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Articles by Myron R. Szewczuk in JoVE
JoVE Immunology and Infection
Neu1 Sialidase गतिविधि के टॉल की तरह रिसेप्टर सक्रियण विनियमन में जांच
Department of Microbiology and Immunology, Queen's University - Kingston, Ontario
sialidase परख एक साधारण तकनीकी दृष्टिकोण है कि माइक्रोबियल संक्रमण और जीना मैक्रोफेज की कोशिका की सतह पर रिसेप्टर स्तर पर भड़काऊ रोगों में भागीदारी के TLR सेंसरों के उपन्यास आणविक तंत्र (ओं) को स्पष्ट करेंगे.
Other articles by Myron R. Szewczuk on PubMed
Trypanosome Trans-sialidase Targets TrkA Tyrosine Kinase Receptor and Induces Receptor Internalization and Activation
Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme that hydrolyzes alpha2,3-linked sialic acids and transfers them to acceptor molecules. Here we show that a highly purified recombinant TS derived from T. cruzi parasites targets TrkA receptors on TrkA-expressing PC12 cells and colocalizes with TrkA internalization and phosphorylation (pTrkA). Maackia amurensis lectin II (MAL-II) and Sambucus nigra lectin (SNA) block TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA. Cells treated with lectins alone do not express pTrkA. The catalytically inactive mutant TSDeltaAsp98-Glu also binds to TrkA-expressing cells, but is unable to induce pTrkA. TrkA-PC12 cells treated with a purified recombinant alpha2,3-neuraminidase (Streptococcus pneumoniae) express pTrkA. Wild-type TS but not the mutant TSDeltaAsp98-Glu promotes neurite outgrowth in TrkA-expressing PC12 cells. In contrast, these effects are not observed in TrkA deficient PC12nnr5 cells but are reestablished in PC12nnr5 cells stably transfected with TrkA and are significantly blocked by inhibitors of tyrosine kinase (K-252a) and MAP/MEK protein kinase (PD98059). Together these observations suggest for the first time that hydrolysis of sialyl alpha2,3-linked beta-galactosyl residues of TrkA receptors plays an important role in TrkA receptor activation, sufficient to promote cell differentiation (neurite outgrowth) independent of nerve growth factor.
Dependence of Neurotrophic Factor Activation of Trk Tyrosine Kinase Receptors on Cellular Sialidase
A direct link between receptor glycosylation and activation following natural ligand interaction has not been observed. Here, we discover a membrane sialidase-controlling mechanism that depends on ligand binding to its receptor to induce enzyme activity which targets and desialylates the receptor and, consequently, causes the induction of receptor dimerization and activation. We also identify a specific sialyl alpha-2,3-linked beta-galactosyl sugar residue of TrkA tyrosine kinase receptor, which is rapidly targeted and hydrolyzed by the sialidase. Trk-expressing cells and primary cortical neurons following stimulation with specific neurotrophic growth factors express a vigorous membrane sialidase activity. Neuraminidase inhibitors, Tamiflu, BCX1812, and BCX1827, block sialidase activity induced by nerve growth factor (NGF) in TrkA-PC12 cells and by brain-derived neurotrophic factor (BDNF) in primary cortical neurons. In contrast, the neuraminidase inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, specific for plasma membrane ganglioside Neu3 and Neu2 sialidases has no inhibitory effect on NGF-induced pTrkA. The GM1 ganglioside specific cholera toxin subunit B applied to TrkA-PC12 cells has no inhibitory effect on NGF-induced sialidase activity. Neurite outgrowths induced by NGF-treated TrkA-PC12 and BDNF-treated PC12(nnr5) stably transfected with TrkB receptors (TrkB-nnr5) cells are significantly inhibited by Tamiflu. Our results establish a novel mode of regulation of receptor activation by its natural ligand and define a new function for cellular sialidases.
Trypanosome Trans-sialidase Mediates Neuroprotection Against Oxidative Stress, Serum/glucose Deprivation, and Hypoxia-induced Neurite Retraction in Trk-expressing PC12 Cells
Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme and a novel ligand of tyrosine kinase (TrkA) receptors but not of neurotrophin receptor p75NTR. Here, we show that TS targets TrkB receptors on TrkB-expressing pheochromocytoma PC12 cells and colocalizes with TrkB receptor internalization and phosphorylation (pTrkB). Wild-type TS but not the catalytically inactive mutant TSDeltaAsp98-Glu induces pTrkB and mediates cell survival responses against death caused by oxidative stress in TrkA- and TrkB-expressing cells like those seen with nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). These same effects are not observed in Trk deficient PC12(nnr5) cells, but are re-established in PC12(nnr5) cells stably transfected with TrkA or TrkB, are partially blocked by inhibitors of tyrosine kinase (K-252a), mitogen-activated protein/mitogen-activated kinase (PD98059) and completely blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Both TrkA- and TrkB-expressing cells pretreated with TS or their natural ligands are protected against cell death caused by serum/glucose deprivation or from hypoxia-induced neurite retraction. The cell survival effects of NGF and BDNF against oxidative stress are significantly inhibited by the neuraminidase inhibitor, Tamiflu. Together, these observations suggest that trypanosome TS mimics neurotrophic factors in cell survival responses against oxidative stress, hypoxia-induced neurite retraction and serum/glucose deprivation.
Dependence of Pathogen Molecule-induced Toll-like Receptor Activation and Cell Function on Neu1 Sialidase
The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC(50) of 1.2 microM compared to an IC(50) of 1015 microM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFkappaB and the production of nitric oxide and pro-inflammatory IL-6 and TNFalpha cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.
Neu1 Desialylation of Sialyl Alpha-2,3-linked Beta-galactosyl Residues of TOLL-like Receptor 4 is Essential for Receptor Activation and Cellular Signaling
The ectodomain of TOLL-like receptors (TLR) is highly glycosylated with several N-linked gylcosylation sites located in the inner concave surface. The precise role of these sugar N-glycans in TLR receptor activation is unknown. Recently, we have shown that Neu1 sialidase and not Neu2, -3 and -4 forms a complex with TLR-2, -3 and -4 receptors on the cell-surface membrane of naïve and activated macrophage cells (Glycoconj J DOI 10.1007/s10719-009-9239-8). Activation of Neu1 is induced by TLR ligands binding to their respective receptors. Here, we show that endotoxin lipopolysaccharide (LPS)-induced MyD88/TLR4 complex formation and subsequent NFkappaB activation is dependent on the removal of alpha-2,3-sialyl residue linked to beta-galactoside of TLR4 by the Neu1 activity associated with LPS-stimulated live primary macrophage cells, macrophage and dendritic cell lines but not with primary Neu1-deficient macrophage cells. Exogenous alpha-2,3 sialyl specific neuraminidase (Streptoccocus pneumoniae) and wild-type T. cruzi trans-sialidase (TS) but not the catalytically inactive mutant TSAsp98-Glu mediate TLR4 dimerization to facilitate MyD88/TLR4 complex formation and NFkappaB activation similar to those responses seen with LPS. These same TLR ligand-induced NFkappaB responses are not observed in TLR deficient HEK293 cells, but are re-established in HEK293 cells stably transfected with TLR4/MD2, and are significantly inhibited by alpha-2,3-sialyl specific Maackia amurensis (MAL-2) lectin, alpha-2,3-sialyl specific galectin-1 and neuraminidase inhibitor Tamiflu but not by alpha-2,6-sialyl specific Sambucus nigra lectin (SNA). Taken together, the findings suggest that Neu1 desialylation of alpha-2,3-sialyl residues of TLR receptors enables in removing a steric hinderance to receptor association for TLR activation and cellular signaling.
Regulation of Phagocytosis in Macrophages by Neuraminidase 1
The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA(S190A)) and a double CathA/Neu1 deficiency (CathA(S190A-Neo)). Macrophages of CathA(S190A-Neo) mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA(S190A) mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 degrees C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcgammaR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcgammaR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.
Thymoquinone from Nutraceutical Black Cumin Oil Activates Neu4 Sialidase in Live Macrophage, Dendritic, and Normal and Type I Sialidosis Human Fibroblast Cells Via GPCR Galphai Proteins and Matrix Metalloproteinase-9
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 metalloproteinase-9 activation may be involved in the activation process of Neu4 sialidase on the cell surface.
Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Neurotrophin Activation of Trk Receptors and Cellular Signaling
Neurotrophin-induced Trk tyrosine kinase receptor activation and neuronal cell survival responses have been reported to be under the control of a membrane associated sialidase. Here, we identify an unprecedented membrane sialidase mechanism initiated by nerve growth factor (NGF) binding to TrkA to potentiate GPCR-signaling via membrane Galphai subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live primary neurons and TrkA- and TrkB-expressing cell lines. Central to this process is that Neu1/MMP-9 complex is bound to TrkA on the cell surface of naïve primary neurons and TrkA-expressing cells. Tamiflu completely blocks this sialidase activity in live TrkA-PC12 cells treated with NGF with an IC(50) of 3.876 microM with subsequent inhibition of Trk activation in primary neurons and neurite outgrowth in TrkA-PC12 cells. Our findings uncover a Neu1 and MMP-9 cross-talk on the cell surface that is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and cellular signaling.
Thymoquinone-induced Neu4 Sialidase Activates NFκB in Macrophage Cells and Pro-inflammatory Cytokines in Vivo
Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling via membrane targeting of Gαi subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NFκB activation are induced by the Neu4 activity associated with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate), pertussis toxin (PTX), a specific inhibitor of Gαi proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4 complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NFκB activation induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NFκB activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1 cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with subsequent NFκB activation and pro-inflammatory cytokine production in vivo.
Neu1 Sialidase and Matrix Metalloproteinase-9 Cross-talk is Essential for Toll-like Receptor Activation and Cellular Signaling
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 α-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 Gα(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 Gα(i)-signaling proteins blocks LPS-induced Neu1 activity and NFκB 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 NFκB 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.
