Nova Biotechnol Chim (2019) 18(2): 115-123 DOI: 10.2478/nbec-2019-0014 Corresponding author: baquralhilly_79@yahoo.com; mohammed79@agre.uoqasim.edu.iq Nova Biotechnologica et Chimica Deleterious amino acid substitutions with a series of putative damaging effects on egg components are revealed in the ovalbumin gene family; an in silico approach Mohammed Baqur S. Al-Shuhaib Department of Animal Production, College of Agriculture, Al-Qasim Green University, 8-Al Qasim, Babil 51001, Iraq Article info Article history: Received: 18th January 2019 Accepted: 14th April 2019 Keywords: Chickens Egg production Oviduct Prediction Single Nucleotide Polymorphism Abstract This study was conducted to identify the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) in the ovalbumin gene family, including OVALX, OVALY, and OVAL genes, which are involved in the synthesis of the most important components in the chickens’ eggs using a comprehensive in silico approach. Ten different computational servers were utilized to prioritize the possible deleterious effects of the retrieved nsSNPs in terms of structure, function, and stability. Results indicated entirely damaging effects of H365P in OVALX, I167T in OVALY, and V209G, L231P, F307C, and S317P in OVAL proteins. Further prediction tools showed that all of these deleterious nsSNPs were positioned in variable locations within several α-helix motifs in all studied ovalbumin proteins. Furthermore, all witnessed nsSNPs were predicted to be resided in the receptors binding sites, signifying remarkable involvement of such nsSNPs in damaging of the altered proteins. In conclusion, the present study provides the first inclusive data with regard to the most deleterious nsSNPs in OVALX, OVALY and OVAL genes in chickens. The present bioinformatics data may be useful for breeders who intend to raise chickens for egg production, in such a way the presence of any of these deleterious nsSNPs in any selected breed may possess several damaging effects on the egg components, which may impair egg production. Therefore, it can be stated that breeders have to confirm the absence of any of these deleterious nsSNPs before being proceeded further for large-scale egg-production purposes. University of SS. Cyril and Methodius in Trnava Introduction The ovalbumin gene family in Gallus gallus is composed of three homologous genes located on chromosome 2, namely ovalbumin (OVAL), ovalbumin-related protein X (OVALX), and ovalbumin-related protein Y (OVALY). OVAL, OVALX, and OVALY proteins belong to the serine protease inhibitor (serpin) family whose members share the same overall tertiary structure of eight to nine α-helices and three beta-sheets. All these three genes are mainly expressed in the oviduct (Sugimoto et al. 2001), and more specifically by tubular gland cells of the chicken’s magnum (Rehault-Godbert et al. 2013). Sequences alignment of these three genes have shown a sequence identity of 73 % between OVAL and OVALX, of 72 % between OVAL and OVALY, and 82 % between OVALX and OVALY genes (Da Silva et al. 2015). Although OVAL, OVALY, and OVALX proteins have exerted high homology in their primary sequences, distinct and subtle physicochemical and structural differences have exhibited among them that might be associated Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC mailto:baquralhilly_79@yahoo.com Nova Biotechnol Chim (2019) 18(2): 115-123 116 with specific function on each one. In addition to their major localization in the egg white (Hashim et al. 2019), OVAL, OVALX, and OVALY proteins have also been detected in all other egg compartments, including eggshell, egg yolk, and vitelline membrane (Mann et al. 2008). OVAL is the major egg white protein and might be a source of amino acids for the developing embryo (Liu et al. 2015). OVAL is synthesized in the tubular gland cells in the hen’s oviduct. It is well known that such a synthesis mechanism is under hormonal control with a consequent effect on egg white formation. OVAL accounts for about 54 % of the total proteins of egg white and more than 80 % of the total proteins of egg yolk (Rehault- Godbert et al. 2018). However, the current hypothesis is that OVAL serves as a source of amino acids for the developing embryo. The synthesis of OVAL is upregulated in the magnum in the early onset of the post- ovulation period (Zhao et al. 2016). OVALX gene and its closest neighbour OVALY gene, are highly similar genes and have a close relation with OVAL. Both have likely arisen by duplication events from a common ancestral OVAL gene because the two gene-coding sequences share highly significant similarity with that of the OVAL (Da Silva et al. 2015). Though OVALX and OVALY properties have been described recently, their biological functions remain to be undefined. With regard to OVALX, it has been shown to exhibit antimicrobial activities against pathogens via its positively charged heparin- binding domain exposed at its surface (Rehault- Godbert et al. 2013). These activities suggest considerable participation for OVALX in egg innate defense mechanisms (Dombre et al. 2017; DaSilva et al. 2019). Meanwhile, the role of OVALY has not been explored yet but because its physiological function is not known. However, increasing data in the literature have shown that its abundance was affected during incubation, suggesting a potential involvement of OVALY in some aspects of fertilization or embryonic development (Qiu et al. 2012). Thus, any deleterious amino acid substitution in these proteins may have a series of damaging effects on their biological activity leading to a series of negative consequences on eggs formation. Irrespective of ovalbumin genes, the recent computations of amino acid substitutions have been applied on several genetic markers to predict a variety of physiological effects in birds, such as chickens (Yakubu et al. 2017), ostriches (Al- Shuhaib et al. 2018), and quails (Al-Shuhaib et al. 2019). Up to date, no comprehensive analyses have presented to highlight this aspect in OVAL, OVALX, and OVALY genes and their altered proteins. Therefore, this study aims to exploit the advent of computational biology to present the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) that may be involved in preventing of OVAL, OVALX, and OVALY proteins in undergoing their roles in the proper egg white formation. Experimental Molecular modelling The uniprotKB codes for OVAL, OVALX, and OVALY proteins were A0A2H4Y7U9, A0A1D5PI58, and E1BTF4, respectively. No tertiary structures were found in the deposited protein databank to cover the full studied proteins. Thus, the 3D structure of the involved OVAL, OVALX, and OVALY proteins were generated using the PhyRe2 (Protein Homology/analogY Recognition Engine) software (Kelley et al. 2015). The generated models were validated using Ramachandran plot (Laskowski et al. 2001) and QMEAN (Qualitative Model Energy ANalysis) Z-score generated by Swiss model structure assessment validation tools (Waterhouse et al. 2018). Protein Data Bank formats of the generated models were used as a template for predictions. SNPs retrieval All the amino acid substitutions of the three ovalbumin genes of chickens were comprehensively investigated in this study, namely OVAL (gene ID: 396058), OVALX (gene ID: 420898), and OVALY (gene ID: 420897) genes. A total of 30 nsSNPs were retrieved were retrieved from ensemble genome browser 97 (https://asia.ensembl.org/index.html), including 6 nsSNPs for OVAL, 15 nsSNPs for OVALX, and 9 nsSNPs for OVALY genes. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC https://asia.ensembl.org/index.html Nova Biotechnol Chim (2019) 18(2): 115-123 117 Prediction of nsSNPs effects on proteins structure, function, and stability All nsSNPs were prioritized in terms of their effects on protein structure, function, and stability using ten different in silico tools. SIFT (Sorting Intolerant from Tolerant SNPs) (Pauline et al. 2003), PROVEAN (Protein Variation Effect Analyzer), Predict SNP (Bendl et al. 2014), MAPP (Multivariate Analysis of Protein Polymorphism) (Chao et al. 2008), PolyPhen (Polymorphism Phenotyping) (Adzhubei et al. 2013), and SNAP2 (Screening for Non-Acceptable Polymorphisms 2) (Smigielski et al. 2000) were utilized to assess the effect of each nsSNP on protein structure and biological activity. Meanwhile, mCSM (Pires et al. 2012), SDM (Worth et al. 2012), I-Mutant2 (Capriotti et al. 2005), and CUPSAT (Parthiban et al. 2006) tools were used to evaluate the effects of the same nsSNPs on proteins stability upon mutations. Prediction of ligand binding sites of the most deleterious nsSNPs The potential effect of each nsSNP on altering protein binding capability was predicted using set of protein-receptors or protein-ligand binding tools, including COACH (Wu et al. 2018), TM-SITE (Zhang et al. 2005), S-SITE (Yang et al. 2013), COFACTOR (Zhang et al. 2017), FINSIDTE (Brylinski et al. 2008), and ConCavity (Capra et al. 2009) prediction tools. Energy analyses of proteins after being mutated with deleterious nsSNPs Two sets of parameters were utilized to assess energy changes upon mutation. The total energy after minimization parameters and RMSD (root mean square deviation) values were calculated by DeepView/Swiss-PdbViewer (Johansson et al. 2012) and YASARA (Krieger et al. 2014) tools, respectively. Results The generation of optimal tertiary models of OVAL family genes was performed via PhyRe2 server. The validation of the generated models that carried out by PROCHECK and QMEAN tools indicated that the generated models were being within the accepted limits (Table 1). Considering the Ramachandran plot, models were considered qualified when they showed 90 % or more within the favoured regions. Results showed 91.3 % of the amino acid residues of OVALX and OVALY were suited in the favoured regions, while the amino acid residues of OVAL exhibited 90.0 % in the same region. Likewise, both OVALX and OVALY did not show any amino acid residues in the disallowed region, while OVAL showed only 2 amino acid residues (0.6 %) in the same region. QMEAN Z-score outputs were further proven the current models by giving -2.60, -2.20, and -1.92 for OVALX, OVALY, and OVAL, which, however, they were under the accepted standards. Table 1. Three-dimensional structures validations for the PhyRe2 generated models of OVALY, OVALX, and OVAL proteins in chickens using PROCHECK tool and Swiss model assessments server. Characteristics OVALX OVALY OVAL Residues in most favoured regions 324 (91.3 %) 324 (91.3 %) 320 (90.9 %) Residues in additional allowed regions 27 (7.6 %) 27 (7.6 %) 29 (8.2 %) Residues in generously allowed regions 4 (1.1 %) 4 (1.1 %) 1 (0.3 %) Residues in disallowed regions 0 (0.0 %) 0 (0.0 %) 2 (0.6 %) Number of non-glycine and non-proline residues 355 (100 %) 355 (100 %) 352 (100 %) Number of end-residues 1 1 1 Number of glycine residues 20 20 19 Number of proline residues 12 12 14 Total number of residues 388 388 386 QMEAN Z-score -2.60 -2.20 -1.92 Cβ -1.49 -1.51 -0.77 All atoms -2.32 -1.92 -1.60 Solvation 0.23 0.48 0.53 Torsion -2.47 -2.15 -2.02 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC Nova Biotechnol Chim (2019) 18(2): 115-123 118 Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC Nova Biotechnol Chim (2019) 18(2): 115-123 119 Table 3. Predictions of ligand binding sites for the most deleterious missense SNPs in the OVALX, OVALY, and OVAL proteins in chickens. Protein Name SNP ID AA Change COACH TM-SITE S-SITE CO- FACTOR FINDSITE ConCavity OVALX rs738624565 H365P Peptide (pdb id: 4au2D) – Multiple ligands – – – OVALY rs1057687043 I167T – – – – Ligand (pdb id: 1BR8B00) – OVAL rs733410745 V209G Peptide (pdb id: 3dy0A) Ligand (1jrrA_BS01) – – – – rs734546333 L231P Peptide (pdb id: 3dy0A) Ligand (1lq8E_BS01) – Ligand (pdb id: 1iz2A) – – rs731320904 F307C – – – – – Binding residue rs733774347 S317P – – Multiple ligands – Ligand (pdb id: 1AZXA00) – In order to evaluate the effect of each nsSNP on protein structure, biological activity, and stability, a variety of computational tools were utilized, including SIFT, PROVEAN, Predict SNP, MAPP, PolyPhen, SNAP2, mCSM, SDM, I-Mutant2, and CUPSAT. Cumulative results showed an entirely deleterious effect for a total six nsSNPs for the ovalbumin gene family, including H365P (rs738624565) for the OVALX gene, I167T (rs1057687043) for the OVALY gene, and V209G (rs733410745), L231P (rs734546333), F307C (rs731320904), and S317P (rs733774347) for the OVAL gene (Table 2). More analyses were conducted on these most deleterious nsSNPs to explore their role to cause such extreme alteration in the mutant proteins. The ability of these risky nsSNPs to change the binding activity with other proteins was investigated by utilizing six prediction tools, including COACH, TM-SITE, S-SITE, COFACTOR, FINDSITE, and ConCavity. All the utilized prediction tools revealed obvious participation of all the included risky nsSNPs in several anticipated intensities with respect to their binding with ligands or receptors (Table 3). These findings may be due to the critical positioning taken by most of these nsSNPs in their proteins. Considering H365P and I167T, their positioning in the α-helix of the OVALX and OVALY, respectively may be involved in their deleterious effects (Fig. 1 A and B). The 3D visualizations of the normal and mutant OVAL proteins were added another layer of confirmation of such deleterious effects caused by V209G, L231P, F307C, and S317P. This was due to their positioning within variable locations in the α-helix (Fig. 2). The total energy values for the native OVALX, OVALY, and OVAL structures and their mutant structures were performed. Both H365P and I167T Table 4. The total energy after minimization parameters and RMSD values for wild type and the most deleterious mutant forms of OVALY, OVALX, and OVAL proteins in chickens. The total energy after minimization and RMSD values were calculated by DeepView/Swiss-PdbViewer and YASARA tools, respectively. Protein name NsSNP ID Amino acid change Total energy after minimization RMSD OVALX Wild type - -15699.480 - rs738624565 H365P -15431.578 0.0219 A OVALY Wild type - -13697.646 - rs1057687043 I167T -13707.831 0.0068 A OVAL Wild type - -17478.184 - rs733410745 V209G -17381.457 0.0218 A rs734546333 L231P -17443.225 0.0085 A rs731320904 F307C -15665.894 0.0309 A rs733774347 S317P -15635.917 0.0095 A Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000043252;r=2:67714111-67721182;t=ENSGALT00000067317;vf=15238280 https://zhanglab.ccmb.med.umich.edu/BioLiP/qsearch_pdb.cgi?pdbid=4au2 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000019551;r=2:67700354-67711715;vf=15237987 https://zhanglab.ccmb.med.umich.edu/COACH/output/CH051932/protein/findsite/complex5.pdb https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237550 https://zhanglab.ccmb.med.umich.edu/BioLiP/qsearch_pdb.cgi?pdbid=3dy0 https://zhanglab.ccmb.med.umich.edu/COACH/output/CH051940/protein/tmsite/complex2_1jrrA_BS01_III.pdb https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237548 https://zhanglab.ccmb.med.umich.edu/BioLiP/qsearch_pdb.cgi?pdbid=3dy0 https://zhanglab.ccmb.med.umich.edu/COACH/output/CH051940/protein/tmsite/complex3_1lq8E_BS01_III.pdb https://zhanglab.ccmb.med.umich.edu/BioLiP/qsearch_pdb.cgi?pdbid=1iz2 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237524 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237522 https://zhanglab.ccmb.med.umich.edu/COACH/output/CH051940/protein/findsite/complex3.pdb https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000043252;r=2:67714111-67721182;t=ENSGALT00000067317;vf=15238280 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000019551;r=2:67700354-67711715;vf=15237987 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237550 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237548 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237524 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237522 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000043252;r=2:67714111-67721182;t=ENSGALT00000067317;vf=15238280 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000019551;r=2:67700354-67711715;vf=15237987 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237550 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237548 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237524 https://asia.ensembl.org/Gallus_gallus/Variation/Summary?db=core;g=ENSGALG00000012869;r=2:67682465-67690037;t=ENSGALT00000037195;vf=15237522 Nova Biotechnol Chim (2019) 18(2): 115-123 120 Fig. 1. Three-dimensional structure of the most deleterious nonsynonymous SNPs in the OVALX and OVALY proteins, namely I90M, F109S, and Y127F. A. positioning of the amino acid substitution H365P in the OVALX protein. B. positioning of the amino acid substitution I167T in the OVALY protein. The generated structures were visualized by PyMol software. did not show a noticeable deviation in the total energy minimization values compared with their wild type counterparts. Considering OVAL deleterious mutations, both F307C and S317P mutant models showed an increase in energy minimization than the native structure (Table 4). These less favourable changes indicated more deleterious nature of both F307C and S317P models than the V209G and L231P counterpart. Discussion Ovalbumin family are the most essential proteins in hens’ reproductive systems and are an ideal source for embryo development. Though several data have increasingly been provided with regard to OVAL, OVALX, and OVALY DNA sequences and its translation (Ren et al. 2017; DaSilva et al. 2019), no comprehensive study has been conducted to analyze the consequences of their nsSNPs on ovalbumin family. In this study, the retrieved nsSNPs of the ovalbumin gene family were comprehensively highlighted in terms of their effects on structure, biological activity, and stability. The present study witnessed six entirely deleterious nsSNPs in the ovalbumin gene family by recruiting up to 10 different in silico tools. However, all of these deleterious amino acid substitutions were found to be localized within several positions in the α-helix motif of OVALX, OVALY, and OVAL proteins. This localization may reflect a similar pattern of these substituted amino acid residues to induce these deleterious effects (Abrusán and Marsh 2016). In order to explore the possible roles of the observed nsSNPs in such deleterious effects, several in silico algorithms were used to perform this task. Moreover, the possible mechanisms of these nsSNPs in the interruption of these mutant proteins with regard to its binding with receptors were analyzed. Noticeable positioning of all these risky nsSNPs in binding sites with their corresponding receptors was notified. These findings indicated that the observed amino acid substitutions were involved in the binding network of the OVALX, OVALY, and OVAL protein, which may provide a clear modification in the affinity of interaction with corresponding receptors. Whatever the pattern each nsSNP takes in the interruption of the ovalbumin family, the current finding Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 28.02.20 12:28 UTC Nova Biotechnol Chim (2019) 18(2): 115-123 121 Fig. 2. Three-dimensional structure of the most deleterious nonsynonymous SNPs in the OVAL protein, namely V209G, L231P, F307C, and S317P. The generated structures were visualized by PyMol software. indicated that these nsSNPs seriously affect the structure, function, and stability of ovalbumin family, which have a series of deleterious consequences on the biological pathways in which these proteins involved, such as egg defense activity, embryonic fertilization and development (Liu et al. 2015; Akazawa et al. 2019). Accordingly, its noteworthy to distinguish that both H365P and I167T, being the most deleterious nsSNPs in OVALX and OVALY, respectively, should take priority in terms of reducing antimicrobial activity in case of OVALX (Guyot et al. 2016), or damaging embryonic development in case of OVALY (Da Silva et al. 2015). This observation is particularly interesting, knowing that once these amino acid substitutions detected in a particular breed, serious precautions should be taken for breeders with respect to the handling of this breed. This is due to the presence of these SNPs in OVALX and its OVALY homolog, which may hamper the eggs production in this mutant population. The same precautions could also be applied to the V209G, L231P, F307C, and S317P mutant OVAL proteins. However, these amino acid substitutions deserve more attention as embryo development and the synthesis of many egg components are sensitive to alterations in OVAL architecture (Lin et al. 2016). Keeping in mind the wide availability of these proteins in all the eggs compartments (Dombre et al. 2017), the altered structure, biological activity, stability, or binding characteristics of ovalbumin proteins family seem to disrupt several biological pathways undertaken by chicks’ embryos to take their schedules tasks of development and proper formation. It can be stated that the following nsSNPs H365P, I167T, V209G, L231P, F307C, and S317P should be absent in ovalbumin family in any chicken populations desired to be raised for higher egg production purposes. Conclusions The present cumulative in silico approach indicated the presence of entirely deleterious six nsSNPs in ovalbumin family, including H365P in OVALX, I167T in OVALY, and V209G, L231P, F307C, and S317P in OVAL. 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