Structural and Functional Analyses of the Human Toll-like Receptor 3

Abstract

Proteins that recognize pathogen-associated molecular patterns are key factors in the cascade of events from the detection to the elimination of an invading organism. This form of innate immunity is conserved in eukaryotes. For example, the Drosophila melanogaster Toll protein is responsible for resistance to fungal and bacterial infections (3, 4), and plants can encode disease-resistance proteins that are important in determining the outcome of infection (5). The vertebrate pathogendetecting proteins called Toll-like receptors (TLRs) 5 are key players in the activation of both the innate and adaptive arms of the immune system (6 -9).The TLRs and related pathogen sensors contain leucine-rich repeat motifs that form docking sites for pathogen ligands or adaptors that bind pathogen ligands, the binding of which will activate signal transduction pathway(s) (10 -12). TLR3 recognizes double-stranded RNA and may be a part of a redundant sensor system to detect viral infections (13-15). Although specific features in the ligands required to interact with TLR3 remain to be identified, TLR3 is activated by polyinosinepolycytidylic acid (poly(I:C)) and has been reported to be activated by RNAs extracted from necrotic cells (16).A number of issues concerning TLR3 structure and function remain to be elucidated. For example, TLR3 can apparently act both on the surface of the plasma membrane, as it does in fibroblasts, and by attaching to the membranes of intracellular vacuoles, where it is proposed to act in immature dendritic cells (17,18). The trafficking of TLRs should be influenced by glycosylation in general, and N-linked glycosylation of TLR2 and TLR4 has been shown to play essential roles in its localization (19,20). A significant recent advance in TLR3 was the elucidation of a 2.1 Å structure of the soluble ectodomain by Choe et al. (1). Bell et al. (2) independently elucidated a highly similar structure. In both studies, the crystallized ectodomains were produced in a baculovirus expression system and formed a horseshoe-shaped solenoid structure that was extensively decorated with glycosyl modifications, some of which were partially resolved in the structure. Whether the glycosylations are important in TLR3 localization and/or function were not directly addressed in these works (1, 2). However, de Bouteiller et al. (21) showed that a change of Asn-247 to an arginine in TLR3 negatively affected TLR3 activity.We expressed the extracellular domain (ECD) of the human TLR3 in human embryonic kidney cells (HEK 293T) and demonstrated that it was modified with N-linked glycosylations. Using the GlcNAc-transferase inhibitor tunicamycin, a concentration-dependent inhibition of TLR3 activity was observed. Systematic mutational analysis of the predicted N-linked glycosylation sites identified two asparagine residues in leucine-rich repeats 8 and 15 that are important for TLR3 activity. The mutant proteins remain expressed at levels similar to wild type. In addition, because our ectodomain was produced in human cells as opp...

The dsRNA binding site of human Toll-like receptor 3

“…The glycan-free face is characteristic of TLR3, because most other human TLRs contain fewer glycosylation sites than TLR3 and, according to their predicted placement on the LRRs (19), do not have an identifiable interaction surface. It has been reported that mutation of 2 of the 15 glycosylation sites on TLR3 (N247 and N413) results in a complete loss of function (16,20), although we failed to detect a significant effect on activity resulting from an Asp-for-Asn replacement at any single glycosylation site (Fig. 8).…”

The Role of UNC93B1 Protein in Surface Localization of TLR3 Receptor and in Cell Priming to Nucleic Acid Agonists

“…This different glycosylation could be due to the addition of N-linked glycans or to the introduction of complex type N-linked glycans (43). TLR3 glycosylation is important for its bioactivity as the inhibition of glycosylation by tunicamycin prevents TLR3-induced signaling (13,44). However, it is unlikely that glycosylation affects ligand binding as glycosylation sites are positioned on the surface of TLR3 ectodomain that does not interfere with the ligand (5).…”

Structural Insight into the Mechanism of Activation of the Toll Receptor by the Dimeric Ligand Spätzle

“…Electron Microscopy-Three-dimensional structures were reconstructed using transmission electron microscopy and single particle analysis as described by Sun et al (13). Briefly, the proteins were diluted into a solution of 20 mM Tris-HCl, pH 7.5, and 150 mM NaCl and then adsorbed to freshly glow-discharged carbon-coated copper grids.…”