Characterization and Quantification of Selenoprotein P: Challenges to Mass Spectrometry.

Selenoprotein P (SELENOP) is an emerging marker of the nutritional status of selenium and of various diseases, however, its chemical characteristics still need to be investigated and methods for its accurate quantitation improved. SELENOP is unique among selenoproteins, as it contains multiple genetically encoded SeCys residues, whereas all the other characterized selenoproteins contain just one. SELENOP occurs in the form of multiple isoforms, truncated species and post-translationally modified variants which are relatively poorly characterized. The accurate quantification of SELENOP is contingent on the availability of specific primary standards and reference methods. Before recombinant SELENOP becomes available to be used as a primary standard, careful investigation of the characteristics of the SELENOP measured by electrospray MS and strict control of the recoveries at the various steps of the analytical procedures are strongly recommended. This review critically discusses the state-of-the-art of analytical approaches to the characterization and quantification of SELENOP. While immunoassays remain the standard for the determination of human and animal health status, because of their speed and simplicity, mass spectrometry techniques offer many attractive and complementary features that are highlighted and critically evaluated.

Processive Recoding and Metazoan Evolution of Selenoprotein P: Up to 132 UGAs in Molluscs.

Selenoproteins typically contain a single selenocysteine, the 21st amino acid, encoded by a context-redefined UGA. However, human selenoprotein P (SelenoP) has a redox-functioning selenocysteine in its N-terminal domain and nine selenium transporter-functioning selenocysteines in its C-terminal domain. Here we show that diverse SelenoP genes are present across metazoa with highly variable numbers of Sec-UGAs, ranging from a single UGA in certain insects, to 9 in common spider, and up to 132 in bivalve molluscs. SelenoP genes were shaped by a dynamic evolutionary process linked to selenium usage. Gene evolution featured modular expansions of an ancestral multi-Sec domain, which led to particularly Sec-rich SelenoP proteins in many aquatic organisms. We focused on molluscs, and chose Pacific oyster Magallana gigas as experimental model. We show that oyster SelenoP mRNA with 46 UGAs is translated full-length in vivo. Ribosome profiling indicates that selenocysteine specification occurs with ∼5% efficiency at UGA1 and approaches 100% efficiency at distal 3' UGAs. We report genetic elements relevant to its expression, including a leader open reading frame and an RNA structure overlapping the initiation codon that modulates ribosome progression in a selenium-dependent manner. Unlike their mammalian counterparts, the two SECIS elements in oyster SelenoP (3'UTR recoding elements) do not show functional differentiation in vitro. Oysters can increase their tissue selenium level up to 50-fold upon supplementation, which also results in extensive changes in selenoprotein expression.

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.

Selenoprotein P-neutralizing antibodies improve insulin secretion and glucose sensitivity in type 2 diabetes mouse models.

Selenoprotein P (SeP) functions as a selenium (Se)-supply protein. SeP is identified as a hepatokine, promoting insulin resistance in type 2 diabetes. Thus, the suppression of Se-supply activity of SeP might improve glucose metabolism. Here, we develop an anti-human SeP monoclonal antibody AE2 as with neutralizing activity against SeP. Administration of AE2 to mice significantly improves glucose intolerance and insulin resistance that are induced by human SeP administration. Furthermore, excess SeP administration significantly decreases pancreas insulin levels and high glucose-induced insulin secretion, which are improved by AE2 administration. Epitope mapping reveals that AE2 recognizes a region of human SeP adjacent to the first histidine-rich region (FHR). A polyclonal antibody against the mouse SeP FHR improves glucose intolerance and insulin secretion in a mouse model of diabetes. This report describes a novel molecular strategy for the development of type 2 diabetes therapeutics targeting SeP.

Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine.

Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7-15 Sec residues depending on species. Assessing an individual's selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys.

Direct interaction between selenoprotein P and tubulin.

Selenium (Se), an essential trace element for human health, mainly exerts its biological function via selenoproteins. Among the 25 selenoproteins identified in human, selenoprotein P (SelP) is the only one that contains multiple selenocysteines (Sec) in the sequence, and has been suggested to function as a Se transporter. Upon feeding a selenium-deficient diet, mice lacking SelP develop severe neurological dysfunction and exhibit widespread brainstem neurodegeneration, indicating an important role of SelP in normal brain function. To further elucidate the function of SelP in the brain, SelP was screened by the yeast two-hybrid system from a human fetal brain cDNA library for interactive proteins. Our results demonstrated that SelP interacts with tubulin, alpha 1a (TUBA1A). The interaction between SelP and tubulin was verified by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assays. We further found that SelP interacts with the C-terminus of tubulin by its His-rich domain, as demonstrated by FRET and Isothermal Titration Calorimetry (ITC) assays. The implications of the interaction between SelP and tubulin in the brain and in Alzheimer's disease are discussed.

Isoform-specific binding of selenoprotein P to the ß-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 ß-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.

Inhibitory effect of selenoprotein P on Cu(+)/Cu(2+)-induced Aß42 aggregation and toxicity.

It has been suggested that the aggregation and cytotoxicity of amyloid-ß (Aß) peptide with transition-metal ions in neuronal cells is involved in the progression of Alzheimer's disease (AD). Selenoproteins are a group of special proteins that contain the 21st amino acid selenocysteine in their sequence, and they are found to be involved in the onset and progression of AD. Here, we report that the histidine-rich domain of selenoprotein P (SelP-H) is capable of binding Cu ions in both oxidation states of Cu(+) and Cu(2+) with high affinity and of modulating Cu(+) and Cu(2+)-mediated Aß aggregation, reactive oxygen species (ROS) production, and neurotoxicity. SelP-H was found to coordinate 1 and 2 mol equiv of Cu(+) and Cu(2+) with sub-picomolar and nanomolar affinities, respectively. Cu(+)/Cu(2+) binding to Aß42 inhibited the fibrillization of Aß42 but induced it to form amorphous aggregates, which could be significantly restored by SelP-H, as observed by thioflavin T fluorescence and transmission electron microscopy. Interestingly, SelP-H inhibited Cu(+)/Cu(2+)-Aß42-induced neurotoxicity and the intracellular ROS production in living cells. These studies suggest that SelP may play certain roles in regulating redox balance as well as metal homeostasis.

Selenoprotein P and selenoprotein M block Zn2+ -mediated Aß42 aggregation and toxicity.

Aggregation and cytotoxicity of the amyloid-ß (Aß) peptide with transition metal ions in neuronal cells have been suggested to be involved in the progression of Alzheimer's disease (AD). A therapeutic strategy to combat this incurable disease is to design chemical agents to target metal-Aß species. Selenoproteins are a group of special proteins that contain the 21st amino acid Sec in their sequence. Due to the presence of Sec, studies of this group of proteins are basically focused on their roles in regulating redox potential and scavenging reactive oxygen species. Here, we reported that the His-rich domain of selenoprotein P (SelP-H) and the Sec-to-Cys mutant selenoprotein M (SelM') are capable of binding transition metal ions and modulating the Zn(2+)-mediated Aß aggregation, ROS production and neurotoxicity. SelM' (U48C) and SelP-H were found to coordinate 0.5 and 2 molar equivalents of Zn(2+)/Cd(2+) with micromolar and submicromolar affinities, respectively. Metal binding induced the structural changes in SelP-H and SelM' according to the circular dichorism spectra. Zn(2+) binding to Aß42 almost completely suppressed Aß42 fibrillization, which could be significantly restored by SelP-H and SelM', as observed by thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM). Interestingly, both SelP-H and SelM' inhibited Zn(2+)-Aß42-induced neurotoxicity and the intracellular ROS production in living cells. These studies suggest that SelP and SelM may play certain roles in regulating redox balance as well as metal homeostasis.

Polymorphisms in thioredoxin reductase and selenoprotein K genes and selenium status modulate risk of prostate cancer.

Increased dietary intake of Selenium (Se) has been suggested to lower prostate cancer mortality, but supplementation trials have produced conflicting results. Se is incorporated into 25 selenoproteins. The aim of this work was to assess whether risk of prostate cancer is affected by genetic variants in genes coding for selenoproteins, either alone or in combination with Se status. 248 cases and 492 controls from an EPIC-Heidelberg nested case-control study were subjected to two-stage genotyping with an initial screening phase in which 384 tagging-SNPs covering 72 Se-related genes were determined in 94 cases and 94 controls using the Illumina Goldengate methodology. This analysis was followed by a second phase in which genotyping for candidate SNPs identified in the first phase was carried out in the full study using Sequenom. Risk of high-grade or advanced stage prostate cancer was modified by interactions between serum markers of Se status and genotypes for rs9880056 in SELK, rs9605030 and rs9605031 in TXNRD2, and rs7310505 in TXNRD1. No significant effects of SNPs on prostate cancer risk were observed when grade or Se status was not taken into account. In conclusion, the risk of high-grade or advanced-stage prostate cancer is significantly altered by a combination of genotype for SNPs in selenoprotein genes and Se status. The findings contribute to explaining the biological effects of selenium intake and genetic factors in prostate cancer development and highlight potential roles of thioredoxin reductases and selenoprotein K in tumour progression.

Multiple forms of selenoprotein P in a candidate human plasma standard reference material.

Selenoprotein P (SePP) is a unique selenium-containing protein responsible for the transport and distribution of the essential trace element selenium (Se) through the human body with the concentration of SePP in human blood representing the most useful marker of Se nutritional status. Although SePP has been extensively studied, the structure of SePP in human plasma remains unresolved. Two potential isoforms of SePP have been identified by Western blot analyses distinguished principally by differences in migration (51 kDa and 61 kDa). The biological relevance of the smaller isoform has been called into question by several studies reporting only one major SePP form (69 kDa) suggesting that the shorter 51 kDa is an artifact of protease activity during the SePP purification process. A deficiency of these Western blot analyses is that no information can be gleaned regarding the Se content of the potential isoforms. This study reports a characterization of SePP isoforms in a human plasma Standard Reference Material representative of a healthy US population. Following immunoprecipitation, three SePP isoforms were unequivocally identified at 45 kDa, 49 kDa and 57 kDa (termed as SePP45, SePP49 and SePP57) by LC-MS/MS analyses from a spectral searching approach. Selenium (Se) was detected by gel electrophoresis LA-ICP-MS in SePP49 and SePP57 which was confirmed by the identification of three selenopeptides covering the SePP sequence from residues 312-346 by LC-MS/MS analyses utilizing a sequence searching approach. Conversely, neither Se nor peptides covering SePP sequence from residues 306-346 was identified in SePP45 which suggests that SePP45 is a truncated isoform transcriptionally terminated at the 2nd in-frame UGA codon thereby terminating the protein with the Ser residue at position 299. An additional band at 23 kDa was found to contain Se but no peptides of SePP. Instead, glutathione peroxidase 3 (GPx3) was unequivocally identified within the band presumably being co-immunoprecipitated with the SePP providing preliminary evidence that SePP and GPx3 interaction may take place in vivo.

High-throughput quantification of selenium in individual serum proteins from a healthy human population using HPLC on-line with isotope dilution inductively coupled plasma-MS.

In this study, a method, based on dual column affinity chromatography hyphenated to isotope dilution inductively coupled plasma-quadrupole MS, was developed for selenium determination in selenoprotein P, glutathione peroxidase, and selenoalbumin in human serum samples from a group of healthy volunteers (n=399). Method improvement was achieved using methanol-enhanced isotope dilution which resulted in improved sensitivity and removal of isobaric interferences. Although no human serum reference materials are currently certified for their selenium species levels, method development was conducted using human serum reference material BCR 637 and 639 as their Se species content has been reported in the previous studies, and thus comparisons were possible. The mean selenium concentrations determined for the 399 healthy volunteer serum samples were 23 ± 10 ng Se mL(-1) for glutathione peroxidase, 49 ± 15 ng Se mL(-1) for selenoprotein P and 11 ± 4 ng Se mL(-1) for selenoalbumin. These values are found to be in close agreement with published values for a limited number of healthy volunteer samples, and to establish baseline Se levels in serum proteins for an apparently healthy group of individuals, thus allowing for subsequent comparisons with respective values determined for groups of individuals with selenium related health issues, as well as assist in the discovery of potential selenium biomarkers. Also, the relationship between Se serum protein levels and some anthropometric characteristics of the volunteer population were investigated. Additionally, further development of the analytical method used in this study was achieved by adding a size exclusion chromatography column after the two affinity columns via a switching valve. This allowed for the separation of small selenium-containing molecules from glutathione peroxidase and thus enhanced the overall confidence in its identification.

Reduced reliance on the trace element selenium during evolution of mammals.

BACKGROUND: Selenium (Se) is an essential trace element that occurs in proteins in the form of selenocysteine (Sec). It is transported throughout the body in the form of Sec residues in Selenoprotein P (SelP), a plasma protein of unclear origin recently proposed as an experimental marker of dietary Se status. RESULTS: Here, we report that the amino-terminal domain of SelP is distantly related to ancestral bacterial thiol oxidoreductases of the thioredoxin superfamily, and that its carboxy-terminal Se transport domain may have originated in early metazoan evolution by de novo accumulation of Sec residues. Reconstruction of evolutionary changes in the Se transport domain indicates a decrease in Sec content of SelP specifically in the mammalian lineage via replacement of Sec with cysteine (Cys). Sec content of mammalian SelPs varies more than two-fold and is lowest in rodents and primates. Compared to mammals, fish show higher Sec content of SelP, larger selenoproteomes, elevated SelP gene expression, and higher levels of tissue Se. In addition, mammals replaced Sec with Cys in several proteins and lost several selenoproteins altogether, whereas such events are not found in fish. CONCLUSION: These data suggest that evolution from fish to mammals was accompanied by decreased use of Sec and that analyses of SelP, selenoproteomes and Sec/Cys transitions provide a genetic marker of utilization of this trace element in vertebrates. The evolved reduced reliance on Se raises questions regarding the need to maximize selenoprotein expression by Se dietary supplements in situations when pathology is not imminent, a currently accepted practice.

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium.

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.

The role of selenium in thyroid autoimmunity and cancer.

The essential micronutrient selenium (Se) occurs in the form of the amino acid selenocysteine in selenoproteins which exert various effects, while maintaining the cell reduction-oxidation balance. The discovery that all three deiodinases that convert thyroxine (T4) into triiodothyronine (T3) contain selenocysteine illustrates how the production of the active thyroid hormone is dependent on Se status. The selenoenzyme families of glutathione peroxidases (GPx) and thioredoxin reductases (TRx) possess powerful antioxidant properties and form a complex defense system that protects thyrocytes from oxidative damage. Se supplementation in patients with autoimmune thyroiditis seems to modify the immune response, probably by enhancing plasma GPx activity and decreasing excess levels of hydrogen peroxide. However, the enhancement of immunocompetence may also be the result of the synergistic action of various selenoproteins and not exclusively of GPx. There is evidence supporting considerable oxidative stress in Graves' disease where Se supplementation, because of its free radical scavenging properties, may increase the enzymatic antioxidant activity. TRx has been found significantly elevated in GD revealing its involvement in the pathogenesis of this condition and representing a potential future target for therapeutical intervention. Low Se serum levels have also been associated with increased risk of thyroid cancer and may play a role in carcinogenesis. It is noteworthy, that the Food and Drug Administration has recently determined that there is sufficient evidence to warrant a qualified health claim for Se and cancer. Furthermore, the recent discovery that defects in the SECIS-binding protein 2 (SBP2), which is an indispensable protein for the incorporation of Se into the selenoproteins, result in thyroid dysfunction, together with the recognition of the many roles of selenoprotein P in Se distribution and storage in the human body, reveal not only the indispensability of Se and the selenoproteins as essential factors in thyroid metabolism and pathogenesis, but open up new prospects for enhanced treatment.

Chemical forms of selenium for cancer prevention.

Cancer is becoming an increasingly significant disease worldwide. Currently, more than 7 million people die each year from cancer. With the existing knowledge, at least one-third of worldwide cancer cases could be prevented. Searching for naturally occurring agents in routinely consumed foods that may inhibit cancer development, although challenging, constitutes a valuable and plausible approach to the control and prevention of cancer. To date, the use of the micronutrient selenium (Se) in human clinical trials is limited, but the outcome indicates that Se is among the most promising agents. Although it is convenient to describe the effects of Se in terms of the element, it must always be kept in mind that the chemical form of Se and the dose are determinants of its biological activities. Hyphenated techniques based on coupling chromatographic separation with inductively coupled plasma mass spectrometric (ICP-MS) detection are now established as the most realistic and potent analytical tools available for real-life speciation analysis. These speciation investigations provide evidence that the Se compounds, which can generate monomethylated Se (e.g., Se-methylselenocysteine and methylseleninic acid), are more efficacious than other Se compounds because of their chemoprevention activity.

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery.

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.

[Prediction of structure and function of human selenoprotein P].

OBJECTIVE: To probe the function and structure of human selenoprotein P (SELP). METHODS: Several bioinformatic server are used to analyze exons, hydrophobic nature, transmembrane helices and the bonding state of cysteines respectively, as well as the secondary structure and relative solvent accessibility. The homology analysis and searching for motifs or domains in SELP are also carried out. RESULTS: Two transmembrane helices are found, as is consistent with the hydrophobic nature of SELP. Matured SELP consists of four modules: M1 (Glu20-Ser67/Lys68), M2 (Ser67/Lys68-Asp138/Arg139), M3(Asp138/Arg139-Thr178), M4 (Thr179-Asn381). A coiled coil exists in the second module whose regular secondary structure is higher than that of other three. The percentage of loop structure is higher in the first, third and fourth modules, so they may be three reactive center. The fourth module is the most active one which has a histidine-rich region (His204-His257) on the surface of SELP. Because most of the residues are the E type of solvent accessibility, SELP appears not to be globular. SELP consists of two kinds of domain, so it more probably has more than one function. HUNB_SCAAL, ZP12_BRARE, BR11_BRARE, BRN1_HUMAN which play roles in the development of brain are found have sequence similarity with SELP and the similarity may be the specificity to brain. Other two similar proteins are KNOB_PLAFG and HPN_ HELPY which anchor host thrombospondin or a parasite analog in a binding complex with the endothelial cell receptor and strongly binds nickel and zinc respectively. CONCLUSION: The results showed suggest that SELP may have metal binding and endothelial cell membrane anchoring activity which are backed up not only by homology analysis but also the prediction of structure. Moreover, the results also suggest the specificity of SELP to brain.