Selenoprotein P expression, purification, and immunochemical characterization

Most selenoproteins contain a single selenocysteine residue per polypeptide chain, encoded by an in-frame UGA codon. Selenoprotein P is unique in that its mRNA encodes 10-12 selenocysteine residues, depending on species. In addition to the high number of selenocysteines, the protein is cysteine- and histidine-rich. The function of selenoprotein P has remained elusive, in part due to the inability to express the recombinant protein. This has been attributed to presumed inefficient translation through the selenocysteine/stop codons. Herein, we report for the first time the expression of recombinant rat selenoprotein P in a transiently transfected human epithelial kidney cell line, as well as the endogenously expressed protein from HepG2 and Chinese hamster ovary cells. The majority of the expressed protein migrates with the predicted 57-kDa size of full-length glycosylated selenoprotein P. Based on the histidine-rich nature of selenoprotein P, we have purified the recombinant and endogenously expressed proteins using nickel-agarose affinity chromatography. We show that the recombinant rat and endogenous human proteins react in Western blotting and immunoprecipitation assays with commercial anti-histidine antibodies. The ability to express, purify, and immunochemically detect the recombinant protein provides a foundation for investigating the functions and efficiency of expression of this intriguing protein.     

Selenoprotein P—Expression,functions, and roles in mammals

Selenoprotein P (Sepp1) is a secreted protein that is made up of 2 domains. The larger N-terminal domain contains 1 selenocysteine residue in a redox motif and the smaller C-terminal domain contains the other 9 selenocysteines. Sepp1 isoforms of varying lengths occur but quantitation of them has not been achieved. Hepatic synthesis of Sepp1 affects whole-body selenium content and the liver is the source of most plasma Sepp1. ApoER2, a member of the lipoprotein receptor family, binds Sepp1 and facilitates its uptake into the testis and retention of its selenium by the brain. Megalin, another lipoprotein receptor, facilitates uptake of filtered Sepp1 into proximal tubule cells of the kidney. Thus, Sepp1 serves in homeostasis and distribution of selenium. Mice with deletion of Sepp1 suffer greater morbidity and mortality from infection with Trypanosoma congolense than do wild-type mice. Mice that express only the N-terminal domain of Sepp1 have the same severity of illness as wild-type mice, indicating that the protective function of Sepp1 against the infection resides in the N-terminal (redox) domain. Thus, Sepp1 has several functions. In addition, plasma Sepp1 concentration falls in selenium deficiency and, therefore, it can be used as an index of selenium nutritional status.     

Selenoprotein synthesis and regulation in Archaea

Background

The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism.

Plant aquaporins: novel functions and regulation properties

Aquaporins are water channel proteins of intracellular and plasma membranes that play a crucial role in plant water relations. The present review focuses on the most recent findings concerning the molecular and cellular properties of plant aquaporins. The mechanisms of transport selectivity and gating (i.e. pore opening and closing) have recently been described, based on aquaporin structures at atomic resolution. Novel dynamic aspects of aquaporin subcellular localisation have been uncovered. Also, some aquaporin isoforms can transport, besides water, physiologically important molecules such as CO(2), H(2)O(2), boron or silicon. Thus, aquaporins are involved in many great functions of plants, including nutrient acquisition, carbon fixation, cell signalling and stress responses.     

Selenoprotein P receptor from rat

Radioreceptor assay technology was used to show the presence in the rat of a receptor that binds selenoprotein P, a selenocysteine-containing rat plasma protein. 75Se-labeled selenoprotein P bound to testis, kidney, and liver membranes. The binding was specific in that increasing amounts of partially-fractionated rat plasma specifically displaced the binding of 75Se-labeled selenoprotein P to testis membrane in a competitive manner. 75Se-labeled selenoprotein P binding was saturable in the presence of increasing amounts of testis membranes. The binding of 75Se-labeled selenoprotein P was optimal at about pH 4.2. Several proteins and blood fractions had little or no significant effect on binding of 75Se-labeled selenoprotein P to testis membranes. All plasma sources tested specifically displaced 75Se-labeled selenoprotein P from testis membrane, indicating that selenoprotein P-related proteins may be widespread in nature. The study indicated that selenoprotein P has a receptor and is involved in selenium transport.     

Selenoprotein P analysis in human plasma

Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P.     

ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowP _open at physiological [ATP]_i. The unitary conductance is 15–20 pS at physiological [K⁺] _o , and the channels are highly selective for K⁺. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. K_ATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pH_i. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K⁺ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.     

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.     

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…     

Selenoprotein P protects low-density lipoprotein against oxidation

Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl₂, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl₂- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl₂- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species.     

Selenoprotein P Controls Oxidative Stress in Cornea

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.     

Telomerase: Structure,functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.     

Nectar: generation, regulation and ecological functions

Nectar contains water, sugars and amino acids to attract pollinators and defenders and is protected from nectar robbers and microorganisms by secondary compounds and antimicrobial proteins. Floral and extrafloral nectar secretion can be induced by jasmonic acid, it is often adjusted to consumer identity and consumption rate and depends on invertase activity. Invertases are likely to play at least three roles: the uploading of sucrose from the phloem, carbohydrate mobilization during active secretion and the postsecretory adjustment of the sucrose:hexose ratio of nectar. However, it remains to be studied how plants produce and secrete non-carbohydrate components. More research is needed to understand how plants produce nectar, the most important mediator of their interactions with mutualistic animals.     

Selenoprotein P is required for mouse sperm development

Selenoprotein P (SEPP1), an extracellular glycoprotein of unknown function, is a unique member of the selenoprotein family that, depending on species, contains 10-17 selenocysteines in its primary structure; in contrast, all other family members contain a single selenocysteine residue. The SEPP1-null (Sepp1(-/-)) male but not the female mice are infertile, but the cellular basis of this male phenotype has not been defined. In this study, we demonstrate that mature spermatozoa of Sepp1(-/-) males display a specific set of flagellar structural defects that develop temporally during spermiogenesis and after testicular maturation in the epididymis. The flagellar defects include a development of a truncated mitochondrial sheath, an extrusion of a specific set of axonemal microtubules and outer dense fibers from the principal piece, and ultimately a hairpin-like bend formation at the midpiece-principal piece junction. The sperm defects found in Sepp1(-/-) males appear to be the same as those observed in wild-type (Sepp1(+/+)) males fed a low selenium diet. Supplementation of dietary selenium levels for Sepp1(-/-) males neither reverses the development of sperm defects nor restores fertility. These data demonstrate that SEPP1 is required for development of functional spermatozoa and indicate that it is an essential component of the selenium delivery pathway for developing germ cells.     

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.     

Ankyrin: structure, properties, and functions

Recent data on characteristics of the structure, functions, and main properties of ankyrins (proteins that are linkers between the spectrin-based cytoskeleton and integral membrane proteins) are summarized. The interactions of ankyrins with band-3 protein, P-type ATPases, ion channels, receptors, and protein kinase C are considered. The structure of ankyrin repeats that are often contained in other proteins (which are not classified with the ankyrin family) and ensure protein-protein interactions as well as interactions between proteins and nucleic acids is described in details. The mechanisms of regulation of the ability of ankyrins to interact with other proteins (alternative splicing and post-translational modification, including phosphorylation) are also considered.     

Rap1 GTPase: functions, regulation, and malignancy

Rap1 is a member of the Ras family of small GTPases that is activated by diverse extracellular stimuli in many cell types. It is activated by distinct types of Rap1 guanine nucleotide exchange factors coupled with various receptors or second messengers, while activated Rap1 is down-regulated by Rap1 GTPase-activating proteins, through which Rap1 activation is controlled spatio-temporally. Functionally, Rap1 either interferes with Ras-mediated ERK activation or activates ERK independently of Ras in a cell-context dependent manner. Accumulating evidence also indicates that Rap1 is a major activator of integrins, playing important roles in the regulation of a variety of integrin-dependent cellular functions. Most recently, significant evidence has emerged that dysregulation of Rap1 activation is responsible for the development of malignancy. Recent extensive research has begun to unveil the roles of this controversial small G protein in physiology and diseases.