Molecular cloning and biochemical characterization of two novel Neu3 sialidases, neu3a and neu3b, from medaka (Oryzias latipes)

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.     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus

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.     

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

Sialyl-linkage specificity of sialidases of the human influenza A virus strains, A/Aichi/2/68 (H3N2) and A/PR/8/34 (H1N1) were studied using natural and synthetic gangliosides. The sialidase of the A/Aichi/2/68 strain hydrolyzed the terminal Neu5Acalpha2-3Gal sequence but not the Neu5Acalpha2-3 linkage on the inner Gal of GM1a, which is a ganglioside that has the gangliotetraose chain (Galbeta1-3GalNAcbeta1-4- (Neu5Acalpha2-3)Galbeta1++ +-4Glcbeta1-Cer). The sialidase hydrolyzed the Neu5Ac on the inner Gal of GM2, which had a shorter gangliotriose chain. GM4, which had the shortest chain (Neu5Acalpha2-3Galbeta1-Cer) of the gangliosides, had a lower substrate specificity. The N1 and N2 sialidase subtypes of the human influenza A virus had no significant variation in their substrate specificity for the gangliosides. Analysis of 11 synthetic gangliosides, which contained various ceramide or sialic acid moieties, demonstrated that A/Aichi/2/68 (H3N2) sialidase recognized the ceramide and sialic acid moiety and the length and structure of the sialyl sugar chain.      

Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli

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.     

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.     

Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells

Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phosphate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.     

Identifying selective inhibitors against the human cytosolic sialidase NEU2 by substrate specificity studies

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.     

Sialidases on Mammalian Sperm Mediate Deciduous Sialylation during Capacitation

Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.     

NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia: NEU1 restrains endothelial cell migration, whereas NEU3 does not

The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.     

Plasma membrane-associated sialidase as a crucial regulator of transmembrane signalling

Mammalian sialidases, glycosidases responsible for the removal of sialic acids from glycoproteins and glycolipids, has been implicated to participate in many biological processes as well as in lysosomal catabolism. Among those forms identified to date, plasma membrane-associated sialidase, Neu3, is a key enzyme in degradation of gangliosides, for which it exhibits a special substrate preference. This sialidase has been shown to control transmembrane signalling for many cellular processes, including cell differentiation, cell growth and apoptosis, and human orthologue NEU3 is markedly up-regulated in various cancers. It is known to suppress apoptosis in cancer cells. Furthermore, its overexpression causes impaired glucose tolerance and hyper-insulinaemia together with overproduction of insulin in enlarged islets in the transgenic mice. The present review primarily summarizes our recent results, focusing on Neu3 as a regulator of transmembrane signalling.     

Modulation of Neuritogenesis by Ganglioside-Specific Sialidase (Neu 3) in Human Neuroblastoma NB-1 Cells

Plasma membrane-associated sialidase (Neu 3), which specifically hydrolyzes gangliosides, is relatively abundantly present in the nervous system. To understand the role of Neu 3 in neuronal differentiation, we studied the relationship between neurite outgrowth and Neu 3 expression in human neuroblastoma NB-1 cells. The expression of Neu 3 in NB-1 cells increased when neurite outgrowth in these cells was induced by dibutyryl cAMP. While treatment with dibutyryl cAMP alone enhanced the outgrowth of dendrite-like processes, transfection of the Neu 3 gave rise to a more prominent outgrowth of neurites with axon-like characteristics, even in the absence of dibutyryl cAMP. Neu 3 induction by dibutyryl cAMP is probably attributable, in part, to transactivation of the Neu 3 gene through cAMP responsive elements in the 5-upstream region, as revealed by the promotor activity assay using Neu 3 promotor expression plasmid. These results indicate that Neu 3 regulates neurite formation in NB-1 cells, and suggest that this effect may be enhanced by dibutyryl cAMP via a cAMP-dependent pathway.     

Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.     

Biochemical and immunological studies on two distinct ganglioside-hydrolyzing sialidases from the particulate fraction of rat brain

Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.     

A close association of the ganglioside-specific sialidase Neu3 with caveolin in membrane microdomains

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.