title: Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in Oplegnathus fasciatus that responds to flagellin challenge and activates NF-κB.
abstract: Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, Oplegnathus fasciatus. Both Oftlr5m (6.7 kb) and Ofmyd88 (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the tlr5m gene has evolved with extensive rearrangement; whereas, the myd88 gene has maintained a stable structure throughout the evolution. Inspection of 5' flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected Oftlr5m mRNA in eleven tissues with the highest abundance in liver. In vivo flagellin administration strongly induced the transcripts of both Oftlr5m and Ofmyd88 in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with Oftlr5m and Ofmyd88 demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa, 252-8570, Japan.
authors.forename: Navaneethaiyer
authors.initials: N
authors.lastname: Umasuthan
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: S D N K
authors.initials: SDNK
authors.lastname: Bathige
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: William Shanthakumar
authors.initials: WS
authors.lastname: Thulasitha
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea.
authors.forename: R G P T
authors.initials: RGPT
authors.lastname: Jayasooriya
authors.affiliation: Insilicogen Inc., Giheung-gu, Yongin-si, Gyeonggi-do, 16954, Republic of Korea. Electronic address: myhshin@insilicogen.com.
authors.forename: Younhee
authors.initials: Y
authors.lastname: Shin
authors.affiliation: Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea; Fish Vaccine Development Center, Jeju National University, Jeju Self-Governing Province, 63243, Republic of Korea. Electronic address: jehee@jejunu.ac.kr.
authors.forename: Jehee
authors.initials: J
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005408:Flagellin / Q000494:pharmacology*; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D010473:Perciformes / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000502:physiology; D016415:Sequence Alignment / Q000662:veterinary; D051198:Toll-Like Receptor 5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D012333:RNA, Messenger; D051198:Toll-Like Receptor 5; D005408:Flagellin
keywords: Flagellin (FLA) sensing; Genomic evolution; Membrane-anchored toll-like receptor 5 (TLR5M); Myeloid differentiation primary response gene 88 (MyD88); NF-κB activation