Shubham Goyal

Structural and dynamic investigation of bovine folate receptor alpha (FOLR1), and role of ultra-high temperature processing on conformational and thermodynamic characteristics of FOLR1–folate complex

The folate receptor alpha (FOLR1) present in milk has widely been studied to investigate the effects of pasteurization, ultra-high temperature (UHT) processing and fermentation on net folate concentration. However, the folate binding mechanism with FOLR1, and effect of temperature on FOLR1-folate complex is poorly explored till now in bovine milk which is a chief resource of folate. Despite of enormous importance of folic acid and the routine intake of bovine milk, folic acid deficiency diseases are common in human race. To understand the folate deficiency in milk after processing, in absence of experimental structure, 3D model of bovine FOLR1 (bvFOLR1) was built followed by 40ns molecular dynamics (MD) simulation. The folate and its derivatives binding sites in bvFOLR1 were anticipated by molecular docking using AutoDock 4.2. Essential MD studies suggested the presence of a longer signal peptide (22 residues) and a short propeptide (7 residues) at the C-terminus that may cleaved during post-translational modification. MD analysis of bvFOLR1-folate complex at 298, 323, 353, 373 and 408K followed by binding energy (BE) calculation showed maximum binding affinity at ∼353K. However, at 373K and UHT (408K), the folate BE is significantly decreased with substantial conformational alteration. Heating at UHT followed by cooling within 298-408K range demoed no structural reformation with temperature reduction, and the folate was displaced from the active site. This study presented the disintegration of folate from bvFOLR1 during high temperature processing and revealed a lower folate concentration in UHT milk and dairy products.

Exploration of the binding modes of buffalo PGRP1 receptor complexed with meso-diaminopimelic acid and lysine-type peptidoglycans by molecular dynamics simulation and free energy calculation.

The peptidoglycan recognition proteins (PGRPs) are the key components of innate-immunity, and are highly specific for the recognition of bacterial peptidoglycans (PGN). Among different mammalian PGRPs, the PGRP1 binds to murein PGN of Gram-positive bacteria (lysine-type) and also have bactericidal activity towards Gram-negative bacteria (diaminopimelic acid or Dap-type). Buffaloes are the major sources of milk and meat in Asian sub-continents and are highly exposed to bacterial infections. The PGRP activates the innate-immune signaling, but their studies has been confined to limited species due to lack of structural and functional information. So, to understand the structural constituents, 3D model of buffalo PGRP1 (bfPGRP1) was constructed and conformational and dynamics properties of bfPGRP1 was studied. The bfPGRP1 model highly resembled human and camel PGRP structure, and shared a highly flexible N-terminus and centrally placed L-shaped cleft. Docking simulation of muramyl-tripeptide, tetrapeptide, pentapeptide-Dap-(MTP-Dap, MTrP-Dap and MPP-Dap) and lysine-type (MTP-Lys, MTrP-Lys and MPP-Lys) in AutoDock 4.2 and ArgusLab 4.0.1 anticipated β1, α2, α4, β4, and loops connecting β1-α2, α2-β2, β3-β4 and α4-α5 as the key interacting domains. The bfPGRP1-ligand complex molecular dynamics simulation followed by free binding energy (BE) computation conceded BE values of -18.30, -35.53, -41.80, -25.03, -24.62 and -22.30 kJ mol(-1) for MTP-Dap, MTrP-Dap, MPP-Dap, MTP-Lys, MTrP-Lys and MPP-Lys, respectively. The groove-surface and key binding residues involved in PGN-Dap and Lys-type interaction intended by the molecular docking, and were also accompanied by significant BE values directed their importance in pharmacogenomics, and warrants further in vivo studies for drug targeting and immune signaling pathways exploration.

Expression Analysis of Genes Associated with Prolificacy in FecB Carrier and Noncarrier Indian Sheep

ABSTRACT The effect of FecB mutation on the gene expression in FecB carrier and noncarrier estrous synchronized ewes, has been analyzed. For this study the whole ovarian tissues and Graafian follicles were collected from estrus synchronized FecB carrier Garole, and non-carrier Deccani Indian sheep, showing remarkable differences in the numbers of preovulatory follicles among two groups. Eleven potential candidate genes (BMP15, GDF9, BMP4, BMP7, BMPR1B, BMPR1A, SMAD9, LHCGR, FSHR, IGF1R, and STAT5) were selected for their expression analysis by SybrGreen based real-time PCR, across ovaries and Graafian follicles of different fecundity groups, for having better insights into the effect of FecB genotypes on follicular development. Variable expression was observed for almost all the genes included in the present study among high and low fecundity groups that was most significant for the BMP7, BMP4, LHCGR, and FSHR transcripts in the ovarian follicles of high and low fecundity ewes, indicating their importance in governing the fecundity in FecB carrier, Indian Garole sheep. BMP4 expression among the genes studied was significantly higher in FecB carrier Garole sheep. This study confirms the changes in mRNA expression of the genes implicated in follicular development in FecB carrier and noncarrier Indian sheep breeds.

Identification of polymorphism in fatty acid binding protein 3 (FABP3) gene and its association with milk fat traits in riverine buffalo (Bubalus bubalis)

The fatty acid binding protein 3 (FABP3) gene, known to be associated with fat percentage of milk and meat in bovines, was screened among swamp and riverine buffaloes for polymorphism detection and further association with milk fat contents. An SNP g.307C > T was identified in the intron 2 (+53 exon 2) region of FABP3 gene of Indian buffaloes. The SNP identified was genotyped in 692 animals belonging to 15 riverine, swamp and hybrid (riverine × swamp) buffalo populations of diverse phenotypes and utilities, by PCR-RFLP. A marked contrast was observed between the C and T allele frequencies in three types of buffaloes. The frequency of C allele ranged from 0.67 to 0.96 in pure swamp buffalo populations, with the highest in Mizoram (0.96). Whereas the frequency of T allele was high across all the Indian riverine buffalo breeds, ranging from 0.57 to 0.96. None of the genotypes at FABP3 g.307C > T locus was found to have significant association with milk fat and other production traits in Mehsana dairy buffalo breed. Our study revealed marked differences in the allele frequencies between riverine and swamp buffaloes at FABP3 g.307C > T locus, without any significant association with different milk traits in riverine buffaloes.

Genetic diversity analysis of buffalo fatty acid synthase (FASN) gene and its differential expression among bovines

Fatty Acid Synthase (FASN) gene seems to be structurally and functionally different in bovines in view of their distinctive fatty acid synthesis process. Structural variation and differential expression of FASN gene is reported in buffalo (Bubalus bubalis), a bovine species close to cattle, in this study. Amino acid sequence and phylogenetic analysis of functionally important thioesterase (TE) domain of FASN revealed its conserved nature across mammals. Amino acid residues at TE domain, responsible for substrate binding and processing, were found to be invariant in all the mammalian species. A total of seven polymorphic nucleotide sites, including two in coding region of TE domain were identified across the 10 buffalo populations of riverine and swamp types. G and C alleles were found almost fixed at g18996 and g19056 loci, respectively in riverine buffaloes. Principal component analysis of three SNPs (g18433, g18996 and g19056) revealed distinct classification of riverine and swamp buffalo populations. Reverse Transcription-PCR amplification of mRNA corresponding to exon 8-10 region of buffalo FASN helped in identification of two transcript variants; one transcript of 565 nucleotides and another alternate transcript of 207 nucleotides, seems to have arisen through alternative splicing. Both the transcripts were found to be expressed in most of the vital tissues of buffalo with the highest expression in mammary gland. Semi-quantitative and real-time expression analysis across 13 different buffalo tissues revealed its highest expression in lactating mammary gland. When compared, expression of FASN was also found to be higher in liver, adipose and skeletal muscle of buffalo tissues, than cattle. However, the FASN expression was highest in adipose among the three tissues in both the species. Results indicate structural and functional distinctiveness of bovine FASN. Presence of alternate splicing in buffalo FASN also seems to be a unique phenomenon to the bovines, probably associated with mRNA based regulation of the biological functions of FASN in these species.

Molecular Characterization of Buffalo Haptoglobin: Sequence Based Structural Comparison Indicates Convergent Evolution Between Ruminants and Human

ABSTRACT Haptoglobin (Hp) protein has high affinity for hemoglobin (Hb) binding during intravascular hemolysis and scavenges the hemoglobin induced free radicals. Earlier reports indicate about uniqueness of Hp molecule in human and cattle, but in other animals, it is not much studied. In this paper, we characterized buffalo Hp molecule and determined its molecular structure, evolutionary importance, and tissue expression. Comparative analysis and predicted domain structure indicated that the buffalo Hp has an internal duplicated region in α-chain only similar to an alternate Hp2 allele in human. This duplicated part encoded for an extra complement control protein CCP domain. Phylogenetic analysis revealed that buffalo and other ruminants were found to group together separated from all other non-ruminants, including human. The key amino acid residues involved in Hp and Hb as well as Hp and macrophage scavenger receptor, CD163 interactions in buffalo, depicted a significant variation in comparison to other non-ruminant species. Constitutive expression of Hp was also confirmed across all the vital tissues of buffalo, for the first time. Results revealed that buffalo Hp is both structurally and functionally conserved, having internal duplication in α-chain similar to human Hp2 and other ruminant species, which might have evolved separately as a convergent evolutionary process. Furthermore, the presence of extra Hp CCP domain possibly in all ruminants may have an effect during dimerization of molecule in these species.

Sequence characterization of S100A8 gene reveals structural differences of protein and transcriptional factor binding sites in water buffalo and yak.

The present study was undertaken to characterize the structure of S100A8 gene and its promoter in water buffalo and yak. Sequence data of 2.067 kb, 2.071 kb, and 2.052 kb with respect to complete S100A8 gene including 5′ flanking region was generated in river buffalo, swamp buffalo, and yak, respectively. BLAST analysis of coding DNA sequences (CDS) of S100A8 gene revealed 95% homology of buffalo sequence with cattle, 85% with pig and horse, 83% with dog, 72–73% with murines, and around 79% with primates and humans. Phylogenetic analysis of predicted CDS revealed distinct clustering of murines, primates, and domestic animals with bovines and bubalines forming a subcluster among farm animals. In silico translation of predicted CDS revealed a sequence of 89 amino acids with 7 amino acid changes between cattle and buffalo and 2 changes between cattle and yak. The search for Pfam family revealed the N-terminal calcium binding domain and the noncanonical EF hand domain in the carboxy terminus, with more variations being observed in the N-terminal domain among different species. Two amino acid changes observed in carboxy terminal EF hand domain resulted in altered secondary structure of yak S100A8 protein. Analysis of S100A8 gene promoter revealed 14 putative motifs for transcriptional factor binding sites. Two putative motifs viz. C/EBP and v-Myb were found to be absent in swamp buffalo as compared to river buffalo and cattle. Differences in the structure of S100A8 protein and the transcriptional factor binding sites identified in the present study need to be analyzed further for their functional significance in yak and swamp buffalo respectively.