Overview: The rapid initiation of an immune response is key to mammals' ability to overcome potential infections. This initiation depends upon the recognition, at the molecular level, of invading pathogens by specific pathogen recognition proteins called Toll-like receptors (TLRs). The structure of these receptor proteins is unknown and little is understood of the mechanisms of their activation. The overall objective of this program is to characterize biomolecular interactions relevant to innate immune responses, leading us to the creation of more effective therapeutic molecules.
Background: The determination of the presence of pathogens through the recognition of pathogen-associated molecular patterns (PAMPs) represents the first step in the initiation of the innate immune responses. Activation of the innate immune system serves a dual function in eliciting primary defensive responses, as well as representing a prerequisite step in the induction of acquired immunity. The ability of the host to initiate a conserved response against a diverse and dynamic spectrum of potential pathogens is accomplished through the recognition of specific biomolecular structures that are common and conserved within the pathogen but absent from the host. These PAMPs are specifically recognized and bound by germ-line encoded pattern recognition proteins known as the Toll-like receptors (TLRs). The TLRs are conserved across a wide range of species with 11 mammalian homologues described to date.
TLRs are type I integral membrane proteins that display a high degree of intra- and extracellular conservation. Extracellularly, the ligand-binding domains of TLRs are characterized by varying number of leucine-rich repeat (LRR) motifs. The LRR motif is present in a large number of eukaryotic proteins, which of diverse function, typically share a unifying characteristic of involvement in recognition and interaction processes. Indeed the primary function of the LRR motif appears to be in providing a versatile structural matrix for the formation of biomolecular interactions; the versatility of which is best exemplified by the vast and structurally diverse ligands recognized by different TLR receptors, as well as the multiple ligands often recognized by individual TLR receptors.
While the diversity of ligands recognized by the TLR family, and individual TLR receptors, is likely reflective of an appropriate physiological redundancy, it may also be indicative of the intrinsic binding propensities of the LRR motif. For example is has been reported that phosphorothioate oligodeoxynucleotides (PTOs) bind to TLR9 receptors in a sequence-independent fashion but mediate sequence-dependent activation; highlighting the distinction between TLR9 interaction and activation. Receptor activation is therefore likely dependent upon structural events that occur subsequent to ligand binding.
While there is considerable substantiation that unmethylated CpG-containing nucleic acid molecules signal through TLR9, there is limited information on the specifics of this interaction and of the structural mechanisms of activation that occur subsequent to ligand binding. Previous investigations of the TLR9-ligand interaction have been performed through surface plasmon resonance, which while effective in monitoring biomolecular interactions, has limited capacity to discriminate non-activating interactions that result from the inherently sticky TLR ectodomains, from specific interactions that result in receptor activation.
Based on homology to a known CpG-DNA binding protein, a region of TLR9 was hypothesized, and confirmed through mutational analysis, to be responsible for the interaction with DNA. Interestingly this region is predicted to be devoid of glycosylation sites, leading to our hypothesis that the sugar modifications of TLR9 may be of greater importance to cellular trafficking rather than ligand binding. Based on this hypothesis, we examined the potential for over-expression of the TLR9 extracellular domain, as well as the full-length protein, in a prokaryotic expression system. This expression system generated sufficient quantities of TLR9 protein for biochemical characterization through a modified agarose electrophoretic mobility shift assay. A significant advantage of this system over surface plasmon resonance is that it allows the visualization and discrimination of protein-DNA complexes of different stoichiometries and conformations.
Objectives:
1. Determine the necessity for additional host cell protein factors required for ligand binding.
2. Investigate the sequence specificity of TLR9s from different species.
3. Determine the influence of nucleic acid structure and topology on the ability to serve as a TLR9 ligand.
Progress: 1. The extracellular domain of bovine TLR9 has been expressed and purified from Escherichia coli.
2. The functional capabilities of the protein have been established through a modified agarose shift assay.
3. The ability of TLR9 to bind double-stranded plasmid DNA has been characterized and a preferential association with supercoiled species has been observed.
4. The ability for TLR9 to bind bacterial ribsosomes has been established through a proteomic investigation and the ability of the bacterial ribosomal complex to function as a PAMP has been investigated.
Project Leader:
