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

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. 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...

A novel G-protein-coupled receptor-signaling platform and its targeted translation in human disease

“…89,90 This membrane-signaling paradigm suggests a Neu1 and MMP-9 cross talk in alliance with GPCR signaling through Gα i proteins and TLRs on the cell surface to mediate receptor activation. 84 Our other reports 21,34,35,63,69 support a receptor glycosylation model in corroborating the importance of activated Neu1 hydrolysis of sialyl α-2,3-linked β-galactosyl residues of TLR in the initial stages of ligand-induced receptor activation. This desialylation process is proposed to remove steric hindrance to TLR dimerization, MyD88/TLR complex recruitment, NF-κB activation, and proinflammatory cytokine response.…”

“…by the hydrolytic metabolite oseltamivir carboxylate form, 68 we have actually observed the opposite effect in our livecell assay system. 35,69 To further elucidate the inhibitory capacity of Tamiflu and its hydrolytic metabolite oseltamivir carboxylate, the 50% inhibitory concentration (IC 50 ) of each compound was determined by plotting the decrease in sialidase activity against the log of the agent concentration. Tamiflu was found to have an IC 50 of 1.175 µM compared to an IC 50 of 1015 µM for oseltamivir carboxylate.…”

Sialylation facilitates self-assembly of 3D multicellular prostaspheres by using cyclo-RGDfK(TPP) peptide

“…Activated Neu1 hydrolyzes α2,3-sialyl residues of the receptors, which allows the removal of steric hindrance between receptors, allowing subsequent kinase activation and cellular signaling. To determine whether the PCa cells express sialidase activity on the cell surface, a sialidase assay was performed as previously established and optimized 52,60,61. Sialidase activity is measured in the periphery surrounding the live cells due to the lysis of fluorogenic substrate 4-MUNANA into 4-methylumbelliferone product which has an emission wavelength of 450 nm.…”

“…The sialidase activity assay was performed as described previously 52,53. Briefly, cells were incubated overnight on 12 mm sterile circular glass slides in a conditioned medium in a 24-well plate.…”

Neuraminidase activity mediates IL-6 production through TLR4 and p38/ERK MAPK signaling in MRL/lpr mesangial cells

“…The only other NEU detectable in the kidney is NEU3 with message levels of Neu1 expressed approximately 40-fold higher than Neu3 (19) and NEU1 and NEU3 proteins are readily detected in the glomeruli of renal tissue sections of MRL/lpr lupus mice (12). While NEU1 was largely observed to desialylate glycoproteins, NEU3 showed a preference for gangliosides (25)(26)(27)(28)(29) and their activity at the plasma membrane impacts receptor activation and signaling in a variety of cell types (29)(30)(31)(32)(33)(34)(35).…”