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      • HARVEST
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      Expression and characterization of ligand binding by the ectodomain of toll-like receptor 9

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      Date
      2007
      Author
      Potter, Jean Elizabeth Anore
      Type
      Thesis
      Degree Level
      Masters
      Metadata
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      Abstract
      Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is ‘primed’ for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.
      Degree
      Master of Science (M.Sc.)
      Department
      Biochemistry
      Program
      Biochemistry
      Supervisor
      Napper, Scott
      Committee
      Warrington, Rob C.; Moore, Stanley A.; Khandelwal, Ramji L.; Griebel, Philip J.
      Copyright Date
      2007
      URI
      http://hdl.handle.net/10388/etd-08302007-140139
      Subject
      ODN
      CpG
      TLR
      innate immunity
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