RESUMEN
Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp, Labeo rohita. The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.
Asunto(s)
Carpas/genética , Simulación por Computador , Poli I-C/metabolismo , Polimorfismo de Nucleótido Simple/genética , Receptores Toll-Like/genética , Animales , Simulación del Acoplamiento Molecular , Proteínas Mutantes/química , Mutación Missense/genética , Unión Proteica , Homología Estructural de Proteína , Receptores Toll-Like/químicaRESUMEN
The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.