Mass spectrometric determination of selenenylsulfide linkages in rat selenoprotein P

The reversible formation of a selenenylsulfide linkage in mammalian thioredoxin reductase was identified as having a key role in its activity. Identification of selenenylsulfide and/or diselenide linkages is therefore critical to the determination of the structure and function of selenoproteins. A selenopeptide, (298)SGSAITUQCAENLPSLCSUQGLFAEEK(324) (U=selenocysteine), was isolated from a tryptic digest of rat selenoprotein P. Its two cysteine residues and two selenocysteine (Sec) residues were determined to be present in oxidized form by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The selenopeptide was subjected to partial reduction by dithiothreitol with immediate alkylation by iodoacetamide. This process was monitored by MALDI-TOFMS to determine the number of alkylations that had taken place. The partially reduced and alkylated peptides were then analyzed by nano-electrospray ionization tandem mass spectrometry and the results indicated that selenenylsulfide linkages Sec304-Cys314 and Cys306-Sec316 were present. It is concluded that selenoprotein P contains these two selenenylsulfide bonds.     

Fractionation of selenium-containing proteins in serum by multiaffinity liquid chromatography before size-exclusion chromatography–ICPMS

Immunoaffinity chromatography has been investigated for fractionation of serum into selenoalbumin and true selenoproteins. Among several albumin-depletion kits tested, a multiaffinity column specifically binding albumin and five other major serum proteins provided the best results. It extracted ca 95% of both albumin and selenoalbumin, which enabled interference-free determination of glutathione peroxidase, selenoprotein P, and selenoalbumin by size-exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC–ICPMS). The efficiency of the multiaffinity column did not vary over a period of 18 months. The purity of fractions separated by immunoaffinity LC was confirmed by elution-volume matching with standards in SEC–ICPMS and by selenopeptide mapping in capillary HPLC–ICPMS. Quantification of the selenium distribution among the different proteins in human serum from a control group and from a person on a selenium-rich diet revealed that 67% of the supplemented selenium was incorporated into albumin, 30% into glutathione peroxidase, and 3% into selenoprotein P.     

Selenoproteins: the key factor in selenium essentiality. State of the art analytical techniques for selenoprotein studies

Selenium is an essential element for human health. The benefits of selenium are many including protection against cancer, heart diseases and other cardiovascular and muscle disorders. Selenium is also helpful in controlling gastrointestinal disorders, enhancing immunity of the human body and reducing age-related diseases. The health-promoting properties of Se are due to vital functions of selenoproteins in which selenium is present as selenocysteine, the 21st amino acid. To date, dozens of selenoprotein families have been described though many have roles that have not been fully elucidated. Selenoproteins research has attracted tremendous interest from different scientific areas. Analytical chemists have not remained indifferent to the attractive features of these unique proteins. Different analytical techniques, such as multidimensional chromatography–inductively coupled plasma mass spectrometry (ICPMS), electrospray (tandem) mass spectrometry (ESI-MS/MS), matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis–laser ablation inductively coupled plasma mass spectrometry (SDS-PAGE-LA-ICPMS), have been applied to the determination of selenoproteins and selenium-containing proteins. This review describes the best-characterized selenoproteins to date in addition to the major contributions of analytical chemistry to the field of selenoproteins. The article also highlights the challenges of combining elemental and molecular mass spectrometry for the determination of selenoproteins and selenium-containing proteins.     

Post-transcriptional control of selenoprotein biosynthesis

Selenoproteins are defined as proteins containing the 21st proteinogenic amino acid, selenocysteine (Sec). Sec is encoded by UGA (STOP) codons which are re-coded to Sec by the presence of a selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region of selenoprotein mRNAs. The SECIS element is bound by several proteins, including SECIS-binding protein 2 (SBP2). Translation of selenoproteins critically depends on the integrity of the SECIS element - SBP2 interaction. Mutations in a SECIS element can abrogate expression of the respective selenoprotein. Mutations in SBP2 impinge on biosynthesis of a subset of selenoproteins and lead to a syndrome including hormonal, neurological, immunological symptoms as well as myopathy. Several other RNA-binding proteins are involved in selenoprotein translation and mediate the hierarchical response of selenoproteins to selenium deficiency. Global inhibition of selenoprotein translation is lethal in the mouse and hypomorphic mutations in selenocysteine synthase in humans leads to Progressive Cerebello Cerebral Atrophy, a neurodevelopmental and neurodegenerative disease in pediatric patients.     

仿硒酶研究进展

硒是人体中必需的微量元素,它与各种疾病和人类健康息息相关。硒在生物体内以硒代半胱氨酸形式表现其生理活性和功能。为了探索硒在硒蛋白中结构和功能关系并可能发展成硒相关的适用药物,人们付出许多努力来发展硒蛋白模拟化学。由于硒酶—谷胱甘肽过氧化物酶（GPX）重要的抗氧化作用以及潜在的药用价值,国际上广泛开展了对它的人工模拟研究。本文对近年来硒酶模拟化学和生物学相关研究进展进行了综述。     

硒蛋白的分子生物学及与疾病的关系*

硒蛋白是微量元素硒在体内存在和发挥生物功能的主要形式。因硒蛋白的活性中心硒代半胱氨酸由传统终止码TGA编码,故从基因组中预测硒蛋白以及用基因工程技术表达硒蛋白均很困难。有关硒抗氧化、对癌症、神经退行性疾病和病毒作用的报导较多,但结论并不一致。本文综述了硒蛋白基因预测、蛋白质表达调控以及硒和硒蛋白对癌症、神经退行性疾病和病毒的作用及机制等方面的近期进展,研究提高硒蛋白生物信息学预测准确率和基因工程表达量的方法,分析了解硒蛋白与疾病发生发展的关系和机制,探索不同硒蛋白作为预防药物开发、作为癌症治疗和药物筛选靶标的可能性。     

Selenocysteine‐Mediated Native Chemical Ligation

C‐Terminal peptide thioesters are shown to react efficiently with peptide fragments containing an N‐terminal selenocysteine to give selenoproteins. In analogy to the native chemical ligation of thioesters and peptides containing N‐terminal cysteines, the selenol presumably attacks the thioester nucleophilically to give a selenoester intermediate that subsequently rearranges to give a native chemical bond. The utility of this procedure was demonstrated by the synthesis of a selenium‐containing derivative of bovine pancreatic trypsin inhibitor (BPTI) in which Cys³⁸ is replaced by selenocysteine. The artificial selenoprotein folds into a conformation similar to that of wild‐type BPTI and inhibits trypsin and chymotrypsin with unaltered affinity.     

A systematic approach to the accurate quantification of selenium in serum selenoalbumin by HPLC-ICP-MS

In this paper, two different methods are for the first time systematically compared for the determination of selenium in human serum selenoalbumin (SeAlb). Firstly, SeAlb was enzymatically hydrolyzed and the resulting selenomethionine (SeMet) was quantified using species-specific isotope dilution (SSID) with reversed phase-HPLC (RP-HPLC) hyphenated to (collision/reaction cell) inductively coupled plasma-quadrupole mass spectrometry (CRC ICP-QMS). In order to assess the enzymatic hydrolysis yield, SeAlb was determined as an intact protein by affinity-HPLC (AF-HPLC) coupled to CRC ICP-QMS. Using this approach, glutathione peroxidase (GPx) and selenoprotein P (SelP) (the two selenoproteins present in serum) were also determined within the same chromatographic run. The levels of selenium associated with SeAlb in three serum materials, namely BCR-637, Seronorm level 1 and Seronorm level 2, obtained using both methods were in a good agreement. Verification of the absence of free SeMet, which interferes with the SeAlb determination (down to the amino acid level), in such materials was addressed by analyzing the fraction of GPx, partially purified by AF-HPLC, using RP-HPLC (GPx only) and size exclusion-HPLC (SE-HPLC) coupled to CRC ICP-QMS. The latter methodology was also used for the investigation of the presence of selenium species other than the selenoproteins in the (AF-HPLC) SelP and SeAlb fractions; the same selenium peaks were detected in both control and BCR-637 serum with a difference in age of ca. 12 years. It is also for the first time that the concentrations of selenium associated with SeAlb, GPx and SelP species in such commercially available serums (only certified or having indicative levels of total selenium content) are reported. Such indicative values can be used for reference purposes in future validation of speciation methods for selenium in human serum and/or inter-laboratory comparisons.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2-10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 +/- 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma.     

Direct determination of selenoproteins in polyvinylidene difluoride membranes by electrothermal atomic absorption spectrometry

A method for the direct determination of selenoproteins in plastic membranes after protein separation by gel electrophoresis was developed. Quantification was based on the determination of the selenium content of the proteins by electrothermal atomic absorption spectrometry (ET-AAS) after manual introduction of membrane pieces into the graphite furnace. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane by semi-dry electroblotting. After staining the membrane, the protein bands were excised and chemical modifier was added on top of the excised membrane prior to atomic absorption measurement. Acceptable linearity was achieved in the range 2– 10 ng Se, corresponding to selenium concentrations close to 1 mg/L, when aqueous solutions of selenomethionine standard as well as selenoprotein standard were applied to the membrane. A characteristic mass of 54 ± 4 pg/0.0044 s was obtained for the selenoprotein standard. Protein transfer from polyacrylamide gel to the membrane was quantitative and no interferences were introduced. The method was used for identification of selenoprotein P after enrichment of the protein from human plasma. Received: 28 June 1999 / Revised: 14 September 1999 / Accepted: 16 September 1999     

Modelling the Inhibition of Selenoproteins by Small Molecules Using Cysteine and Selenocysteine Derivatives

Small molecule-based electrophilic compounds such as 1-chloro-2,4-dinitrobenzene (CDNB) and 1-chloro-4-nitrobenzene (CNB) are currently being used as inhibitors of cysteine- and selenocysteine-containing proteins. CDNB has been used extensively to determine the activity of glutathione S-transferase and to deplete glutathione (GSH) in mammalian cells. Also, CDNB has been shown to irreversibly inhibit thioredoxin reductase (TrxR), a selenoenzyme that catalyses the reduction of thioredoxin (Trx). Mammalian TrxR has a C-terminal active site motif, Gly-Cys-Sec-Gly, and both the cysteine and selenocysteine residues could be the targets of the electrophilic reagents. In this paper we report on the stability of a series of cysteine and selenocysteine derivatives that can be considered as models for the selenoenzyme–inhibitor complexes. We show that these derivatives react with H₂O₂ to generate the corresponding selenoxides, which undergo spontaneous elimination to produce dehydroalanine. In contrast, the cysteine derivatives are stable towards such elimination reactions. We also demonstrate, for the first time, that the arylselenium species eliminated from the selenocysteine derivatives exhibit significant redox activity by catalysing the reduction of H₂O₂ in the presence of GSH (GPx (glutathione peroxidase)-like activity), which suggests that such redox modulatory activity of selenium compounds may have a significant effect on the cellular redox state during the inhibition of selenoproteins.     

Protein factors mediating selenoprotein synthesis

The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.     

以蛋白质为基础模拟谷胱甘肽过氧化物酶

硒元素作为一种生命体中必须的微量元素,与人类的健康和疾病息息相关.硒元素主要以硒代半胱氨酸的形式存在于至少25种硒蛋白中,执行着多种生物功能.在这20多种硒蛋白中,谷胱甘肽过氧化物酶(GPx)作为一种主要的抗氧化酶,能够有效地利用谷胱甘肽还原氢过氧化物以防止机体的氧化损伤.这里,我们主要介绍以蛋白质为骨架构筑GPx模拟物的一些策略和方法,以期望于能够更好的理解硒蛋白的生物学性质,甚至开拓更为有效的技术去模拟这种抗氧化酶.     

Detection of selenocompounds in a tryptic digest of yeast selenoprotein by MALDI time-of-flight MS prior to their structural analysis by electrospray ionization triple quadrupole MS

MALDI-TOFMS was proposed as a key technique to a novel generic approach for the speciation analysis of selenium in yeast supplements. Owing to a lower detection limit and superior matrix tolerance to electrospray MS it allowed a successful detection of selenocompounds in samples for which electrospray MS had failed. The analytical approach developed was applied to the identification of a previously unreported selenopentapeptide (m/z 596) in the tryptic digest of a water-soluble selenoprotein fraction isolated by size-exclusion chromatography. The information on the mass of the protonated molecular ion obtained from MALDI allowed the optimization of the conditions for collision induced dissociation MS using a triple quadrupole spectrometer that enabled the determination of the amino acid sequence SeMet-Asn-Ala-Gly-Arg of the selenopeptide.     

Novel approaches for selenium speciation in foodstuffs and biological specimens: A review

Selenium is an essential element for human health. It has been recognized as an antioxidant and chemopreventive agent in cancer. Selenium is known to develop its biological activity via selenocysteine residue in the catalytically active centre of selenoproteins. The main source of selenium in human beings is the diet. However, in several regions of the world the content of selenium in diet has been estimated insufficient for a correct expression of the proteins. The beneficial effects of selenium on human health are strongly dependent on its chemical form and concentration. This review critically evaluated the state-of-the art of selenium speciation in biological matrices mainly focused in nutritional and food products. Besides the number of publications related to selenium speciation, isolation and accurate characterization and quantification of selenium species is still a challenge. Hyphenated techniques based on coupling chromatography separation with inductively coupled plasma spectrometry (ICP-MS) and its combination with molecular mass spectrometry (ESI-MS, ESI-MS-MS and MALDI-TOF) and isotopic dilution allow identification, quantification and structural characterization of selenium species. Particular attention is paid in the development of Se-enriched food and nutritional products and how the application of the techniques mentioned above is mandatory to get reliable results on selenium metabolisms in these particular matrices.     

Trends in selenium biochemistry

The biochemistry of selenium-containing natural products, including selenoproteins, is reviewed up to May 2002. Particular emphasis is placed on the assimilation of selenium from inorganic and organic selenium sources for selenoprotein synthesis, the catalytic role of selenium in enzymes, and medical implications of an unbalanced selenium supply. The review contains 393 references on key discoveries and recent progress.     

Characterization of selenium species in extract from Niboshi (a processed Japanese anchovy)

Fish are selenium rich foodstuffs and a major selenium source for the Japanese population. Niboshi is processed from Japanese anchovy (Engraulis japonicus) and commonly used to prepare soup stock for Japanese dishes. In this study, we characterized selenium species in the Niboshi extract by ultrafiltration, ion-exchange chromatography and mass spectrometry. Selenium species in the Niboshi were more extractable by polar solvents (water and ethanol) than an apolar one (hexane) along with amino acids and proteinous species. Selenium in the water-extract from the Niboshi was mostly ascribed to organoselenium compounds with a molecular mass less than 5?kDa. Although selenoamino acids and selenoproteins and their fragments were involved in the extract, a large portion of the selenium species appeared to be low-molecular-mass organoselenium compounds other than selenoamino acids and their derivatives. Ion-exchange chromatographic separations revealed that most of the selenium species in the extract possess anionic and/or amphoteric characteristics. One of these selenium species from the Niboshi extract was detected at m/z 577 for 80Se by mass spectrometry subsequent to ion-pair extraction.     

Screening for disulfide-rich peptides in biological sources by carboxyamidomethylation in combination with differential matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptides with biological functions often contain disulfide bridges connecting two cysteine residues. In an attempt to screen biological fluids for peptides containing cysteine residues, we have developed a sensitive and specific method to label cysteines selectively and detect the resulting molecular mass shift by differential mass spectrometry. First, reduction of disulfide bridges and carboxyamidomethylation of free thiols is adjusted to quantitatively achieve cysteine alkylation for complex peptide extracts. In a second step, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) before and after chemical derivatization is performed, followed by differential analysis to determine shifted peaks; shifted peaks belong to cysteine-containing peptides, other peaks remain unchanged. The number of cysteines can then be determined by the resulting molecular mass shift. Free, reduced cysteines are shifted by 57 u, two oxidized cysteines involved in disulfide bridges (cystine) result in a shift to higher mass per disulfide bridge of 116 u. Disulfide bridges connecting different amino acid chains like insulin break up during reduction. In this case, two peaks with lower molecular masses result from a single one in the unmodified sample. With this technique, we were able to identify cysteine-containing peptides and short fragments of proteins present in human blood filtrate.     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

Isolation of selenoprotein-P and determination of Se concentration incorporated in proteins in human and mouse plasma by tandem heparin affinity and size-exclusion column HPLC-ICPMS

Sel-P is considered to be the most important selenoprotein for evaluating the selenium (Se) status in the body. To isolate and determine Sel-P in plasma, we have developed an analytical method combining heparin affinity (AF) and size-exclusion column (SEC) high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). We used this method to validate the adsorption efficiency of selenoproteins on a heparin AF column, and to then determine the Se concentrations incorporated in proteins in human and mouse plasma. The adsorption efficiency of Sel-P on a heparin column was more than 90% for both human and mouse plasma. Tandem AF and SEC separation proved to be useful for determining the Se concentrations incorporated in Sel-P in mouse plasma, but not in human plasma, because of nonspecific adsorption of plasma-extracellular glutathione peroxidase (eGPx) and albumin on the heparin AF column. Ultimately, we used the tandem AF and SEC separation method for mouse plasma and SEC separation alone for human plasma. The Se concentration incorporated in selenoproteins determined by our method showed good agreements with the total Se concentration determined following acid digestion.