ArticleLiterature Review

Recent Development in Mammalian Sialidase Molecular Biology

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

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.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... 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. ...
Article
Full-text available
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.
... 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]. ...
Article
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.
... 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. ...
Article
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.
... 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. ...
Article
Full-text available
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.
... Because of their terminal location and negative charge, sialic acid residues are strategically positioned to influence intermolecular and cellcell interactions through steric hindrance and/or electrostatic repulsion. The sialylation state of glycoproteins and glycolipids is dynamically and coordinately regulated through the opposing catalytic activities of sialyltransferases and sialidase/ neuraminidase (NEU) (3,4). NEUs hydrolyze the glycosidic linkage between terminal sialic acid and the adjacent subterminal sugar within glycan chains. ...
... A dense glycocalyx, which comprised numerous, terminally sialylated glycoproteins and glycolipids, covers the surface of all mammalian cells (1,2). Although these many sialylated molecules are regulated, in part, by sialidases, only 4 mammalian sialidases (NEU1-NEU4) have been identified (3)(4)(5). NEU1 reportedly associates with and desialylates the EDs of at least 14 integral membrane glycoproteins, including MUC1, EGFR, CD5, CD31, CD36, CD44, TLR4, TLR7, TLR9, integrins β2 and β4, insulin receptor, insulin-like growth factor receptor 1, and neurotrophin (11,33,44,(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57). In fact, a recent proteomic analysis cataloged 71 newly identified, putative NEU1-binding partners (53). ...
Article
Full-text available
The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal co-immunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD), but not with the MUC1-ED. Prior pharmacologic inhibition of NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-BA-DANA, did not diminish NEU1-MUC1-CD association. In addition, glutathione S-transferase (GST) pull-down assays utilizing deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 amino acids of the MUC1-CD. In a cell-free system, we found that purified NEU1 interacted with immobilized GST-MUC1-CD, and purified MUC1-CD associated with immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either wild-type NEU1 or a catalytically-dead NEU1 G68V mutant diminished association of the established MUC1-CD binding partner, phosphoinositide 3-kinase (PI3K), to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.
... 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. ...
Article
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). ...
Article
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.
... Although mammalian sialidases have different substrate specificities and properties, they share a common genomic organization. 93,94 However, the overall amino acid identity of Neu1 compared to the other mammalian sialidases is about 19− 24%, whereas Neu2, Neu3, and Neu4 show 34−40% homology to each other. 6 X-ray structures were reported for human Neu2 in free form and in a complex with 2-deoxy-2,3-dehydro-Nacetylneuraminic acid (DANA) inhibitor. ...
Article
Full-text available
Sialidases, or neuraminidases, are enzymes that catalyze the hydrolysis of sialic acid (Sia)-containing molecules, mostly removal of the terminal Sia (desialylation). By desialylation, sialidase can modulate the functionality of the target compound and is thus often involved in biological pathways. Inhibition of sialidases with inhibitors is an important approach for understanding sialidase function and the underlying mechanisms and could serve as a therapeutic approach as well. Transition-state analogues, such as anti-influenza drugs oseltamivir and zanamivir, are major sialidase inhibitors. In addition, difluoro-sialic acids were developed as mechanism-based sialidase inhibitors. Further, fluorinated quinone methide-based suicide substrates were reported. Sialidase product analogue inhibitors were also explored. Finally, natural products have shown competitive inhibiton against viral, bacterial, and human sialidases. This Perspective describes sialidase inhibitors with different mechanisms and their activities and future potential, which include transition-state analogue inhibitors, mechanism-based inhibitors, suicide substrate inhibitors, product analogue inhibitors, and natural product inhibitors.
... Currently, four kinds of sialidases NEU1, NEU2, NEU3 and NEU4 have been found in mammals. The former three are distributed in lysosome, cytoplasm, and plasma membrane, respectively, while NEU4 is located in lysosome, mitochondria and cell inner membrane [13]. From an evolutionary perspective, NEU1 has the highest conservation and expression level in all mammalian tissues. ...
Article
Full-text available
Background: Melanoma is a kind of malignant tumor with high mortality originating from melanocytes. It is urgent to find new molecular biomarkers for prognosis and new treatment methods for melanoma. As an important molecule of sialidase family, neuraminidase-1 (NEU1) has been found to play an important role in regulating the occurrence and progression of tumors, but the role of NEU1 in melanoma is not sure. Methods: The expression level of NEU1 in melanoma and normal tissues was evaluated by analyzing the expression data from ONCOMINE, UALCAN and GEPIA database. The mutation, copy number alteration and gene correlation of NEU1 in melanoma were evaluated by analyzing the melanoma data from cBioPortal database The protein expression levels of NEU1 were further validated by immunohistochemical (IHC) staining data from The Human Protein Atlas database. The melanoma data in TIMER 2.0 database were used to analyze the correlation between NEU1 expression and immune cell infiltration. The proliferative and migratory abilities of melanoma cells were examined by cell proliferation and migration assay in vitro and nude mice. Results: We discovered that NEU1 was highly expressed in melanoma samples compared with normal samples. The alteration frequency of NEU1 in melanoma patients reached 18%, and most of them were "mutation" type. The expression of NEU1 was positively correlated with the overall survival of patients with melanoma. The expression of NEU1 was positively correlated with the expression of proliferation marker CDK2 and epithelial-mesenchymal transition marker CD44 and negatively correlated with the expression of apoptosis marker CASP3 and CASP8. Moreover, the expression level of NEU1 was related to the infiltration of immune cells in melanoma. Knockdown of NEU1 attenuated the in vitro proliferative and migratory abilities of melanoma cells, as well as in vivo tumor progression of melanoma cells. Conclusions: These findings suggest that NEU1 may play a key role in the development of melanoma and may be used as a prognostic target of melanoma.
... The sialylation state of a specific molecule is dynamically and coordinately regulated through the opposing catalytic activities of STs 24,25 and NEUs 26-28 . STs catalyze the transfer of SA residues to glycan chains 24,25 , while NEUs hydrolyse the linkage between terminal SAs and their adjacent subterminal sugars [26][27][28] . ...
Article
Full-text available
To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.
... Membrane localization allows NEUs to act as structural and functional modulators of extracellular soluble and membrane-bound molecules (17). Further information on the subcellular, cellular, and tissue distribution, substrate specificity, catalytic properties, and amino acid homologies of the four mammalian NEUs can be found in prior review articles (12,15,(81)(82)(83)(84)(85)(86)(87). ...
Article
Full-text available
Mammalian neuraminidases (NEUs), also known as sialidases, are enzymes that cleave off the terminal neuraminic, or sialic, acid resides from the carbohydrate moieties of glycolipids and glycoproteins. A rapidly growing body of literature indicates that in addition to their metabolic functions, NEUs also regulate the activity of their glycoprotein targets. The simple post-translational modification of NEU protein targets—removal of the highly electronegative sialic acid—affects protein folding, alters protein interactions with their ligands, and exposes or covers proteolytic sites. Through such effects, NEUs regulate the downstream processes in which their glycoprotein targets participate. A major target of desialylation by NEUs are mucins (MUCs), and such post-translational modification contributes to regulation of disease processes. In this review, we focus on the regulatory roles of NEU-modified MUCs as coordinators of disease pathogenesis in fibrotic, inflammatory, infectious, and autoimmune diseases. Special attention is placed on the most abundant and best studied NEU1, and its recently discovered important target, mucin-1 (MUC1). The role of the NEU1 - MUC1 axis in disease pathogenesis is discussed, along with regulatory contributions from other MUCs and other pathophysiologically important NEU targets.
... Although the common sequence identity between the four sialidases is low, their catalytic domains have a common "six-blade β-propeller fold" architecture. Each of these enzymes selectively hydrolyzes specific glycosidic bonds between sialic acid molecules and subterminal sugar of glycoconjugates [42,43]. ...
Article
The most typical feature of atherogenesis in humans at its early stage is the formation of foam cells in subendothelial arterial intima, which occurs as the consequence of intracellular cholesterol deposition. The main source of lipids accumulating in the arterial wall are circulating low-density lipoprotein (LDL). However, LDL particles should undergo proatherogenic modification to acquire atherogenic properties. One of the known types of atherogenic modification of LDL is enzymatic deglycosilation, namely, desialylation, which is the earliest change in the cascade of following multiple LDL modifications. The accumulating data make sialidases an intriguing and plausible therapeutic target, since pharmacological modulation of activity of these enzymes may have beneficial effects in several pathologies, including atherosclerosis. The hypothesis exists that decreasing LDL enzymatic desialylation may result in prevention of lipid accumulation in arterial wall, thus breaking down one of the key players in atherogenesis at the cellular level. Several drugs acting as glycomimetics and inhibiting sialidase enzymatic activity already exist, but the concept of sialidase inhibition as an anti-atherosclerosis strategy remains unexplored to date. This review is focused on the potential possibilities of the repurposing of sialidase inhibitors for pathogenetic anti-atherosclerotic therapy.
... 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. ...
Article
Full-text available
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]. ...
Article
Full-text available
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.
... 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. ...
Article
Full-text available
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.
... 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. ...
Article
Full-text available
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. ...
Article
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.
... 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). ...
Article
Full-text available
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.
... 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. ...
Article
Full-text available
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.
... 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). ...
Article
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.
... 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]. ...
... 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 . ...
Article
Full-text available
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.
... 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). ...
Article
Full-text available
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.
... 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. ...
Article
Full-text available
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.
... 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]. ...
Article
Full-text available
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.
... 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. ...
... 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). ...
Article
Full-text available
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.
... 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). ...
Article
Full-text available
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.
... 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). ...
Article
Full-text available
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.
... 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. ...
Article
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.
... 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). ...
Article
Full-text available
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.
... 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. ...
... [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. ...
Article
Full-text available
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.
Chapter
Gangliosides are a large group of complex lipids found predominantly in the outer layer of the plasma membrane of cells, particularly abundant in nerve endings. Their half-life in the nervous system is short, and their membrane composition and content are strictly connected to their metabolism. The neobiosynthesis of gangliosides starts in the endoplasmic reticulum and is completed in the Golgi apparatus, whereas catabolism occurs primarily in lysosomes. However, the final content of gangliosides in the plasma membrane is defined by other cellular processes. This chapter will discuss structural changes in the oligosaccharideOligosaccharides chains of gangliosidesGangliosides, induced by the activity of plasma membrane-associated glycohydrolasesGlycohydrolases and glycosyltransferasesGlycosyltransferases (GTs). Some of the plasma membranePlasma membranes enzymes originate from fusion processes between intracellular fractions and the plasma membranePlasma membranes, while, others display a different structureStructures. Several of these plasma membranePlasma membranes enzymes have been characterized and some of them seem to have a specific role in the nervous system.
Chapter
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.
Chapter
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.
Article
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.
Article
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.
Article
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.
Thesis
Full-text available
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.
Conference Paper
Full-text available
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.
Article
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
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.
Article
Full-text available
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.
Article
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] /* */
Article
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Article
Full-text available
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.
Chapter
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).
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.
Article
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.