Journal of Genetics (2020)99:25 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-020-1183-1 (0123456789().,-volV)(0123456789().,-volV)

RESEARCH NOTE

An integrative bioinformatics analysis of caprine GNAI3 gene

CHINMOY MISHRA1* and SWAGAT MOHAPATRA2

1Department of Animal Breeding and Genetics, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar 751 003, India 2Department of Veterinary Physiology, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar 751 003, India *For correspondence. E-mail: [email protected].

Received 23 August 2019; revised 6 December 2019; accepted 8 January 2020

Abstract. Goat is the most preferred domesticated animal in Indian subcontinent. However, the climatic change-induced heat stress causes a formidable challenge for maintaining optimum productivity. G protein subunit alpha i3 (GNAI3) is one of the genes that may have significant role in heat tolerance mechanism in goats. The caprine GNAI3 gene was searched for homology analysis and its three dimensional protein structure was predicted followed by its validation through in silico approach. Nucleotide sequence-based phylogenetic tree analysis showed that the caprine GNAI3 gene has close evolutionary relationship with that of Ovis aries. Homology modelling of caprine GNAI3 protein was done in MODELLER 9.18 (P1), PHYRE2 (P2), GENO3D (P3) and SWISS MODEL (P4). The modelled structures were further validated after observing the Ramachandran and hydrophobicity plots. In the best of three dimensional protein structure (P4 as produced by SWISS MODEL), 330 (98.8%), three (0.9%) and one (0.3%) amino acid residues were found in favoured region, allowed region and outlier region, respectively. Degree of hydrophobicity of the generated protein structures revealed the presence of alternate hydrophobic and hydrophilic regions. The ligand receptor interaction site of the predicted 3D model was traced out using Discovery Studio 3.5. STRING database revealed protein interactions with Plcb1, Plcb2, Plcb3 and other proteins of G family such as Gnb1, Gnb2, Gnb3,Gnb4, Gng2, Gng4 and Gpsm1. KEGG pathway maps revealed interaction with eNOS, iNOS, VEGF and MAPK, which are reported to be transcribed in response to heat stress. Thus, caprine GNAI3 can be used as a possible biomarker for studying heat tolerance mechanism in goats.

Keywords. gene; GNAI3 gene; goat; nucleotide; protein.

Introduction demonstrated usefulness in many areas of biomedicine ranging from approximate family assignments to drug Goat is the most preferred domesticated animal due to low-cost designing (Zhang 2009). The protein sequence of interest rearing, less complex management practices (Jyotiranjan et al. (the target) can be modelled with reasonable accuracy on a 2017a) and prolific birth rate (Mishra et al. 2017). In Indian very distantly related sequence of known structure (the geographical conditions, goats are mainly used for chevon template; Eswar et al. 2006). production. A thermoneutral environment is an important G protein subunit alpha i3 (GNAI3) is one of the genes aspect in maintaining sustainable productivity in goats similar that have a potential role in the heat tolerance mechanism in to other animals like pig (Jyotiranjan et al. 2017b). goats which can be used as a genomic signature for selection Proteins are the essential components of animal body and (Kim et al. 2016). Guanine nucleotide-binding proteins (G virtually participate in every process with in a cell. Studying proteins) are involved as modulators or transducers in vari- the protein functions demand a detailed knowledge of the ous transmembrane signalling pathways (Offermanns 2003). protein structure (Gruber et al. 2008). The structure of a The multigene family of G protein a subunits, which interact protein is three to 10 times more conserved during evolution with receptors and effectors, exhibit a high level of sequence than its amino acid sequence (Illerga˚rd et al. 2009). Com- diversity (Wilkie et al. 1992). In mammals, 15 G a subunit putationally predicting 3D structure of a protein had genes can be grouped by sequence and functional 25 Page 2 of 7 Chinmoy Mishra and Swagat Mohapatra similarities into four classes. The caprine GNAI3 gene is The web-based server, GENO3D studies the distance mapped to its third autosome. However, 3D structure of the geometry of protein modelling to build its 3D model. It is GNAI3 protein formed as a result of gene expression has not able to compare protein structure modelling by spatial yet been reported and only the predicted nucleotide sequence restraints satisfaction independently of experimental deter- is available in National Centre for Biotechnology Informa- mination (Combet et al. 2002). SWISS MODEL template tion (NCBI). Thus, in the present study, an attempt has been library provides annotation of quaternary structure and made to accurately predict the 3D structure of caprine essential ligands to allow for building of complete structural GNAI3 protein and it possible interactions. models, including their oligomeric structure (Biasini et al. 2014). These software generate a refined 3D model of the protein based on sequence alignment from the suitable tem- Materials and methods plates. The structures were further polished in CHARMM and DISCOVERY STUDIO 3.5 by performing an optimized The nucleotide-coding sequence of goat GNAI3 gene was geometry calculation. The protein structures were further collected from NCBI (accession number XM_018045873) validated by evaluation of residues in Ramachandran plot and nucleotide BLAST was carried out. Any full-length produced by RAMPAGE (http://mordred.bioc.cam.ac.uk/ published sequence was not available in the NCBI database. *rapper/rampage.php) and hydrophobicity plot (Wang Therefore, the predicted sequence of exotic goat was inclu- et al. 2016). Ramachandran plot was originally developed to ded in the present study. Sequences producing significant alignments from other species were selected for creating phylogenetic tree in MEGA X using maximum likelihood method. Here, the general reversible chloroplast model with gamma distribution was used. The functional association between the proteins can be indicated by the occurrence of genes in each other’s neigh- bourhood on genomes. Protein–protein interaction of GNAI3 in ruminants was carried out using STRING data- base with a cut of score of 0.7 which includes both direct (physical) and indirect (functional) associations (Szklarczyk et al. 2017). The KEGG pathway map representing molec- ular pathways for metabolism, genetic information process- ing, environmental information processing, cellular processes, organismal systems was constructed which pro- vide a web of interactions through which the GNAI3 gene can modulate the heat stress (Kanehisa and Goto 2000). The amino acid sequence of GNAI3 (accession num- Figure 1. Phylogenic tree showing evolutionary relationships of ber XP_017901362) was retrieved from the protein database GNAI3 gene with other closely related species. of the NCBI. The FASTA sequence was searched from the protein data bank (PDB) using BLASTP to find a suit- able template for homology modelling. Since, any goat specific protein did not show homology, heterodimeric complex human Rqs8 (2ODE A) was taken as the template on the basis of maximum identity among the amino acids and query coverage. Of the two transcript variants, transcript variant 2 showed maximum homology with 2ODE a protein. The three dimensional models were generated using MODELLER 9.18 (https://salilab.org/modeller/), PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=Index), GENO3D (https://geno3dprabi.ibcp.fr/cgibin/geno3d_automat. pl?page=/GENO3D/geno3d_home.html) and SWISS MODEL (https://swissmodel.expasy.org/). In MODELLER 9.18, five types of 3D structures were generated using 2ODE_A as the template (Marti Renom et al. 2000).Thebestpossiblestructure was selected considering the discrete optimized protein energy (DOPE) score. The PHYRE2 is a web-based service for protein prediction which takes into account the secondary structure for Figure 2. STRING database representation showing protein–pro- disorder prediction and domain analysis (Kelley et al. 2015). tein interaction of GNAI3 gene. In silico analysis of caprine GNAI3 gene Page 3 of 7 25

Figure 3. KEGG pathway depicting mechanism of heat stress modulation by GNAI3 gene. visualize the energetically allowed regions for back- appropriate protein model was then viewed in RASMOL. bone dihedral angles w against u of amino acid residues The hydrophobicity plots of the obtained 3D-protein struc- in protein structure (Perdih et al. 2012; Mortazavi et al. tures were formulated using DISCOVERY STUDIO 3.5 2016). Structural validation was done by evaluating the (http://accelrys.com/resourcecenter/downloads/updates/discovery- number of amino acid residues in favoured region, allowed studio/dstudio35/latest.html). The plot has amino acid region and in outlier region of Ramachandran plot. The most sequence of a protein on its x axis, and degree of 25 Page 4 of 7 Chinmoy Mishra and Swagat Mohapatra

Figure 4. Predicted 3D protein models of GNAI3 gene. (Clockwise) P1 is the predicted structure from MODELLER9.18, P2 predicted from PHYRE2, P3 predicted from GENO3D and P4 predicted from SWISS MODEL. hydrophobicity and hydrophilicity on its y axis. The for survival in heat stress as studied in mouse embryo hydropathy index model can be applied to predict a pro- cells (Bai et al. 2002). It interacts with G protein subunit tein’s folding patterns (Sarkar and Kellogg 2010). To cal- beta genes such as Gnb1, Gnb2, Gnb3, Gnb4 present in culate the hydropathy index for a protein, the individual caprine chromosome numbers 16, 25, 5 and 3, respec- hydropathy values for an arbitrary number of amino acids tively. It also interacts with G protein subunit gamma are averaged starting at the N terminus. Plotting the proteins such as Gng2 and Gng4 present in chromosome hydropathic index versus the position of the amino acid numbers 10 and 28, respectively. G protein coupled gives a graphical representation of the region hydrophobic receptors (GPCRs) perceive many extracellular signals and and hydrophilic regions inside the protein. Sharp peaks transduce them to heterotrimeric G proteins, which further with positive values in the hydropathy plot correlates well transduce these signals intracellular to appropriate down- with the hydrophobic regions that will span the membrane stream effectors and thereby play an important role in (Ciborowski and Silberring 2016). various signalling pathways. G protein signalling modu- lator 1 (Gpsm1) also works in close co-ordination with GNAI3 gene. Results and discussion The KEGG map (figure 3) suggests that GNAI3 takes part in relaxin signalling pathway (RFXP1 signalling) to interact The phylogenetic tree (figure 1) with the highest log likeli- with phosphatidylinositol 4, 5 bisphosphate 3 kinase cat- hood (–538.3763) was generated based on the Tamura 3 alytic (PI3K) subunit alpha/beta/delta and distantly with parameter model (Tamura 1992). Initial tree(s) for the nitric oxide synthase, endothelial (eNOS), nitric oxide syn- heuristic search was obtained automatically by applying thase, inducible (iNOS) which result in vasodilation. The neighbour-joining (NJ) and BioNJ algorithms to a matrix of GNAI3 also distantly interact with vascular endothelial pairwise distances estimated using the maximum composite growth factor A (VEGF) which results in angiogenesis likelihood approach, and then selecting the topology with through VEGF signalling pathway. The genes eNOS and superior log likelihood value (Kumar et al. 2016). VEGF are involved in regulation of heat stress in goats The GNAI3 gene was predicted to interact with the (Jyotiranjan et al. 2017a). Through RFXP3 signalling, other genes of the G protein family (figure 2). It interacts GNAI3 interacts with the mitogen activated protein kinase with phospholipase C beta subtypes such as Plcb1, Plcb2 (MAPK) signalling pathway which modulates the response and Plcb3 present in the caprine chromosome numbers 13, to stress. GNAI3 gene interaction also results in direct and 10 and 29, respectively. Phospholipase C beta is required indirect effects like increase in lung perfusion, gas exchange In silico analysis of caprine GNAI3 gene Page 5 of 7 25

Figure 5. Ramachandran plots obtained from DISCOVERY STUDIO 3.5 (clockwise) of P1 to P4. P1 predicted structure from MODELLER9.18, P2 predicted from PHYRE2, P3 predicted from GENO3D and P4 predicted from SWISS MODEL.

Table 1. Evaluation of residues in Ramachandran Plot as prepared The best possible structure obtained from MODELLER from RAMPAGE. 9.18, PHYRE 2, GENO3D and SWISS MODEL were des- ignated as P1, P2, P3, P4, respectively (figure 4). The Protein Favoured region Allowed region Outlier region Ramachandran plots were deduced for each of the predicted structure (%) (%) (%) proteins generated from the four homology modelling soft- P1 97.4 2.6 0.0 wares (figure 5) and residues were evaluated (table 1). The P2 93.1 5.8 1.1 Ramachandran plot of P4 which showed 330 (98.8%) resi- P3 67.6 23.9 8.5 dues in favoured region, three (0.9%) residues in allowed P4 98.8 0.9 0.3 region and one (0.3%) in outlier region was considered as the most suitable structure for GNAI3 (Lovell et al. 2003) and SWISS MODEL was found to be the best model for protein structure prediction of GNAI3. and relaxation on bronchi in lungs, increase in atrial con- Hydrophobicity plots for all the 3D structures of protein traction and force in heart, increase in effective renal plasma of GNAI3 were generated (figure 6). The presence of alter- flow, GFR and sodium excretion in kidneys and reduction of nating hydrophobic and hydrophilic waves in the graph of all fibrosis in heart, kidneys, liver and lungs. the four predicted 3D structures clearly indicates that there is 25 Page 6 of 7 Chinmoy Mishra and Swagat Mohapatra

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