Suguru Kurokawa

Selenoprotein P and apolipoprotein E receptor-2 interact at the blood-brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration

Selenoprotein P (Sepp1) and its receptor, apolipoprotein E receptor 2 (apoER2), account for brain retaining selenium better than other tissues. The primary sources of Sepp1 in plasma and brain are hepatocytes and astrocytes, respectively. ApoER2 is expressed in varying amounts by tissues; within the brain it is expressed primarily by neurons. Knockout of Sepp1 or apoER2 lowers brain selenium from ~120 to ~50 ng/g and leads to severe neurodegeneration and death in mild selenium deficiency. Interactions of Sepp1 and apoER2 that protect against this injury have not been characterized. We studied Sepp1, apoER2, and brain selenium in knockout mice. Immunocytochemistry showed that apoER2 mediates Sepp1 uptake at the blood‐brain barrier. When Sepp1–/–or apoER2–/– mice developed severe neurodegeneration caused by mild selenium deficiency, brain selenium was ~35 ng/g. In extreme selenium deficiency, however, brain selenium of ~12 ng/g was tolerated when both Sepp1 and apoER2 were intact in the brain. These findings indicate that tandem Sepp1‐apoER2 interactions supply selenium for maintenance of brain neurons. One interaction is at the blood‐brain barrier, and the other is within the brain. We postulate that Sepp1 inside the blood‐brain barrier is taken up by neurons via apoER2, concentrating brain selenium in them.—Burk, R. F., Hill, K. E., Motley, A. K., Winfrey, V. P., Kurokawa, S., Mitchell, S. L., Zhang, W. Selenoprotein P and apolipoprotein E receptor‐2 interact at the blood‐brain barrier and also within the brain to maintain an essential selenium pool that protects against neurodegeneration. FASEB J. 28, 3579–3588 (2014). www.fasebj.org

Maternal-fetal transfer of selenium in the mouse

Selenoprotein P (Sepp1) is taken up by receptor‐mediated endocytosis for its selenium. The other extracellular selenoprotein, glutathione peroxidase‐3 (Gpx3), has not been shown to transport selenium. Mice with genetic alterations of Sepp1, the Sepp1 receptors apolipoprotein E receptor‐2 (apoER2) and megalin, and Gpx3 were used to investigate maternalfetal selenium transfer. Immunocytochemistry (ICC) showed receptor‐independent uptake of Sepp1 and Gpx3 in the same vesicles of d‐13 visceral yolk sac cells, suggesting uptake by pinocytosis. ICC also showed apoER2‐mediated uptake of maternal Sepp1 in the d‐18 placenta. Thus, two selenoprotein‐dependent maternalfetal selenium transfer mechanisms were identified. Selenium was quantified in d‐18 fetuses with the mechanisms disrupted. Maternal Sepp1 deletion, which lowers maternal whole‐body selenium, decreased fetal selenium under selenium‐adequate conditions but deletion of fetal apoER2 did not. Fetal apoER2 deletion did decrease fetal selenium, by 51%, under selenium‐deficient conditions, verifying function of the placental Sepp1‐apoER2 mechanism. Maternal Gpx3 deletion decreased fetal selenium, by 13%, but only under selenium‐deficient conditions. These findings indicate that the selenoprotein uptake mechanisms ensure selenium transfer to the fetus under selenium‐deficient conditions. The failure of their disruptions (apoER2 deletion, Gpx3 deletion) to affect fetal selenium under selenium‐adequate conditions indicates the existence of an additional maternal‐fetal selenium transfer mechanism.—Burk, R. F., Olson, G. E., Hill, K. E., Winfrey, V. P., Motley, A. K., and Kurokawa, S., Maternal‐fetal transfer of selenium in the mouse. FASEB J. 27, 3249–3256 (2013). www.fasebj.org

Long Isoform Mouse Selenoprotein P (Sepp1) Supplies Rat Myoblast L8 Cells with Selenium via Endocytosis Mediated by Heparin Binding Properties and Apolipoprotein E Receptor-2 (ApoER2)

Background: ApoER2 endocytosis of Sepp1 supplies testis and brain with selenium, but the mechanism of supply to other tissues is not known. Results: Sepp1 supplies selenium to heart and skeletal muscle cell lines via apoER2 and many tissues express apoER2. Conclusion: ApoER2 endocytosis of Sepp1 supplies selenium to many tissues. Significance: ApoER2 uptake of Sepp1 likely regulates selenium distribution in the whole body. In vivo studies have shown that selenium is supplied to testis and brain by apoER2-mediated endocytosis of Sepp1. Although cultured cell lines have been shown to utilize selenium from Sepp1 added to the medium, the mechanism of uptake and utilization has not been characterized. Rat L8 myoblast cells were studied. They took up mouse Sepp1 from the medium and used its selenium to increase their glutathione peroxidase (Gpx) activity. L8 cells did not utilize selenium from Gpx3, the other plasma selenoprotein. Neither did they utilize it from Sepp1Δ240–361, the isoform of Sepp1 that lacks the selenium-rich C-terminal domain. To identify Sepp1 receptors, a solubilized membrane fraction was passed over a Sepp1 column. The receptors apoER2 and Lrp1 were identified in the eluate by mass spectrometry. siRNA experiments showed that knockdown of apoER2, but not of Lrp1, inhibited 75Se uptake from 75Se-labeled Sepp1. The addition of protamine to the medium or treatment of the cells with chlorate also inhibited 75Se uptake. Blockage of lysosome acidification did not inhibit uptake of Sepp1 but did prevent its digestion and thereby utilization of its selenium. These results indicate that L8 cells take up Sepp1 by an apoER2-mediated mechanism requiring binding to heparin sulfate proteoglycans. The presence of at least part of the selenium-rich C-terminal domain of Sepp1 is required for uptake. RT-PCR showed that mouse tissues express apoER2 in varying amounts. It is postulated that apoER2-mediated uptake of long isoform Sepp1 is responsible for selenium distribution to tissues throughout the body.

Sepp1UF forms are N-terminal selenoprotein P truncations that have peroxidase activity when coupled with thioredoxin reductase-1

Mouse selenoprotein P (Sepp1) consists of an N-terminal domain (residues 1-239) that contains one selenocysteine (U) as residue 40 in a proposed redox-active motif (-UYLC-) and a C-terminal domain (residues 240-361) that contains nine selenocysteines. Sepp1 transports selenium from the liver to other tissues by receptor-mediated endocytosis. It also reduces oxidative stress in vivo by an unknown mechanism. A previously uncharacterized plasma form of Sepp1 is filtered in the glomerulus and taken up by renal proximal convoluted tubule (PCT) cells via megalin-mediated endocytosis. We purified Sepp1 forms from the urine of megalin(-/-) mice using a monoclonal antibody to the N-terminal domain. Mass spectrometry revealed that the purified urinary Sepp1 consisted of N-terminal fragments terminating at 11 sites between residues 183 and 208. They were therefore designated Sepp1(UF). Because the N-terminal domain of Sepp1 has a thioredoxin fold, Sepp1(UF) were compared with full-length Sepp1, Sepp1(Δ240-361), and Sepp1(U40S) as a substrate of thioredoxin reductase-1 (TrxR1). All forms of Sepp1 except Sepp1(U40S), which contains serine in place of the selenocysteine, were TrxR1 substrates, catalyzing NADPH oxidation when coupled with H2O2 or tert-butylhydroperoxide as the terminal electron acceptor. These results are compatible with proteolytic cleavage freeing Sepp1(UF) from full-length Sepp1, the form that has the role of selenium transport, allowing Sepp1(UF) to function by itself as a peroxidase. Ultimately, plasma Sepp1(UF) and small selenium-containing proteins are filtered by the glomerulus and taken up by PCT cells via megalin-mediated endocytosis, preventing loss of selenium in the urine and providing selenium for the synthesis of glutathione peroxidase-3.

Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.

Selenite Assimilation into Formate Dehydrogenase H Depends on Thioredoxin Reductase in Escherichia coli

Escherichia coli growing under anaerobic conditions produce H(2) and CO(2) by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH H), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma 54-dependent transcriptional activator FhlA that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDH H activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDH H even without formate and d-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH H activity and also reductive assimilation of selenite ( SeO 3(2-)) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH H production and for labelling the FDH H polypeptide with 75Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

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

Background: ApoER2 facilitates uptake of Sepp1, but the binding mechanism has not been elucidated. Results: The two longest isoforms of Sepp1 bind to the YWTD β-propeller domain of apoER2, which functions as a Sepp1 receptor. Conclusion: Only longer Sepp1 isoforms with six or more selenocysteine residues can interact with a unique binding site of apoER2. Significance: ApoER2 takes up long isoform Sepp1 through its YWTD β-propeller domain. 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.