Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon (Salmo salar)

Abstract

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes (cc motif chemokine 19 precursor [ccl19], integrin β2 (itβ2, also named cd18), glutathione transferase omega-1 [gsto-1], heat shock 70 KDa protein [hsp70], major histocompatibility complex class I [mhc-I]) were analyzed to identify SNPs by data mining. These genes were chosen based on their previously reported expression on infectious pancreatic necrosis virus (IPNV)-infected Atlantic salmon phenotype. The available EST sequences for these genes were obtained from the Unigene database. Twenty-eight SNPs were found in the genes evaluated and identified most of them as transition base changes. The effect of the SNPs located on the 5'-untranslated region (UTR) or 3'-UTR upon transcription factor binding sites and alternative splicing regulatory motifs was assessed and ranked with a low-medium predicted FASTSNP score risk. Synonymous SNPs were found on itβ2 (c.2275G > A), gsto-1 (c.558G > A), and hsp70 (c.1950C > T) with low FASTSNP predicted score risk. The difference in the relative synonymous codon usage (RSCU) value between the variant codons and the wild-type codon (ΔRSCU) showed one negative (hsp70 c.1950C > T) and two positive ΔRSCU values (itβ2 c.2275G > A; gsto-1 c.558G > A), suggesting that these synonymous SNPs (sSNPs) may be associated to differences in the local rate of elongation. Nonsynonymous SNPs (nsSNPs) in the gsto-1 translatable gene region were ranked, using SIFT and POLYPHEN web-tools, with the second highest (c.205A > G; c484T > C) and the highest (c.499T > C; c.769A > C) predicted score risk possible. Using homology modeling to predict the effect of these nonsynonymous SNPs, the most relevant nucleotide changes for gsto-1 were observed for the nsSNPs c.205A > G, c484T > C, and c.769A > C. Molecular dynamics was assessed to analyze if these GSTO-1 variants have significant differences in their conformational dynamics, suggesting these SNPs could have allosteric effects modulating its catalysis. Altogether, these results suggest that candidate SNPs identified may play a crucial potential role in the immune response of Atlantic salmon.

Keywords: 3D protein structure; Salmo salar; homology modeling; immune response; molecular dynamics simulation; nonsynonymous SNP; single-nucleotide polymorphism; synonymous SNP.

Figure 1

Homology modeling for predicted S. salar GSTO-1 based on H. sapiens GSTO-1 structure. (A) Tridimensional H. sapiens GSTO-1 (hGSTO-1) structure. (B) Predicted tridimensional S. salar GSTO-1 (sGSTO-1) structure. The differences on the secondary structure in the sGSTO-1 compared to hGSTO-1 are shown (arrow lines). (C) hGSTO-1 and sGSTO-1 overlay. (D) Ramachandran plot for the predicted sGSTO-1 structure. The amino acid distribution into most favored regions (A,B,L), additional allowed regions (a,b,l,p), generously allowed regions (~a,~b,~l,~p), and disallowed regions (GLU83) is indicated. In the protein structures, α-helices (α1- α8), β-sheets (β1-4) are indicated. The reduced glutathione (GSH) molecule is represented.

Figure 2

Evaluation of single-nucleotide polymorphism (SNP) c.205A > G effect on the predicted S. salar GSTO-1 (sGSTO-1) structure by homology modeling. (A) Predicted tridimensional sGSTO-1 including the S26G substitution (sGSTO-1 S26G). (B) sGSTO-1 and sGSTO-1 S26P overlay. The punctual amino acid substitution (dotted arrow line) and the region of the secondary structure affected (β2 sheet, circle) are indicated. (C) Enlarged view of the sGSTO-1 S26P substitution shown in (B). sGSTO-1 (transparent) and sGSTO-1 (colored) are shown. The reduced glutathione (GSH) molecule is represented. (D) Ramachandran plot for the predicted sGSTO-1 S26G structure. The amino acid distribution into most favored regions (A,B,L), additional allowed regions (a,b,l,p), generously allowed regions (~a,~b,~l,~p), and disallowed regions (GLU83, ASP97) is indicated.

Figure 3

Evaluation of single-nucleotide polymorphism (SNP) c.484T > C effect on the predicted S. salar GSTO-1 (sGSTO-1) structure by homology modeling. (A) Predicted tridimensional sGSTO-1 including the S119P substitution (sGSTO-1 S119P). (B) sGSTO-1 and sGSTO-1 S119P overlay. The punctual amino acid substitution (dotted arrow line) and the structural region (α4 helix kink, circle) are indicated. (C) Enlarged view of the sGSTO-1 S119P substitution shown in (B). sGSTO-1 (transparent) and sGSTO-1 (colored) are shown. (D) Ramachandran plot for the predicted sGSTO-1 S119P structure. The amino acid distribution into most favored regions (A,B,L), additional allowed regions (a,b,l,p), generously allowed regions (~a,~b,~l,~p), and disallowed regions (GLU83) is indicated.

Figure 4

Evaluation of single-nucleotide polymorphism (SNP) c.499T > C effect on the predicted S. salar GSTO-1 (sGSTO-1) structure by homology modeling. (A) Predicted tridimensional sGSTO-1 including the Y124H substitution (sGSTO-1 Y124H). (B) sGSTO-1 and sGSTO-1 Y124H overlay. The punctual amino acid substitution (dotted arrow line) is indicated. (C) Enlarged view of the sGSTO-1 Y124H substitution shown in (B). sGSTO-1 (transparent), sGSTO-1 (colored), and the reduced glutathione (GSH) molecule are shown. (D) Ramachandran plot for the predicted sGSTO-1 Y124H structure. The amino acid distribution into most favored regions (A,B,L), additional allowed regions (a,b,l,p), generously allowed regions (~a,~b,~l,~p), and disallowed regions (GLU83, ASP97) is represented.

Figure 5

Evaluation of single-nucleotide polymorphism (SNP) c.769A > C effect on the predicted S. salar GSTO-1 (sGSTO-1) structure by homology modeling. (A) Predicted tridimensional sGSTO-1 including the T214P substitution (sGSTO-1 T214P). (B) sGSTO-1 and sGSTO-1 T214P overlay. The punctual amino acid substitution (dotted arrow line) and the region of the secondary structure affected (α7 helix, circle) are indicated. (C) Enlarged view of the sGSTO-1 T214P substitution shown in (B). (D) Ramachandran plot for the predicted sGSTO-1 structure. The amino acid distribution into most favored regions (A,B,L), additional allowed regions (a,b,l,p), generously allowed regions (~a,~b,~l,~p), and disallowed regions (GLU83) is indicated. In the protein structures, α-helices (α1- α8), β-sheets (β1-4) are indicated. The reduced glutathione (GSH) molecule is represented.

Figure 6

Structure of the GSTO-1 homology model with labeled secondary structure elements. Colored spheres show the position of each punctual variant: red (S26G), yellow (S119P), blue (T214P).

Figure 7

Differences in conformational dynamics between GSTO-1 wild-type (WT), and the variants S26G, S119P, and T214P. The radius of the secondary protein structure and the gradient color (from 0.5 to 4.5 Å) are functions of the RMSF values calculated. As the most affected protein regions, arrowheads highlight the helix α10 (black) and the loop between helixes α6 and α7 (white), respectively.