The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.
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.
1. Resurrecting Frozen Macrophage Cells
2. Plating Cells for the Sialidase Assay
3. Sialidase Assay
1. Making the Control
2. Making the Positive Test
3. Making the Positive Test together with Neuraminidase inhibitor Tamiflu
4. Determination of the Concentration of Inhibitor needed to Inhibit 50% of the Sialidase Activity (IC50)
5. Secrets to Success
6. Representative Results
See animated protocol of the sialidase assay with representative results in the attached Powerpoint file.
Using the newly developed assay to detect sialidase activity in live macrophage cells 2, we used this technology to detect sialidase activity in ligand-induced 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. Neuraminidase inhibitors like Tamiflu (oseltamivir phosphate) inhibited thymoquinone-induced sialidase activity in live BMC-2 cells with an IC50 of 0.0194 μM compared to an IC50 of 19.17 μM for neuraminidase inhibitor DANA (2-deoxy-2,3-dehydro-D-N-acetylneuraminic acid) 5. We have also reported that other applications such as specific 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 an application of the sialidase activity to detect 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 1,2,5. In addition, pertussis toxin (PTX), a specific inhibitor of Gαi 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 5. 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 5.
Taken together, the sialidase assay can be used to provide powerful insights to the molecular mechanisms of ligand-induced receptor activation involving sialidases like Neu1 or Neu4 depending on the nature of the ligand. The rapidity of the ligand-induced sialidase activity mediated by the ligand-bound receptor suggests that glycosylated receptors like NGF TrkA, BDNF TrkB and TOLL-like receptors form a signaling paradigm on the cell surface membrane involving a molecular organizational platform of ligand-bound receptor, Gαi proteins, MMP-9 and Neu1 or Neu4 sialidase. Ligand binding respective receptors induces sialidase activity through GPCR-signaling via membrane Gαi proteins and MMP-9 activation. Neu1 or Neu4 and MMP-9 cross-talk in complex with the receptor on the cell surface enables a rapid activation of the sialidase to remove sialic acid-steric hinderance to receptor association in generating a functional receptor.
The authors have nothing to disclose.
Partial support by grants to MRS is from Natural Sciences and Engineering Research Council of Canada (NSERC), the Harry Botterell Foundation for Neuroscience Research, ARC, and the Garfield Kelly Cardiovascular Research and Development Fund. S.R.A. is a recipient of the Queen’s University Research Award and the Robert J. Wilson Fellowship. P.J. is a recipient of the Queen’s Graduate Award and the Robert J. Wilson Fellowship. A.G. and S.A. are recipients of the Queen’s Graduate Award. S.F. was the recipient of the Ontario Graduate Scholarship in Science and Technology (OGSST). A.G. is the recipient of the Queen’s Franklin Bracken Graduate Scholarship. S.A. is the recipient of the Queen’s R.S. McLaughlin Graduate Scholarship.
Material Name | Tip | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM | Gibco, Rockville, MD | |||
4-MUNANA | Biosynth International Inc., Itasca, IL, USA | |||
Fetal calf serum | HyClone, Logan, Utah, USA | |||
DakoCytomation, Fluorescent Mounting Media | DAKO North America, Inc., Carpintenia, CA | S3023 | 15 mL | |
Tamiflu (Oseltamivir Phosphate) | Hoffman La Roche |