Analysis of the interaction between human TLR3 ectodomain and nucleic acids.
Toll-like receptor 3 (TLR3) recognizes dsRNA of viral origin and polyriboinosine-polyribocytidylic acid (poly (I:C)). TLR3 mediates the activation of IRF-3 and NF-kappaB and thereby the secretion of type I interferons and inflammatory cytokines. However, the mechanism of this activation is poorly understood. To study the molecular recognition events and biochemical interactions between TLR3 and dsRNA, human TLR3 ectodomain (ECD) fused with a signal peptide at the N-terminus and 6 x His-tag at the C-terminus (TLR3-ECD) was obtained by the baculovirus expression system. To examine the various nucleic acids binding to TLR3-ECD in vitro, a filter binding assay was carried out at pH 4.2-7.6. Interaction of TLR3-ECD with various nucleic acids (particularly dsRNA, in vitro transcripts of tRNA and HCV NS3 aptamer) required an acidic pH.
Fukuda
Kotaro
K
Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan.
Tsujita
Tadayuki
T
Matsumoto
Misako
M
Seya
Tsukasa
T
Sakiyama
Hiroshi
H
Nishikawa
Fumiko
F
Nishikawa
Satoshi
S
Hasegawa
Tsunemi
T
D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D051196:Toll-Like Receptor 3 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D012330:RNA, Double-Stranded; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D012343:RNA, Transfer