4 ǀ DISCUSSION
To the best knowledge, the present study is the first instance in which OAS1 was amplified, sequenced, characterized, analyzed by integrated Bioinformatics, and expression pattern was also studied in the endometrium of local goats (Anjori goats).
Caprine OAS1 displayed the highest sequence homology with the predictedCapra hircus and Ovis aries sequences followed by other species. This analysis indicated that the cpOAS1 sequence is conserved in mammals. But some nucleotide differences lead to changes in amino acid composition which can lead to different properties with other species. OAS1 protein has been characterized as an innate immunity gene in different species60,61,62 but no report has been there on its structural and functional prediction in goats.
In the present study, the instability index of cpOAS1 is predicted to a very unstable as the value is greater than 40, which indicates that it is not suitable for in vitro heterologous expression system30. The calculated AI of the cpOAS1 protein is 81.96 which indicates its stability at higher temperature31. The negative GRAVY value of cpOAS1 protein indicates that it is a protein consisting of more hydrophilic residues which may be a clue towards its secretory nature. The theoretical pI value of cpOAS1 indicates that it is basic and useful for wet lab extraction (through chromatographic methods). The half-life of cpOAS1 is 1.3 hours which will be helpful in the estimation of the residual time of this protein after its expression in any system. These structural and physicochemical properties of the cpOAS1 may be used for predicting protein structural and functional classes63,64, protein-protein interactions65, subcellular locations66, peptides of specific properties67, microarray data68 and protein secondary structure69.
Membrane proteins usually have one or more transmembrane segments, which are very hydrophobic making the chances for crystallization of membrane proteins small70,71. The absence of transmembrane helix and signal peptides in the cpOAS1 protein sequence suggests that the cpOAS1 gene encodes a hydrophilic, secretory protein. This also suggests that the protein is not destined to be transported across the membrane and hence localized in the cytoplasm. Results of ProtScale also additionally showed that many hydrophilic amino acids are present in the cpOAS1 protein.
cpOAS1 protein was predicted as multi-located but dominantly in the cytoplasm. Moreover, its subcellular localization was additionally confirmed to be the cytoplasmic protein. Furthermore, the different variants of OAS protein were also found in the cytoplasm and mitochondria72,73 which also supports the currentin silico analysis of this protein to its subcellular localization. The secondary structure of cpOAS1 revealed the domination of alpha-helix in cpOAS1 which indicates folding energy for right-handed is more favorable. Therefore, primary and secondary structure prediction was very useful in predicting the interaction of OAS1 with other molecules like RNAase L (innate immunity role) and IFNT which leads to its release during viral infection as well as conceptus implantation74. Solvent exposed vs. non-exposed amino acid residues have an important role in determining the protein structure75. The presence of buried and exposed amino acid residues in the cpOAS1 signifies the presence of transmembrane segments in this protein. Surface accessibility (SA) or solvent accessible surface area (SASA) of proteins has always been considered the main feature for determining protein folding, packaging and stability76. The current study revealed the different immunogenic B cell epitopes and conformational epitopes in the cpOAS1. These regions represent a strong indicator of the antigenic nature of these proteins which can be used for vaccine/antibody and immunotherapy development77,78,79.
Many eukaryotic proteins require multiple protein translational modifications (PTMs) to reach a native, biologically active conformation and should be considered as the major determinants for successful protein synthesis in addition to the physio-chemical and structural features of amino acid sequences80. In the present study, different PTMs were predicted. The presence of two mucin type GalNAc O-glycosylation sites in the cpOAS1 indicates its role in protein sorting, immune recognition, receptor binding, folding, stability as well as serving regulatory functions81,82. The identification of nine glycation sites in the cpOAS1 protein may be helpful to understand the biological function of protein glycation and identification of the associated disease treatment43,83. The presence of 21 phosphorylated sites in the cpOAS1 indicates its roles in the regulation of many cellular processes, including cell cycle, growth, apoptosis and signal transduction pathways84. The presence of one arginine and lysine propeptide cleavage site in the cpOAS1 indicates that this protein undergoes for post‐translational processing to become biologically active polypeptides45. The presence of internal acetylation sites in the cpOAS1 protein orchestrates a variety of cellular processes, including transcription regulation85,86, DNA repair87, apoptosis88,89, cytokine signaling90, and nuclear import91, etc. The presence of two SUMOylation sites in the cpOAS1 indicates that it is involved in the regulation of gene expression, cellular signaling, immune responses and the maintenance of genomic integrity92. The presence of cysteine disulfide bridges in the cpOAS1 provides proper folding, stability and more suitability for eukaryotic expression system than prokaryotic system93.
The tertiary structure of proteins refers to the overall three-dimensional shape and this included polar, nonpolar, acidic, and basic R groups that exist on the protein. In the present study, the Ramachandran plot shows that residues fell in the most favored, allowed and disallowed regions in the model (P2) generated by SWISS-MODEL were the most favored cpOAS1 3-D structure. It indicated that the refined model may be a true reflection of an experimentally-obtained OAS1 protein structure of goat. This computationally predicting 3D structure of cpOAS1 protein may be helpful for drug screening and drug design, designing mutagenesis experiments, detecting active sites, solving the phase problem by molecular replacement, and understanding the effect of disease-associated mutations74,94. This 3-D structure is also useful to evaluate the conformational differences with other species, its cross-reactivity and deciphering the activity of protein during different interactions with molecules95.
Protein domains are the fundamental units of protein structure, folding, function, evolution and design96,97. Out of various domains in cpOAS1, the OAS1_C domain is one of the important domain and is the largely alpha-helical, C-terminal half of 2’-5’-oligoadenylate synthetase 1, being described as domain 2 of the enzyme and homologous to a tandem ubiquitin repeat. It carries the region of enzymic activity between 320 and 344 at the extreme C-terminal end98. Oligoadenylate synthetases are antiviral enzymes that counteract viral attacks by degrading viral RNA. The enzyme uses ATP in 2’-specific nucleotidyl transfer reactions to synthesize 2’-5’-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. The 2’-5’ OASs are also implicated in cell growth control, differentiation, and apoptosis99,100. This domain is often associated with the NTP_transf_2 domain. In vertebrates, 2’-5’ OASs (2’-5’-oligoadenylate synthetases) are induced by interferon during the innate immune response to protect against RNA virus infections. Nuclear factors NF90 and NF45 dimerize through their common DZF domain and form a protein complex that is involved in a variety of cellular processes and affects the replication of several viruses through direct interactions with viral RNA. Eukaryotic linear motifs (ELMs) are compact protein interaction sites and these provide a wide range of functionality to proteins101,102. The presence and characterization of ELM in cpOAS1 may be useful to our understanding of both the physiological and disease states of the cell. Recognition of 51 different types of motifs and 120 instances in the cpOAS1 may be helpful in the identification of different parts of cpOAS1 protein which is involved in cell signaling processing and its interaction with other molecules.
In the present study, cpOAS1 interacted with different molecules. All the 10 predicted interactions indicate a reliable interaction among them (Table 11). The proteins DDX58, RSAD2, ISG17, IRF7, IFITI and IFIH were known to have directly or indirectly in antiviral activity and immunity development like OAS1, and the rest were either uncharacterized or had different functions (Table 11). Such networks may be useful for filtering and assessing functional genomics data and for providing an intuitive platform for annotating structural, functional and evolutionary properties of cpOAS1 proteins. These predicted interaction networks can also suggest new directions for future experimental research and provide cross-species predictions for efficient interaction mapping103.
In the present study, the presence of basal cpOAS1 transcript and protein in cyclic endometrium of does is harmonious with the expression of OAS gene in the endometrium of bovine9,11 and ovine11,23. The presence of the OAS1 in non-pregnant endometrium indicates that it may be involved in normal uterine development by creating an anti-viral environment during the estrous cycle. It has been found that during the estrous cycle, the endometrial cellular content of 2-5(A) synthetase may have a role in regulating luteolysis10 and also have an inverse relationship with PGF2 secretion in IFNT-treated bovine uterine explants104.
In agreement with our study, a higher level of cpOAS mRNA and protein was also observed in the endometrium during the early stage of pregnancy (13 to 25 days) in bovine9,10 and ovine11,23. The expression of OAS1 mRNA is parallel with conceptus elongation and IFNT secretion during early pregnancy between 16 to 21 days in does105,106, and 11 to 21 d in ewes and cows107. Some studies have also found that intrauterine infusion of IFNT in cyclic ewes and cows, andin-vitro culture of different types of endometrial cells supplementation with IFNT increases the OAS expression or its activity10,11. Moreover, different ovine endometrial cell lines expressed the different isoforms of OAS (40/46, 69/71 and 100-kDa). Three distinct classes of 2-5A synthetases have been described that correspond to proteins of 40-46 kDa, 69-71 kDa, and 100 kDa. The 40 and 46 kDa forms are produced by alternative splicing of the same gene. The 69 and 71 kDa forms are also produced by alternative splicing of a gene and consist of two adjacent homologous domains whose sequences are highly similar to that of the 40/46 kDa forms19,99. Because the three major OAS forms are generated from independent genes, each OAS gene likely has different response elements in its promoter, which may account for the differential effects of types I and II IFNs on the OAS gene transcription15,16,17,108. Various forms especially the 40/ 46- and 69/71-kDa isoforms of OAS induced by IFNT are likely to have a physiological role in the endometrium during early pregnancy in sheep and cows10,11. The exact mechanism of endometrial OAS in the establishment of pregnancy in ruminants is still clearly unknown. But it is believed that the OAS system is implicated in the antiviral response and regulation of ISG expression in response to IFNs or viral infection as well as cell growth, differentiation, and apoptosis12,14,23,108. Present findings and other studies advocate that various OAS-mediated cellular events could contribute to endometrial function during the establishment of pregnancy in ruminants109, 110.