title: Essential role for Toll-like receptor 7 (TLR7)-unique cysteines in an intramolecular disulfide bond, proteolytic cleavage and RNA sensing.
abstract: Toll-like receptor 7 (TLR7) an innate immune sensor for microbial RNA, erroneously responds to self-derived RNA. To avoid autoimmune responses, TLR7 is suggested to be silenced until the N-terminal half of the TLR7 ectodomain (TLR7N) is cleaved off. Resultant truncated TLR7 (TLR7C) is thought to signal microbial RNA. We here show that TLR7N remains associated with TLR7C through a disulfide bond. By N-terminal amino acid sequencing, TLR7C was found to start at 461E or 462A. The newly established monoclonal anti-TLR7N showed that endogenous TLR7 in bone marrow-derived dendritic cells was almost all cleaved and cleaved TLR7N remained in endolysosomes. TLR7N in endolysosomes was linked with TLR7C by a disulfide bond. In contrast, TLR9 did not have a disulfide bond between TLR9N and TLR9C fragments. Among the cysteines unique to the ectodomain of TLR7 but not TLR9 (Cys98, Cys445, Cys475 and Cys722), Cys98 in TLR7N and Cys475 in TLR7C were required for an intramolecular disulfide bond. These cysteines were also needed for proteolytic cleavage of and RNA sensing by TLR7, but not for TLR7 trafficking from endoplasmic reticulum to endosomes. No response was seen in TLR7 mutants lacking the proteolytic cleavage site or TLR7C alone. These results demonstrate requirement for proteolytic cleavage and TLR7N in TLR7 responses and indicate RNA sensing by TLR7N + TLR7C.
authors: Division of Innate Immunity, Department of Microbiology and Immunology, University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan.
authors: Atsuo
authors: A
authors: Kanno
authors: Chikako
authors: C
authors: Yamamoto
authors: Masahiro
authors: M
authors: Onji
authors: Ryutaro
authors: R
authors: Fukui
authors: Shin-Ichiroh
authors: S
authors: Saitoh
authors: Yuji
authors: Y
authors: Motoi
authors: Takuma
authors: T
authors: Shibata
authors: Fumi
authors: F
authors: Matsumoto
authors: Tatsushi
authors: T
authors: Muta
authors: Kensuke
authors: K
authors: Miyake
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry*; D003713:Dendritic Cells; D004220:Disulfides / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D059748:Proteolysis; D012313:RNA / Q000378:metabolism*; D051199:Toll-Like Receptor 7 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D008562:Membrane Glycoproteins; C477038:Tlr7 protein, mouse; D051199:Toll-Like Receptor 7; D012313:RNA; D003545:Cysteine
keywords: TLR7; innate immunity; monoclonal antibody