Discussion
Manis javanica's selenoproteins were characterized by analyzing its homology against Homo sapiens selenoproteins. Since both are placental mammals and they are closely related, Manis javanica’s genome was only compared to the Homo sapiens’ genome. Moreover, Homo Sapiens genome is well-sequenced, containing 54 different proteins described in SelenoDB database.
In human, from these 54 proteins sequenced, 25 are described as selenoproteins and 7 are involved in the selenoproteins machinery. The rest of proteins are considered as proteins involved in the Selenium metabolism. Although, few selenoproteins, concretely 6, are now considered as homologous for Cys. Due to the evolution, in homologous for Cys proteins, the Sec residue has changed into a Cys residue.[15,17,20,23]
In Manis javanica all 54 proteins were analyzed and 21 could be defined as selenoproteins. The ones from selenoprotein machinery and Selenium metabolism could also be defined. Regarding to the other proteins, 4 of them described as selenoproteins in human seem to have lost the Sec residue in Manis javanica. Finally, according to the 6 proteins considered as homologous for Cys in human, only 5 of them are conserved in Manis javanica.
Selenoproteins in Manis javanica
15-kDa selenoprotein (Sel15)
Selenoprotein 15 (Sel15) is one of the ancestral selenoproteins found in all vertebrates. Sel15 is proposed to mediate the cancer prevention effect of dietary Se. Due to its redox activity in ER homeostasis, it may also act as an antioxidant. Sel15 might also have a role in disulphide bond formation and quality control of some glycoproteins. It is highly expressed in prostate, liver, brain, kidneys and testis.[16,20,23]
In Manis javanica Sel15 was well-predicted. The resultant protein contains a Sec residue and one SECIS element correctly described in the 3’-UTR region of the gene. Even though, Seblastian could not be predicted. Analyzing the t-coffee output, a huge conservation between both sequences was observed. Although Sel15’s function is not clear, the great conservation of this protein points out the importance of this selenoprotein.
Glutathione peroxidases (GPx)
The glutathione peroxidase family was the first family of selenoproteins described and one of the largest families in vertebrates. In human, there are eight GPx paralogues, from which five are selenoproteins (GPx1 - 4 and GPx6) and the other three are GPx homologous for Cys (GPx5 and GPx7 - 8). It is known that these three homologues have evolved from the ancestor selenoproteins. The seleno-peroxidases (Sec-GPx) are prevalent in vertebrates, while GPx homologs are found in terrestrial plants, yeast, protozoa, and bacteria.
It has been described an accurate relation between their activity and the concentration of Se in the enterocytes. They are found in the cytosol, where they play a wide role in the physiological functions in organisms; hydrogen peroxide (H2O2) signalling, detoxification of hydroperoxides and cellular redox homeostasis maintaining. They are also a vehicle to collect Se. Concretely, the principal function of the glutathione peroxidases (GPx) is to protect the organism from oxidative damage.[17,20,25]
Almost the same hits for each GPx were found while blasting the two sequences, indicating the elevated intrafamiliar relationship in this family.
GPx1
As it is known, GPx1 in humans is a selenoprotein. Even though, in Manis javanica no selenocysteine residue was found, since Sec residue in human aligns with a Ser in Manis javanica, so it could not be predicted as a selenoprotein. Analyzing the t-coffee output, just a few part of the sequences could be aligned. The lack of alignment shows that the protein is not conserved, which differs from the information found, that classifies GPx1 as a really conserved protein.
GPx2
GPx2 is a tetrameric selenoprotein in human which was also predicted in Manis javanica. The resultant protein contains a Sec residue, one SECIS element correctly described in the 3’-UTR region of the gene and Seblastian prediction was also found. The sequence obtained in the Seblastian prediction was exactly the same obtained by bioinformatics tools. Analyzing the t-coffee output, a huge conservation between both sequences was observed, which means a great conservation between the sequences in this protein.
GPx3
Another tetrameric selenoprotein in human is GPx3, which is secreted primarily from kidney and is the major GPx form in plasma. As GPx2, GPx3 was also predicted in Manis javanica. The resultant protein contains a Sec residue and one SECIS correctly described in the 3’-UTR region of the gene. Even though, Seblastian could not be predicted. Analyzing the t-coffee output, a huge conservation between both sequences was observed, which means a great conservation between the sequences in this protein.
GPx4
Differently, GPx4 is a monomeric selenoprotein expressed in a wide range of cell types and tissues in human, and it was also predicted in Manis javanica. The resultant protein contains a Sec residue, one SECIS correctly described in the 3’-UTR region of the gene and Seblastian prediction was also found. For GPx4 the Seblastian prediction showed one exon less than the t-coffee output. This fact is due to the length of the scaffold used to predict the protein; in the manual prediction a longest sequence was analyzed so the protein in this case is longer and it has an extra exon, the first one.
By analyzing the t-coffee output, a huge conservation between both sequences was observed, which indicates a great conservation between the sequences in this protein. This information accords to the one referred in the literature which defines GPx4 as one of the most conserved Sec-containing selenoproteins.
GPx5 and GPx6
It is known that the most recently evolved GPx are GPx5 and GPx6, due to a tandem duplication of GPx3 in placental mammals. The phylogeny reflects a displacement Sec-to-Cys in a GPx3 duplication as a possible consequence of the origin of GPx5. In contrast, for GPx6, several independent Cys conversions were observed.
In humans, GPx5 is known to be an homologous for Cys protein. In contrast, in Manis javanica the Cys residue seems to be replaced by a Leu. Due to that fact found in Manis javanica, GPx5 is not an homologous for Cys, so GPx5 in this specie is neither a selenoprotein nor an homologous for Cys protein. This may be consequence of a mutation and the evolution of the different proteins.
GPx6 is a selenoprotein in humans only found in the olfactory epithelium during embryonic development. In contrast, GPx6 could not be predicted in Manis javanica. The selenocysteine residue seems to be replaced by a cysteine in this case, which leads to the possibility of GPx6 being an homologous for Cys. A SECIS element was found for GPx6, which strengthens the theory of GPx6 being an homologous for Cys protein. Having a SECIS element in 3’UTR region indicates that GPx6 used to be a selenoprotein but it has evolved into an homologous for Cys.
GPx7 and GPx8
Before the separation of mammals and fishes, GPx7 and GPx8 evolved from a GPx4 ancestor becoming Cys-containing proteins. In human, both proteins are considered homologous for Cys and it also happens in Manis javanica. In both proteins, any selenocysteine residue was found and all the cysteine residues were conserved between Homo sapiens and Manis javanica, which indicates a great conservation among species also observed in the t-coffee outputs.
As it was said before, GPx family proteins have an elevated intrafamiliar relationship between all of them. That’s why almost the same hits were found, and this lead to some problems during the selection of the best scaffolds for each protein. This could be a reason to justify some incoherences found in the analysis of the different proteins such as the phylogeny tree described in results, in which both GPx5 and GPx6 appear in an incorrect position. GPx5 in Manis javanica appears to be correlated with GPx1 instead of appearing correlated with the human GPx5. In the same way, GPx6 appears to be correlated with GPx8 instead of appearing with the human GPx6. For the rest of the phylogenetic tree the information agrees with the one obtained from the literature; GPx7 and GPx8 came from the same ancestor as GPx4 and GPx5 and GPx6 in human came from the same ancestor as GPx3.
The correlation of GPx6 with GPx8 is due to an error in the selection of the scaffold. GPx6 was the last protein of the family analyzed and since all its scaffolds were already selected for other proteins, there was no alternative to use the same scaffold as for GPx8. Even though this is not correct there were only three Sec residues among all the scaffolds which means that in Manis javanica there are only three selenoproteins in the GPx family.
Iodothyronine deiodinases (DIO)
DIO family is the second most important family of selenoproteins. Three DIO members have been identified with a tissue a subcellular localization, which are involved in the regulation of the thyroid hormone activity. DIO1 and DIO2 catalyze the deiodination of T4 into T3, the active hormone. DIO3 converts T4 into reverse T3 and also T3 into 3,3’-diiodothyronine. All three DIOs are integral membrane proteins and share significant structural similarity. As it happened for the GPx family the structural similarity brings to the finding of the same hits in all the proteins, so it was necessary to determine the best scaffolds for each protein regarding in not to use the same.[15,19,20]
DIO1
As it is known, DIO1 is a selenoprotein in humans, and the analysis of Manis javanica’s genome showed that it is also a selenoprotein in this specie. A Sec residue, a SECIS element in the 3’-UTR region and a Seblastian prediction were correctly found, determining the existence of the selenoprotein in Manis javanica. Moreover, the manual analysis perfectly correlates with the Seblastian prediction. The conservation grade of the alignment between the two sequences was not as great as in other proteins, which involves a lower conservation of the protein in Manis javanica.
DIO2
As in humans, DIO2 was determined as a selenoprotein in Manis javanica. A Sec residue and a SECIS element in the 3’-UTR region were found, determining the existence of the selenoprotein. Even though, Seblastian prediction could not be found. Analyzing the t-coffee alignment, a great conservation between sequences was found, which implies a great conservation of this protein between these two species.
DIO3
Finally, DIO3 was also predicted as a selenoprotein in both human and Manis javanica. A Sec residue, a SECIS element in the 3’-UTR region and a Seblastian prediction were found, determining the existence of the selenoprotein. As it happened with DIO1, the manual analysis correlates perfectly with the Seblastian prediction. Similar to what happened with DIO2, a great conservation between sequences while analyzing the t-coffee output was found, which implies a great conservation of this protein between species.
Selenoprotein H
Selenoprotein H (SelenoH) is another ancestral selenoprotein found in all the vertebrates, although its function is not clear. This protein is located specifically in the nucleoli and it is sensitive to dietary Se intake. As in humans, in Manis javanica SelenoH was predicted as a selenoprotein. A Sec residue, a SECIS element in the 3’-UTR region and a Seblastian prediction were found, determining the existence of the selenoprotein.[15,17,19,20,23]
In the t-coffee output, a great conservation between both sequences was observed. As said previously, SelenoH is an ancestral selenoprotein, and this correlates with the fact that it is perfectly conserved among the species. Although their function is not completely known, it seems to be important for the species survival due to its high grade of conservation.
Selenoprotein I
Selenoprotein I is also an ancestral selenoprotein found in all vertebrates. It is one of the least studied selenoproteins since it evolved recently and is exclusively found in vertebrates. This protein is involved in the formation and maintenance of vesicular membranes, regulation of lipid metabolism and protein folding.[15,17,19,20,23]
In Manis javanica SelenoI was predicted as a selenoprotein with a Sec residue in the last part of the protein sequence. Seblastian prediction and a SECIS element of grade A in the 3’-UTR region were also found in order to confirm the existence of the selenoprotein. The manual analysis correlates perfectly with the Seblastian prediction.
Finally, by analyzing the t-coffee output a perfect alignment between the two sequences was found. This leads to a huge conservation of the protein sequence in both species and it correlates with the fact that SelenoI is an ancestral selenoprotein of all vertebrates.
Selenoprotein K and Selenoprotein S
Selenoprotein K is an ancestral selenoprotein found in all vertebrates. SelenoK and SelenoS are the most widespread eukaryotic selenoproteins since they are present in nearly all the organisms that use Se, from unicellular eukaryotes to humans.[15,17,19,20,23]
According to that fact, in Manis javanica SelenoK was predicted as a selenoprotein with a Sec residue also in the last part of the protein sequence, as in SelenoI. Seblastian prediction and a SECIS element of grade A in the 3’-UTR region were also found in order to confirm the existence of selenoprotein. Moreover, the manual analysis correlates almost perfectly with the Seblastian prediction.
The t-coffee output showed a perfect alignment between the two sequences which leads to a huge conservation of the protein sequence in both species. Regarding the fact that SelenoK is an ancestral selenoprotein present in all the organisms, this result perfectly agrees with this statement.
As it is said, this family also contains SelenoS. In this case, the t-coffee output also showed a highly conserved alignment between both species. Seblastian prediction gave the same results as the ones obtained by bioinformatics tools. Sec residue is near the COOH-terminal and SECIS element of grade A is well-located in 3’-UTR region of the gene. This both results confirm that in Manis javanica SelenoS is established as a selenoprotein.
Selenoprotein M
As it is known, SelenoM belongs to the same family as Sel15. Both proteins have two different motifs, Cys-X-X-Sec and Cys-X-Sec. Among these two selenoproteins, there is a 31% of homology between sequences. SelenoM is expressed in brain and it might have a neuroprotective action. Furthermore, it is related with Ca2+ regulation in response to H2O2 although its function is unclear.[15,17,19,20,23]
Analysing the t-coffee, it is observed the Cys-X-X-Sec motif pointed in the literature and it could also be observed a high conservation between both species. The obtained results fit perfectly with the Seblastian prediction including the SECIS element that was also predicted by SECISearch3. That’s why it can be concluded that SelenoM is a selenoprotein with a SECIS element located in 3’-UTR in Manis javanica as the same way as in human.
Selenoprotein N
Selenoprotein N is one of the ancestral vertebrate selenoproteins and it is really conserved among different lineages. It is highly expressed during embryonic development and to a lesser extent in adult tissues including skeletal muscle. It might play an important role in the maintenance of satellite cells and is required in the regeneration of skeletal muscle tissue following stress or injury.[15,17,19,20,23]
In this case, SelenoN in human has two different Sec residues whether in Manis javanica it has only a Sec residue in the exon 7. The alignment between both species seems to be incomplete in the first part of the Manis javanica protein since the scaffold used was not complete. Even though, no more scaffolds were available in blast prediction. Nevertheless, the second Sec residue was well-aligned. Observing the results obtained, it is unclear of what happened with the first Sec, because its localization is in the middle of the non-well-codified part in Manis javanica. This may suggest that Pangolin lost one part of this gene, including the first Sec residue.
Contrarily to many other selenoproteins, no Seblastian prediction was found, but a SECIS element of grade A was predicted in the right place, leading to consider this SelenoN as a selenoprotein in Manis javanica.
Selenoprotein O
Selenoprotein O is also one of the selenoproteins found in the ancestral vertebrate selenoproteome although it is one of the least characterised. The Sec residue is located near the COOH-terminal and most of the homologous replaced this Sec into a Cys. However, Manis javanica has this Sec residue in the same position as humans.[15,17,19,20,23]
In this case, Seblastian is predicted. By comparing Seblastian prediction and the results obtained manually, it has been found that they only differ in the position of the first exon, being the first exon larger in the Seblastian prediction. Also, in the Seblastian output, the first part of the protein is almost conserved whether in t-coffee, the alignment of the first part is incomplete. Blast has a second scaffold, which was not selected since there was no Sec residue in the sequence. Nevertheless, the last part of the protein is well-conserved in both predictions, correlating with the fact that the Sec residue is well-located in Manis javanica and confirming that this specie does not contain an homologous for Cys in this selenoprotein, as it happens with other species. Regarding SECIS element, our results predicted more than one SECIS element, being the selected one the same as in the Seblastian prediction.
All this results confirm that SelenoO belongs to the group of selenoproteins in Manis javanica.
Selenoprotein P
Selenoprotein P is one of the ancestral selenoproteins in vertebrates and is abundantly expressed and secreted in mammals. It accounts for almost 50% of the total Se in plasma. It is known that this protein has recently evolved in vertebrates. It has multiple Sec residues; for example, in human it has 10 in-frame Sec residues and two SECIS elements located in the 3’-UTR region. For this reason, SelenoP might function as a Se supplier to peripheral tissues, particularly brain and testis.[17,19,20,29]
By comparing human against Manis javanica, it can be observed more Sec residues in Manis javanica than in human, concretely 14 against 10 in human. In this case, Seblastian is also predicted. The 10 Sec residues found in human are located in the same position in Manis javanica, suggesting that in Manis javanica some duplications in Sec residues have occurred during evolution. Regarding the two SECIS elements that are described in the literature, only one SECIS element is selected in Manis javanica since the other SECIS element predicted was not located in 3’-UTR.
Methionine sulfoxide reductases B or Selenoprotein R (MSRB)
This family is formed by two different proteins, MSRA and MSRB. Only one of them, MSRB, known also as SelenoR, is a selenoprotein, suggesting that these two proteins have different origins. MSRB has three different isoforms, MSRB1-3, being MSRB1 one of the ancestral vertebrate selenoproteins. This family catalyzes the conversion of methionine sulfoxide to methionine.[1,17]
Analysing the different isoforms of MSRB it has been found that only MSRB1 has a Sec residue in its sequence. As the selected scaffold for this protein was too short, it was impossible to find out any SECIS element. Moreover, the first part of the human protein is not aligned with the selected scaffold, being this scaffold incomplete. The best scaffold given by blast could not be selected because there was no Sec in the sequence. Despite the SECIS element could not be found, MSRB1 is considered as a selenoprotein.
The other two isoforms are homologous for Cys in both species, human and Manis javanica. There is more than one Cys residue in each human isoform conserved in Manis javanica. As it happens with MSRB1 isoform, these alignments are not complete in the first part of the protein. This suggests that Manis javanica has lost some parts of these three isoforms during evolution. Seblastian prediction could not be obtained for any of the three isoforms.
Selenoprotein T
As other proteins, SelenoT has a Cys-X-X-Sec motif, which is characteristic in many selenoproteins, such as SelenoM. It is thought that SelenoT has a role in the regulation of Ca2+ homeostasis and a neuroendocrine function. It is also known that SelenoT has different isoforms.[15,17,19,20,23]
Contrarily to many selenoproteins, Seblastian could not be predicted. Nevertheless, t-coffee output showed a perfect alignment between both sequences, differing only in one amino acid. This suggests that SelenoT could be one of the most conserved selenoproteins between these two species. Again, these results confirm that SelenoT is another selenoprotein in Manis javanica.
Selenoprotein V
SelenoV is similar to SelenoT, SelenoW and SelenoH. They have a characteristic motif in which the Sec residue is located. It is one of the least characterised selenoproteins in human and it is known that it is an evolution from a duplication of SelenoW.[15,17,20]
Contrarily as it was expected, in Manis javanica SelenoV is not a selenoprotein since no Sec residue could be found in its sequence. Analysing the blast, three scaffolds were available. After doing the hall process in all of the scaffolds, only one was selected. This selection was done in order to obtain the best final alignment, as the hall alignments were too poor. T-coffee output showed that the majority of the human protein could not be found in Manis javanica. Moreover, Sec residue in human, is aligned with Thr in Manis javanica, resulting to a loss of Sec in the specie. Neither Seblastian nor SECIS element could be predicted.
Taking into account all this information, SelenoV is one of the selenoproteins in humans that is not present in Manis javanica. This result suggests that SelenoV is an inactive duplication of SelenoW, that SelenoV in Manis javanica has a different origin or that an aleatory mutation has caused a substitution of the Sec residue.
Selenoprotein W
As it is known, SelenoW was one of the first identified Sec-containing proteins and it is one of the most abundant selenoproteins in mammals. Its expression is highly regulated by Se. There are two SelenoW proteins, SelenoW1 and SelenoW2, both members of the ancestral proteasome. SelenoW1 is highly conserved in different lineages while SelenoW2 is not, only being a selenoprotein in fishes. According to this information, SelenoW1 is a selenoprotein in humans whether SelenoW2 is not. Concretely, SelenoW2 is an homologous for Cys in human.[15,17,19,20,23]
By analysing the t-coffee output of SelenoW1, it can be concluded that the selected scaffold is incomplete because there are some regions that are aligned with gaps. Although one Sec residue is well-aligned, there is another Sec residue only found in Manis javanica's sequence. The SECIS element is not well-predicted because it is located in the 5’-UTR extreme. That fact converts SelenoW1 into a non-containing Sec protein. In this case, there are two different hypothesis; SelenoW1 is a pseudogene and the second Sec residue appeared due to a mutation which correlates with the fact that the SECIS element is not in the functional region or there is an ensemble error, as the sequence found next to the Sec residue is mostly conserved although the last part of the human protein is not found in Manis javanica. For all these reasons, SelenoW1 could not be assumed as a selenoprotein in Manis javanica.
In the other hand, Manis javanica t-coffee output in SelenoW2 showed a highly conserved sequence related to human. Moreover, the homologous Cys appears also in the Manis javanica sequence. SelenoW2 is an homologous for Cys protein as it is in human.
Thioredoxin reductases (TXNRD)
TXNRD family contains three proteins that are selenoproteins in human. They are oxidoreductase so they comprise the major disulphide reduction system of the cell. It is known that this family is conserved in different lineages such as fish, frog and different mammals, being the placentals whose interest us. Correlating with the literature, in Manis javanica all the selenoproteins seem to be present.[15,17,20]
Analyzing the three t-coffee outputs, it was observed that the most well-aligned member of this family is TXNRD2 whereas TXNRD3 is the most incompletely alignment. All of them have a Seblastian prediction that differs from the results obtained. Concretely, TXNRD3 Seblastian differs from the results obtained, specifically on the length of the first exon. However, SECIS element was predicted in the same location as the other results. Moreover, TXNRD1-2 Seblastian differ in the number of exons, as the results obtained by bioinformatic tools contain more exons than in the Seblastian predictions, concretely 6 exons more in the beginning of the gene in TXNRD1 and 3 exons in TXNRD2, located also in the beginning. In contrast, SECIS elements were well-predicted by Seblastian.
The three diferents Sec residues are well-aligned with the human sequence in all of the proteins. Altogether, it can be concluded that TXNRD family is conserved in Manis javanica.
Selenoprotein machinery in Manis javanica
Selenocysteine synthase (SecS)
Selenocysteine synthase (SecS) is a pyridoxal phosphate-dependent enzyme that converts the Ser of the tRNA into selenocysteyl-tRNA[Ser]Sec by incorporating a selenophosphate, the active form of selenium, into the amino acid backbone to form Sec-tRNA[Ser]Sec. That’s why it is considered as a protein involved in selenoprotein machinery and therefore, there is no selenocysteine in the sequence.[17,21]
After analysing the t-coffee output, Manis javanica’s sequence seems to be conserved after the linage separation. The high conservation of its structure is really important for its function suggesting that it does not accept mutations in many sites. Contrarily of what was expected, a SECIS element was predicted by SECISearch3 although it has no sense since there is no selenocysteine. Seblastian was not predicted.
SECIS binding protein 2 (SBP2)
SECIS-binding protein 2 is the enzyme that promotes the incorporation of Sec by association with SECIS elements and recruiting the eEFsec-selenocysteyl-tRNA[Ser]Secs complex to the ribosome. SBP2 has three domains; an NH2-terminal with unknown function, a Sec incorporation domain (SID) and a COOH-terminal RNA-binding domain that it is stably associated with ribosomes and contains a distinct L7Ae RNA-binding domain. This domain works as a suppressor of translation termination. In conclusion, SBP2 is known to bind SECIS elements with high affinity and specificity.[17,23]
After analysing the t-coffee output, Manis javanica’s sequence seems to be conserved especially in the final part. The mid-part of the sequence is least conserved and the scaffold chosen could be a possible cause of that bad alignment. As expected, neither SECIS elements nor selenocysteine were found.
Phosphoseryl-tRNA kinase (PSTK)
Phosphoseryl-tRNA[Ser]Sec kinase is the enzyme that phosphorylates the precursor Serine of the Sec joined to the tRNA. Therefore, this enzyme makes possible the formation of Sec, the 21st amino acid.[17]
Looking at the results obtained, Manis javanica’s sequence seems to be conserved in the most part of the scaffold, even though the last part is less conserved. This agrees with the fact that the function and homology of this protein is conserved across Archaea and Eukarya, which suggests that it plays an important role in selenoprotein biosynthesis and regulation. As a protein involved in selenoprotein machinery, there is no selenocysteine in the sequence. Contrarily of what it would be expected, a SECIS element is predicted by SECISearch3 although it has no sense as there is no selenocysteine. Seblastian is not predicted.
Eukaryotic elongation factor (eEFsec)
eEFsec is the elongation factor that recruits the tRNA with the Sec and binds to SBP2 in order to insert the Sec into nascent polypeptides in response to UGA. Concretely, it is a GTP-dependent RNA binding protein that contains two different domains; an N-terminal elongation factor specific to selenocysteyl-tRNA[Ser]Sec and a C-terminal selenocysteine insertion (SECIS) binding domain.[17]
After analysing the whole results obtained for different scaffolds, the most conserved sequence was selected. In this case, the best three possible scaffolds were analysed in order to obtain the best alignment. Different scaffolds predicted different parts of the protein, being each one different from the others. Moreover, the protein sequences obtained could be positioned one next to the other, as if the protein was broken in three parts in Manis javanica. By analysing these different parts, the second best scaffold was selected in order to give just one result. This alignment shows nearly half of the protein.
Selenophosphate synthetases (SEPHS)
Selenophosphate synthetase family is composed by two different proteins that are involved in selenoprotein machinery and one of them, SEPSH2, is a selenoprotein while SEPSH1 is not. SEPSH2 is the unique protein involved in machinery that is a selenoprotein by itself. These enzymes catalyse the synthesis of selenophosphate from selenide and ATP, generating the Se donor component needed for the synthesis of Sec. It is conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. The fact that SEPSH1 hasn’t got a Sec seems to result to the abolishment of the selenophosphate synthetase function.[17,23]
Analysing the t-coffee output of SEPSH1, it has been found that the sequence is highly conserved as there are only two amino acids that differ between Manis javanica and human sequences. As it is said, no selenocysteine was found in the sequence. SECIS and Seblastian were also not predicted by the in silico program.
In contrast, in SEPSH2’s t-coffee output, a less conservation of the protein was observed, especially at the last part. Two selenocysteines could be found in both human and Manis javanica sequences, resulting on the high conservation of this family.
tRNA Sec 1 associated protein 1 (SECp43)
When SECp43 was discovered, it was characterized as RNA-binding protein that formed a complex with Sec tRNA[Ser]Sec. It was also found to have two RNA binding sites and to interact with a 48-kDa protein. New studies revealed that SECp43 and SLA have a role in regulation of selenoprotein expression and firmly linked these proteins to the pathway of selenoprotein biosynthesis. Moreover, a subcellular localization analysis of SECp43 suggested that it may regulate shuttling of the SecS-selenocysteyl-tRNA[Ser]Sec complex between the nucleus and cytoplasm.[33]
SECp43 is a protein involved in selenoprotein’s machinery found in both species, human and Manis javanica. Analyzing the t-coffee output of this protein, the sequence is not as conserved between species as in other proteins. This fact could be due to the scaffold selected or to a bad annotation of the genome. Since it is a protein involved in the selenoproteins machinery, there is not a selenocysteine residue in the proteic sequence.
In humans, SECp43 is an homologous for Cys protein and it is located in the in-between-region of the machinery and the homologues. In contrast, in Manis javanica the Cys residue of the protein is replaced for a stop codon, suggesting that in Manis javanica the Cys residue is replaced for a Sec.
Selenium metabolism in Manis javanica
ELAV-like RNA binding protein 1 (ELAVL1)
This protein contains three RNA recognition domains and binds short sequences with a high amount of Adenine and Uracil nucleic acids in order to stabilize mRNAs for gene expression regulation. ELAVL1 is also implicated in the selenocysteine regulation of Selenoprotein I.[13]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
ELAV-like family member 1 (CELF1)
CELF1 structure consists of two N-terminal RNA recognition motif domains and another one for C-terminal. The amino acids contained in the middle are variable depending on the tissue. Its main function is to regulate the alternative splicing of pre-mRNA in order to obtain different transcripts encoding many isoforms. This protein is also involved in the selenocysteine regulation in proteins of DIO family, by the reported mechanism.[13]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Eukaryotic translation initiation factor 4A3 (EIF4A3)
This nuclear matrix protein owns to the DEAD box protein family, which contains the conserved motif Asp-Glu-Ala-Asp (DEAD). They all are RNA helicases involved in many cellular processes that alter RNA secondary structure, such as nuclear and mitochondrial splicing and translation initiation. It also acts as a repressor of selenoprotein synthesis when selenium levels are decreased. EIF4A3 binds SECIS elements from non-essential selenoproteins in order to avoid selenocysteine addition.[6]
As it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica. In fact, in this alignment every single amino acid was perfectly conserved.
Exportin 1 (XPO1)
XPO1 mediates nuclear export signalling of proteins with leucine-rich sequences. It is also involved in the control of many cellular processes by regulation of cyclin B, MAPK and MAPKAP kinase 2. It has been recently elucidated that XPO1 also regulates the selenocysteines of Selenoprotein R1.[13]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Glucose-6-phosphate dehydrogenase (G6PD)
It is a cytosolic enzyme usually formed by two identical monomers, although it can also exist as a tetramer. Its function is to catalyse the first chemical reaction in the pentose phosphate pathway, which provides NADPH to the cell and pentoses for the nucleic acids synthesis. G6PD deficiency is very common and causes acute hemolytic anemia due to an abnormal oxidation of hemoglobin. It is reported that in order to balance the partial loss of selenoproteins, which function also as antioxidants, certain tissues may upregulate the expression of proteins involved in reduction or peroxidation processes, like G6PD.[13]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Methionine sulfoxide reductase A (MsrA)
MsrA is a protein involved in methionine repair; it catalyses the reduction of methionine sulfoxide to methionine. Through this way, the oxidative damage is repaired in order to restore its biological activity. Apart from an algae species, any selenoprotein MsrA is reported in any organism. They are all proteins containing Cys residues.[1]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica in the final part of the protein.
Ribosomal protein L30 (RPL30)
This ribosomal protein is a component of the 60S subunit of the ribosome. It belongs to the L30E family of ribosomal proteins and is located in the cytoplasm. RPL30 acts as a component of the eukaryotic selenocysteine recoding machinery. It is reported to bind SECIS elements (both in vitro and in vivo), stimulates UGA recoding and competes with SBP2 for SECIS binding.[9]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, the alignment was not good, indicating a low-conservation of this protein in Manis javanica.
Selenium binding protein 1 (SELENBP1)
This is a member of the selenium-binding protein family. This protein may have an important function in ubiquitination and deubiquitination for protein degradation, mediated by selenium. Therefore, a several decrease in this protein expression is associated with many different cancers (colorectal, lung, ovarian) and neurologic diseases.[13]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Low-density lipoprotein receptor-related proteins (LRP)
LRP2
It is a binding receptor found in the plasmatic membrane of many absorptive epithelial cells, which can bind to a wide variety of ligands. It helps to introduce these ligands into the cell by endocytosis, leading to lysosomal degradation. It is also expressed in epithelial thyroid cells, binding with thyroglobulin. It is reported to have an important significance for the clinical, as structural modifications given by some mutations can lead to Donnai-Barrow syndrome, a rare disease with many body parts impairment (hearing loss, intellectual disability, intestinal abnormalities).[14]
In Manis javanica LRP2 could not be predicted as it contains a very long sequence and the outputs of exonerate, t-coffee and genewise could not be obtained. In consequence, there are no results from this protein in Manis javanica.
LRP8
It is a receptor located in the cell surface which owns to the low-density lipoprotein receptor family, as LRP2. Similar function is reported in this case, but other specific ligands such as reelin, produce different effects. It is well-known the mechanism by which LRP8-reelin binding produces embryonic neuronal migration and postnatal LTP. For this reason, a decrease in LRP8 expression is mainly associated with neurological diseases.[14]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica in the last part of the sequence. In the mid-part of the proteic sequence the alignment was not good enough but it could be a consequence of the scaffold used to predict the protein. Even though, the rest of the alignment is almost perfect.
Selenoprotein U
The selenoprotein U family in mammals is composed by three members; SelenoU1, SelenoU2 and SelenoU3. The SelenoU1 selenoprotein is the only member of the family that takes part of the ancestral selenoproteome. Even though SelenoU function is unknown, all of them contain a Prx-like2 structure domain in their structure.
Phylogenetic analysis of Sec- and Cys- containing forms of the SelenoU family suggested that all Sec- containing SelenoU sequences belong to SelenoU1. Mammals contain three Cys-containing SelenoU proteins (SelenoU1-3), whereas some fish have three Sec-containing SelenoU proteins. It was previously thought that the three Cys-containing SelenoU proteins in all mammals evolved from the three Sec-containing SelU sequences in fish. Even though, no evidence was found to determine this Sec-to-Cys early event for SelenoU2 and SelenoU3.[20]
Since any of SelenoU members are selenoproteins in humans neither in any mammal, the analysis of this family in Manis javanica’s genome shows that they are not selenoproteins in this specie, as they contain Cys residues instead of Ser. Moreover, the analysis of the conservation grade of the alignment between the two sequences is good in SelenoU1 and SelenoU2, showing a high-conservation of the protein in Manis javanica. Even though, this conservation is not shown in SelenoU3.
Selenocysteine lyase (SCLY)
SCLY is a pyridoxal (a form of Vitamine B6) 5’-phosphate-dependent enzyme that catalyzes decomposition of L-selenocysteine to form L-alanine and selenide, and an enzyme-bound selenylsulfide intermediate. The ability to produce selenide suggests a role for this protein in Se recycling. It is mainly expressed in the liver, as it is a key organ in management of Se availability to the entire body.[7]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Sep (O-phosphoserine) tRNA:Sec t-RNA synthase (SEPSECS)
SEPSECS is an enzyme that performs the conversion of O-phosphoseryl-tRNA[Sec] to selenocysteinyl-tRNA[Sec] by using selenophosphate as the selenium donor, a critical step for Sec synthesis. It forms a stable tetramer, acting on phosphoserine that is linked to tRNA[Sec] and not on free phosphoserine or Ser-tRNA[Sec]. However, the molecular basis for substrate discrimination is not clear. This enzyme is essential in the development of organisms, since its deficiency causes abortion of the embryo.[24,35]
Since it is a protein involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Seryl-tRNA synthetase mithocondrial 2 (SARS2)
The SARS2 enzyme catalyzes the ligation of Serine to tRNASer in mitochondria. The enzyme contains an N-terminal tRNA binding domain and a core catalytic domain. It functions in a homodimeric form, which is stabilized by tRNA binding.[24]
Since it is an enzyme involved in the selenium metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.
Tocopherol (alpha) transfer protein (TTPA)
TTPA or Tocopherol (alpha) transfer protein is expressed exclusively in the liver, although it is distributed in different tissues. This protein determinates the incorporation of alpha- or gamma-tocopherol (Vitamin E) into the nascent VLDL particles during their assembly and secretion from the liver.[13]
Since it is an protein involved in the Se metabolism, no selenocysteine was found during the analysis neither in human nor in Manis javanica. By analyzing the conservation grade of this protein, it is shown a good alignment, indicating a high-conservation of this protein in Manis javanica.