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1.
The ganglioside-specific sialidase Neu3 has been suggested to play essential roles in regulation of cell surface functions because of its major localization in the plasma membrane and strict substrate preference for gangliosides involved in signal transduction. Here we show that human Neu3 sialidase is enriched in caveolae microdomains and closely associates with caveolin like other caveolin-binding signaling molecules. Using HeLa cells and Neu3-transfected COS-1 cells, endogenous and exogenous Neu3 was found to co-concentrate caveolin-1 in low density Triton X-100-insoluble membrane fractions on sucrose density gradients of the respective cell extracts, as assessed by enzyme activity assays and immunoblotting with a monoclonal antibody to human Neu3. The presence of a putative caveolin-binding motif within Neu3 prompted us to determine whether Neu3 binds to caveolin-1. In transfectants expressing a polyhistidine-tagged form of Neu3, caveolin-1 co-eluted with Neu3 on affinity column chromatography. A mutation with a single amino acid change in the caveolin-binding motif led to inhibition of recruitment of the sialidase to the microdomain, accompanied by reduction of the enzyme activity. Neu3 also failed to associate with caveolin-enriched microdomains by cholesterol depletion with beta-cyclodextrin (with concomitant decrease of the sialidase activity), whereas Neu3 was activated by increased caveolin-1 expression. The tight association of Neu3 with caveolin-1 was supported further by co-immunoprecipitation of Neu3 by anti-caveolin-1 antibody. These results strongly suggest that Neu3 functions as a caveolin-related signaling molecule within caveolin-rich microdomains.  相似文献   

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Sialidases are enzymes that influence cellular activity by removing terminal sialic acid from glycolipids and glycoproteins. Four genetically distinct sialidases have been identified in mammalian cells. In this study, we demonstrate that three of these sialidases, lysosomal Neu1 and Neu4 and plasma membrane-associated Neu3, are expressed in human monocytes. When measured using the artificial substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (4-MU-NANA), sialidase activity of monocytes increased up to 14-fold per milligram of total protein after cells had differentiated into macrophages. In these same cells, the specific activity of other cellular proteins (e.g. beta-galactosidase, cathepsin A and alkaline phosphatase) increased only two- to fourfold during differentiation of monocytes. Sialidase activity measured with 4-MU-NANA resulted from increased expression of Neu1, as removal of Neu1 from the cell lysate by immunoprecipitation eliminated more than 99% of detectable sialidase activity. When exogenous mixed bovine gangliosides were used as substrates, there was a twofold increase in sialidase activity per milligram of total protein in monocyte-derived macrophages in comparison to monocytes. The increased activity measured with mixed gangliosides was not affected by removal of Neu1, suggesting that the expression of a sialidase other than Neu1 was present in macrophages. The amount of Neu1 and Neu3 RNAs detected by real time RT-PCR increased as monocytes differentiated into macrophages, whereas the amount of Neu4 RNA decreased. No RNA encoding the cytosolic sialidase (Neu2) was detected in monocytes or macrophages. Western blot analysis using specific antibodies showed that the amount of Neu1 and Neu3 proteins increased during monocyte differentiation. Thus, the differentiation of monocytes into macrophages is associated with regulation of the expression of at least three distinct cellular sialidases, with specific up-regulation of the enzyme activity of only Neu1.  相似文献   

5.
Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade beta-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr(334), conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu(111) and the catalytic aspartate Asp(46) are disordered in the apo form but upon binding of DANA become ordered to adopt two short alpha-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.  相似文献   

6.
Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady-state levels and in the cytosolic sialidase enzymatic activity. To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin-positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was on the contrary observed in parental and mock-transfected cells under the same experimental conditions. The results indicate that Neu2 upregulation per se is sufficient to trigger myoblast differentiation in C2C12 cells.  相似文献   

7.
Mammalian Neu3 sialidases are involved in various biological processes, such as cell death and differentiation, through desialylation of gangliosides. The enzymatic profile of Neu3 seems to be highly conserved from birds to mammals. In fish, the functional properties of Neu3 sialidase are not clearly understood, with the partial exception of the zebrafish form. To cast further light on the molecular evolution of Neu3 sialidase, we identified the encoding genes in the medaka Oryzias latipes and investigated the properties of the enzyme. PCR amplification using medaka brain cDNA allowed identification of two novel medaka Neu3 genes, neu3a and neu3b. The YRIP, VGPG motif and Asp-Box, characteristic of consensus motifs of sialidases, were well conserved in the both medaka Neu3 sialidases. When each gene was transfected into HEK293 to allow cell lysates for the use of enzymatic characterization, two Neu3 sialidases showed strict substrate specificity toward gangliosides, similar to mammalian Neu3. The optimal pH values were at pH 4.2 and pH 4.0, respectively, and neu3b in particular showed a broad optimum. Immunofluorescence assays indicated neu3a localization at plasma membranes, while neu3b was found in cytosol. The tissue distribution of two genes was then investigated by estimation of mRNA expression and sialidase activity, both being dominantly expressed in the brain. In neu3a gene-transfected neuroblastoma cells, the enzyme was found to positively regulate retinoic acid-induced differentiation with the elongation of axon length. On the other hand, neu3b did not affect neurite formation. These results and phylogenetic analysis suggested that the medaka neu3a is an evolutionally conserved sialidase with regard to enzymatic properties, whereas neu3b is likely to have originally evolved in medaka.  相似文献   

8.
Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli. The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Müthing, J. Carbohydr. Res. 2001, 330, 335-346). The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay. Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies. The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc). A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer. The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V. cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase. The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.  相似文献   

9.
The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.  相似文献   

10.
Mammalian sialidases are key enzymes in the degradation of glycoconjugates. Neu4L sialidase is localized to mitochondria and specifically expressed in brain. To elucidate the pathophysiological roles of Neu4L in the nervous system, we investigated the possible involvement of Neu4L in the apoptotic neurodegeneration under the existence of catechol metabolites generated by tyrosinase. We demonstrated that: (i) the expression level of Neu4L was dramatically decreased prior to apoptosis; (ii) the apoptotic phenotype was characterized by cytochrome c release into cytosol concomitant with the trafficking of ganglioside GD3 to mitochondria; and (iii) the inhibitor of glucosylceramide synthase partially recovered cell viability. Neu4L and its substrate GD3 may act as key molecules in the mitochondrial apoptotic pathway in neuronal cells.  相似文献   

11.
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 IC50 of 1.2?μM compared to an IC50 of 1015?μM 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 NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα 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.  相似文献   

12.
Ha KT  Lee YC  Cho SH  Kim JK  Kim CH 《Molecules and cells》2004,17(2):267-273
Endogenous expression of human membrane type ganglioside sialidase (Neu3) was examined in various cell lines including NB-1, U87MG, SK-MEL-2, SK-N-MC, HepG2, Hep3B, Jurkat, HL-60, K562, ECV304, Hela and MCF-7. Expression was detected in the neuroblastoma cell lines NB-1 and SK-N-MC, and also in erythroleukemia K562 cells, but not in any other cells. We isolated a Neu3 cDNA from K562 cells and expressed a His-tagged derivative in a bacterial expression system. The purified recombinant product of approximately 48 kDa had sialidase activity toward 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (4MU-NeuAc). The optimal pH of the purified Neu3 protein for GD3 ganglioside was 4.5. The enzyme also efficiently hydrolyzed GD3, GD1a, GD1b and GM3 whereas sialyllactose, 4MU-NeuAc, GM1 and GM2 were poor substrates, and it had no activity against sialylated glycoproteins such as fetuin, transferrin and orosomucoid. We conclude that the sialidase activity of Neu3 is specific for gangliosides.  相似文献   

13.
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.Download video file.(57M, mov)  相似文献   

14.
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 Gαi 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 IC50 of 3.876 μM 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.  相似文献   

15.
《Cellular signalling》2014,26(6):1355-1368
Insulin-induced insulin receptor (IR) tyrosine kinase activation and insulin cell survival responses have been reported to be under the regulation of a membrane associated mammalian neuraminidase-1 (Neu1). The molecular mechanism(s) behind this process is unknown. Here, we uncover a novel Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B G-protein coupled receptor (GPCR), which is essential for insulin-induced IR activation and cellular signaling. Neu1, MMP-9 and neuromedin B GPCR form a complex with IRβ subunit on the cell surface. Oseltamivir phosphate (Tamiflu®), anti-Neu1 antibodies, broad range MMP inhibitors piperazine and galardin (GM6001), MMP-9 specific inhibitor (MMP-9i), and GPCR neuromedin B specific antagonist BIM-23127 dose-dependently inhibited Neu1 activity associated with insulin stimulated rat hepatoma cells (HTCs) that overly express human IRs (HTC-IR). Tamiflu, anti-Neu1 antibodies and MMP-9i attenuated phosphorylation of IRβ and insulin receptor substrate-1 (IRS1) associated with insulin-stimulated cells. Olanzapine, an antipsychotic agent associated with insulin resistance, induced Neu3 sialidase activity in WG544 or 1140F01 human sialidosis fibroblast cells genetically defective in Neu1. Neu3 antagonist 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and anti-Neu3 antibodies inhibited sialidase activity associated with olanzapine treated murine Neu4 knockout macrophage cells. Olanzapine attenuated phosphorylation of IGF-R and IRS1 associated with insulin-stimulated human wild-type fibroblast cells. Our findings identify a novel insulin receptor-signaling platform that is critically essential for insulin-induced IRβ tyrosine kinase activation and cellular signaling. Olanzapine-induced Neu3 sialidase activity attenuated insulin-induced IGF-R and IRS1 phosphorylation contributing to insulin resistance.  相似文献   

16.
Altered sialylation of glycosphingolipids is observed in cancer as a ubiquitous phenotype, leading to the appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signaling. To understand the pathological significance of aberrant alterations of gangliosides in cancer, our studies have been focused on sialidase, which is responsible for the removal of sialic acids from glycoproteins and glycolipids. Among human sialidases so far identified, sialidase NEU3 is a key enzyme for ganglioside degradation because of its uniqueness both in its localization in the plasma membrane and in specifically hydrolyzing gangliosides. NEU3 is markedly up-regulated in many types of cancers including colon and renal carcinomas and suppresses apoptosis of cancer cells. The present paper briefly summarizes our recent results on the sialidase alterations and their significance in cancer. NEU3 is indeed closely related to malignancy and thus may be a potential target for cancer diagnosis and therapy.  相似文献   

17.
Li Y  Cao H  Yu H  Chen Y  Lau K  Qu J  Thon V  Sugiarto G  Chen X 《Molecular bioSystems》2011,7(4):1060-1072
Aberrant expression of human sialidases has been shown to associate with various pathological conditions. Despite the effort in the sialidase inhibitor design, less attention has been paid to designing specific inhibitors against human sialidases and characterizing the substrate specificity of different sialidases regarding diverse terminal sialic acid forms and sialyl linkages. This is mainly due to the lack of sialoside probes and efficient screening methods, as well as limited access to human sialidases. A low cellular expression level of the human sialidase NEU2 hampers its functional and inhibitory studies. Here we report the successful cloning and expression of the human sialidase NEU2 in E. coli. About 11 mg of soluble active NEU2 was routinely obtained from 1 L of E. coli cell culture. Substrate specificity studies of the recombinant human NEU2 using twenty p-nitrophenol (pNP)-tagged α2-3- or α2-6-linked sialyl galactosides containing different terminal sialic acid forms including common N-acetylneuraminic acid (Neu5Ac), non-human N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn), or their C5-derivatives in a microtiter plate-based high-throughput colorimetric assay identified a unique structural feature specifically recognized by the human NEU2 but not two bacterial sialidases. The results obtained from substrate specificity studies were used to guide the design of a sialidase inhibitor that was selective against human NEU2. The selectivity of the inhibitor was revealed by the comparison of sialidase crystal structures and inhibitor docking studies.  相似文献   

18.
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.  相似文献   

19.
Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.  相似文献   

20.
Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.  相似文献   

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