Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Summary

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.

Abstract

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.

Protocol

1. Resurrecting Frozen Macrophage Cells 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. For one vial of frozen cells, one adds 4 mL, 1 mL of the cultured medium to a 25 cm2 …

Discussion

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 i…

Materials

Material Name	Tipo	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