Selenium is an essential micronutrient which plays different roles in live organisms. The biological effects of selenium are largely mediated by selenoproteins, that contain at least one selenium-containing amino acid, selenocysteine (Sec, U). Sec is described as the 21st naturally occurring amino acid in the genetic code.[1,2]

Selenoproteins occur in organisms representing the three domains of life as well as viruses. It is known that approximately half of eukaryotes have them while only about 25% of bacteria and 15% of archea have conserved these proteins during evolution. Eukaryotes have highly variable sets of selenoproteins (selenoproteomes) that show a mosaic occurrence with some organisms, such vertebrates and algae, having dozens of these proteins whereas other organisms, such as higher plants and fungy, having lost all of them during evolution. In the ones that do not posses selenoproteïns, cysteine-containing homologs of some selenoproteins are utilized instead. The only structural difference between selenocystein and cysteine (Cys) is the presence of a selenium atom instead of a sulphur radical. [1,2,3]

However, it has been reported a higher effectiveness in catalysis that would explain why a large number of organisms have developed and conserved Se-dependent pathways and the specialized machinery required for Se insertion into proteins. Selenoproteins are mostly involved in oxidation-reduction reactions.[4]

After sequencing selenoprotein genes, it was discovered that Sec is encoded by the usually-known stop codon TGA meaning that UGA directs its insertion and requires the presence of a conserved stem-loop structure known as the Sec insertion sequence (SECIS) element.[1,2,3]

Owing to fast development of sequencing methods and genomic tools, new organisms are being genotyped constantly. There is the need to well-annotate its selenoproteome in order to provide a wide and accurate information about phylogenetic evolution.

Synthesis of selenoproteins

The introduction of Sec into proteins require several genes and a complex machinery. In eukariotes, that control of selenoprotein expression at the level of the UGA-selenocysteine recoding process is possible due to a set of dedicated cis-acting factors (the SECIS element and the in-farme UGA codon), trans-acting factors (SPS1,SPS2, SecS, Pstk, eEFsec, SBP2 and the tRNA[Ser]Sec) as well as a variety of regulatory mechanisms and elements, that enables a high grade of translational control. [4]

The incorporation of Sec is dictated by the UGA codon, as previously described. This represents a challenge for the ribosome because UGA is usually a termination signal. [2,4]

The genetic code illustrating the dual function of the UGA codon and that Sec is the 21st amino acid that is encoded by UGA. (Labunskyy VM, et al. 2014)

Fortunately, there are three unique aspects of selenoprotein mRNA translation that enable the clarification of this duality:

•      Sec is an amino acid synthesized on its tRNA

•      Decodification of UGA as Sec depends on the cis- elements

•      Standard elongation factors do not recognise the mRNA and Sec-tRNASec is delivered to the ribosome by trans-acting proteins

Scheme of the Se-proteins biosynthesis pathway.(Roman M, et al. 2014)

Interestingly, Sec is the only aminoacid synthesized directly on the tRNA, and isn’t produced from a cysteine but from a serine[5,6]. tRNA Pstk phosphorylates the tRNA[Ser]Sec , which will allow the next reaction. Selenophosphate synthetase 2 SPS2 prepares the Selenium to be incorporated, and selenocysteine synthase (SecS) binds the atom to the serine, completing the synthesis of the tRNA. This tRNA is not recognized by usual elongation factors, and is instead bound to the specific factor eEFSec.[5,7,8]

tRNA Selenocysteine 1 associated protein 1 (Secp43) is also involved in the synthesis of the tRNA[Ser]Sec and selenoproteins.

Mechanism of Sec insertion in eukaryotes.(Labunskyy VM, et al. 2014)

At this point, the selenocysteine insertion sequence (SECIS) element within selenoprotein mRNA, which is a conserved stem-loop structure encoded in the 3’-UTR of the selenoprotein mRNA in eukaryotes, plays an important role in order to recodify the UGA codon. [5,6]

The protein SBP2 (SECIS Binding Protein 2), that is stably associated with ribosomes, binds the 3D SECIS structure and allows the recruitment of the eEFSec that reclutes the tRNAsec. This is essentialy needed because this specific tRNA is not recognized by typical elongation factors. The incorporation of the selenocysteine residue into the growing polypeptide occurs because the tRNA[Ser]Sec has an anticodon complementary to the UGA. After that, the decoding process continues to complete the selenoprotein translation.[5,9]

There are other proteins that play a role in selenoproteins biosynthesis such as the ribosomal protein L30, which is part of the basal Sec insertion machinery; nucleolin and eIF4A3, that are regulatory proteins. [2]

Eukaryotes have a size of the selenoproteome significantly different. The highest content of selenoproteins is observed in aquatic organisms, whether they are animals (e.g., fish) or plants (e.g., algae). The selenoprotein evolution is influenced by the environment in which organisms live and the availability of Se. On one hand, the availability of constant supplies of Se in the sea water could have made it possible to increase the use of this element for various oxidoreductases. On the other hand, selenoproteins are more susceptible to oxidative damage, so in higher oxygen levels environments, selenoproteins may be replaced by cysteine homologs.[3,10]

Although fish selenoproteomes are larger than those of mammals, the same core selenoprotein families are found in mammals and fish. In addition, fish have several selenoproteins (Fep15, SelJ, SelL) that are missing in mammals, as well as several Sec-containing copies of Selenoproteins T, U and W, and two forms of SelP.[3,10]

Several selenoproteins genes are found duplicated in bony fishes probably owing to the whole genome duplication in the early evolution of ray-finned fishes. In zebrafish there are only additional copies of SelO, SelT1 and SelW2, named respectively SelO2, SelT1b, and SelW2b.[3,10]

Gpx – Gluthatione peroxidase

Glutathione peroxidase is one of the largest and best-known selenoprotein family. The members of this group catalyze a reacction which forms gluthathione disulfide from two molecules of reduced gluthatione. Thus, they function in the detoxification of hydrogen peroxide.

Four Gpx have been described in fishes: classical GPx1, gastrointestinal GPx2, plasma GPx3 and phospholipid hydroperoxide GPx4. Each type of GPx has a physiological localization and substrate specificity, so collectively they provide a wide spectrum of antioxidant protection.[11]

Dio – Iodothyronine deiodinase

This selenoprotein family consists in three enzyms (types 1, 2 and 3) which are anchored on the membrane and share substantial sequence homology and catalytic properties.[11]

Iodothyronine deiodinase enzymes control activation and inactivation of thyroid hormones. The types DIO1 and DIO2 catalyze the 5'5-mono-deiodination of the pro-hormone thyroxin (T4) to the active thyroid hormone 3,3'5-triiodothyronine (T3). And the type DIO3 degrades both hormones by inner ring deiodination (IRD). Thus, thyroid hormone metabolism is dependent upon the combined actions of the three deiodinases.[11]

Sep15/Sel15 – 15 kDa Selenoprotein

The specific function of this selenoprotein is not known yet. Sel15 levels differentially respond to selenium supplementation. Studies in mouse suggest that this selenoprotein may have redox function and may be involved in the quality control of protein folding.[11]

Fp15 – Fish 15-kDa selenoprotein-like

The function of this protein is not clarified yet. But it is known that Fep15 is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec.[12]

MsrB – Methionine sulfoxide reductase B

It’s also named Selenoprotein R (SelR). This selenoprotein is part of the methionine sulfoxide reductase (Msr) family, which protect cells by repairing oxidatively damaged methionine residues in proteins. MsrB is widely expressed in a variety of tissues and its distribution is both perinuclear and cytosolic.[9]

SPS/SelD – Selenophosphate synthetase

This selenoprotein is required for Sec synthesis and it’s conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. SPS is itself a selenoprotein in many species, although it’s common to find functionally equivalent homologs that replace the Sec site with cysteine (Cys). [8]

SPS2 is used as a marker for Sec utilization in eukaryotes This image shows SPS genes and predicted selenoproteins found in a representative set of eukaryotic genomes. The presence of SPS2 genes (defined as those with Sec or Cys) correlates perfectly with the presence of selenoproteins.[8]

Phylogenetic profile of SPS genes and approximate selenoproteome size of eukaryotes. (Mariotti M., et al; 2015)

SepH/SelH – Selenoprotein H

Selenoprotein H is an antioxidant protein strongly expressed in the developing CNS of vertebrates and is essential for embryonic development in zebrafish, since SelH induce MeHg downregulation in zebrafish and, consequently reduce Me HG toxicity.[13]

SepI/SelI – Selenoprotein I

It’s localized in the membrane (as SelK, S, T andN) and forms selenysulfide bonds leading to the formation and stabilization of protein complexes required for protein trafficking. Its particular function is not known. There is no relevant information about the expression in different tissues.[10]

SepJ/SelJ – Selenoprotein J

In contrast to known selenoproteins, the expression of SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. This protein is expressed preferably in the eye lens in early stages of zebrafish development. [14]

This image shows the expression pattern of the SelJ gene during development in zebrafish embryos. Shown are a gastrula (A), an embryo at middle somitogenesis (B), dorsoventral (C), or transversal (D) views of the 20-somite stage, a whole embryo 24 h after fertilization (E) and a closer view of the eyes (F), a whole embryo 36 h after fertilization (G), and an embryo 48 h after fertilization (H). All views are lateral, except C and D.(C) Territories in which SelJ is expressed are indicated in black, and the position of the midbrain–hindbrain boundary is shown in red.[14]

SepK/SelK – Selenoprotein K

Selenoprotein K is a small (16kDa) protein localized in the endoplasmatic reticulum membrane and some evidences suggest that it’s involved in endoplasmatic reticulum associated-degradation (ERAD) of soluble glycosylated proteins and plays a role in the protection of cells from ER stressed-induced apoptosis and from oxidative stress. Finally, it’s described that SepK is required for Ca+2 flux in immune cells.[10]

SepL/SelL – Selenoprotein L

This selenoprotein contains two Sec separated by two other residues, forming a UxxU motif. SelL proteins show an unusual occurrence, being present in diverse aquatic organisms, including fish, invertebrates, and marine bacteria and they aren’t present in mammals. Some evidences suggest that SelL has a redox function.[15]

SepM/SelM – Selenoprotein M

This selenoprotein is 15kDa and shares 31% sequence identity with Sel15. Both of them are localized at ER. Different studies suggest it as thiol-disulfide oxidoreductase, and also play a role in ER protein-folding.[10]

SepN/SelN – Selenoprotein N

It’s a transmembrane protein localized at ER membrane. In humans it’s seen that mutations in the gene coding for SelN cause different forms of congenital muscular dystrophy. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene causes muscle architecture disorganization and greatly reduced motility. Therefore, SelN has an important role for muscle organization during early development. [16]

SepO/SelO – Selenoprotein O

Using bioinformatics tools, it’s predicted that SelO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, SelO kinases might have retained catalytic phosphotransferase activity. Lastly, the role of the selenocysteine residue in its Cys-X-X-Sec motive suggest the possibility of an oxidoreductase-regulated kinase function for SelO. [17]

SepS/SelS – Selenoprotein S

This is a transmembrane protein located in the ER and plasma membranes and it’s widely expressed in a variety of tissues. SelS is involved in the degradation process of misfolded endoplasmic reticulum (ER) luminal proteins. It also participates in the transfer of misfolded proteins from the ER to the cytosol, where they are destroyed by the proteasome in a ubiquitin-dependent manner. Finally it’s been suggested to protect cells from oxidative damage and ER stress-induced apoptosis. Expression of SelS has been shown to be modulated by glucose metabolism and ER stress.[10]

SepT/SelT – Selenoprotein T

Selenoprotein T (SelT) is a member of a subfamily of selenoproteins (also including SelW, SelH and SelV) that share sequence similarity containing a thioredoxin-like fold and a conserved Cys-X-X-Sec motif. The expression of SelT is proposed to be similar to those selenoproteins involved in stress-related phenomena. SelT has shown to have a biological role in calcium mobilization.[10]

SepP/SelP – Selenoprotein P

In the eukaryotic kingdom, selenoprotein P (SelP) is the selenoprotein family that contains the most Sec residues. There are 10 Sec residues in human SelP and up to 17 in that of zebrafish.[18]

The function of SelP is not known. Several recent reports suggested that this protein may have antioxidant properties. It can attach to epithelial cells and protect against diquat-induced oxidative damage (Isolated SelP was reported to have phospholipid hydroperoxide glutathione peroxidase activity. In addition, SelP, owing to its high Se content, was implicated in the selenium delivery systems, such as selenium intercellular transport and storage.[18]

SepU/SelU – Selenoprotein U

Selenoprotein U (SelU) was firstly found in fish and also reported in birds and unicellular eukaryotes. In high mammalian species, such as humans and mice, all SelU proteins exist in Cys form. Its function is not known.[10]

SepW/SelW – Selenoprotein W

This selenoprotein interacts with glutathione and evidence suggest that it plays a role as a glutathione (GSH)-dependent antioxidant. It may be involved in a redox-related process and protects the developing of myoblasts from oxidative stress. It is also involved in the myopathies of selenium deficiency.[10]

TR – Thioredoxin reductase

Thioredoxin reductase (TR) enzymes are oxidoreductases that use NADPH to catalyze the reduction of oxidized thioredoxin (Trx). Trx is in turn used by several cellular enzymes as a cofactor in dithiol-disulfide exchange reactions and this is a major mechanism by which a reduced environment is maintained within cells, particularly serving to maintain reduced cysteine groups. There are three mammalian TRs: cytoplasmic/nuclear TR1, mitochondrial TR2, and testes-specific thioredoxin-glutathione reductase TR3.[10]

Periophthalmodon schlosseri, also known as the giant mudskipper, is a fish of the subfamily Oxudercinae, that represent the most land-adapted subfamily of fish. It is included in the family of globiidae (gobies).[19,20]

The giant mudskipper is an amphibious air-breathing teleost fish. It has convergent evolution of some amphibious traits.

• Kingdom: Animalia
• Phylum: Chordata
• Subphylum: Vertebrata
• Superclass: Osteichthyes
• Class: Actinopterygii
• Order: Perciformes
• Family: Globiidae
• Genre: Periophthalmodon
• Specie: P. schlosseri

Morphological traits

Periophthalmodon schlosseri’s length is between 8 and 20 cm but some males can reach 27 cm. The fish has a pale to dark brown body colour with two black lines from eye to tail. It has bluish-white speckles in the cheeks and the flanks. The front dorsal fin is reddish-brown and the rear dorsal is dark, with a pale baser. All of them have a white-cream margin. [19,21]

A distinctive character in mudskippers are the huge eyes at the top of their heads. When swimming with their head above the water, they have a 360º view.[19,21]

Mudskippers have arm-like pectorals. They make little hops by keeping their body rigid and jerking forwards on their pectoral fins. They leave typical trails on the mud. Some of these fishes have an adapted pelvic fins, that can be used to hold them in vertical surface. In addition, if they use the their arm-like pectoral, they can creep up roots and rocks. Curiously, they move faster in land and in the water surface than in the water. [19] Its gill chambers are locked when the animal is in the land. [19]

Feeding

It is known that P. schlosseri is a carnivorous fish that includes insects, worms, crabs an bivalves in its diet. [19,20]

Distribution and habitat

This fish is found in Indo-West Pacific: Singapore, Bedok, Changi, Pulu Obi, Pulu ayer Merbau, Banka, Sumatra, Java, Jakarta, Madura, Borneo, Celebes, Ambon, Waigeu, New Guinea, India, Peninsular Malaysia, Sarawak, and Thailand. [20,22]

The mudskipper inhabits the sea, estuaries and lower reaches of rivers. It excavates burrows in mudflats by mouth and then sitting mud pellets onto de surface.[20,22]

Digging the burrow supposes a high energetic expenditure. However, it is necessary to remain protected against predators, to avoid desiccation in extreme temperatures as well as for eggs incubation. Interestingly, burrows usually have a specialized chamber that contains eggs.[20,22]

Other characteristics

Respiration

P. schlosseri has the capability to breathe both, underwater and in land, which is not typical in fish.[24] Underwater, it breathes through gills as the other fishes. When in land, it has the ability to absorb oxygenated air through blood-rich membranes at the back of the mouth and buccopharyngeal cavity but also its skin, rich in blood capillaries, can absorb air as long as it is moist. To make this possible, the enlarged gill chambers that can retain water are locked. However, this chambers need to be replenished with fresh water. In conclusion, the fish cannot stay far from water but it can neither stay indefinitely in there.[19,22]

Interestingly, P. schlosseri can cope with oxygen-depleted water due to its capability to accumulate air in its burrows. Their burrows (approximately 125 cm depth) are filled with water. Immediately before entering a burrow, the mudskipper inflates its buccopaharyngeal cavity with air and is deflated when the fish emerges. Then, the animal takes immediately another gulp and often re-enters the burrow. The accumulation of air in there provides a reservoir of oxygen for burrow-dwelling fish and for developing embryos, that could not survive in hypoxic water.[22,23]

It is known that, as long as air is accessible, P. schlosseri rarely uses aquatic gill ventilation. Oxygen sensors on the epithelial surface for air breathing have a predominant role compared with oxygen gill sensors or oxygen levels in blood.[23]


Regulation of ammonia levels

The burrow water may be hypoxic and hypercapnic and have high ammonia levels. However, this organism has developed traits to maintain ammonia concentrations in its tissues: a highly vascularized epithelium, with small diffusion distances between air and blood and a gill epithelium densely packed with mitochondria-rich cells. The objective is probably accomplished by active ammonium ion transport across the mitochondria-rich cells via an apical Na/H+(NH4+) exchanger and a basolateral Na/K+(NH4+) ATPase.[25]

CO2 production as well as well as proton excretion cause acidification of the burrow water that are useful to reduce ammonia toxicity.[25] In addition, the skin of the fish has high concentration of saturated fatty acids and cholesterol that decrease membrane fluidity and gas (i.e. ammonia) permeability. [25]

In conclusion, the ammonia influx is reduced due to the acidification of the environment and the low NH3 skin permeability so that the fish can maintain tissue ammonia levels by active ammonium ion excretion, even in water containing high levels of this ion.[25]

For more information: Wikipedia