhosphorImage analysis. (B) (C) HeLa cells were labeled with ⁷⁵Se selenite or ⁷⁵Se-SELENOP in the presence or absence of 10% fetal bovine serum as indicated. Note that the relative intensity of endogenous selenoproteins labeled by ⁷⁵Se-selenite or ⁷⁵Se-SELENOP varied due to the use of two different batches of ⁷⁵Se that differed in specific activity.

Figure 5. Degradation of SELENOP occurs under acidic and reducing conditions

(A) HepG2 derived conditioned media containing ⁷⁵Se-SELENOP was incubated at 37 °C for 30 min in varying concentrations of acetic acid and/or 2 mM DTT as indicated. (B) ⁷⁵Se-SELENOP was purified by nickel affinity chromatography and incubated at 37 °C for 30 min.

Figure 6. Fish SELENOP can also be taken up and processed

Conditioned medium derived from HEK-293 cells stably expressing C-terminally FLAG tagged zebrafish SELENOP were labeled with equal ⁷⁵Se-selenite and the labeled protein purified by anti-FLAG immunoaffinity purification. The resulting purified protein was analyzed by SDS-PAGE followed by PhosphorImage analysis. (B) The purified SELENOP shown in (A) was applied to naive HEK-293 cells and lysates were analyzed by SDS-PAGE after 24 hours. The labeling of HEK 293 cells with an equal of ⁷⁵Se-selenite is shown as a control as indicated.