Microbial recognition by TLR2 and CD36

Abstract

The discovery of the first Toll-like receptor in 1998 was a crucial step towards understanding how the innate immune response is established. Although there have been significant advances in understanding the molecular pathways activated by Toll-like receptors, still little is known about the molecular mechanism leading to the recognition of specific ligands by Toll-like receptors. In particular, Toll-like receptor 2 (TLR2) has the broadest range ofligand recognition among Toll-like receptors. In order to study the interaction ofTLR2 with its ligands, the ectodomain ofTLR2 was expressed in insect cells. An analysis of its N-linked glycosylation showed the presence ofhigh-mannose-type glycans. Circular dichroism showed that TLR2 has a predominantly p-sheet secondary structure and that its Tm point lies between 45°C and 55 °C. A direct interaction between the TLR2 ectodomain and its ligands was demonstrated using native PAGE. I concluded that the diacylglycerol is a relevant ligand moiety for interacting with TLR2. I also demonstrated that the TLR2 ectodomain is able to bind its ligands at physiological conditions and independently of its association with co-receptors TLR6 and TLRI. Recently CD36, a member of the scavenger receptor family, was reported as a coreceptor ofTLR2. In order to investigate the mechanism by which CD36 enhances TLR2 activation, the CD36 ectodomain was expressed in insect eells. Binding assays demonstrated a selective binding towards negatively charged ligands at physiological conditions. The selective binding explains the fact that CD36 is a selective co-receptor ofTLR2. Furthermore, the activation ofTLR2 in macrophages from CD36 deficient (CD36 Obl·/Obl-) mice was restored by addition ofthe CD36 ectodomain. This result clearly demonstrated that the ectodomain, but not the transmembrane or intracellular domain ofCD36, is the essential domain for the activity of CD36 as TLR2 co-receptor. Moreover, the inability of soluble CD36 to restore TNF-a secretion from CD14 deficient macrophages, shows that CD14 is a necessary molecule for the function of CD36 as a TLR2 co-receptor. Finally, we expressed the LRR domain of 18-Wheeler and Windpipe, two transmembrane LRR proteins ofDrosophila melanogaster. 18-Wheeler belongs to the Toll receptor family, while Windpipe is a novel transmembrane protein with a predicted LRR domain in its extracellular domain. Both receptors are involved in the morphogenesis ofDrosophila melanogaster. Although crystallisation conditions for both the LRR domain ofWindpipe and the ectodomain of 18-Wheeler were found, optimisation ofthese crystallisation conditions are required to determine the crystal structure ofthese LRR proteins.EThOS - Electronic Theses Online ServiceGBUnited Kingdo