Identification and prediction of the consequences of nonsynonymous SNPs in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of zebrafish Danio rerio

Abstract

The recent progress in DNA sequencing and computational algorithms dramatically heightened the value of single nucleotide polymorphism (SNP) databases. The experimental studies of mutation for the particular gene of interest are laborious and timeconsuming. In this study, we used several computational algorithms to investigate the structural and functional consequences of SNPs on the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of zebrafish (Danio rerio). The GAPDH gene is involved in multiple cellular activities, in addition to its association with classical glycolytic pathway. It has most commonly been used as the housekeeping gene for differential gene expression profiling, including western blots. The computational analysis, which used sequence-based tools such as SIFT, PANTHER, PROVEAN, and I-Mutant2.0, predicted deleterious nonsynonymous SNPs (nsSNPs) at p.W194R in the GAPDH gene. The SWISS Model tool generated a homology-modeled 3D structure of GAPDH by applying homologous protein structures retrieved from the RCSB PDB databank (PDB ID: 1U8F_O, 1ZNQ_O, 1J0X_O, 3GPD_R, and 1SZJ_G). The sequence identities of the above selected models were 86%, 86%, 86%, 83%, and 75%, respectively. The Procheck quality assessment in the Ramachandran plot showed that the incorporated mutation (p.W194R) led to a shifting of amino acid residues from the most favored regions towards the disallowed region, as compared to the native counterpart. The lowered ERRAT and PROSA Z scores in the mutant confirmed the overall deteriorating quality of mutant GAPDH. We also predicted an effect of mutation on increased H-bonding patterns due to mutant residue. The molecular docking analysis, while binding GAPDH to its ligand (NAD +), also revealed elevated global energy in the mutant. Altogether, this study demonstrated the impact of nsSNPs on the protein structure and function. The results of work provide an insight/ roadmap for future investigations with regard to the pathological consequences of nsSNPs using in vivo methods.

Molecular characterization and expression patterns of Nanog gene validating its involvement in the embryonic development and maintenance of spermatogonial stem cells of farmed carp, Labeo rohita

“…Extracted RNAs were verified by PCR using β - actin house-keeping gene primers. Genomic DNA was isolated from the liver of rohu via the phenol-chloroform extraction method [ 30 – 32 ]. The quantity and quality of extracted total RNA and genomic DNA were determined by agarose gel electrophoresis and Nanodrop readings.…”

Status of genetic and genomic approaches for delineating biological information and improving aquaculture production of farmed rohu, Labeo rohita (Ham, 1822)

“…The earlier studies reported that SIFT, I‐Mutant, PROVEAN and PANTHER algorithms were used for investigating the effect of non‐synonymous mutation (Rasal et al, 2017). In zebrafish and farmed rohu, impact of nsSNPs were analysed in different key genes such as GAPDH (Rasal et al, 2015), TLR22 (Chakrapani et al, 2018) and MSTN (Rasal et al, 2016a) using computational tools. Thus, recently generated genomic data and SNPs in rohu will be utilized for predicting structural and functional impact of missense variants on protein structure and functions.…”

Identification of Deleterious Mutations inMyostatinGene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches

“…Direct experimental studies linked to incorporating mutation in a particular gene of interests have been laborious and time-consuming. The computational study can efficiently produce useful information to rationalize and guide in undertaking further experimental studies [ 28 – 30 ]. These point mutations affect gene expressions and their products by altering change in their functional activities [ 31 ].…”