The Effects Of Selenoprotein K On T Lymphocyte Immune Response

Selenium is an essential trace element in humans and animals,which is physically of great importance and is involved in protections against various diseases such as cancers, neurodegenerative diseases and cardiovascular diseases as well as in the regulation of immune response. Selenium are mainly incorporated into selenoproteins in the form of selenocysteine, the 21 st amino acid, to exert its biological effects.There have been 25 selenoproteins identified in humans, functions of these selenoproteins have not explored except the glutathione peroxidase and thioredoxin reductase. Selenoprotein K(Sel K) is a small selenoprotein with unknown functions and first identified by the Gladyshev laboratory in 2003. There have been some breakthroughs about the biological functions of Sel K in recent years. Sel K can protect cells against oxidative stress in cardiomyocytesand ER stress in Hep G2 cells. Sel K may also play an important role in keeping ER homeostasis by eliminating misfolded proteins from the ER. Besides, Sel K is found to be able to regulate immune response in mice, and contribute to foam cell formation and atherogenesis by adjusting palmitoylation of CD36 in macrophages.Tissue distribution or subcellular localization of one protein have relations to its functions. The expression of Sel K was detected in the different tissues of mice by Real time-PCR, results showed that there were significantly higher expression of Sel K in the spleen than other tissues.The result of Western blotting was basically consistent with the Real time-PCR. Immunohistochemistry assays were performed to detected Sel K in mouse spleen and exhibited a perinuclear distribution consistent with ER localization. Immunocytochemistry were analyzed by confocal microscope to observe the subcellular localization of Sel K after incubation with two antibodies(Sel K and ER maker, KDEL) in mouse T lymphocytes. Result showd that Sel K were co-localized with KDEL, which illustrated Sel K localized to the ER in mouse T lymphocytes.To explore more functions of Sel K, we constructed three recombinant plasmids(p GPU-Sel K-mus-222; p GPU-Sel K-mus-102 and p GPU-Sel K-mus-101) used for down-regulation of Sel K in mouse lymphocytes(T cells). Select one sh RNA which can specifically and efficiently interfered with the intracellular expression of Sel K at the m RNA level.Sel K-knockdown cells showed immunity-decreased features after activation of CD3. The concentration of intracellular free Ca2+ of T cell was significantly decreased in sh Sel K-treated T cells after CD3 stimulation. Further studies showed that Sel K interference inhibit the expression of endoplasmic reticulum calcium homeostasis protein(CHERP), which illustrated Sel K may through of CHERP to regulate the concentration of intracellular free Ca2+ during the activation of T cell. The m RNA level of IL-2Rα was down-regulated, and the IL-4 level in cell culture supernates was significantly downregulated. These result indicate that Sel K is involved in regulating the concentration of intracellular free Ca2+ during T cell activation and also involved in regulating the expression of IL-2Rα and the secretion of cytokines such as IL-4 during T cell activation. As these factors are very important in the process of T cell activation and proliferation, we presume Sel K may play important roles in the proliferation and differentiation of T cell by TCR stimulation.Selenomethionine was used to test the protection effect on cells. We used different concentrations(1μM, 3μM, 5μM, 10μM, 50μM,100μM) of Selenomethionine to treat cells, and then used Cell the Counting Kit-8(CCK-8) to detect the best drug concentration of Selenomethionine effect the cells proliferation. Results showed that there was a “Low to promote high suppression” phenomenon of cell proliferation after Selenomethionine stimulated T cells. 5μM Selenomethionine simulated T cells for 24 h had the best effect in promoting cell proliferation. The results showed that Sel K expression level has raised in T cells after 5μM Selenomethionine stimμLation. These results suggest that the right concentration of selenium can increase the Sel K expression and promote proliferation of T lymphocytes, or thereby increasing the immunity of organism.These results indicate that Sel K were high expressed in spleen and localized to the ER in mouse T lymphocytes. Further more, Sel K may through of CHERP to regulate the concentration of intracellular free Ca2+ during the activation of T cell by regulation of CHERP, and Sel K also involved in regulating the expression of IL-2Rα and the secretion of cytokines such as IL-4 during T cell activation. In addition, the right amount of selenium can increase the expression Sel K and promote proliferation of T lymphocytes, or thereby increasing the immunity of organism.