Regulation of Selenocysteine Incorporation into the Selenium Transport Protein,Selenoprotein P

Selenoproteins are unique as they contain selenium in their active site in the form of the 21st amino acid selenocysteine (Sec), which is encoded by an in-frame UGA stop codon. Sec incorporation requires both cis- and trans-acting factors, which are known to be sufficient for Sec incorporation in vitro, albeit with low efficiency. However, the abundance of the naturally occurring selenoprotein that contains 10 Sec residues (SEPP1) suggests that processive and efficient Sec incorporation occurs in vivo. Here, we set out to study native SEPP1 synthesis in vitro to identify factors that regulate processivity and efficiency. Deletion analysis of the long and conserved 3′-UTR has revealed that the incorporation of multiple Sec residues is inherently processive requiring only the SECIS elements but surprisingly responsive to the selenium concentration. We provide evidence that processive Sec incorporation is linked to selenium utilization and that reconstitution of known Sec incorporation factors in a wheat germ lysate does not permit multiple Sec incorporation events, thus suggesting a role for yet unidentified mammalian-specific processes or factors. The relationship between our findings and the channeling theory of translational efficiency is discussed.     

The Selenium Transport Protein,Selenoprotein P,Requires Coding Sequence Determinants to Promote Efficient Selenocysteine Incorporation

Selenoproteins are an essential and unique group of proteins in which selenocysteine (Sec) is incorporated in response to a stop codon (UGA). Reprograming of UGA for Sec insertion in eukaryotes requires a cis-acting stem–loop structure in the 3′ untranslated region of selenoprotein mRNA and several trans-acting factors. Together these factors are sufficient for Sec incorporation in vitro, but the process is highly inefficient. An additional challenge is the synthesis of selenoprotein P (SELENOP), which uniquely contains multiple UGA codons. Full-length SELENOP expression requires processive Sec incorporation, the mechanism for which is not understood. In this study, we identify core coding region sequence determinants within the SELENOP mRNA that govern SELENOP synthesis. Using ⁷⁵Se labeling in cells, we determined that the N-terminal coding sequence (upstream of the second UGA) and C-terminal coding sequence context are two independent determinants for efficient synthesis of full-length SELENOP. In addition, the distance between the first UGA and the consensus signal peptide is also critical for efficiency.     

Uptake and Utilization of Selenium from Selenoprotein P

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.     

Selenoprotein P Is the Major Selenium Transport Protein in Mouse Milk

Selenium is transferred from the mouse dam to its neonate via milk. Milk contains selenium in selenoprotein form as selenoprotein P (Sepp1) and glutathione peroxidase-3 (Gpx3) as well as in non-specific protein form as selenomethionine. Selenium is also present in milk in uncharacterized small-molecule form. We eliminated selenomethionine from the mice in these experiments by feeding a diet that contained sodium selenite as the source of selenium. Selenium-replete dams with deletion of Sepp1 or Gpx3 were studied to assess the effects of these genes on selenium transfer to the neonate. Sepp1 knockout caused a drop in milk selenium to 27% of the value in wild-type milk and a drop in selenium acquisition by the neonates to 35%. In addition to decreasing milk selenium by eliminating Sepp1, deletion of Sepp1 causes a decline in whole-body selenium, which likely also contributes to the decreased transfer of selenium to the neonate. Deletion of Gpx3 did not decrease milk selenium content or neonate selenium acquisition by measurable amounts. Thus, when the dam is fed selenium-adequate diet (0.25 mg selenium/kg diet), milk Sepp1 transfers a large amount of selenium to neonates but the transfer of selenium by Gpx3 is below detection by our methods.     

Effects of Dietary Selenium on Selenoprotein W Gene Expression in the Chicken Immune Organs

Selenoprotein W (SelW) is expressed in the immune systems of mammals. However, its pattern of expression in the immune organs of birds is still unclear. To investigate the distribution of SelW and effects of dietary Se levels on the SelW mRNA expression in the immune organs of birds, 1-day-old male chickens were fed either a commercial diet or an Se-supplemented diet containing 0.601, 1.058, 1.514, or 2.427?mg Se per kilogram, and 1.0, 2.0, 3.0 or 5.0?mg sodium selenite per kilogram for 90?days. The immune organs (spleen, thymus, and bursa of Fabricius) were collected and examined for Se content and SelW mRNA levels. The mRNA expression of SelW was detected in all the tissues. Although Se content was the highest in the spleen, the remarkable stability of the SelW mRNA level was observed in this organ during different times of dietary Se supplementation. Se-supplemented diet can make the SelW expression levels higher within a certain range in thymus and bursa of Fabricius. The present study demonstrates that SelW is widely expressed in immune organs of birds and that Se-supplementation of the feed increases SelW expression in the thymus and the bursa of Fabricius.     

Mice Lacking Selenoprotein P and Selenocysteine Lyase Exhibit Severe Neurological Dysfunction,Neurodegeneration, and Audiogenic Seizures

Selenoproteins are a unique family of proteins, characterized by the co-translational incorporation of selenium as selenocysteine, which play key roles in antioxidant defense. Among selenoproteins, selenoprotein P (Sepp1) is particularly distinctive due to the fact that it contains multiple selenocysteine residues and has been postulated to act in selenium transport. Within the brain, Sepp1 delivers selenium to neurons by binding to the ApoER2 receptor. Upon feeding a selenium-deficient diet, mice lacking ApoER2 or Sepp1 develop severe neurological dysfunction and exhibit widespread brainstem neurodegeneration, indicating an important role for ApoER2-mediated Sepp1 uptake in normal brain function. Selenocysteine lyase (Scly) is an enzyme that plays an important role in selenium homeostasis, in that it catalyzes the decomposition of selenocysteine and allows selenium to be recycled for additional selenoprotein synthesis. We previously reported that constitutive deletion of Scly results in neurological deficits only when mice are challenged with a low selenium diet. To gain insight into the relationship between Sepp1 and Scly in selenium metabolism, we created novel transgenic mice constitutively lacking both genes (Scly^−/−Sepp1^−/−) and characterized the neurobehavioral phenotype. We report that deletion of Scly in conjunction with Sepp1 further aggravates the phenotype of Sepp1^−/− mice, as these mice needed supraphysiological selenium supplementation to survive, and surviving mice exhibited impaired motor coordination, audiogenic seizures, and brainstem neurodegeneration. These findings provide the first in vivo evidence that Scly and Sepp1 work cooperatively to maintain selenoprotein function in the mammalian brain.     

Production of Selenoprotein P (Sepp1) by Hepatocytes Is Central to Selenium Homeostasis

Sepp1 is a widely expressed extracellular protein that in humans and mice contains 10 selenocysteine residues in its primary structure. Extra-hepatic tissues take up plasma Sepp1 for its selenium via apolipoprotein E receptor-2 (apoER2)-mediated endocytosis. The role of Sepp1 in the transport of selenium from liver, a rich source of the element, to peripheral tissues was studied using mice with selective deletion of Sepp1 in hepatocytes (Sepp1^c/c/alb-cre^+/− mice). Deletion of Sepp1 in hepatocytes lowered plasma Sepp1 concentration to 10% of that in Sepp1^c/c mice (controls) and increased urinary selenium excretion, decreasing whole-body and tissue selenium concentrations. Under selenium-deficient conditions, Sepp1^c/c/alb-cre^+/− mice accumulated selenium in the liver at the expense of extra-hepatic tissues, severely worsening clinical manifestations of dietary selenium deficiency. These findings are consistent with there being competition for metabolically available hepatocyte selenium between the synthesis of selenoproteins and the synthesis of selenium excretory metabolites. In addition, selenium deficiency down-regulated the mRNA of the most abundant hepatic selenoprotein, glutathione peroxidase-1 (Gpx1), to 15% of the selenium-replete value, while reducing Sepp1 mRNA, the most abundant hepatic selenoprotein mRNA, only to 61%. This strongly suggests that Sepp1 synthesis is favored in the liver over Gpx1 synthesis when selenium supply is limited, directing hepatocyte selenium to peripheral tissues in selenium deficiency. We conclude that production of Sepp1 by hepatocytes is central to selenium homeostasis in the organism because it promotes retention of selenium in the body and effects selenium distribution from the liver to extra-hepatic tissues, especially under selenium-deficient conditions.     

Effects of Dietary Selenium Deficiency or Excess on Gene Expression of Selenoprotein N in Chicken Muscle Tissues

Previous studies have determined the effects of dietary selenium (Se) supplementation on selenoprotein N (SelN, SEPN1), selenophosphate synthetase-1 (SPS1), and selenocysteine-synthase (SecS) mRNA abundance in chicken skeletal and cardiac muscles. To investigate collective responses of these genes to dietary Se concentrations ranging from deficiency to moderately high level in muscle tissues of chicken, 1-day-old chickens were exposed to a diet of deficient Se and supplemented with Se (0.15 mg Se/kg and 1.50 mg Se/kg) as sodium selenite in the feed for 35 days. Muscle tissues (flight, breast, leg, and cardiac muscles) were collected and examined for Se content and mRNA levels of SelN on days 1, 15, 25, and 35 days, respectively. Moreover, SPS1 and SecS mRNA levels were analyzed. The results showed that the expression of SelN gene in cardiac muscle responded to dietary Se concentrations. SelN gene was downregulated in the Se deficiency group (L group), and upregulated in the Se excess group (H group) compared with the moderate Se group (M group) (P?<?0.05) in cardiac muscle. Se deficiency mainly unregulated SelN mRNA level in skeletal muscles compared with M group. Excess dietary Se mainly resulted in the upregulation of SelN mRNA level in skeletal muscles compared with the M group. SecS mRNA levels responded to dietary Se concentrations showed a similar change compared with SelN in cardiac muscle. SPS1 mRNA levels responded to dietary Se concentrations showed a downregulation in L group and upregulation in H group. However, SelN mRNA levels displayed a different expression pattern in different skeletal and cardiac muscles. Moreover, Se also regulated the levels of SPS1 and SecS mRNAs. In summary, Se regulated the expression of SelN gene and affected the mRNA levels of SecS and SPS1. The level of Se in the feed may regulate SelN biosynthesis by affecting the levels of SPS1 and SecS mRNA.     

Effects of Dietary Selenium Deficiency on mRNA Levels of Twenty-One Selenoprotein Genes in the Liver of Layer Chicken

Selenium (Se) is an essential trace element in many life forms due to its occurrence as selenocysteine (Sec) residue in selenoproteins. However, little is known about the expression pattern of selenoproteins in the liver of layer chicken. To investigate the effects of Se deficiency on the mRNA expressions of selenoproteins in the liver tissue of layer chickens, 1-day-old layer chickens were randomly allocated into two groups (n?=?120/group). The Se-deficient group (?Se) was fed a Se-deficient corn–soy basal diet; the Se-adequate group as control (+Se) was fed the same basal diet supplemented with Se at 0.15 mg/kg (sodium selenite). The liver tissue was collected and examined for mRNA levels of 21 selenoprotein genes at 15, 25, 35, 45, 55, and 65 days old. The data indicated that the mRNA expressions of Gpx1, Gpx2, Gpx3, Gpx4, Sepn1, Sepp1, Selo, Sepx1, Selu, Txnrd1, Txnrd2, Txnrd3, Dio1, Dio2, SPS2, Selm, SelPb, Sep15, and Sels were decreased (p?<?0.05), but not the levels of Dio3 and Seli (p?>?0.05). The results showed that the mRNA levels of 19 selenoprotein (except Seli and Dio3) genes in the layer chicken liver were regulated by diet Se level. The present study provided some compensated data about the roles of Se in the regulation of selenoproteins.     

Interplay between Selenium Levels,Selenoprotein Expression,and Replicative Senescence in WI-38 Human Fibroblasts

Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence.     

Selenoprotein N Was Required for the Regulation of Selenium on the Uterine Smooth Muscle Contraction in Mice

Selenium (Se) is an essential micronutrient affecting various aspects of health. The balance of the Se concentration has an important protective and promoter effect on physiological function in inducing muscular disorders in smooth muscle. Selenoprotein N (SelN) is closely related to Ca²⁺ release. The present study aimed to determine the effects and mechanism of action of dietary Se on uterine smooth muscle contraction via SelN using a mouse model. Quantitative polymerase chain reaction (qPCR) analysis was performed to detect mRNA levels. Western blotting was performed to detect protein levels. The results of the immunohistochemical analysis showed that Se had an effect on the uterine smooth muscle. The Se-supplement increased the release of Ca²⁺, Ca²⁺-calmodulin (CaM) expression, myosin light chain kinase (MLCK) expression, and myosin light chain (MLC) phosphorylation but did not affect ROCK and RhoA in uterine smooth muscle. Furthermore, the lack of Se showed an opposite impact. The effects of Se regulation were closely related to SelN. The interference of mouse SelN was performed on the uterine smooth muscle cell. Additionally, the results displayed the regulation of Se on the release of Ca²⁺, CaM expression, MLCK expression, and MLC phosphorylation were significant inhibited, and there was no effect on ROCK and RhoA. In conclusion, Se played an important role in regulating the process of contraction in uterine smooth muscle with SelN.     

Thyroid Hormones Regulate Selenoprotein Expression and Selenium Status in Mice

Impaired expression of selenium-containing proteins leads to perturbed thyroid hormone (TH) levels, indicating the central importance of selenium for TH homeostasis. Moreover, critically ill patients with declining serum selenium develop a syndrome of low circulating TH and a central downregulation of the hypothalamus-pituitary-thyroid axis. This prompted us to test the reciprocal effect, i.e., if TH status would also regulate selenoprotein expression and selenium levels. To investigate the TH dependency of selenium metabolism, we analyzed mice expressing a mutant TH receptor α1 (TRα1+m) that confers a receptor-mediated hypothyroidism. Serum selenium was reduced in these animals, which was a direct consequence of the mutant TRα1 and not related to their metabolic alterations. Accordingly, hyperthyroidism, genetically caused by the inactivation of TRβ or by oral TH treatment of adult mice, increased serum selenium levels in TRα1+m and controls, thus demonstrating a novel and specific role for TRα1 in selenium metabolism. Furthermore, TH affected the mRNA levels for several enzymes involved in selenoprotein biosynthesis as well as serum selenoprotein P concentrations and the expression of other antioxidative selenoproteins. Taken together, our results show that TH positively affects the serum selenium status and regulates the expression of several selenoproteins. This demonstrates that selenium and TH metabolism are interconnected through a feed-forward regulation, which can in part explain the rapid parallel downregulation of both systems in critical illness.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Privileged Incorporation of Selenium as Selenocysteine in Lactobacillus reuteri Proteins Demonstrated by Selenium-specific Imaging and Proteomics

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS (⁷⁸Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.In recent years selenium (Se)¹ has received considerable attention as an essential element for human health. Severe Se deficiency is linked to oxidative stress and aging (1), elevated mortality with HIV (2), and irreversible brain injury (seizures, Parkinson''s disease) (3). Se occurs in nature principally in four inorganic chemical forms: the highly toxic selenide (Se²⁻⁻) (4), the moderately toxic selenate (SeO₄²⁻⁻) and selenite (SeO₃²⁻⁻), and elemental selenium (Se⁰) which is essentially nontoxic and can be stored by several bacterial species as nanoparticles on the cell surface (5, 6, 7). Inorganic selenium can be converted by biological systems (microorganisms, plants, and mammals) into seleno-amino acids, which are then incorporated into proteins. The two most common seleno-amino acids are selenomethionine (SeMet) and selenocysteine (SeCys). The former is synthesized via a route similar to the sulfur metabolic pathway in which selenium substitutes sulfur with no alteration of the protein structure (8, 9). The insertion of SeCys is genetically encoded by the UGA (TGA) codon and requires a SECIS element downstream of such a codon, a specific tRNA^[Ser]Sec and accessory proteins (10).Selenoproteins containing genetically encoded SeCys are known to be synthesized by several bacteria. Among Gram-negative ones, E. coli produces three forms of selenated formate dehydrogenase (FdhN, FdhO, FdhH) (11). Among Gram-positive bacteria, all the selenoproteins experimentally known were found exclusively in anaerobic bacteria belonging to the clostridial clade. Examples include glycine reductase from Clostridium sticklandii (12) and Eubacterium acidaminophilum (13), proline reductase in C. sticklandii (14), xanthine dehydrogenase in C. acidiurici (15), and several antioxidant defense proteins (16). Enterococcus faecalis is the only member of the Firmicutes/Lactobacillales subdivision containing a SeCys-decoding trait (SelD) (17).Lactobacillus reuteri species has been widely described as a probiotic: it produces antimicrobial compounds, such as reuterin, with a broad spectrum of action (18), it is effective against diarrhea in children (19) and possesses immunomodulatory (potent TNF-inhibitory activity) effects in humans (20). Because L. reuteri species are native inhabitants of human microbiota, the association of the probiotic feature L. reuteri Lb2 BM-DSM 16143 with its ability to fix selenium into proteins, offers an innovative approach to combat human selenium deficiency.The objectives of this study were to investigate the ability of Firmicutes/Lactobacillales subdivision, Lactobacillus reuteri Lb2 BM-DSM 16143to incorporate selenium into proteins, and to investigate, for the first time, its speciation in order to identify the pathway(s) of this process (SeMet or SeCys). For this purpose an analytical approach based on laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) of Se-containing proteins in 2D gel electrophoresis, followed by their identification by capillary HPLC - electrospray Orbitrap MS/MS assisted by the quantitative control of selenium elution by ICP MS, was developed.     

硒蛋白P的研究进展

微量元素硒 (Ｓｅ)作为许多具有重要生物功能的硒酶的活性中心 ,不但与机体的免疫应答及抗氧化作用等生理功能密切相关 ,而且能够降低癌症的发生率[1,2 ] 。在流行病学和临床研究中 ,常用血浆或全血中Ｓｅ浓度作为衡量Ｓｅ状态的指标 ,而且血浆浓度能比全血浓度更迅速地反映Ｓｅ状态的变化。在哺乳动物血浆中 ,Ｓｅ主要结合在 3种蛋白质中 :硒蛋白Ｐ、胞外谷胱甘肽过氧化物酶和清蛋白。其中硒蛋白Ｐ所含Ｓｅ大约占血浆中全部Ｓｅ浓度的 5 0 %。硒蛋白Ｐ不同于目前所鉴定的所有其他硒蛋白 ,因为它含有 10～ 12个硒代半胱氨酸 (ＳｅＣｙｓ)残…     

Selenium Regulates Gene Expression of Selenoprotein W in Chicken Gastrointestinal Tract

Selenoprotein W (SelW) is an existing form of selenium (Se). Se influences the levels of SelW in mammals. However, little is known about the pattern of SelW expression in the gastrointestinal tract tissue of bird. The present paper describes the effects of different dietary levels of Se on the SelW mRNA expression in the gastrointestinal tract tissue of chicken. The expression levels of SelW mRNA and the Se contents in the gastrointestinal tract tissues (glandular stomach, gizzard, duodenum, small intestine, and rectum) were determined on days 15, 25, 35, 45, and 55, respectively. The results showed that the Se contents and the SelW mRNA expression were significantly higher (p < 0.05) in the high-Se group, and the Se contents and SelW mRNA expression in the low-Se group were significantly lower (p < 0.05) than in the controls. The Se contents were the highest in the duodenum and the lowest in the rectum, while the SelW mRNA expression was the highest in the gizzard and the lowest in the rectum. In addition, the SelW mRNA levels in the gastrointestinal tract tissue were found to increase in a time-dependent manner with increasing feeding time. Furthermore, the expression of the SelW mRNA in the gastrointestinal tract tissues of chickens was found to correlate with the dietary Se concentrations, but not with the tissue Se contents.     

Isoform-specific Binding of Selenoprotein P to the β-Propeller Domain of Apolipoprotein E Receptor 2 Mediates Selenium Supply

Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery.