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.