Expression and characterization of ligand binding by the ectodomain of toll-like receptor 9
| dc.contributor.advisor | Napper, Scott | en_US |
| dc.contributor.committeeMember | Warrington, Rob C. | en_US |
| dc.contributor.committeeMember | Moore, Stanley A. | en_US |
| dc.contributor.committeeMember | Khandelwal, Ramji L. | en_US |
| dc.contributor.committeeMember | Griebel, Philip J. | en_US |
| dc.creator | Potter, Jean Elizabeth Anore | en_US |
| dc.date.accessioned | 2007-08-30T14:01:39Z | en_US |
| dc.date.accessioned | 2013-01-04T04:55:53Z | |
| dc.date.available | 2008-09-04T08:00:00Z | en_US |
| dc.date.available | 2013-01-04T04:55:53Z | |
| dc.date.created | 2007 | en_US |
| dc.date.issued | 2007 | en_US |
| dc.date.submitted | 2007 | en_US |
| dc.description.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. | en_US |
| dc.identifier.uri | http://hdl.handle.net/10388/etd-08302007-140139 | en_US |
| dc.language.iso | en_US | en_US |
| dc.subject | ODN | en_US |
| dc.subject | CpG | en_US |
| dc.subject | TLR | en_US |
| dc.subject | innate immunity | en_US |
| dc.title | Expression and characterization of ligand binding by the ectodomain of toll-like receptor 9 | en_US |
| dc.type.genre | Thesis | en_US |
| dc.type.material | text | en_US |
| thesis.degree.department | Biochemistry | en_US |
| thesis.degree.discipline | Biochemistry | en_US |
| thesis.degree.grantor | University of Saskatchewan | en_US |
| thesis.degree.level | Masters | en_US |
| thesis.degree.name | Master of Science (M.Sc.) | en_US |