Abstract
Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. Although cell stimulation experiments demonstrate the preferential activation of TLR9 by CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions with respect to the ability of this receptor to bind nucleic acids in a sequence-specific manner. To address this apparent discrepancy, we report the purification of the soluble ectodomain of human TLR9 with characterization of its ligand binding properties. We observe that TLR9 has a high degree of specificity in its ability to bind nucleic acids that contain CpG dinucleotides as well as higher order motifs that mediate species-specific activation. However, TLR9 is also functionally influenced by nucleic acids in a sequence-independent fashion as both stimulatory and nonstimulatory nucleic acids sensitize TLR9 for in vitro ligand binding as well as in vivo activation. We propose a model in which receptor activation is achieved in a sequence-dependent manner, and sensitivity is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. This model bears resemblance to that recently proposed for Toll in that activation is a two-step process in which formation of a ligand-bound monomer precedes formation of the activated dimer. In each model receptor sensitivity is determined within the second step with the crucial distinction that Toll undergoes negative cooperativity, whereas TLR9 is sensitized through a positive cooperative effect.
Highlights
The mammalian innate immune system initiates conserved responses against an array of microbial challenges by targeting biomolecules that are conserved within microbes but largely absent from the host
The mass of this reactive band is consistent with that reported by others for the glycosylated form of comparable fragments of the receptor [25]. This protein was not present in media collected from cells that were not transfected with the Toll-like receptor 9 (TLR9)(LBD) expression vector (Fig. 1A)
Treatment of TLR9(LBD) with the deglycosylation enzyme PNGase F resulted in a protein species of ϳ100 kDa, which is consistent with the predicted mass of the nonglycosylated polypeptide, as well as the observations of others [25] (Fig. 1B)
Summary
The mammalian innate immune system initiates conserved responses against an array of microbial challenges by targeting biomolecules that are conserved within microbes but largely absent from the host. These pathogen-associated molecular patterns (PAMPs) are recognized by germ line-encoded, pat-. The ligand-binding regions of the TLRs consist primarily of repeating elements of a leucine-rich repeat motif [3]. This motif is present in a large number of eukaryotic proteins that often share the unifying characteristic of mediating biomolecular interactions [4]. There have been numerous human trials examining the immunotherapeutic potential of mononuclear cell; PNGase F, peptide N-glycosidase F; LBD, ligand-binding domain
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