title: Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.
abstract: TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853; and.
authors.forename: Siddhartha S
authors.identifier: 0000-0002-7320-6320
authors.initials: SS
authors.lastname: Sinha
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853; and.
authors.forename: Jody
authors.initials: J
authors.lastname: Cameron
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853; and Glycobia Inc., Ithaca, NY 14853.
authors.forename: James C
authors.initials: JC
authors.lastname: Brooks
authors.affiliation: Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853; and cal59@cornell.edu.
authors.forename: Cynthia A
authors.identifier: 0000-0003-0987-9747
authors.initials: CA
authors.lastname: Leifer
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D047468:Immunoprecipitation; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D059748:Proteolysis; D051217:Toll-Like Receptor 9 / Q000276:immunology* / Q000378:metabolism*
chemical_list: C495370:Tlr9 protein, mouse; D051217:Toll-Like Receptor 9