The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429

Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743