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The signaling pathways of mammalian Toll-like receptors (TLRs) are well characterized, but the precise mechanism(s) by which TLRs are activated upon ligand binding remains poorly defined. Recently, we reported a novel membrane sialidase-controlling mechanism that depends on ligand binding to its TLR to induce mammalian neuraminidase-1 (Neu1) activity, to influence receptor desialylation, and subsequently to induce TLR receptor activation and the production of nitric oxide and proinflammatory cytokines in dendritic and macrophage cells. The ␣-2,3-sialyl residue of TLR was identified as the specific target for hydrolysis by Neu1. Here, we report a membrane signaling paradigm initiated by endotoxin lipopolysaccharide (LPS) binding to TLR4 to potentiate G protein-coupled receptor (GPCR) signaling via membrane G␣ i subunit proteins and matrix metalloproteinase-9 (MMP9) activation to induce Neu1. Central to this process is that a Neu1-MMP9 complex is bound to TLR4 on the cell surface of naive macrophage cells. Specific inhibition of MMP9 and GPCR G␣ i -signaling proteins blocks LPS-induced Neu1 activity and NFB activation. Silencing MMP9 mRNA using lentivirus MMP9 shRNA transduction or siRNA transfection of macrophage cells and MMP9 knock-out primary macrophage cells significantly reduced Neu1 activity and NFB activation associated with LPS-treated cells. These findings uncover a molecular organizational signaling platform of a novel Neu1 and MMP9 cross-talk in alliance with TLR4 on the cell surface that is essential for ligand activation of TLRs and subsequent cellular signaling.The mammalian Toll-like receptors (TLRs) 11 are one of the families of sensor receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial infections for innate immune cells; they play important roles in the pathophysiology of infectious, inflammatory, and autoimmune diseases. Thus, the intensity and duration of TLR responses with these diseases must be tightly controlled. It follows that the structural integrity of TLR receptors, their ligand interactions, and their signaling components are important for our understanding of subsequent immunological responses.Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between TLRs and their ligands have remained poorly defined until now. We have recently identified a novel paradigm of TLR activation by its natural ligand, which has not been observed previously (1). This paradigm suggests that ligand-induced TLR activation is tightly controlled by Neu1 activation. The data indicate that Neu1 is already in complex with either TLR2, -3, or -4 receptors and is induced upon ligand binding to their respective receptors. In addition, activated Neu1 specifically hydrolyzes ␣-2,3-sialyl residues linked to ␤-galactosides, which are distant from ligand binding. This desialylation process is proposed to remove steric hindrance to TLR4 dimerization, MyD88-TLR4 complex recruitment, NFB activation, and p...

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2 . It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3 , which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4 . The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 5 . For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6 . Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways. Before resurrecting frozen cells from the -80°C freezer, one needs to prepare culture medium using sterile filtered Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5 μg/mL of plasmocin. Plasmocin is an antibiotic solution used in our research to eliminate and prevent mycoplasma contamination of cell cultures. 2. For one vial of frozen cells, one adds 4 mL, 1 mL of the cultured medium to a 25 cm 2 cell culture flask in a sterile biohazard containment hood. 3. When the vial of frozen cells are taken from the -80°C freezer, they are quickly thawed by hand warming in the...

Anti-inflammatory activities of thymoquinone (TQ) have been demonstrated in in vitro and in vivo studies. However, the precise mechanism(s) of TQ in these anti-inflammatory activities is not well understood. Using a newly developed assay to detect sialidase activity in live macrophage cells (Glycoconj J doi: 10.1007/s10719-009-9239-8 ), here we show that TQ has no inhibitory effect on endotoxin lipopolysaccharide (LPS) induced sialidase activity in live BMC-2 macrophage cells. In contrast, the parent black seed oil (BSO) and another constituent of BSO para-cymene (p-CY) completely block LPS induced sialidase activity. All of these compounds had no effect on cell viability. On the other hand, TQ induces a vigorous sialidase activity in live BMC-2 macrophage cells in a dose dependent manner as well in live DC-2.4 dendritic cells, HEK-TLR4/MD2, HEK293, SP1 mammary adenocarcinoma cells, human WT and 1140F01 and WG0544 type I sialidosis fibroblast cells. Tamiflu (oseltamivir phosphate) inhibits TQ-induced sialidase activity in live BMC-2 cells with an IC(50) of 0.0194 microM compared to an IC(50) of 19.1 microM for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid). Anti-Neu1, -2 and -3 antibodies have no inhibition of TQ-induced sialidase activity in live BMC-2 and human THP-1 macrophage cells but anti-Neu4 antibodies completely block this activity. There is a vigorous sialidase activity associated with TQ treated live primary bone marrow (BM) macrophage cells derived from WT and hypomorphic cathepsin A mice with a secondary Neu1 deficiency (NeuI KD), but not from Neu4 knockout (Neu4 KO) mice. Pertussis toxin (PTX), a specific inhibitor of Galphai proteins of G-protein coupled receptor (GPCR) and the broad range inhibitors of matrix metalloproteinase (MMP) galardin and piperazine applied to live BMC-2, THP-1 and primary BM macrophage cells completely block TQ-induced sialidase activity. These same inhibitory effects are not observed with the GM1 ganglioside specific cholera toxin subunit B (CTXB) as well as with CTX, tyrosine kinase inhibitor K252a, and the broad range GPCR inhibitor suramin. The specific inhibitor of MMP-9, anti-MMP-9 antibody and anti-Neu4 antibody, but not the specific inhibitor of MMP-3 completely block TQ-induced sialidase activity in live THP-1 cells, which express Neu4 and MMP-9 on the cell surface. Neu4 sialidase activity in cell lysates from TQ-treated live THP-1 cells desialylates natural gangliosides and mucin substrates. RT-PCR and western blot analyses reveal no correlation between mRNA and protein values for Neu3 and Neu4 in human monocytic THP-1 cells, suggesting for the first time a varied post-transcriptional mechanism for these two mammalian sialidases independent of TQ activation. Our findings establish an unprecedented activation of Neu4 sialidase on the cell surface by thymoquinone, which is derived from the nutraceutical black cumin oil. The potentiation of GPCR-signaling by TQ via membrane targeting of Galphai subunit proteins and matrix metalloproteinas...