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Recent Development in Mammalian Sialidase Molecular Biology
Abstract
This review summarizes the recent research development on mammalian sialidase molecular cloning. Sialic acid-containing compounds are involved in several physiological processes, and sialidases, as glycohydrolytic enzymes that remove sialic acid residues, play a pivotal role as well. Sialidases hydrolyze the nonreducing, terminal sialic acid linkage in various natural substrates, such as glycoproteins, glycolipids, gangliosides, and polysaccharides. Mammalian sialidases are present in several tissues/organs and cells with a typical subcellular distribution: they are the lysosomal, the cytosolic, and the plasma membrane-associated sialidases. Starting in 1993, 12 different mammalian sialidases have been cloned and sequenced. A comparison of their amino acid sequences revealed the presence of highly conserved regions. These conserved regions are shared with viral and microbial sialidases that have been characterized at three-dimensional structural level, allowing us to perform the molecular modeling of the mammalian proteins and suggesting a monophyletic origin of the sialidase enzymes. Overall, the availability of the cDNA species encoding mammalian sialidases is an important step leading toward a comprehensive picture of the relationships between the structure and biological function of these enzymes.
... The levels and linkages of Sias, known as sialylation status, are critical to regulate various biological processes [4]. Despite the importance of sialylation, the hydrolytic removal of Sias (desialylation) from glycoproteins or glycolipids is also imperative in regulating various physiological and pathological pathways [5][6][7]. Desialylation of glycoconjugates influences cell signaling, adhesion, and apoptosis, receptor activation for adhesion, phagocytosis, cell migration, regulation of cell transformation, differentiation and migration, neuritogenesis, carcinogenesis, also insulin signaling [5][6][7]. Therefore, sialidases regulate many cellular processes in both physiological and pathological pathways by removing Sias from glycoconjugates. ...
... Despite the importance of sialylation, the hydrolytic removal of Sias (desialylation) from glycoproteins or glycolipids is also imperative in regulating various physiological and pathological pathways [5][6][7]. Desialylation of glycoconjugates influences cell signaling, adhesion, and apoptosis, receptor activation for adhesion, phagocytosis, cell migration, regulation of cell transformation, differentiation and migration, neuritogenesis, carcinogenesis, also insulin signaling [5][6][7]. Therefore, sialidases regulate many cellular processes in both physiological and pathological pathways by removing Sias from glycoconjugates. Sialidases (Neuraminidases, NAs) are glycosidases responsible for the removal of Sia residues (desialylation) from glycan portions of either glycoproteins or glycolipids. ...
... Sialidases (Neuraminidases, NAs) are glycosidases responsible for the removal of Sia residues (desialylation) from glycan portions of either glycoproteins or glycolipids. Based on their hydrolytic action on sialyl glycosidic linkages, they are classified into two major classes, exo-sialidases and endosialidases [5][6][7]. Exo-sialidases catalyze the hydrolysis of α-2,3-, α2,6-, α2,8-glycosidic linkages of terminal Sia residues in glycoproteins and glycolipids [8], while endo-sialidases catalyze the hydrolysis of α2,8-sialyl linkages in oligo-or poly (sialic) acids and are assigned to family GH58 [9]. Most of the sialidases are exo-sialidases. ...
Sialidases are glycosidases responsible for the removal of sialic acid (Sia) residues (desialylation) from glycan portions of either glycoproteins or glycolipids. By desialylation, sialidases are able to modulate the functionality and stability of the Sia-containing molecules and are involved in both physiological and pathological pathways. Therefore, evaluation of sialidase activity and specificity is important for understanding the biological significance of desialylation by sialidases and its function and the related molecular mechanisms of the physiological and pathological pathways. In addition, it is essential for developing novel mechanisms and approaches for disease treatment and diagnosis and pathogen detection as well. This review summarizes the most recent sialidase substrates for evaluating sialidase activity and specificity and screening sialidase inhibitors, including (i) general sialidase substrates, (ii) specific sialidase substrates, (iii) native sialidase substrates and (iv) cellular sialidase substrates. This review also provides a brief introduction of recent instrumental methods for quantifying the sialidase activity, such as UV, fluorescence, HPLC and LC-MS methods.
... Glycoproteins and glycolipids expressed on the cell surface contain oligosaccharide chains whose outermost positions can terminate with sialic acid. The sialylation state of these surface structures is regulated, at least in part, through sialidase catalytic activity (Monti et al. 2002;Monti et al. 2010;Miyagi and Yamaguchi 2012). At least four human sialidases, also referred to as neuraminidases, have been identified, NEU1, -2, -3 and -4 (Pshezhetsky et al. 1997;Miyagi et al. 1999;Monti et al. 1999;Comelli et al. 2003). ...
... NEU1 participates in multiple cellular functions (Monti et al. 2002(Monti et al. , 2010Miyagi and Yamaguchi 2012). In most human cells, NEU1 is co-expressed with NEU2, -3 and -4 (Monti et al. 2002(Monti et al. , 2010Miyagi and Yamaguchi 2012). ...
... NEU1 participates in multiple cellular functions (Monti et al. 2002(Monti et al. , 2010Miyagi and Yamaguchi 2012). In most human cells, NEU1 is co-expressed with NEU2, -3 and -4 (Monti et al. 2002(Monti et al. , 2010Miyagi and Yamaguchi 2012). Selective inhibition of NEU1 without off-target cross-inhibition of any of the other three isoforms could provide insight into NEU1 function and/or therapeutic possibilities for clinical conditions in which NEU1 might be overexpressed and/or activated. ...
NEU1 is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates
multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic
acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia, and lung fibroblasts
to be 3.74 µM, 13.0 µM, and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced,
NEU1-mediated MUC1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa, and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound
and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A
(PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase
activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity
for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the
seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other
enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment
of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically-expressed
sialidase activity and established NEU-1-mediated bioactivities in human airway epithelia, lung microvascular endothelia,
and fibroblasts, in vitro, and murine lungs, in vivo.
... All human sialidases contain Asp boxes from Ser/Thr-X-Asp-X-Gly-X-X-Trp/Phe, where X represents the variable residues [58,59] and the Y/FRIP motif composed of Tyr/Phe-Arg-Ile-Pro sequences located near the N-terminal part, which were also described for bacterial sialidases [20,27]. The Asp box motif is located far from the active site of human sialidases, specifically at the turn between third and fourth β-strand and may be repeated up to five times in the protein sequence [51]. ...
... The NEU1 shows higher levels of expression in human tissues than NEU3 and NEU4, while NEU2 expression level is extremely low [50]. Although, human neuraminidases are different in subcellular localization and substrate specificity, they share a common genomic organization including the Arg triad, Asp boxes and RIP motif [58,59]. The overall amino acid identity of NEU1 to the other human sialidases is relatively low (19-24%), whereas NEU2, NEU3 and NEU4 show 34-40% homology to each other [17]. ...
... Cytosolic NEU2 hydrolyses a wide range of glycoproteins, oligosaccharides and gangliosides [71,75]. The NEU2 is expressed predominantly in the skeletal muscle and foetal liver [59]. Chavas et al. reported the first high resolution X-ray structures of human NEU2 in its free form and the complex with an inhibitor of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) [76]. ...
Sialidases are enzymes essential for numerous organisms including humans. Hydrolytic sialidases (EC 3.2.1.18), trans-sialidases and anhydrosialidases (intramolecular trans-sialidases, EC 4.2.2.15) are glycoside hydrolase enzymes that cleave the glycosidic linkage and release sialic acid residues from sialyl substrates. The paper summarizes diverse sialidases present in the human body and their potential impact on development of antiviral compounds - inhibitors of viral neuraminidases. It includes a brief overview of catalytic mechanisms of action of sialidases and describes the origin of sialidases in the human body. This is followed by description of the structure and function of sialidase families with a special focus on the GH33 and GH34 families. Various effects of sialidases on human body are also briefly described. Modulation of sialidase activity may be considered a useful tool for effective treatment of various diseases. In some cases, it is desired to completely suppress the activity of sialidases by suitable inhibitors. Specific sialidase inhibitors are useful for the treatment of influenza, epilepsy, Alzheimer's disease, diabetes, different types of cancer, or heart defects. Challenges and future directions are shortly depicted in the final part of the paper.
... The sialylation state of glycoconjugates is dynamically and coordinately regulated through the opposing catalytic activities of sialyltransferases (7,17,18) and sialidases (1,35,42,55). Sialidases hydrolyze the glycosidic linkage between SA and the subterminal sugar of glycoconjugates. Four mammalian neuraminidase/sialidases (NEUs) have been identified: NEU1, 2, 3, and 4 (6,10,36,39,41,47). ...
... Four mammalian neuraminidase/sialidases (NEUs) have been identified: NEU1, 2, 3, and 4 (6,10,36,39,41,47). NEU1 is localized to lysosomes and is only active in association with cathepsin A and -galactosidase (1,6,35,39,42,47,55). NEU2 is found in the cytosol, NEU3 is associated with the plasma membrane, and NEU4 is located in mitochondria (1,35,39,42,55). ...
... Four mammalian neuraminidase/sialidases (NEUs) have been identified: NEU1, 2, 3, and 4 (6,10,36,39,41,47). NEU1 is localized to lysosomes and is only active in association with cathepsin A and -galactosidase (1,6,35,39,42,47,55). NEU2 is found in the cytosol, NEU3 is associated with the plasma membrane, and NEU4 is located in mitochondria (1,35,39,42,55). As members of the neuraminidase/sialidase superfamily, each of these enzymes contains one or more conserved Asp boxes, an amino acid sequence comprised of -S-X-D-X-G-X-T-Wwhere X represents variable residues, together with the -F/Y-R-I-P-motif (1,35,39,42,55). ...
Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically-active NEU3 sialidase.
... Although these chemical and genetic approaches are effective in improving sialylation of rhEPO, these approaches still do not allow for rhEPO production in fed-batch cultures. CHO cells have three active sialidases, Neu 1, 2, and 3, located in different organelles within the cells (Monti et al., 2002). Sialidases, which are secreted from viable cells and/or released from dead cells, accumulate during CHO cell culture Park et al., 2017Park et al., , 2017a. ...
... Four sialidases (Neu1, 2, 3, and 4) remove terminal sialic acids from glycans; these are known to be expressed at different locations in mammalian cells (Monti et al., 2002). Only Neu1, 2, and 3 are expressed in CHO-EPO cells, as reported for other CHO cell lines (Monti et al., 2002;Zhang et al., 2010). ...
... Four sialidases (Neu1, 2, 3, and 4) remove terminal sialic acids from glycans; these are known to be expressed at different locations in mammalian cells (Monti et al., 2002). Only Neu1, 2, and 3 are expressed in CHO-EPO cells, as reported for other CHO cell lines (Monti et al., 2002;Zhang et al., 2010). Sialidase activity in culture supernatants continued to increase during both batch and fed-batch cultures of CHO-EPO cells, which reduced rhEPO sialylation. ...
Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2-44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.
... Sialidases, or neuraminidases, are glycohydrolytic enzymes that remove sialic acid residues from the terminal end of glycoproteins, oligosaccharides, and glycolipids. [1][2][3] There are four known mammalian sialidases and they vary in their subcellular patterns of expression, enzymatic properties, and chromosomal localization of the encoding genes. Neu1, Neu2, Neu3, and Neu4 classical subcellular localizations are lysosomes, cytosol, plasma membranes, and the lysosomal or mitochondria lumina and intracellular membranes, respectively. ...
... Their involvement in such phenomena could be either direct or secondary to desialylation of many different substrates. 1 In cancer, sialidases are reported to be present in several different types of malignancies, although not well characterized. 5 Sialidase activity, for instance, was found to be highly elevated in the sera of breast cancer patients. ...
Sialidases are enzymes that catalyze the removal of sialic acids from glycoproteins and glycolipids. Previously, we have studied the effect of sialidase inhibition as a modulator of sialylation-related mechanisms of invasion and found that it induces aggressiveness in canine mammary tumors (CMTs). In this study, we aimed to assess the expression of glycoprotein-acting sialidases, Neu1, Neu2, and Neu4, in the complex multistage process of cancer metastasis. Thus, we examined their expression in a series of spontaneous malignant CMTs, CMT cell lines, and nude mice xenografts. All malignant CMT lesions expressed mammalian sialidases, although overall decreased when compared to normal adjacent mammary tissues. This difference was statistically significant regarding Neu4. In accordance, CMA07 adenoma cell line expressed higher levels of sialidase protein expression when compared with the CMT-U27 carcinoma cell line. Finally, with few tumor subpopulation exceptions, Neu1 and Neu4 expression was also overall low in primary and metastatic CMT xenografts. Thus, overall loss of sialidases seems to be an important feature for CMT progression and invasion.
... These highly electronegative sugars influence protein tertiary conformation and, in their terminal location, are strategically positioned to influence intermolecular and cell-cell interactions through steric hindrance and/or electrostatic repulsion. The sialylation state of a specific glycoprotein is dynamically and coordinately regulated through the opposing catalytic activities of sialyltransferases (24,36) and neuraminidases/sialidases (NEU) (55)(56)(57). Sialyltransferases catalyze transfer of SA to specific glycans in specific linkages, whereas NEUs hydrolyze the glycosidic linkage between SAs and their underlying subterminal sugars. Four human NEUs have now been identified: NEU1, -2, -3, and -4 (55)(56)(57). ...
... Sialyltransferases catalyze transfer of SA to specific glycans in specific linkages, whereas NEUs hydrolyze the glycosidic linkage between SAs and their underlying subterminal sugars. Four human NEUs have now been identified: NEU1, -2, -3, and -4 (55)(56)(57). Changes in the sialylation state of a glycoprotein may mask or unmask cryptic binding sites, leading to functional consequences (36,55). ...
Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase 14. Intratracheal instillation of NEU1-encoding but not control adenovirus induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues, and elevations of pulmonary transforming growth factor β and collagen. The lymphocytes were predominantly T cells, with CD8+ cells exceeding CD4+ cells by nearly 2-fold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo consistent with mechanisms implicated in lung fibrosis.
... In contrast, NEUs counterregulate sialylation through hydrolysis of the linkage between terminal SAs and their subterminal sugars (28 -30). Although the numerous prokaryote and eukaryote members of the NEU superfamily share little amino acid (aa) sequence homology, they contain conserved motifs, including the Asp box (an aa sequence composed of SXDXGXTW) and the (F/Y)RIP motif (28). More recently, bioinformatic analysis has revealed high conservation of 6 residues essential for catalytic activity, including an Arg triad (Arg-21, Arg-237, and Arg-304), a Tyr/Glu nucleophile pair (Tyr-334 and Glu-218), and an Asp that functions as an acid/base catalyst (Asp-46) (31). ...
... NEU1 is a ϳ45.5-kDa (415-aa) protein that contains three conserved and two degenerate Asp boxes and one (F/Y)RIP motif (28). In human tissues, it is the most widely expressed and most abundant NEU (29). ...
The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, AS et al; J Biol Chem 287:15966-15980, 2012). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular EC (HPMEC)s, prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing MOIs of an adenovirus (Ad) encoding for catalytically-active wild-type (WT) NEU1 dose-dependently impaired tube formation whereas overexpression of either a catalytically-dead NEU1 mutant, NEU1-G68V, or another sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin (SNA) lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin (PNA) lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase (ST) 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counter-acted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically-active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.
... There are four known human sialidases with various cellular locations and substrate specificities; 5 thus, activation of these specific enzymes can cause remodeling of glycoproteins in the cellular environment. [6][7][8] Regulation of protein-ligand interactions by glycans, and specifically by terminal sialic acids, has been noted in a number of glycoproteins. For example, CD44 can be an E-Selectin ligand when sialylated and fucosylated to display the sialyl-Lewis X tetrasaccharide on the non-reducing end of the glycan. ...
Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein-ligand binding involved in cell-cell and cell-matrix interactions. CD44 is a single-pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N-terminal extracellular hyaluronan-binding domain (HABD); specifically, sialic acid capped N-glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N-glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously-formed charge-paired hydrogen bonding interactions between the negatively-charged sialic acid residues and positively-charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
... [19][20][21][22][23] Sialidases (EC 3.2.1.18) catalytically remove sialic acid 24 and, among many crucial cell events, in the nervous system they are involved in neuronal differentiation, neuritogenesis, and axonal growth. [25][26][27] Among the four sialidases described so far, that is, NEU1, NEU2, NEU3, and NEU4, the latter appeared to be intriguingly related to the fate of neural cells. ...
The human sialidase, NEU4, has emerged as a possible regulator of neuronal differentiation and its overexpression has been demonstrated to promote the acquisition of a stem cell-like phenotype in neuroblastoma cells. In this paper, we demonstrated that glioblastoma stem cells (GSCs) isolated from glioblastoma multiforme (GBM) cell lines and patients' specimens as neurospheres are specifically marked by the upregulation of NEU4; in contrast, the expression of NEU4 is very low in non-neurosphere-differentiated GBM cells. We showed that NEU4 silencing by miRNA or a chemical inhibitor of its catalytic activity triggered key events in GSCs, including (a) the activation of the glycogen synthase kinase 3β, with the consequent inhibition of Sonic Hedgehog and Wnt/β-catenin signalling pathways; (b) the decrease of the stem cell-like gene expression and marker signatures, evidenced by the reduction of NANOG, OCT-4, SOX-2, CD133 expression, ganglioside GD3 synthesis, and an altered protein glycosylation profile; and (c) a significant decrease in GSCs survival. Consistent with this finding, increased NEU4 activity and expression induced in the more differentiated GBM cells by the NEU4 agonist thymoquinone increased the expression of OCT-4 and GLI-1. Thus, NEU4 expression and activity appeared to help to determine the molecular signature of GSCs and to be closely connected with their survival properties. Given the pivotal role played by GSCs in GBM lethality, our results strongly suggest that NEU4 inhibition could significantly improve current therapies against this tumour.
... It has been reported that in mice, sialidase deficiency impairs the activation of macrophages and T lymphocytes. In fact, in a NEU1 deficiency mouse model, no cell surface expression of sialidase was observed in T cells, indicating that the lysosomal and the membrane-associated forms of Neu1 are the product of the same gene (222). Certainly, further elucidation of the kinetics of this interaction would lend mechanistic insight to the regulation of TLR activation. ...
... Individual isoenzymes have been found to vary in both subcellular and tissue localization. [1][2][3][4] NEU1 is part of a membrane complex, and is found primarily in the lysosome and plasma membrane. 5,6 Murine NEU1 has four sites of N-link glycosylation, several of which are critical for enzyme function. ...
The human neuraminidase enzymes (hNEU) are a family of four isoenzymes that hydrolyze sialosides, including gangliosides and glycoproteins. These enzymes are proposed to play roles in several important signaling pathways and are implicated in diseases such as diabetes and cancer. Despite their importance, only a limited number of studies have sought to identify potent inhibitors for these enzymes. This review summarizes the substrate specificity and known inhibitors of the hNEU isoenzymes, as well as the emerging work on development of isoenzyme-specific inhibitors.
... 117 Galectin-3 also regulates p21 stability in human prostate cancer cells 68 and ablation of galectin-3-induced p27KIP1dependent premature senescence without oncogenic stress. 118 In another model, immortalized corneal epithelial cells, galectin-3 activated the focal adhesion kinase (FAK), a key regulator of integrin-dependent cell signaling, and a member of Rho GTPases, Rac1 GTPase, which is known to play an important role in reorganizing the actin skeleton and the formation of lamellipodial extensions. The role of galectin-3 in promoting lamellipodia formation in this model was dependent on the N-glycosylation of the α3β1-integrin. ...
Galectins are a family of proteins that contain a canonical carbohydrate-recognition domain (CRD) with affinity for beta-galactosides. Within this family, an unique member, the chimeric, galectin-3, may be found in the cytoplasm and nucleus, and on the cell surface, besides being released into the extracellular space. Galectin-3 interactions with certain glycans and extracellular matrix (ECM) proteins have been described to promote and/or antagonize tumor cell apoptosis, to induce endothelial cell proliferation and angiogenesis, and to promote tumor cell adhesion and invasion, thus both potentially facilitating and hindering metastasis. Moreover, although galectin-3 is expressed in several types of malignancies and its expression has been correlated with transformation and metastasis-related events, its downregulation has also been associated with malignancy and tumor progression. These apparently conflicting data demonstrate that the role of galectin-3 in metastasis remains to be fully understood. Of course in nature, different cancer progression phenomena are simultaneously occurring in the many instances, where the patient has primary tumor and blood-borne and distant metastatic cells. This makes it all the more interesting to overview the role of galectins in cancer metastasis, especially galectin-3, since these and their related molecules are more than probable disease marker candidates and/or therapeutic targets.
... Sialylated N-glycans on the epithelial cells lining the airways are targets for HA-mediated viral adhesion, and promote the subsequent clathrin-dependent or independent internalization of the virus (Lakadamyali et al., 2004;de Vries et al., 2011). The abundant sialylation of these glycans is dynamically regulated through the complementing activities of endogenous sialyltransferases (Harduin-Lepers et al., 2001) and sialidases (Monti et al., 2002;Schwerdtfeger and Melzig, 2010). The viral NA cleaves the terminal sialic acid residues from both the newly synthesized virion glycoproteins as well as those from the host cell surface, enabling the cell-surface aggregated virion progeny to elute away from the host cell and spread the infection (von Itzstein, 2007). ...
The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence motif, with critical regulatory roles in development and immune homeostasis. In this study, we examined the possibility that galectins expressed in the airway epithelial cells might play a significant role in viral or pneumococcal adhesion to airway epithelial cells. Our results in a mouse model for influenza and pneumococcal infection revealed that the murine lung expresses a diverse galectin repertoire, from which selected galectins, including galectin 1 (Gal1) and galectin 3 (Gal3), are released to the bronchoalveolar space. Further, the results showed that influenza and subsequent S. pneumoniae infections significantly alter the glycosylation patterns of the airway epithelial surface and modulate galectin expression. In vitro studies on the human airway epithelial cell line A549 were consistent with the observations made in the mouse model, and further revealed that both Gal1 and Gal3 bind strongly to IAV and S. pneumoniae, and that exposure of the cells to viral neuraminidase or influenza infection increased galectin-mediated S. pneumoniae adhesion to the cell surface. Our results suggest that upon influenza infection, pneumococcal adhesion to the airway epithelial surface is enhanced by an interplay among the host galectins and viral and pneumococcal neuraminidases. The observed enhancement of pneumococcal adhesion may be a contributing factor to the observed hypersusceptibility to pneumonia of influenza patients.
... Among the glycosylation processes, sialylation is crucial for a variety of cellular functions such as cell adhesion signal recognition, and biological stability of glycoproteins. Sialylation of glycoproteins is regulated by two opposing enzymatic activities: sialyltransferases and sialidases [16,17]. It is interesting to mention that NEU1, a well-known lysosome sialidase, has been proposed to regulate EGFR and MUC1 signalling (ref Lillehoj et al). ...
Triple-negative breast carcinoma (TN) is a heterogeneous cancer type expressing EGFR in 75% of cases. MUC1 is a large type I sialylated glycoprotein comprising two subunits (α and β chains, also called respectively MUC1-VNTR and MUC1-CT), which was found to regulate EGFR activity through endocytic internalisation. Endocytosis and autophagy use the lysosome pathway involving NEU1. Recently, a molecular EGFR-MUC1-NEU1 complex was suggested to play a role in EGFR pathway. In the aim to understand the relationship between EGFR-MUC1-NEU1 complex and autophagy in breast carcinoma, we compared triple negative (TN) showing a high-EGFR expression with luminal (LUM) presenting low-EGFR level. We studied the expression of MUC1-VNTR, MUC1-CT and NEU1 in comparison with those of two molecular actors of autophagy, PI3K (p110β) and Beclin1. A total of 87 breast cancers were split in two groups following the immunohistochemical classification of breast carcinoma: 48 TN and 39 LUM. Our results showed that TN presented a high expression of EGFR and a low expression of MUC1-VNTR, MUC1-CT, NEU1, Beclin-1 and PI3Kp110β. Moreover, in TN, a positive statistical correlation was observed between Beclin-1 or PI3Kp110β and MUC1-VNTR or NEU1, but not with EGFR. In conclusion, our data suggest that autophagy is reduced in TN leading likely to the deregulation of EGFR-MUC1-NEU1 complex and its associated cellular pathways.
... The extent of glycoprotein sialylation can be negatively regulated by sialidases, which catalyze the hydrolysis of terminal sialic acid residues (Monti et al., 2002). At least four mammalian sialidase homologs have been described in the human genome (Neu1, Neu2, Neu3, Neu4). ...
Unlabelled:
The UT-A1 urea transporter is crucial to the kidney's ability to generate concentrated urine. Native UT-A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycosylation forms of 97 and 117 kDa. In diabetes, UT-A1 protein abundance, particularly the 117 kD isoform, is significantly increased corresponding to an increased urea permeability in perfused IM collecting ducts, which plays an important role in preventing the osmotic diuresis caused by glucosuria. However, how the glycan carbohydrate structure change and the glycan related enzymes regulate kidney urea transport activity, particularly under diabetic condition, is largely unknown. In this study, using sugar-specific binding lectins, we found that the carbohydrate structure of UT-A1 is changed with increased amounts of sialic acid, fucose, and increased glycan branching under diabetic conditions. These changes were accompanied by altered UT-A1 association with the galectin proteins, β-galactoside glycan binding proteins. To explore the molecular basis of the alterations of glycan structures, the highly sensitive next generation sequencing (NGS) technology, Illumina RNA-seq, was employed to analyze genes involved in the process of UT-A1 glycosylation using streptozotocin (STZ)-induced diabetic rat kidney. Differential gene expression analysis combining with quantitative PCR revealed that expression of a number of important glycosylation related genes were changed under diabetic conditions. These genes include the glycosyltransferase genes Mgat4a, the sialylation enzymes St3gal1 and St3gal4 and glycan binding protein galectin-3, -5, -8, and -9. In contrast, although highly expressed in kidney IM, the glycosyltransferase genes Mgat1, Mgat2, and fucosyltransferase Fut8, did not show any changes.
Conclusions:
In diabetes, not only is UT-A1 protein abundance increased but the protein's glycan structure is also significantly changed. UT-A1 protein becomes highly sialylated, fucosylated and branched. Consistently, a number of crucial glycosylation related genes are changed under diabetic conditions. The alteration of these genes may contribute to changes in the UT-A1 glycan structure and therefore modulate kidney urea transport activity and alleviate osmotic diuresis caused by glucosuria in diabetes.
... b0095 b1065 b1070 b1075 b1080 b108519,[213][214][215][216][217] In microorganisms, sialidase is likely to function for nutritional purposes and in the processes of adhesion and invasion to host cells with pathogens. ...
... This protein sequence contains the sialidase domain. Sialidases (neuraminidases) hydrolyse the nonreducing, terminal sialic acid linkage in various natural substrates such as glycoproteins, glycolipids, gangliosides and polysacharides [33]. In viruses, sialidases enable the transport of the virus through mucin, the release of the virus from the infected host cell and prevent self-aggregation of virus particles [34]. ...
A lytic bacteriophage 812 ranks among promising candidates for phage therapy, which emerges as an alternative to antibiotics for treatment of staphylococcal infections. Proteome of bacteriophage 812 was analyzed using various mass spectrometry techniques. Eleven proteins were identified, of which seven for the first time. Gel electrophoresis with peptide mass fingerprinting was found the most efficient for overall analysis; HPLC-ESI MS was used for confirmation in dubious cases. Seemingly abnormal migration of main tail sheath protein (812_mtsp) was explained after adjustment of gel electrophoresis conditions and peptide mass fingerprinting combined with MALDI TOF/TOF MS results. Three similarly-sized 812_mtsp fragments, which originally migrated in one band, were separated and identified. Proteome characterization of bacteriophage 812 is a prerequisite for future comparison of the standard-type phage 812 with its mutants and related phages to correlate proteome changes with their different host range.
... The mechanism of how PSA is cleaved from a neuron is unclear. We examined the role of the intrinsic neuraminidase/ sialidases, which initiate the catabolism of sialo-glycoconjugates by removing their terminal sialic acid residues [21,22], in PSA degradation. We cultured the slices in the presence of NeuAc2en, a neuraminidase inhibitor. ...
Hippocampal granule cells (GCs) are generated throughout the lifetime and are properly incorporated into the innermost region of the granule cell layer (GCL). Hypotheses for the well-regulated lamination of newly generated GCs suggest that polysialic acid (PSA) is present on the GC surface to modulate GC-to-GC interactions, regulating the process of GC migration; however, direct evidence of this involvement is lacking. We show that PSA facilitates the migration of newly generated GCs and that the activity of N-acetyl-α-neuraminidase 1 (NEU1, sialidase 1) cleaves PSA from immature GCs, terminating their migration in the innermost GCL. Developing a migration assay of immature GCs in vitro, we found that the pharmacological depletion of PSA prevents the migration of GCs, whereas the inhibition of PSA degradation with a neuraminidase inhibitor accelerates this migration. We found that NEU1 is highly expressed in immature GCs. The knockdown of NEU1 in newly generated GCs in vivo increased PSA presence on these cells, and attenuated the proper termination of GC migration in the innermost GCL. In conclusion, this study identifies a novel mechanism that underlies the proper lamination of newly generated GCs through the modulation of PSA presence by neuronal NEU1.
... Although NEU3 sialidase has been often referred to as the "plasma-membrane associated" member of the sialidase family (31), the effects of NEU3 activity on key cellular processes, including cancerogenesis and cell differentiation, are not limited to its ability of modulating sialic acids content, but they can be also the result of a direct interaction of the enzyme with signalling molecules, such as caveolin-1, Rac-1, integrin β4, Grb-2 and EGFR (11,(19)(20)(21)(22). Moreover, although NEU3 is mainly localized on the plasma membrane, it was reported that the enzyme is also present in the endosomal compartment (2). ...
NEU3 sialidase has been shown to be a key player in many physio- and patho-logical processes, including cell differentiation,
cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma
membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating
cell-to-cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the
plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under
opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity.
Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in
each cell compartment. They were identified by mass spectrometry and some selected ones also confirmed by cross-immunoprecipitation
with the enzyme, supporting NEU3 involvement in the cell-stress response, protein folding, and intracellular trafficking.
... In particular, this toxin in enterococci has been characterized as a heat-labile, oxygen-stable molecule (35), suggesting that it is available to induce damage in vivo even after the atmosphere switches. Regarding mucin, it is a heavily glycosylated cysteine-rich protein that covers the epithelium of the gastrointestinal tract and is a potential natural source of amino acids and sugars for enterococci (36)(37)(38)(39). ...
Negligible in vivo growth of enterococci and high-level dispersion of data have led to inaccurate estimations of antibiotic pharmacodynamics
(PD). Here we improved an in vivo model apt for PD studies by optimizing the in vitro culture conditions for enterococci. The PD of vancomycin (VAN), ampicillin-sulbactam (SAM), and piperacillin-tazobactam (TZP)
against enterococci were determined in vivo, comparing the following different conditions of inoculum preparation: aerobiosis, aerobiosis plus mucin, and anaerobiosis
plus mucin. Drug exposure was expressed as the ratio of the area under the concentration-time curve for the free, unbound
fraction of the drug to the MIC (fAUC/MIC) (VAN) or the time in a 24-h period that the drug concentration for the free, unbound fraction exceeded the MIC under
steady-state pharmacokinetic conditions (fT>MIC) (SAM and TZP) and linked to the change in log10 CFU/thigh. Only anaerobiosis plus mucin enhanced the in vivo growth, yielding significant PD parameters with all antibiotics. In conclusion, robust in vivo growth of enterococci was crucial for better determining the PD of tested antibacterial agents, and this was achieved by
optimizing the procedure for preparing the inoculum.
... The sialidase Neu3 is one of the first enzymes involved in the ganglioside catabolism found to be preferentially at the cell surface level. Neu3 can be considered an ubiquitous enzyme being expressed at different levels in the plasma membrane of several normal and pathological human tissues, such as brain [43], normal and colon rectal carcinoma tissue, hepatic tumor and kidney carcinoma [44][45][46][47][48]. In addition, its expression and activity were also found in normal and pathological cell lines such as erythroid and erytroleukemic cells [49][50][51], fibroblasts [52], neurons, neuroblastoma cells [53], breast ductal cancer T47D cells, colon carcinoma CaCo2 cells, colorectal adenocarcinoma HT29 cells, different types of ovarian cancer cells and cervix adenocarcinoma HeLa cells [48]. ...
Glycosphingolipids are a large group of complex lipids particularly abundant in the outer layer of the neuronal plasma membranes. Qualitative and quantitative changes in glycosphingolipids have been reported along neuronal differentiation and aging. Their half-life is short in the nervous system and their membrane composition and content are the result of a complex network of metabolic pathways involving both the de novo synthesis in the Golgi apparatus and the lysosomal catabolism. In particular, most of the enzymes of glycosphingolipid biosynthesis and catabolism have been found also at the plasma membrane level. Their action could be responsible for the fine tuning of the plasma membrane glycosphingolipid composition allowing the formation of highly specialized membrane areas, such as the synapses and the axonal growth cones. While the correlation between the changes of GSL pattern and the modulation of the expression/activity of different glycosyltransferases during the neuronal differentiation has been widely discussed, the role of the glycohydrolytic enzymes in this process is still little explored. For this reason, in the present review, we focus on the main glycolipid catabolic enzymes β-hexosaminidases, sialidases, β-galactosidases, and β-glucocerebrosidases in the process of the neuronal differentiation.
... Since that report, four sialidases with distinct cellular localizations and likely different substrate preferences and cellular functions have been identified in humans and mice (8)(9)(10)(11). The most abundant, lysosomal sialidase (NEU1), associates with other proteins to form a multienzyme complex (9,12). Membrane-associated sialidase (NEU3) is a protein that preferentially desialylates gangliosides (13,14) and perhaps selected surface glycoproteins (15). ...
Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA “superfamily” has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN) sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s), presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo. We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously described microbial NA superfamily extends to human sialidases. Strategies designed to therapeutically inhibit microbial NA may need to consider potential compromising effects on human sialidases, particularly those expressed in cells of the immune system.
... Sialidase (Enzyme entry: EC 3.2.1.18), also called neuraminidase (NA), belongs to the family of exo-glycosidases, and hydrolyzes terminal sialic acid from cell surface glycoproteins [5,6]. Viruses, microorganisms, and vertebrate synthesize NA. ...
Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 μM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.
... The levels and linkages of Sias, known as sialylation status, vary upon cell activation related to both physiological and pathological processes [4]. Despite importance of sialylation of cell surface to regulate various biological processes, the hydrolytic removal of Sias (desialylation) catalyzed by sialidases from glycoproteins or glycolipids is also important in regulating various physiological and pathological processes [5][6][7][8][9]. Desialylation of glycoconjugates influences cell signaling, adhesion, and apoptosis, receptor activation for adhesion, phagocytosis, cell migration, regulation of cell transformation, differentiation and migration, neuritogenesis, carcinogenesis, and also in insulin signaling [6,7]. ...
Sialidases or neuraminidases play important roles in various physiological and pathological processes by cleaving terminal sialic acids (Sias) (desialylation) from the glycans of both glycoproteins and glycolipids. To understand the biological significance of desialylation by sialidases, it is important to investigate enzyme specificity with native substrate in biological membrane of cells. Herein, we report a membrane-mimicking system with liposome ganglioside conjugates containing different lipids for evaluating substrate specificity of sialidase and the lipid effect on the enzyme activity. Briefly, liposomes of phosphatidylcholine (PC) and cholesterol with ganglioside (GM3 or GM1) along with different percentage of phosphatidylserine (PS) or phosphatidylethanolamine (PE) were prepared and characterized. Their desialylation profiles with Arthrobacter ureafaciens (bacterial) sialidase and H1N1 (influenza viral) sialidase were quantified by HPLC method. A diversity of substrate preference was found for both bacterial and viral sialidase to the liposome ganglioside conjugate platform. The apparent Km and Vmax were dependent on the type of lipid. These results indicate that the intrinsic characteristics of the membrane-like system affect the sialidase specificity and activity. This biomimetic substrate provides a better tool for unravelling the substrate specificity and the biological function of sialidases and for screening of functional sialidase inhibitors as well.
... Although four sialidases (Neu1, Neu2, Neu3 and Neu4) have been found in human, mouse, and rat [41], the gene encoding Neu4 could not be amplified from CHO cells in this study. This may echo an earlier report, in which the authors believe that Neu4 is a pseudogene in CHO cells [40]. ...
... Sialidases are not only expressed in humans but also in bacteria, Viruses, as well as fungi species and are involved in the modulation of molecules linked to biological processes [12,13]. Sialic acid is synthesized de novo or obtained exogenously. ...
Humans face a constant threat from pathogens like influenza varieties H1N1, H5N1, and others and there is a need to prevent these from epidemics. The pathogens depend on successful colonization of the host in order to reproduce and multiply. Sialidases are known as neuraminidases are a group of enzymes, the most abundant of these being the exo-sialidases that can catalyze the cleavage of sialic acids from carbohydrates, glycoproteins or glycolipids. Sialidases have been thoroughly studied since their discovery 75 years ago and their occurrence in bacteria and viruses is widespread. They are found in diverse virus families and bacteria and other microbes. Moreover, sialic acids serve as a receptor for various pathogens. This allows bacteria like H1N1 or other influenza viruses, to enter the host cell. There is a need to block sialidases as they release sialic acid that serves as nutrition for the microbes and as well allows them to bind and invade the host cell where they can proliferate. This makes sialidases an interesting target to control pathogenic activity. Metadichol ® is nanoemulsion of long-chain lipid alcohols derived from food ingredients. In rats, it has an LD50 of 5000 mg/kilo and its ingredients are present in many foods we consume on a daily basis. It has antiviral and antibacterial and anti-parasitic properties. We studied inhibition of Sialidases by inducing it with Lipopolysaccharide (LPS) using THP1 cells. Metadichol showed inhibition at 1 picogram per ml to 1 nanogram per/ml. Compared to Prednisone. It is 100 times more active. Previous studies on Metadichol ® showed that it is toxic to cancer cells at higher concentrations. Since it is safer, it has the potential of being directly tested on humans without side effects and could have a potential role in mitigating the pathogens that a burden on the Public health system.
... Among the three a(2,3)-sialyltransferases (ST3s) that place terminal sialic acid modifications in a(2,3)-linkage of type 2 lactosamine units (3), ST3GalIII and ST3GalIV were expressed by both monocytes and B cells, whereas ST3GalVI transcripts were only observed in monocytes. The surprising finding that enzymes that direct synthesis of sialofucosylations of terminal type 2 lactosamines are present on B cells prompted us to evaluate the expression of key enzymes that can influence expression of sLe X by either competing for common acceptors [ST3GalI, a(2,6)-sialyltransferase [ST6]GalI, ST6GalNAcII] (38-40), are requisite to create core glycans carrying terminal sialyllactosaminyl glycans (C2GnT1, MGAT1) (38, 41), or are "trimming" glycosidases (Neu1, Neu 3, and Fuc1) (42,43) that can remove sialic acid or fucose modifications from sLe X (see Supplemental Fig. 2 for details). Additionally, we probed for expression of the nucleotide-fucose transporter (GDP-FucT1) that controls the Golgi availability of the GDP-fucose donor sugar indispensable for fucosylation (44). ...
Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. In this study, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry andWestern blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human PBMCs. Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sialyl Lewis X (sLeX) and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4⁺ and CD8⁺ T cells but no binding by B cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds, including P-selectin glycoprotein ligand-1, CD43, and CD44 (rendering the E-selectin ligands cutaneous lymphocyte Ag, CD43E, and hematopoietic cell E-selectin/L-selectin ligand, respectively), and B cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B cells, and, commensurately, cell surface a(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites.
... The pancreas is one of the first organs to undergo autolysis, with autolysis of this organ occurring within hours after death [61]. Supporting our hypothesis is the fact that the pancreas is highest in expression of Neu1, a neuraminidase that cleaves both α 2-3 and α 2-6 linked sialic acids [62]. Thus, the Philipsen reports of neuraminidase [60] exposing TF-Ag in normal pancreas and the Monti review [63] describing the high Neu1 expression in the pancreas support our hypothesis that TF-Ag could become exposed on these tissues. ...
The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by mi-cro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG 3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untar-getable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarci-noma Antibody and In Vitro Efficacy Analysis" (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124 I-JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.
... CHO cells contain four different sialidases (Neu1-4) distributed in the lysosome (Neu1 and Neu4), cytosol (Neu2), and plasma membrane (Neu3). [71] During glycoprotein biologic manufacture, lowering the activity of these enzymes is desirable, but not completely, due to their important roles in crucial biological functions. [72][73][74][75] The cytosolic sialidiase, Neu2 is released into the supernatant during cells lysis and preferentially removes α-2,3-linked sialic acids from the glycoprotein products. ...
For several decades, glycoprotein biologics have been successfully produced from Chinese hamster ovary (CHO) cells. The therapeutic efficacy and potency of glycoprotein biologics are often dictated by their post translational modifications, particularly glycosylation, which unlike protein synthesis, is a non-templated process. Consequently, both native and recombinant glycoprotein production generate heterogeneous mixtures containing variable amounts of different glycoforms. Stability, potency, plasma half-life, and immunogenicity of the glycoprotein biologic are directly influenced by the glycoforms. Recently, CHO cells have also been explored for production of therapeutic glycosaminoglycans (e.g. heparin), which presents similar challenges as producing glycoproteins biologics. Approaches to controlling heterogeneity in CHO cells and directing the biosynthetic process toward desired glycoforms are not well understood. A systems biology approach combining different technologies is needed for complete understanding of the molecular processes accounting for this variability and to open up new venues in cell line development. In this review, we describe several advances in genetic manipulation, modeling, and glycan and glycoprotein analysis that together will provide new strategies for glycoengineering of CHO cells with desired or enhanced glycosylation capabilities.
... and PSORT II (http://psort.hgc.jp/form2.html) software did not suggest the existence of α-helical transmembrane segments, as previously reported 16,27 . Thus, results from these analyses suggested that Neu3 is not a canonical α-helical transmembrane protein, and instead spans the lipid bilayer by a different mechanism. ...
Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, and plays crucial roles in numerous biological processes. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane proteins. By different experimental approaches, we demonstrated that its C-terminus is exposed to the cytosol while another portion of the protein is exposed to the extracellular space, suggesting that Neu3 possesses the features of a transmembrane protein. However, in silico analysis and homology modeling predicted that the sialidase does not contain any α-helical transmembrane segment and shares the same β-propeller fold typical of viral and bacterial sialidases. Additionally, we found that Neu3 is S-acylated. Since this post-translational modification is restricted to the cytosolic side of membranes, this finding strongly supports the idea that Neu3 may contain a cytosolic-exposed domain. Although it remains to be determined exactly how this sialidase crosses the lipid bilayer, this study provides new insights about membrane association and topology of Neu3.
... Treating platelets by administering neuraminidases in vitro or by direct injecting neuraminidases into animals result in evident platelet desialylation 9,42,43 . Vertebrate neuraminidases are a family of four enzymes that possess differential distribution and particular substrate preferences (Neu1-4) 44,45 . Among them, Neu1 is a lysosomal neuraminidase with narrow substrate specificity that hydrolyzes sialic acids from glycoproteins preferentially 25 . ...
In addition to antiplatelet autoantibodies, CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the increased platelet destruction in immune thrombocytopenia (ITP). Recent studies have highlighted that platelet desialylation leads to platelet clearance via hepatocyte asialoglycoprotein receptors (ASGPRs). Whether CD8+ T cells induce platelet desialylation in ITP remains unclear. Here, we investigated the cytotoxicity of CD8+ T cells towards platelets and platelet desialylation in ITP. We found that the desialylation of fresh platelets was significantly higher in ITP patients with positive cytotoxicity of CD8+ T cells than those without cytotoxicity and controls. In vitro, CD8+ T cells from ITP patients with positive cytotoxicity induced significant platelet desialylation, neuraminidase-1 expression on the platelet surface, and platelet phagocytosis by hepatocytes. To study platelet survival and clearance in vivo, CD61 knockout mice were immunized and their CD8+ splenocytes were used. Platelets co-cultured with these CD8+ splenocytes demonstrated decreased survival in the circulation and increased phagocytosis in the liver. Both neuraminidase inhibitor and ASGPRs competitor significantly improved platelet survival and abrogated platelet clearance caused by CD8+ splenocytes. These findings suggest that CD8+ T cells induce platelet desialylation and platelet clearance in the liver in ITP, which may be a novel mechanism of ITP.
Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g., GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal maltose-binding protein (MBP)-affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the galactose and glucose residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
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IntroductionPolysialic Acid in NaturePSA Biosynthesis and BiodegradationPharmacological Effects of PSAPSA Conjugation: Polysialylation for Therapeutic ApplicationsSummaryReferences
Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated “sialic acid.” The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.
The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.
Gangliosides are a large group of complex lipids found predominantly on the outer layer of the plasma membranes of cells, and they are particularly concentrated in nerve endings. Their half-life in the nervous system is short, and their membrane composition and content are strictly connected to their metabolism. Their neobiosynthesis starts in the endoplasmic reticulum and is completed in the Golgi; catabolism occurs primarily in the lysosomes. However, the final content of gangliosides in the plasma membrane is affected by other cellular processes.
In this chapter structural changes in the oligosaccharide chains of gangliosides induced by the activity of glycohydrolases and in some cases by glycosyltransferases that are associated with plasma membranes are discussed. Some of the plasma membrane enzymes arise from fusion processes between intracellular fractions and the plasma membrane; however, other plasma membrane enzymes display a structure different from that of the intracellular enzymes. Several of these plasma membrane enzymes have been characterized and some of them seem to have a specific role in the nervous system.
Sialidase transition state analog inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en, DANA) has played a leading role in developing clinically used anti-influenza virus drugs. Taking advantage of the Neu5Ac2en-forming catalytic property of Streptococcus pneumoniae sialidase SpNanC, an effective one-pot multienzyme (OPME) strategy has been developed to directly access Neu5Ac2en and its C-5, C-9, and C-7-analogs from N-acetylmannosamine (ManNAc) and analogs. The obtained Neu5Ac2en analogs can be further derivatized at various positions to generate a larger inhibitor library. Inhibition studies demonstrated improved selectivity of several C-5- or C-9-modified Neu5Ac2en derivatives against several bacterial sialidases. The study provides an efficient enzymatic method to access sialidase inhibitors with improved selectivity.
Insulin receptor (IR) signaling plays a key role in the regulation of glucose homeostasis. A dysfunctional and/or unregulated IR activation has been shown to cause a range of clinical manifestations including insulin resistance, type 2 diabetes, obesity, cancer, hypertension, and cardiovascular disorders. The molecular mechanisms mediating IR activation have become an important area of scientific and clinical research. Here, we summarize the current understanding of IR structure, function, and signaling, and highlight the role of glycosylation and sialylation in IR activation. The key interactions that induce IR activation are identified and a novel IR-signaling platform is proposed. A mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B G-protein coupled receptor (GPCR) is uncovered which is essential for insulin-induced IR activation and cellular signaling. Evidence exposing the invisible link connecting insulin-binding to a proposed IR-signaling paradigm will be reviewed in relation to human disease.
When cells undergo oncogenic transformation, the sialylation of cell surface glycoconjugates is altered, which is thought to be associated with malignant phenotype. To elucidate the significance and the molecular mechanism of the alteration, we have been focusing on sialidase that catalyzes the removal of sialic acid residues from glycoproteins and glycolipids. In mammalian cells, four types of sialidases have been identified to date and were found to behave in different manners during carcinogenesis. A sialidase found in lysosomes is decreased in the activity and mRNA level in cancer cells, while a sialidase in plasma membrane is increased as compared with those in the control cells. The former sialidase affects anchor-age-independent growth and metastatic ability and introduction of the sialidase gene leads to reversion of these phenotypes. On the other hand, the latter brings about suppression of apoptosis in cancer cells and knocking down of this gene with short interfering RNA results in acceleration of apoptosis. In this review, we describe and summarize the alteration of sialidases and its possible significance in carcinogenesis.
Complexity and heterogeneity of oligosaccharides present a considerable challenge to the biopharmaceutical industry to manufacture biotherapeutics with reproducible and consistent glycoform profiles. Mammalian cells, especially Chinese hamster ovary cells, are the most widely used platform for the production of biotherapeutics. The glycans produced are predominantly of the complex type, with some differences between human and nonhuman mammalian glycosylation existing. This review briefly summarizes metabolic glyco-engineering strategies used in mammalian cells in order to alter the glycosylation patterns attached to proteins applied for diverse biotechnology applications.
After reports from Japan of neuropsychiatric adverse events (NPAEs) in children taking oseltamivir phosphate (hereafter referred to as oseltamivir [Tamiflu®; F. Hoffmann-La Roche Ltd, Basel, Switzerland]) during and after the 2004–5 influenza season, Roche explored possible reasons for the increase in reporting rate and presented regular updates to the US FDA and other regulatory authorities. This review summarizes the results of a comprehensive assessment of the company’s own preclinical and clinical studies, post-marketing spontaneous adverse event reporting, epidemiological investigations utilizing health claims and medical records databases and an extensive review of the literature, with the aim of answering the following questions: (i) what the types and rates of neuropsychiatric abnormalities reported in patients with influenza are, and whether these differ in patients who have received oseltamivir compared with those who have not; (ii) what levels of oseltamivir and its active metabolite, oseltamivir carboxylate are achieved in the CNS; (iii) whether oseltamivir and oseltamivircarboxylate have pharmacological activity in the CNS; and (iv) whether there are genetic differences between Japanese and Caucasian patients that result in different levels of oseltamivir and/or oseltamivir carboxylate in the CNS, differences in their metabolism or differences in their pharmacological activity in the CNS. In total, 3051 spontaneous reports of NPAEs were received by Roche, involving 2466 patients who received oseltamivir between 1999 and 15 September 2007; 2772 (90.9%) events originated from Japan, 190 (6.2%) from the US and 89 (2.9%) from other countries. During this period, oseltamivir was prescribed to around 48 million people worldwide. Crude NPAE reporting rates (per 1 000 000 prescriptions) in children (aged ≤16 years) and adults, respectively, were 99 and 28 events in Japan and 19 and 8 in the US. NPAEs were more commonly reported in children (2218 events in 1808 children aged >16 years vs 833 in 658 adults) and generally occurred within 48 hours of the onset of influenza illness and initiation of treatment. After categorizing the reported events according to International Classification of Diseases (9th edition) codes, abnormal behaviour (1160 events, 38.0%) and delusions/perceptual disturbances (661 events, 21.7%) were the largest categories of events, and delirium or delirium-like events (as defined by the American Psychiatric Association) were very common in most categories. No difference in NPAE reporting rates between oseltamivir and placebo was found in phase III treatment studies (0.5% vs 0.6%). Analyses of US healthcare claims databases showed the risk of NPAEs in oseltamivir-treated patients (n =159 386) was no higher than those not receiving antivirals (n = 159 386). Analysis of medical records in the UK General Practice Research Database showed that the adjusted relative risk of NPAEs in influenza patients was significantly higher (1.75-fold) than in the general population. Based on literature reports, NPAEs in Japanese and Taiwanese children with influenza have occurred before the initiation of oseltamivir treatment; events were also similar to those occurring after the initiation of oseltamivir therapy. No clinically relevant differences in plasma pharmacokinetics of oseltamivir and its active metabolite oseltamivir carboxylate were noted between Japanese and Caucasian adults or children. Penetration into the CNS of both oseltamivir and oseltamivir carboxylate was low in Japanese and Caucasian adults (cerebrospinal fluid/plasma maximum concentration and area under the plasma concentration-time curve ratios of approximately 0.03), and the capacity for converting oseltamivir to oseltamivir carboxylate in rat and human brains was low. In animal autoradiography and pharmacokinetic studies, brain: plasma radioactivity ratios were generally 20% or lower. Animal studies showed no specific CNS/behavioural effects after administration of doses corresponding to ≥100 times the clinical dose. Oseltamivir or oseltamivir carboxylate did not interact with human neuraminidases or with 155 known molecular targets in radioligand binding and functional assays. A review of the information published to date on functional variations of genes relevant to oseltamivir pharmacokinetics and pharmacodynamics and simulated gene knock-out scenarios did not identify any plausible genetic explanations for the observed NPAEs. The available data do not suggest that the incidence of NPAEs in influenza patients receiving oseltamivir is higher than in those who do not, and no mechanism by which oseltamivir or oseltamivir carboxylate could cause or worsen such events could be identified.
Central arterial blood pressure (BP) is more predictive of future cardiovascular events than is brachial BP because it reflects the BP load imposed on the left ventricle with greater accuracy. However, little is known about the effects of exercise training on central hemodynamic response to acute exercise. The purpose of the present study was to determine the influence of an aerobic exercise regimen on the response of aortic BP after a single aerobic exercise in postmenopausal women. Nine healthy postmenopausal women (age: 61 ± 2 years) participated in a 12-week aerobic exercise training regimen. Before and after the training, each subjects performed a single bout of cycling at ventilatory thresholds for 30 min. We evaluated the post-exercise aortic BP response, which was estimated via the general transfer function from applanation tonometry. After the initial pre-training aerobic exercise session, aortic BP did not change significantly: however, aortic pulse pressure and augmentation pressure were significantly attenuated after the single aerobic exercise session following the 12-week training regimen. The present study demonstrated that a regular aerobic exercise training regimen induced the post-exercise reduction of aortic pulse pressure and augmentation pressure. Regular aerobic exercise training may enhance post-exercise reduction in aortic BP.
Synchrotron radiation reflectometry was used to access the transverse structure of model membranes under the action of the human sialidase NEU2, down to the Ångström length scale. Model membranes were designed to mimic the lipid composition of so-called Glycosphingolipids Enriched Microdomains (GEMs), which are membrane platforms specifically enriched in cholesterol and sphingolipids, where also typical signalling molecules are hosted. Gangliosides, glycosphingolipids containing one or more sialic acid residues, are asymmetrically embedded in GEMs, in the outer membrane leaflet. There, gangliosides are claimed to directly interact with growth-factor receptors, modulating their activation and then the downstream intracellular signalling pathways. Thus, membrane dynamics and signalling could be strongly influenced by the activity of enzymes regulating the membrane ganglioside composition, including sialidases. Our results, concerning the structure of single membranes undergoing in-situ enzymatic digestion, show that the outcome of the sialidase action is not limited to the emergence of lower-sialylated ganglioside species. In fact, membrane reshaping occurs, involving a novel arrangement of the headgroups on its surface. Thus, sialidase activity reveals to be a potential tool to dynamically control the structural properties of the membrane external leaflet of living cells, influencing both the morphology of the close environment and the extent of interaction among active molecules belonging to signalling platforms.
Carbohydrates are one of the major classes of organic compounds found in nature and living organisms, and the diverse roles of carbohydrates are crucial for most life forms. The transformation and degradation of glycans are effectively regulated by carbohydrate-modifying enzymes. This thesis describes the characterization of carbohydrate-modifying enzymes, glycosyltransferases and glycosidases, using electrospray mass spectrometry (ES-MS) to gain a better understanding of these enzymes.
The ES-MS binding assay was applied to quantify the affinities of the human blood group synthesizing glycosyltransferases (GTA and GTB) for their H-antigen substrate in the absence and presence of bound uridine 5′-diphosphate (UDP) and divalent metal cofactor Mn2+. The presence of UDP and Mn2+ in the binding site had a marked influence on the association constant (Ka), enthalpy (ΔHa) and entropy (ΔSa) for the association of H-antigen to GTs. Moreover, the interactions between GT and nucleotide-sugar donor were investigated. Our results revealed that Mn2+ enhances the affinities of donors by 20 ~ 100 times. However, donors undergo enzyme-catalyzed hydrolysis in the presence of Mn2+ resulting in monosaccharide and UDP.
The catalytic mechanism of GTA and GTB was also investigated using ES-MS. To trap the glycosyl-enzyme intermediates in their enzymatic reaction, GT mutants, in which the putative catalytic nucleophile Glu303 was replaced with Cys, were utilized. The formation of intermediates was observed by incubation of GT
mutants with donor substrates. Tandem MS analysis confirmed Cys303 as the site of glycosylation. Incubation of the purified intermediates with H-antigen resulted in the decrease of intermediates and the formation of the trisaccharide products. Our results suggest that the GT mutants could operate by a double displacement mechanism.
The rate of substrate cleavage by the human neuraminidase 3 (NEU3) was measured using ES-MS. The kinetic analysis using synthetic substrates revealed that NEU3 activity depended upon the hydrophobicity of the aglycone. In addition, the substrates with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were cleaved by NEU3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac.
Cells are surrounded with glycosynapses as ganglioside-based microdomain in outer leaflet of plasma membrane. Because GSLs are expressed in eukaryotic plasma membranes, defects of GSL catabolic metabolism are associated with inherited lysosomal storage diseases. Therefore, it is considered that GSLs regulate the membrane receptor-mediated phosphorylation. Glycosynapses interact with transmembrane receptor or signal transducer for cell growth, adhesion, and migration. “Cis” type interactions of gangliosides largely influence the phosphorylation activities of receptor-tyrosine kinases (RTK) associated with assembled molecules in the PM of the cells. The associated receptors are known to contain RTK and regulate cell adhesion, growth, differentiation, interaction, survival, and migration. For example, during influenza virus infection, the fibroblast growth factor receptor (FGFR) is regulated through its RTK [1, 2]. Influenza viral entry utilizes the cell surface levels of GM1 and GM3.
background: Invasive fungal infections are an important cause of morbidity and mortality among patients with hematologic malignancies undergoing intensive chemotherapy with or without autologous or allogeneic hematopoietic stem cell transplantation. In most patients, the diagnosis of invasive aspergillosis triggers prolonged antifungal treatment voriconazole, a fluorinated triazole compound. A long-term voriconazole, as a risk factor for the development of fluoride excess and subsequent painful periostitis and exostoses in
post transplant patients. In this work, we report a case for acute toxicity of voriconazole in a patient transplanted bone marrow who was receiving long-term therapy with this medication.
Methods: A patient (21 years) transplanted bone marrow following a medullary aplasia who complains periostitis and exostoses treated with voriconazole therapy for a pulmonary Aspergillus infection. Bone pain and radiographic evidence of periostitis were exclusively observed .There is no history of rheumatologic disease. To determine whether voriconazole is a cause of fluoride excess, we measured urinary fluoride
levels. Fluorides were analyzed in the biological fluids using potentiometric method based on ion-selective electrodes (ISE) (Jenway).Quantitative determination of fluoride was carried out in the linear concentration range between 6 and 1ppm. The detection limit of this sensor was observed at 4, this sensitivity is sufficient for concentrations which are toxic for humans.
Results: The patient has an elevated alkaline phosphatase of 400UI/l and elevated urine fluoride level (20 ppm). Discontinuation of voriconazole therapy in the patient resulted in an improvement in pain and a reduction in alkaline phosphatase and fluoride.
Conclusion: Voriconazole is associated with painful periostitis, exostoses, and fluoride excess in post-transplant patient with long-term voriconazole use for prophylaxis and treatment of Aspergillus infection.
Sialyltransferase (ST) and sialidase (SA) are the main enzymes responsible for the addition or removal of sialic acids to the terminal of glycan on glycoprotein or glycolipids. In our study, the expression of sialic acids, sialidases and sialyltransferases in normal bladder epithelial cell line HCV29, and two bladder cancer cell lines KK47 and YTS-1 were detected. Our results showed YTS-1 cells, the highly invasive cell line, expressed higher level of sialic acids than other two cell lines. However, the level of Neu1 in YTS-1 cells was the lowest among all the cell lines. The expression of Toll like receptors TLR1,2,3,4 was consistent with neul expression in these three cell lines. When normal bladder cell HCV29 underwent TGF-beta induced epithelial to mesenchymal transition (EMT) process, expression of Neu1 and TLR3 were significantly decreased. When Neu1 expression was inhibited in HCV29 cell line, the level of TLR3 was also reduced. Overexpression of Neu1 in YTS-1cells resulted in TLR3 increase accompanied with the activation of NF-kappa B signaling pathway. Our results indicated that Neu1 shared closed relationship with the expression of TLRs in bladder cancer cells, which may potentially provide therapeutic candidates in treating bladder cancer.
Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.
Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.
Sialidosis is an autosomal recessive disease caused by the genetic deficiency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots and myoclonus (sialidosis type I) or with skeletal dysplasia, Hurler-like phenotype, dysostosis multiplex, mental retardation and hepatosplenomegaly (sialidosis type II). We have analyzed the genomic DNA from nine sialidosis patients of multiple ethnic origin in order to find mutations responsible for the enzyme deficiency. The activity of the identified variants was studied by transgenic expression. One patient had a frameshift mutation (G623delG deletion), which introduced a stop codon, truncating 113 amino acids. All others had missense mutations: G679G→A (Gly227Arg), C893C→T (Ala298Val), G203G→T (Gly68Val), A544A→G (Ser182Gly) C808C→T (Leu270Phe) and G982G→A (Gly328Ser). We have modeled the three-dimensional structure of sialidase based on the atomic coordinates of the homologous bacterial sialidases, located the positions of mutations and estimated their potential effect. This analysis showed that five mutations are clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We hypothesize that this region may be involved in the interface of sialidase binding with lysosomal cathepsin A and/or &bgr;-galactosidase in their high-molecular-weight complex required for the expression of sialidase activity in the lysosome.
Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.
Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R-factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co-ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.
The influenza virus neuraminidase glycoprotein is a tetramer with a box-shaped head, 100 X 100 X 60 A, attached to a slender stalk. The three-dimensional structure of neuraminidase heads shows that each monomer is composed of six topologically identical beta-sheets arranged in a propeller formation. The tetrameric enzyme has circular 4-fold symmetry stabilized in part by metal ions bound on the symmetry axis. Sugar residues are attached to four of the five potential glycosylation sequences, and in one case contribute to the interaction between subunits in the tetramer.
Tay-Sachs and Sandhoff diseases are clinically similar neurodegenerative disorders. These two sphingolipidoses are characterized by a heritable absence of beta-hexosaminidase A resulting in defective GM2 ganglioside degradation. Through disruption of the Hexa and Hexb genes in embryonic stem cells, we have established mouse models corresponding to each disease. Unlike the two human disorders, the two mouse models show very different neurologic phenotypes. Although exhibiting biochemical and pathologic features of the disease, the Tay-Sachs model showed no neurological abnormalities. In contrast, the Sandhoff model was severely affected. The phenotypic difference between the two mouse models is the result of differences in the ganglioside degradation pathway between mice and humans.
The cDNA encoding GM2 activator was expressed in the Escherichia coli/pT7-7 system. The yield of the GM2 activator with greater than 99% purity was about 3 mg per liter culture. The recombinant GM2 activator was found to be as active as that isolated from human kidney. The availability of the recombinant GM2 activator enabled us to critically examine the specificity of this activator protein. Our results show that the specificity of GM2 activator is not as strict as that reported previously. Although GM2 activator stimulates most efficiently the degradation of GM2 carried out by beta-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) conversion of GM2 to GA2 by clostridial sialidase; (b) hydrolysis of GalNAc from dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 by Hex A; and (c) liberation of Gal from GM1 by beta-galactosidase at a high activator concentration. Thus, this activator does not differentiate between GM2 and dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 or between Hex A and clostridial sialidase. The micellar forms of GD2 and GalNAc-GD1a were found to be more readily hydrolyzed by Hex A than GM2 in the absence of GM2 activator. Our results also show that saposin B can enhance the stimulatory activity of GM2 activator, but it cannot promote the stimulatory activity of sodium taurodeoxycholate. Taken together, our results suggest that the mechanism of action of GM2 activator is different from saposin B, and the action of GM2 activator is more than to solubilize lipid substrates. The effectiveness of GM2 activator in stimulating the hydrolysis of GM2 may be due to its ability to recognize the specific trisaccharide structure of the GM2 epitope, GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.
Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases. The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution. The viral enzyme is a tetramer; the bacterial enzyme, a monomer. Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%). The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+. Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues. However, an rms fit of 3.1 A between 264 C alpha atoms of the S. typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold. In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes. The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure. Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed.
We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.
Gangliosides of the plasma membrane are important modulators of cellular functions. Previous work from our laboratory had suggested that a plasma membrane sialidase was involved in growth control and differentiation in cultured human neuroblastoma cells (SK-N-MC), but its substrates had remained obscure. We now performed sialidase specificity studies in subcellular fractions and found ganglioside GM3 desialylating activity in presence of Triton X-100 to be associated with the plasma membrane, but absent in lysosomes. This Triton-activated plasma membrane enzyme desialylated also gangliosides GDla, GD1b, and GT1b, thereby forming GM1; cleavage of GM1 and GM2, however, was not observed. Sialidase activity towards the glycoprotein fetuin with modified C-7 sialic acids and towards 4-methylumbelliferyl neuraminate was solely found in lysosomal, but not in plasma membrane fractions.
The role of the plasma membrane sialidase in ganglioside desialylation of living cells was examined by following the fate of [³H]galactose-labelled individual gangliosides in pulse-chase experiments in absence and presence of the extracellular sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. When the plasma membrane sialidase was inhibited, radioactivity of all gangliosides chased at the same rate. In the absence of inhibitor, GM3, GD1a, GD1b, GD2, GD3 and GT1b were degraded at a considerably faster rate in confluent cultures, whereas the GM1-pool seemed to be filled by the desialylation of higher gangliosides. The results thus suggest that the plasma membrane sialidase causes selective ganglioside desialylation, and that such surface glycolipid modification triggers growth control and differentiation in human neuroblastoma cells.
N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.
Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with beta-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.
Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28-29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.
IL-4 is important in controlling the development of immune responses. Following activation with anti-CD3epsilon under serum-free conditions, splenocytes from most normal (neu-1b) mouse strains directly produced IL-4 and other T cell cytokines. However, splenic T cells from SM/J and B10.SM (H-2v, neu-1a) strain mice, deficient in neu-1 sialidase activity, failed to produce IL-4 but produced normal levels of IL-2 following activation. Moreover, sialidase-deficient mice produced markedly less IgE and IgG1 Abs following immunization with protein Ags than did mouse strains with normal neu-1 sialidase activity. Enriched T cells from neu-1a mice failed to be effectively primed with exogenous murine IL-4 to become IL-4-producing cells. Treatment of splenocytes or enriched T cells from neu-1a mice with bacterial sialidase prior to activation or IL-4 priming promoted their subsequent capacity to produce IL-4. In contrast, activation of T cells from neu-1b mice in the presence of a sialidase inhibitor almost completely blocked subsequent IL-4 production. The presence of IL-4 during priming enhanced T cell expression of neu-1-specific sialidase activity and increased the membrane expression of asialo-G(M1) compared with T cells activated without IL-4. These results suggest that T cell-associated neu-1 sialidase is required for early IL-4 production by splenic T cells and is involved in the IL-4 priming process of conventional T cells to become active IL-4 producers.
Sialidases (EC 3.2.1.18; N-acylneuraminosyl glycohydrolase) are a family of exoglycosidases that catalyze the cleavage of nonreducing sialic acid residues ketosidically linked to mono-or oligosaccharide chains of glycoconjugates. They are widely distributed in viruses, bacteria, fungi, mycoplasma, and protozoa as well as avian and mammalian species (Rosenberg and Schengrund, 1976; Corfield et al., 1981a; Corfield and Schauer, 1982; Conzelmann and Sandhoff, 1987; Corfield, 1992). Among the various sialidase species, viral and bacterial enzymes have been studied extensively; a number of them have been purified to homogeneity and characterized for their properties and structures. Mammalian sialidases are more labile and often are bound tightly to membranes, hindering successful purification of these enzymes. Much attention, however, has been directed toward these enzymes as interest in the metabolism and biological function of sialoglycoconjugates in mammalian cells has grown in recent years (Schauer, 1982, 1985, 1991; Ledeen, 1989; Schengrund, 1990; Varki, 1992). The term “sialidase” was first proposed by Heimer and Meyer (1956), and “neuraminidase” was introduced a year later (Gottschalk, 1957). Both names have been used interchangeably in the literature. Since the enzyme does not usually apply to neuraminic acid itself, but to its derivatives, sialic acid, the term “sialidase” is deemed more appropriate (Rosenberg and Schengrund, 1976).
Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the β3A subunit of the heterotetrameric AP-3 complex. The patients’ fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant β3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 μ3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery.
In mammalian tissues, the pathway known for the catabolism of GM1 [Galβ3GalNAcβ4(Neu5Acα3)Galβ4GlcCer; where Cer is ceramide] is the conversion of this ganglioside into GM2 [GalNAcβ4(Neu5Acα3)Galβ4GlcβCer] by β-galactosidase followed by the conversion of GM2 into GM3 (Neu5Acα3Galβ4GlcβCer) by β-N-acetylhexosaminidase A (Hex A). However, the question of whether or not GM1 and GM2 can also be respectively converted into asialo-GM1 (Galβ3GalNAcβ4Galβ4GlcCer; GA1) and asialo-GM2 (GalNAcβ4Galβ4GlcβCer, GA2) by mammalian sialidases has not been resolved. This is due to the fact that sialidases purified from mammalian tissues always contained detergents that interfered with the in vitro hydrolysis of GM1 and GM2 in the presence of an activator protein. The mouse model of human type B Tay-Sachs disease created by the disruption of the Hexa gene showed no neurological abnormalities, with milder clinical symptoms than the human counterpart, and the accumulation of GM2 in the brains of affected mice was only limited to certain regions [Sango, Yamanaka, Hoffmann, Okuda, Grinberg, Westphal, McDonald, Crawley, Sandhoff, Suzuki and Proia (1995) Nat. Genet. 11, 170-176]. These results suggest the possible presence of an alternative catabolic pathway (the GA2 pathway) in mouse to convert GM2 into GA2 by sialidase. To show the existence of this pathway, we have used recombinant mammalian cytosolic sialidase and membrane-associated sialidase to study the desialylation of GM1 and GM2. We found that the mouse membrane-bound sialidase was able to convert GM1 and GM2 into their respective asialo-derivatives in the presence of human or mouse GM2 activator protein. The cytosolic sialidase did not exhibit this activity. Our results suggest that, in vivo, the stable NeuAc of GM1 and GM2 may be removed by the mammalian membrane-associated sialidase in the presence of GM2 activator protein. They also support the presence of the GA2 pathway for the catabolism of GM2 in mouse.
Cathepsin A [EC 3.4.16.1], so called protective protein, occurs as an enzyme complex with lysosomal β-galactosidase [3.2.1.23] and is involved in the stable enzymic expression of lysosomal sialidase [3.2.1.18]. In this study we investigated the enzymatic properties of cathepsin A in the bovine β-galactosidase complex and how it is involved in the molecular multiplicities of the β-galactosidase and sialidase complexes. Bovine protective protein homologous to the human protein had a molecular weight of 48 kDa on SDS-PAGE and cathepsin A activity optimum around pH 6.0. It hydrolyzed dipeptide substrates composed of hydrophobic amino acids much faster than any other type of substrate tested. This specificity was found to be conserved from human to a non-mammal, chicken.Immunoprecipitation using an anti β-galactosidase antibody demonstrated that cathepsin A is a component of both the sialidase and β-galactosidase complexes. The over 700 kDa sialidase complex depolymerized by a brief incubation at pH 7.5 and the sialidase was inactivated irreversibly via formation of an enzyme active smaller species of sialidase. The 669 kDa β-galactosidase complex dissociated reversibly into a 120 kDa β-galactosidase and a 170 kDa cathepsin A, but the 120 kDa β-galactosidase, free from the cathepsin A, formed a 260 kDa aggregate under the same conditions. Inactivation of cathepsin A by heat treatment did not affect its complex forming activity. The 170 kDa protective protein dissociated into a 50 kDa one at pH 7.5, which no longer formed the complex. These findings indicate that the 170 kDa protective protein could be the minimum unit required for in vitro reconstitution of the complex, and that its complex forming activity is carried in a heat-stable domain. Both β-galactosidase and cathepsin A activities were labile under the dissociated condition, indicating that it physiologically stabilizes not only β-galactosidase but also itself by forming the complex.
Our previous studies have shown that the enzymatic activities of Neu-1, an endogenous sialidase encoded in the murine MHC, are involved in promoting IL-4 synthesis by naive CD4+T cells. Our present studies have characterized responsible sialoconjugate targets of Neu-1 and questioned possible biochemical mechanisms responsible for their regulatory influences on IL-4 gene expression. These studies determined that treatment of T cells with the naturally occurring ganglioside GM3 inhibited the production of IL-4 without affecting the production of IL-2. An analysis of IL-4-primed CD4+T cells further demonstrated that GM3 treatment specifically inhibited the restimulated production of IL-4, IL-5 and IL-13, without inhibiting the production of IL-2 and IFN-γ. The inhibitory effects of GM3 could be overcome by treatment with thapsigargin or ionomycin, suggesting ganglioside regulation occurs upstream of activation-induced calcium mobilization. GM3 treatment attenuated the level of calcium influx following CD3ϵ crosslinking, and CD4+T cells from Neu-1-deficient B10.SM strain mice (neu-1aand IL-4-deficient) expressed reduced levels of intracellular calcium following activation. Our results indicate that activities by membrane gangliosides can influence the cytokine programs in CD4+T cells, possibly through the modulation of calcium responses induced by T cell activation.
To investigate the possibility that deletion en block in the HLA region had caused the combined deficiency of neuraminidase and 21-hydroxylase in a female patient, genetic markers on the short arm of chromosome 6 were examined in the patient and her parents, and 21-hydroxylase genes of the patient were analyzed by the Southern blot technique. The affected "extended haplotype" identical by descent might have been recombined at two sites, between HLA-A and C and between HLA-DQ and GLO. This suggests that the neuraminidase gene is mapped between HLA-A and GLO. Southern blot analysis revealed the existence of two 21-hydroxylase genes, so that we found no evidence to support the possibility that deletion en bloc in the HLA class III region had caused the combined deficiency of neuraminidase and 21-hydroxylase.
Three site-specific mutations were performed in two regions of a sialidase gene fromClostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed inEscherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, asK
M andV
max, as well asK
i measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.
The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.
The role of myelin-associated neuraminidase in ganglioside metabolism was examined using rats of ages ranging from 17 to 97 days. The neuraminidase activity directed toward the ganglioside GM3 in the total myelin fraction was high during the period of active myelination and, thereafter, decreased rapidly to the adult level. The ganglioside composition became simpler during development with an increasing amount of GM1 and decreasing percentages of di- and polysialogangliosides. The decrease in the proportion of GD1a was most prominent, whereas relative amounts of GD1b and GT1b increased transiently before reducing to the adult levels. The heavy myelin subfraction contained higher percentages of di- and polysialo-species compared to the light myelin fraction at young and adult ages. The in vitro incubation of myelin of young rats under an optimal condition for neuraminidase action produced a profile of ganglioside changes similar to that observed in in vivo development. These results strongly suggest that myelin-associated neuraminidase may play a pivotal role in the developmental changes in the ganglioside composition of rat brain myelin.
Treatment of three neuroblastoma cell types in culture with neuraminidase resulted in enhanced neurite outgrowth. These included the mouse Neuro-2A and rat B104 and B50 lines. The morphological changes depended on the presence of exogenous Ca2+ and were accompanied by modest but statistically significant increases in 45Ca2+ influx. Neuraminidase-stimulated neuritogenesis was blocked by the B subunit of cholera toxin (cholera B) and anti-GM1 antibody, a finding suggesting the effect was due to an increased amount of GM1 on the cell surface. Cholera B also blocked the increase in 45Ca2+ influx. The mouse N1A-103 line, previously characterized as "neurite minus," did not respond to neuraminidase with either neurite outgrowth or enhanced Ca2+ influx. These results point to an influence of GM1 on neuritogenesis in cells with differentiation potential and suggest a mechanism involving modulation of Ca2+ flux.
The genes of the bacterial sialidases fromClostridium sordellii G12,C. perfringens A99,Salmonella typhimurium LT-2 andVibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases.
Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein. All these sequences exhibit significant similarities. Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp. Additionally, most of the distances betweeen the four repeated regions are also conserved among the different sialidases. The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.
The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuraminidase-1 (NEU-1) is one of two neuraminidase isozymes which can be detected electrophoretically in mouse liver extracts. The inheritance of variation in NEU-1 and the linkage relationships of the gene controlling this variation were studied through a backcross analysis involving the SM/J and MA/MyJ inbred strains, and by examination of NEU-1 phenotypes in three congenic strains: B10.SM, B10.SM(22R) and B10.RVB. The data indicate that NEU-1 is controlled by Neu-1, a gene previously identified by its effect on total liver neuraminidase activity in whole tissue homogenates. Analysis of the congenic strains revealed identical low activity (SM/J-type: Neu-1a/Neu-1a) NEU-1 phenotypes in all three strains. This indicates that Neu-1 lies in the segment of the SM/J-derived H-2 region that is common to all three strains: H-2E alpha to H-2D. In addition, we examined the relationship between NEU-1 and phenotypic variation in liver acid phosphatase (AP; for which a new typing method is described) and linkage order among several other enzyme-coding genes linked to H-2. In all animals that could be scored confidently for AP, the NEU-1 and AP phenotypes were concordant, adding support to the hypothesis that both phenotypes are controlled by Neu-1. Recombination rates among six H-2-linked marker loci were unexpectedly low, but were sufficient to verify the position of Upg-1 as the telomeric flanking marker relative to Glo-1, H-2 (C4), Neu-1 (Apl), Ce-2 and Pgk-2.
The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well-preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+-K+ ATPase, 5′-nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone-insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5- and 0.7-fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane-bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds-fetuin. The Km values for CMP-NeuNAc were different using lactosylceramide and ds-fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds-fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds-fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane-bound sialyltransferase activity working on lactosylceramide as acceptor substrate.
The low activity of liver neuraminidase that is characteristic of mouse strain SM/J is inherited as a single gene on chromosome 17, near the major histocompatibility complex. This gene, neuraminidase-1 (Neu-1), is represented by the low activity allele Neu-1s in SM/J and the high activity allele Neu-1b in C57BL/6J and most other strains. Previously described variations in the posttranslational processing of acid phosphatase, alpha-mannosidase, arylsulfatase-B, and alpha-glucosidase are attributed to pleiotropic effects of this gene.
The outer surface of mouse B lymphocytes carries constitutive and inducible beta-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher beta-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non-Neu-1) and inducible (Neu-1) sialidase isozymes. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.
Sialidase activity in synaptic plasma membranes (SPM) isolated from C57BL/6 mouse brain was examined using exogenous ganglioside substrates. The enzyme activity directed toward GM3 showed sharp pH dependency with optimal pH of 4.0, and was greatly enhanced by Triton CF-54, Nonidet P-40 or CHAPS. The apparent Km and Vmax values for enzyme activity in SPM were 11 microM and 164 pmol/mg protein/min, respectively. Examination of sialidase activities in subcellular fractions of brain tissues showed the enrichment of enzyme activity in SPM prepared from either young adult or senescent mice. Substrate specificity of SPM sialidase was compared with that of myelin sialidase using delipidated, solubilized enzyme preparations. The SPM sialidase hydrolyzed GD1a more effectively as compared with the myelin enzyme. While SPM sialidase could hydrolyze GM1, the hydrolytic rate by the SPM enzyme was significantly lower than that by the myelin enzyme. The sialidase activity in SPM decreased with increasing age; activity was highest between the ages of 4-7 months, decreased to a relatively constant level between 13-25 months, and reached its lowest level at 31 months. These results demonstrate that SPM contain a distinct sialidase activity which is regulated in an age-dependent manner.
Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin.
We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role.
The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.
A cDNA encoding a soluble sialidase from Chinese hamster ovary (CHO) cells has been cloned and expressed. Completely degenerate oligonucleotide primers, which were based on the amino acid sequence of peptides obtained from the purified sialidase (Warner et al., Glycobiology, 3, 455-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide probe. The unique probe of 93 bp was used for screening a lambda gt 10 CHO cell cDNA library. A single clone, which contained a 1.4 kb insert, was isolated after screening 450,000 recombinants. The complete coding region of the protein, 1137 nucleotides, was contained in the isolated clone and it predicted a protein of 379 amino acids. The insert had a 186 bp 5' non-coding leader sequence and a 40 bp 3' non-coding region. No signal peptide was identified in the insert, suggesting a cytosolic localization for the protein. No significant primary sequence identities were observed when the deduced amino acid sequence of the CHO cell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment similarity with several bacterial sialidases. Two 'Asp box' motifs in the CHO cell sialidase had a remarkable alignment positioning in the protein sequence with the similar motifs of the Salmonella LT2 and Clostridium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa californica nuclear polyhedrosis virus harbouring the sialidase cDNA.
Neuraminidase, one of the two surface glycoproteins of influenza virus, cleaves terminal sialic acid residues from glycolipids or glycoproteins. Its crystal structure is known at high resolution, but the mechanism of glycosyl hydrolysis remains unclear.
We have determined the crystal structure at 1.8 A resolution of two complexes of influenza B/Beijing neuraminidase containing either the reaction product, sialic acid, or the transition state analogue inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). The sialic acid is bound in a distorted 'boat' conformation closely resembling that of bound DANA, stabilized by a conserved tyrosine residue (Tyr408). This distortion also gives rise to a suicidal side reaction that converts sialic acid to DANA at a low rate.
The mechanism of neuraminidase action is distinct from that of other known glycosyl hydrolases. Substrate distortion appears to be the driving force in glycosyl bond hydrolysis and the proton required for catalysis can probably be donated by water, rather than by residues in the active site, thus allowing the enzyme to operate at high pH. The side reaction converting sialic acid to DANA appears reasonably favourable, and it is unclear how this is minimized by the enzyme.
In cultured human neuroblastoma cells (SK-N-MC), a plasma membrane-bound besides a lysosomal ganglioside GM3 sialidase was detected. Both activities can be distinguished by the specific activation with detergents, as well as differential inhibition by Cu++. Plasma membrane and lysosomal sialidase specific activities showed strikingly different behaviour during the growth phase of neuroblastoma cells. Thus, the plasma membrane sialidase increased about 15-fold and mirrored cell growth, it differed from the kinetics of ornithine decarboxylase, an early marker of cell proliferation. The lysosomal sialidase, on the other hand, exhibited constant specific activities during growth of the cells, as did lysosomal and plasma membrane marker enzymes. When the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was included in the culture medium, a profound change in proliferation kinetics was observed, indicating a release from density-dependent control of cell division. Additionally, the inhibitor abolished the increase of the biochemical differentiation marker acetylcholinesterase. The results suggest an important role of the ganglioside sialidase of the plasma membrane in the processes of proliferation control and differentiation in this neuronal cell system.
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Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aK
m value of 2.56 · 10−4
m and an apparentV
max of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface.
Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs are also associated with intracellular organelles. We now report that the subcellular localization of GSLs varies depending on the GSL structure and cell type. GSL localization was determined by indirect immunofluorescence microscopy of fixed permeabilized cells. A single GSL exhibited variable subcellular localization in different cells. For example, antibody to GalCer is localized primarily to the plasma membrane of HaCaT II-3 keratinocytes, but to intracellular organelles in other epithelial cells. GalCer is localized to small vesicles and tubulovesicular structures in MDCK cells, and to the surface of phase-dense lipid droplets in HepG2 hepatoma cells. Furthermore, within a single cell type, individual GSLs were found to exhibit different patterns of subcellular localization. In HepG2 cells, LacCer was associated with small vesicles, which differed from the phase-dense vesicles stained by anti-GalCer, and Gb4Cer was associated with the intermediate filaments of the cytoskeleton. Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides, localized to mitochondria. The distinct subcellular localization patterns of GSLs raise interesting questions about their functions in different organelles. Together with published data on the enrichment of GSLs in specific organelles and in apical plasma membrane, these findings indicate the existence of specific sorting mechanisms that regulate the intracellular transport and localization of GSLs.
We have previously cloned a cDNA of a rat cytosolic sialidase which is strongly expressed in skeletal muscle. Both the highest enzyme activity, as well as the highest mRNA level, are present in this tissue. To understand the basis of the expression of this sialidase, we have cloned and sequenced the rat gene and its 5'-upstream region from a rat genomic library. The gene encoding the 1.8 kb skeletal muscle mRNA was found to span 3.4 kb of genomic DNA, and to consist of two introns and three exons. Exon 1 contains the 5' noncoding region, and exons 2 and 3 encode the regions containing the AUG initiation codon and two Asp-boxes, respectively. In the 5'-upstream sequence, there are a TATA box and two E-box pairs known as consensus binding sites for muscle-specific transcription factors. Analysis of the expression of transfected sialidase enhancer/promoter expression plasmid demonstrated the sialidase enhancer/promoter to be active in rat L6 myogenic cells shown to express this gene, but inactive in rat 3Y1 fibroblasts shown not to express the enzyme. The transcription activity was increased 3-fold after induction of myoblast differentiation by serum depletion. These observations give an account of constitutive expression of the sialidase gene in skeletal muscle.
We have previously observed that the cytosolic sialidase gene is highly expressed in rat skeletal muscle and that it contains an enhancer/promoter region which is transcriptionally active in rat L6 myogenic cells. Here we present evidence for the involvement of cytosolic sialidase in myoblast differentiation. During L6 myoblast differentiation induced by serum depletion, cytosolic sialidase is increased in activity as well as in terms of mRNA level. Sialidase activity is essentially lacking in untreated myoblasts but appears concomitantly with myotube formation after induction of differentiation. The mRNA becomes able to be detected after 3 days at the time which myogenin mRNA reaches a maximum level. Myotube formation can, in fact, be blocked by the addition of an antisense oligodeoxyribonucleotide complementary to the first 8 codons of the cytosolic sialidase.
The structure-based design of a potent inhibitor of the influenza-virus neuraminidase (sialidase) is one of the outstanding successes of rational drug design. Recent clinical trials of the drug have stimulated many companies to seek a share of the potentially huge flu market. Sialidases, however, are involved in the pathogenesis of a whole range of other diseases, so perhaps the knowledge and expertise gained from the influenza story can be used in the design of other drugs, given that they all share certain structural features.
Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.