Toll-Like Receptor 22 in Labeo rohita: Molecular Cloning, Characterization, 3D Modeling, and Expression Analysis Following Ligands Stimulation and Bacterial Infection

• Published in: Applied biochemistry and biotechnology 2014-09, Vol.174 (1), p.309-327
• Main Authors: Samanta, Mrinal, Swain, Banikalyan, Basu, Madhubanti, Mahapatra, Girishbala, Sahoo, Bikash R, Paichha, Mahismita, Lenka, Saswati S, Jayasankar, Pallipuram
• Format: Article
• Language: English
• Subjects: Aeromonas hydrophila; Amino acids; Amphibians; Animals; Bacteria; Bacterial diseases; Bacterial infections; Biochemistry; Biological and medical sciences; Biotechnology; brain; Chemistry; Chemistry and Materials Science; Cloning; Cloning, Molecular; Cyprinidae - genetics; Cyprinidae - immunology; Cyprinidae - metabolism; Cyprinidae - microbiology; Cyprinus carpio; Developmental stages; embryogenesis; Fish Diseases - genetics; Fish Diseases - immunology; Fish Diseases - microbiology; Fish Proteins - biosynthesis; Fish Proteins - chemistry; Fish Proteins - genetics; Fish Proteins - immunology; Freshwater fish; Fundamental and applied biological sciences. Psychology; gene expression; Gene Expression Regulation - genetics; Gene Expression Regulation - immunology; Immune response; Immune system; innate immunity; Labeo rohita; Ligands; lipopolysaccharides; liver; Models, Molecular; molecular cloning; molecular weight; open reading frames; Pathogens; phylogeny; polyinosinic-polycytidylic acid; Protein Structure, Tertiary; quantitative analysis; quantitative polymerase chain reaction; receptors; reverse transcriptase polymerase chain reaction; Ribonucleic acid; RNA; signal peptide; Signal transduction; Teleostei; Toll-Like Receptors - biosynthesis; Toll-Like Receptors - chemistry; Toll-Like Receptors - genetics; Toll-Like Receptors - immunology
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1058-0

Toll-like receptors (TLRs) are a class of innate immune receptors that sense pathogens or their molecular signatures and activate signaling cascades to induce a quick and non-specific immune response in the host. Among various types of TLRs, TLR22 is exclusively present in teleosts and amphibians and is expected to play the distinctive role in innate immunity. This report describes molecular cloning, three-dimensional (3D) modeling, and expression analysis of TLR22 in rohu (Labeo rohita), the most commercially important freshwater fish species in the Indian subcontinent. The open reading frame (ORF) of rohu TLR22 (LrTLR22) comprised of 2,838 nucleotides (nt), encoding 946 amino acid (aa) residues with the molecular mass of ∼107.6 kDa. The secondary structure of deduced LrTLR22 exhibited the presence of signal peptide (1–22 aa), 18 leucine-rich repeat (LRR) regions (79–736 aa), and TIR domain (792–935 aa). The 3D model of LrTLR22-LRR regions together elucidated the horse-shoe-shaped structure having parallel β-strands at the concave surface and few α-helices at the convex surface. The TIR domain structure revealed alternate presence of five α-helices and β-sheets. Phylogenetically, LrTLR22 was closely related to common carp and exhibited significant similarity (92.2 %) and identity (86.1 %) in their amino acids. In rohu, TLR22 was constitutively expressed in all embryonic developmental stages, and tissue-specific analysis illustrated its expression in all examined tissues, highest was in liver and lowest in brain. In vivo modulation of TLR22 gene expression was analyzed by quantitative real-time PCR (qRT-PCR) assay following stimulation with lipopolysaccharide (LPS), synthetic double stranded RNA (polyinosinic-polycytidylic acid), and bacterial (Aeromonas hydrophila) RNA. Among these ligands, bacterial RNA most significantly (p