Selenoproteins—What unique properties can arise with selenocysteine in place of cysteine?

The defining entity of a selenoprotein is the inclusion of at least one selenocysteine (Sec) residue in its sequence. Sec, the 21st naturally occurring genetically encoded amino acid, differs from its significantly more common structural analog cysteine (Cys) by the identity of a single atom: Sec contains selenium instead of the sulfur found in Cys. Selenium clearly has unique chemical properties that differ from sulfur, but more striking are perhaps the similarities between the two elements. Selenium was discovered by Jöns Jacob Berzelius, a renowned Swedish scientist instrumental in establishing the institution that would become Karolinska Institutet. Written at the occasion of the bicentennial anniversary of Karolinska Institutet, this mini review focuses on the unique selenium-derived properties that may potentially arise in a protein upon the inclusion of Sec in place of Cys. With 25 human genes encoding selenoproteins and in total several thousand selenoproteins yet described in nature, it seems likely that the presence of that single selenium atom of Sec should convey some specific feature, thereby explaining the existence of selenoproteins in spite of demanding and energetically costly Sec-specific synthesis machineries. Nonetheless, most, if not all, of the currently known selenoproteins are also found as Cys-containing non-selenoprotein orthologues in other organisms, wherefore any potentially unique properties of selenoproteins are yet a matter of debate. The pK_a of free Sec (approximately 5.2) being significantly lower than that of free Cys (approximately 8.5) has often been proposed as one of the unique features of Sec. However, as discussed herein, this pK_a difference between Sec and Cys can hardly provide an evolutionary pressure for maintenance of selenoproteins. Moreover, the typically 10- to 100-fold lower enzymatic efficiencies of Sec-to-Cys mutants of selenoprotein oxidoreductases, are also weak arguments for the overall existence of selenoproteins. Here, it is however emphasized that the inherent high nucleophilicity of Sec and thereby its higher chemical reaction rate with electrophiles, as compared to Cys, seems to be a truly unique property of Sec that cannot easily be mimicked by the basicity of Cys, even within the microenvironment of a protein. The chemical rate enhancement obtained with Sec can have other consequences than those arising from a low redox potential of some Cys-dependent proteins, typically aiming at maintaining redox equilibria. Another unique aspect of Sec compared to Cys seems to be its efficient potency to support one-electron transfer reactions, which, however, has not yet been unequivocally shown as a Sec-dependent step during the natural catalysis of any known selenoprotein enzyme.     

Nucleotide sequence of the secY gene from Lactococcus lactis and identification of conserved regions by comparison of four SecY proteins

Sec Y is an integral membrane protein which participates in the translocation of proteins through the bacterial cell membrane. We have cloned the sec Y gene of Lactococcus lactis, and found its deduced protein sequence, 439 amino acids long, to be similar in length to the previously determined Sec Y proteins of Escherichia coli, Bacillus subtilis and Mycoplasma capricolum. Comparison of the L. lactis Sec Y to the 3 other Sec Y proteins revealed 90 conserved amino acid residues (21%). Nearly half of the conserved residues are clustered in 2 of the 10 transmembrane segments, and in 2 of the 6 cytoplasmic regions. Some of the conserved regions are apparently responsible for the interactions of Sec Y with signal sequences, and the proteins SecE and SecA.     

Structure and inherent properties of the bacteriophage lambda head shell. V. Amber mutants in gene E

A total of 940 amber mutants in gene E of bacteriophage lambda was isolated to study the structure-function relationship of the gene product, the major capsid protein. The mutants were mapped to 43 mutation sites, most of which have been located, albeit tentatively, at exact points in the known base sequence, by deletion mapping and by the specificity of mutagenesis and the patterns of suppression. The patterns of suppression were interpreted in terms of both the efficiency of insertion of amino acid residues by suppressors and the exchangeability of amino acid residues. The exchangeability seems to be related to the hydrophilicity of the residues themselves and their environment, as well as to the functional similarity between the replaced and the inserted amino acid residues. Suppression of two of the mutations resulted in the production of characteristic aberrant head-related structures, each showing a defect in a different functional site in the protein. This, together with the approximate positions of some specific missense mutations as determined in this study, revealed the distribution of the functional sites along the polypeptide chain of the gene E product.     

Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces

Cysteine (Cys) is an enigmatic amino acid residue. Although one of the least abundant, it often occurs in the functional sites of proteins. Whereas free Cys is a polar amino acid, Cys in proteins is often buried, and its classification on the hydrophobicity scale is ambiguous. We hypothesized that the deviation of Cys residues from the properties of a free amino acid is due to their reactivity and addressed this possibility by examining Cys in large protein structure data sets. Compared to other amino acids, Cys was characterized by the most extreme conservation pattern, with the majority of Cys being either highly conserved or poorly conserved. In addition, clustering of Cys with another Cys residue was associated with high conservation, whereas exposure of Cys on protein surfaces was associated with low conservation. Moreover, although clustered Cys behaved as polar residues, isolated Cys was the most buried residue of all, in disagreement with known chemical properties of Cys. Thus, the anomalous hydrophobic behavior and conservation pattern of Cys can be explained by elimination of isolated Cys from protein surfaces during evolution and by clustering of other Cys residues. These findings indicate that Cys abundance is governed by Cys function in proteins rather than by the sheer chemical-physical properties of free amino acids, and suggest that a high tendency of Cys to be functionally active can considerably limit its abundance on protein surfaces.     

Mutations in the Sec61p channel affecting signal sequence recognition and membrane protein topology

The orientation of most single-spanning membrane proteins obeys the "positive-inside rule", i.e. the flanking region of the transmembrane segment that is more positively charged remains in the cytosol. These membrane proteins are integrated by the Sec61/SecY translocon, but how their orientation is achieved is unknown. We have screened for mutations in yeast Sec61p that alter the orientation of single-spanning membrane proteins. We identified a class of mutants that are less efficient in retaining the positively charged flanking region in the cytosol. Surprisingly, these mutations are located at many different sites in the Sec61/SecY molecule, and they do not only involve charged amino acid residues. All these mutants have a prl phenotype that so far have only been seen in bacteria; they allow proteins with defective signal sequences to be translocated, likely because the Sec61p channel opens more easily. A similar correlation between topology defects and prl phenotype was also seen with previously identified yeast Sec61 mutants. Our results suggest a model in which the regulated opening of the translocon is required for the faithful orientation of membrane proteins.     

The exchangeability of amino acids in proteins

The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of "experimental exchangeability" values EX(ij) derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance.     

Evolution of selenocysteine-containing proteins: significance of identification and functional characterization of selenoproteins

In the genetic code, UGA serves as either a signal for termination or a codon for selenocysteine (Sec). Sec rarely occurs in protein and is different from other amino acids in that much of the biosynthetic machinery governing its incorporation into protein is unique to this amino acid. Sec-containing proteins have diverse functions and lack a common amino acid motif or consensus sequence. Sec has previously been considered to be a relic of the primordial genetic code that was counter-selected by the presence of oxygen in the atmosphere. In the present report, it is proposed that Sec was added to the already existing genetic code and its use has accumulated during evolution of eukaryotes culminating in vertebrates. The more recently evolved selenoproteins appear to take advantage of unique redox properties of Sec that are superior to those of Cys for specific biological functions. Further understanding of the evolution of selenoproteins as well as biological properties and biomedical applications of the trace element selenium requires identification and functional characterization of all mammalian selenoproteins.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.     

Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli.

Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized. The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs. The purified E. coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences. The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed. The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues. This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E. coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.     

A 4-selenocysteine, 2-selenocysteine insertion sequence (SECIS) element methionine sulfoxide reductase from Metridium senile reveals a non-catalytic function of selenocysteines

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-R-sulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic (75)Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions.     

Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Different catalytic mechanisms in mammalian selenocysteine- and cysteine-containing methionine-R-sulfoxide reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Effect of site-directed point mutations on protein misfolding: A simulation study

A Monte Carlo simulation based sequence design method is proposed to investigate the role of site-directed point mutations in protein misfolding. Site-directed point mutations are incorporated in the designed sequences of selected proteins. While most mutated sequences correctly fold to their native conformation, some of them stabilize in other nonnative conformations and thus misfold/unfold. The results suggest that a critical number of hydrophobic amino acid residues must be present in the core of the correctly folded proteins, whereas proteins misfold/unfold if this number of hydrophobic residues falls below the critical limit. A protein can accommodate only a particular number of hydrophobic residues at the surface, provided a large number of hydrophilic residues are present at the surface and critical hydrophobicity of the core is preserved. Some surface sites are observed to be equally sensitive toward site-directed point mutations as the core sites. Point mutations with highly polar and charged amino acids increases the misfold/unfold propensity of proteins. Substitution of natural amino acids at sites with different number of nonbonded contacts suggests that both amino acid identity and its respective site-specificity determine the stability of a protein. A clash-match method is developed to calculate the number of matching and clashing interactions in the mutated protein sequences. While misfolded/unfolded sequences have a higher number of clashing and a lower number of matching interactions, the correctly folded sequences have a lower number of clashing and a higher number of matching interactions. These results are valid for different SCOP classes of proteins.     

Mass spectrometric identification of N- and O-glycosylation sites of full-length rat selenoprotein P and determination of selenide-sulfide and disulfide linkages in the shortest isoform

Rat selenoprotein P is an extracellular glycoprotein of 366 amino acid residues that is rich in cysteine and selenocysteine. Plasma contains four isoforms that differ principally by length at the C-terminal end. Mass spectrometry was used to identify sites of glycosylation on the full-length protein. Of the potential N-glycosylation sites, three located at residues 64, 155, and 169 were occupied, while the two at residues 351 and 356 were not occupied. Threonine 346 was variably O-glycosylated. Thus, full-length selenoprotein P is both N- and O-glycosylated. The shortest isoform of selenoprotein P, which terminates at residue 244, was analyzed for selenide-sulfide and disulfide linkages. In this isoform, a single selenocysteine and seven cysteines are present. Mass spectrometric analysis indicated that a selenide-sulfide bond exists between Sec40 and Cys43. Two disulfides were also detected as Cys149-Cys167 and Cys153-Cys156. The finding of a selenide-sulfide bond in the shortest isoform is compatible with a redox function of this pair that might be analogous to the selenol-thiol pair near the C terminus of animal thioredoxin reductase. The disulfide formed by Cys153-Cys156 also has some characteristics of a redox active pair.     

Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum

The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

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.     

Selenocysteine in proteins-properties and biotechnological use

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.     

Quinone-induced enhancement of DNA cleavage by human topoisomerase IIalpha: adduction of cysteine residues 392 and 405

Several quinone-based metabolites of drugs and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein and appear to increase levels of enzyme-DNA cleavage complexes by at least two potentially independent mechanisms. Treatment of topoisomerase IIalpha with quinones inhibits DNA religation and blocks the N-terminal gate of the protein by cross-linking its two protomer subunits. It is not known whether these two effects result from adduction of quinone to the same amino acid residue(s) in topoisomerase IIalpha or whether they are mediated by modification of separate residues. Therefore, this study identified amino acid residues in human topoisomerase IIalpha that are modified by quinones and determined their role in the actions of these compounds as topoisomerase II poisons. Four cysteine residues were identified by mass spectrometry as sites of quinone adduction: Cys170, Cys392, Cys405, and Cys455. Mutations (Cys --> Ala) were individually generated at each position. Only mutations at Cys392 or Cys405 reduced sensitivity ( approximately 50% resistance) to benzoquinone. Top2alphaC392A and top2alphaC405A displayed faster rates ( approximately 2-fold) of DNA religation than wild-type topoisomerase IIalpha in the presence of the quinone. In contrast, as determined by DNA binding, protein clamp closing, and protomer cross-linking experiments, mutations at Cys392 and Cys405 did not affect the ability of benzoquinone to block the N-terminal gate of topoisomerase IIalpha. These findings indicate that adduction of Cys392 and Cys405 is important for the actions of quinones against the enzyme and increases levels of cleavage complexes primarily by inhibiting DNA religation.