Sequence based structural characterization and genetic diversity analysis across coding and promoter regions of goat Toll-like receptor 5 gene Shubham Goyal a , P.K. Dubey a,1 , B.R. Sahoo b , S.K. Mishra a , S.K. Niranjan a , Sanjeev Singh a , Ritu Mahajan c , R.S. Kataria a, ⁎ a National Bureau of Animal Genetic Resources, G.T. Road By-Pass, Karnal, Haryana 132001 India b Laboratory of Molecular Biophysics, Institute of Protein Research, Osaka University, Osaka Prefecture 5650871, Japan c Department of Biotechnology, Kurukshetra University, Kurukshetra, Haryana, India abstract article info Article history: Accepted 31 January 2014 Available online 12 February 2014 Keywords: Goat, Toll-like receptor 5 Leucine-rich repeats (LRR) Toll/IL-1 receptor (TIR) domain Polymorphism Transcription factor binding sites Molecular dynamics simulation Molecular docking The exploration of candidate immune response genes in goat may be vital in improving further our understand- ing about the species specific response to pathogens specifically among the ruminants. In this study, approxi- mately 3.7 kb long genomic sequence of Toll-like receptor 5 (TLR5) covering the entire coding and 5′upstream regions of the gene, was characterized in the Indian goat breeds. Sequence analysis revealed a 2577-nucleotide long open reading frame (ORF) of goat TLR5, encoding 858 amino acids from single exon, similar to other rumi- nants. The domain structure analysis of goat TLR5 showed the presence of 13 leucine rich repeats (LRRs) in ex- tracellular domain (amino acid position 1–634), single transmembrane domain (position 644–666), and a Toll/ interleukin-1 receptor (position 692–837) in cytoplasmic domain, similar to other species. A total of 87 putative transcription factor binding sites were observed within the 5′ upstream region of TLR5 gene in goat, 106 in cattle, and 103 in buffalo. Sixteen polymorphic sites were observed in goat TLR5 gene, out of which 10 non-synonymous SNPs were in the functionally important regions. However, none of the amino acid substitutions was found to be potentially damaging to the structure and function of the receptor protein. Further, one of the SNPs in the trans- membrane region was genotyped by a TETRA-ARMS PCR in 444 goats of nine breeds from different geographical regions and having different utilities. A significant variation in allelic frequencies was observed across the milch and other types of goat breeds. The comparative modeling of goat TLR5 followed by molecular dynamics simula- tion gave an insight into its 3D structural arrangements. The molecular docking of Salmonella flagellin and TLR5 dimer elucidated LRRNT (N-terminal) to LRR4 as the key flagellin binding domains region in goat TLR5. The study shows that, although being highly conserved among the ruminants, comparatively high variations in goat TLR5 might give an opportunity to host for recognizing the wider spectrum of pathogens. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Toll-like receptors (TLRs) are important pattern recognition mole- cules, responsible for the induction of innate as well as adaptive immu- nity against the wide range of microbial pathogens (Werling and Jungi, 2003). The basic structure of TLRs includes N-terminal extracellular do- main (ECD), composed of leucine-rich repeats (LRR), which recognizes a wide range of pathogen associated molecular patterns (PAMPs) and an intracellular Toll/IL-1 receptor (TIR) domain, important in initiating the signal transduction. TLRs have the ability to recognize the different kinds of ligands, both exogenous and endogenous, the molecules derived from pathogens and processed by the host cell, respectively. Till now, 13 TLRs have been identified in mammals, out of which 10 have been well characterized in human, cattle, sheep, buffalo, and pig (Chang et al., 2009; Dubey et al., 2013). These TLRs are generally divided into two groups depending upon their localization in the host cells (Uenishi and Shinkai, 2009). The first group comprises of TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, expressed on the cell surface and recogniz- ing the compounds derived mainly from bacteria. The second group includes TLR3, TLR7, TLR8, and TLR9, which are expressed on the mem- branes of intracellular organelles, identifying nucleic acids as well as the various derivatives of nucleotides of viral and bacterial origins (Heil et al., 2003). Among the identified TLRs, TLR5 plays an important role in the host defense, specifically recognizing the flagellated bacterial pathogens. TLR5 deficient mice have been found to be more susceptible to spontaneous colitis and Escherichia coli infection of urinary tract (Feuillet et al., 2006; Vijay et al., 2007). The extracellular pattern recog- nition domain (ECD) of TLR5 has been found to be conserved among primates and mammals, which shows the evidence of adaptive positive selection (Smith et al., 2012). Gene 540 (2014) 238–245 Abbreviations: TLR, Toll-like receptor; SNP, Single nucleotide polymorphism; LRR, Leucine rich repeats; TIR, Toll-interleukin1 receptor; Kb, Kilobase; PCR, Polymerase chain reaction; ARMS, Amplification Refractory Mutation System. ⁎ Corresponding author. Fax: +91 184 2267654. E-mail address: katariaranji@yahoo.co.in (R.S. Kataria). 1 Present address: Immune regulation, WPI-IFREC, Osaka University, Osaka Prefecture 5650871, Japan. 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.076 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene