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Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

High-level physical performance in rhythmic gymnastics is influenced by numerous skills and anthropometric factors. In order to understand if genetic predisposition could play a role to define the elite rhythmic gymnast phenotype, we analysed the frequency of common polymorphisms linked to genes correlated with body mass (ADRB2 and FTO), explosive strength (ACTN3 and ACE), and joint mobility (COL5A1), in 42 gymnasts involved in National and International events, and in 42 control girls. Our results demonstrated that high-level rhythmic gymnasts constituted a genetically selected population showing higher frequency of: (a) ADRB2 and FTO alleles linked to low body mass index and low fat mass; (b) COL5A1 CT genotype linked to high joint mobility and to the occurrence of genu recurvatum, but also to a higher incidence of injuries. ACTN3 and ACE polymorphisms did not appear to be connected with the phenotype of high-level rhythmic gymnast. Based on these data, it can be assumed that these polymorphisms could positively affect the phenotype and performance of gymnasts.

Chronic administration of β-alanine has been shown to increase muscle carnosine content and improve anaerobic performance. It is not clear whether acute ingestion of carnosine and beta alanine may have the same effects. With a view to investigating acute effects of carnosine and β-alanine ingestion on anaerobic intermittent running performance and on the responses of Interleukin-6 and -10 to exercise, twelve healthy, young, active participants (age: 21 ± 4 years) underwent the running-based anaerobic test (RAST) twice (with 30 min recovery in between) on two separate occasions (randomized, crossover design). The test consisted of 6 × 35-m sprints interspersed with 10 s rests after acute ingestion (4 hours before the test) of either 2 g L-carnosine + 2 g β-alanine or placebo. The overall performance decreased (RAST1 vs RAST2, carnosine + β-alanine: 32.8 ± 1.3 s, 33.4 ± 1.2 s; Placebo: 32.9 ± 1.0 s, 33.6 ± 1.2 s), pain after RASTs increased (RAST1 vs RAST2, carnosine + β-alanine: 3.0 ± 2.1 a.u., 4.2 ± 1.9 a.u.; Placebo: 3.0 ± 1.8 a.u., 3.4 ± 1.2 a.u.) almost in the same way in both groups, and RPE did not show any difference. IL6 and IL10 gene expression increased and decreased respectively in response to exercise in the same fashion in both conditions. During RAST 2 we found a potentially increased performance in the carnosine + β-alanine group (main effect of condition, p < 0.05). In conclusion these findings suggest that acute administration of carnosine + β-alanine does not influence the cytokine response to exercise but might have a very small enhancing effect on anaerobic sprint performance.

Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell surface glycoconjugates by one or both sialidase promotes cytokine production. Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1–/– and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

Recent advances in stem cell biology have demonstrated that terminally differentiated adult cells can be induced to de-differentiate into progenitor cells (induced stem cells) upon proper stimuli. This has been achieved by the induced expression of key regulatory genes by retro- or lenti-viral systems. On the other hand, synthetic "small molecules" can also induce de-differentiation and may represent a potentially safer approach as compared with genetic manipulation. Along this line, a synthetic purine called "reversine" has been shown to induce the de-differentiation of fibroblasts into mesenchymal stem-cell-like progenitors, which can be successively induced to differentiate into skeletal muscle, smooth muscle and bone cells. The mechanism whereby reversine is able to achieve de-differentiation has yet to be clarified. In this context, we defined the protein changes induced by reversine treatment in murine fibroblasts by 2-D difference gel electrophoresis, coupled with MS. Proteins involved in cytoskeletal and cell shape remodeling, RNA export, degradation, folding, stress control and ATP production were found to be remarkably changed after reversine treatment. Ingenuity pathway analysis (IPA) predicted that these protein pattern changes enabled to propose that about 40 proteins might be associated to several biological functional networks, including cellular assembly, cell signaling and cell death. Altogether our data confirm the intrinsic complexity of the de-differentiation process induced by reversine and suggest more selected approaches to investigate the action mechanism of this small molecule.

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually “switched on” activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45–66 kDa, showed a 50–70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.

Objectives: Hexosaminidase activity is present in lysosomes, plasma membrane and cytosol of many human cells. Plasma membrane and cytosolic hexosaminidase is not well characterized, particularly as regards their isoenzyme forms and their relationship with the lysosomal ones. Design and methods: Erythrocyte hexosaminidase isoforms were chromatographically separated, characterized and compared to those in the plasma of healthy individuals and in the erythrocytes of a Tay–Sachs patient. Results: Hexosaminidase isoenzymes were found in plasma membrane and cytosol and were composed of the same α- and β-subunits as the lysosomal and plasma hexosaminidase A and B isoenzymes, though with some structural and kinetic differences. In addition, the cytosol contained a hexosaminidase that is a specific N -acetyl-β- d -glucosaminidase, the one involved in the removal of N -acetylglucosamine residues O -linked to proteins, named O -GlcNAcase. Conclusions: This work provides an additional step in the characterization of hexosaminidases helping better understand their role in non-lysosomal compartments and their involvement in physiological or pathological situations.

Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc.

In this study, a comparison between elderly (>70 years) and young subjects reveals that elder people are subject to a higher oxidative stress, which causes an increase in plasma hydroperoxide levels (18%) and a decrease in antioxidant defenses (25%). Moreover, the marked decrease of the erythrocyte membrane fluidity observed in elderly subjects was likely to affect the behavior of some membrane glycohydrolases. In fact, a significant decrease of β- d -glucuronidase and neutral sialidase (30 and 50%, respectively) was detected. Activity differences were also observed when erythrocytes were further distinguished according to their biological age. Striking differences between young and elderly subjects were observed for β- d -glucuronidase and neutral sialidase in young and senescent erythrocytes, respectively. Overall β- d -glucuronidase decreases with the subjects' age, while neutral sialidase levels are higher in the elderly. This is presumably due to the localization of these enzymes in distinct plasma membrane micro-domains, which are differently peroxidized. A possible role of these enzymes in signaling praecox membrane alterations has also been evidenced.

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade β-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr334, conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu111 and the catalytic aspartate Asp46 are disordered in the apo form but upon binding of DANA become ordered to adopt two short α-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.

The behavior of the 2 sialidase forms present in the erythrocyte membrane was investigated in 117 subjects with type 2 diabetes mellitus versus 95 healthy controls. A significant increase of the acidic form of sialidase, which is anchored to the membrane by a glycosylphosphatidylinositol bridge, was observed in erythrocyte resealed membranes. On the contrary, the neutral form of the enzyme, the only one capable of removing lipid- and protein-bound sialic acid from endogenous and exogenous sialoderivatives, was significantly reduced with a consequent increase of erythrocyte membrane total sialic acid content. Disease duration, therapy, glycemia, parameters of metabolic control, and presence of complications, except nephropathies, had no influence on the tested enzyme activities. Diabetic subjects showed a different erythrocyte age distribution, with an almost double proportion of young red cells and only one quarter of senescent ones compared with controls. In young erythrocytes, diabetic and control subjects had the same distribution of the 2 enzymes, while in senescent cells the acidic enzyme was increased 3.5-fold and the neutral form was reduced by half in the diabetic subjects. The increase of both acidic sialidase and total membrane-bound sialic acid, together with an overpresence of young red cells in diabetics, suggests that in this pathological condition there might be an altered aging process with a diminished expression of the neutral form of the enzyme and an increase of bound sialic acid. It has been suggested that the expression of the neutral enzyme requires some activation mechanism that is impaired in diabetes.

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.

Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.

Background In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids). Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor. On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients. Methods Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients. Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS. N-glycolyl GM3 was identified employing the 14 F7 antibody. Gene expression was assayed by Real Time PCR. Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation. Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis. Results Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3. Moreover, our data demonstrated that: a) a correlation could be traced between patients’ survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients’ survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins. Conclusions Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness. Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-560) contains supplementary material, which is available to authorized users.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

Acidic and neutral sialidases (pH optimum 4.7 and 7.2, respectively) were assayed on human circulating erythrocytes during ageing. The assays were performed on intact erythrocytes and resealed erythrocyte ghost membranes. From young to senescent erythrocytes the acidic sialidase featured a 2.7-fold and 2.5-fold decrease in specific activity when measured on intact cells or resealed ghost membranes, whereas the neutral sialidase a 5-fold and 7-fold increase, respectively. The Ca2+-loading procedure was employed to mimic the vesiculation process occurring during erythrocyte ageing. Under these conditions the released vesicles displayed an elevated content of acidic sialidase, almost completely linked through a glycan phosphoinositide (GPI) anchor but no neutral sialidase activity, that was completely retained by remnant erythrocytes together with almost all the starting content of sialoglycoconjugates. The loss with vesiculation of acidic sialidase with a concomitant relative increase of neutral sialidase was more marked in young than senescent erythrocytes. The data presented suggest that during ageing erythrocytes loose acidic sialidase, and get enriched in the neutral enzyme, the vesiculation process, possibly involving GPI-anchors-rich membrane microdomains, being likely responsible for these changes. The enhanced neutral sialidase activity might account for the sialic acid loss occurring during erythrocyte ageing.

The feature of intact human erythrocytes and erythrocyte white ghosts is a unique sialidase activity with acidic optimal pH (acidic sialidase). The treatment of white ghosts with mildly alkaline isotonic solutions at 37°C, like that used to produce resealed ghosts, is accompanied by the expression, together with the acidic sialidase, of a novel sialidase with a pH optimum of 7.2 (neutral sialidase) that remained masked in the inside-out vesicles prepared from white ghosts. Exhaustive treatment of resealed ghosts with Bacillus Thuringiensis phosphatidylinositol-specific phospholipase C causes an almost complete release of the acidic sialidase, with the neutral enzyme remaining totally unaffected. The treatment of resealed ghosts with 1.2% Triton X-100 resulted in the solubilization of only the neutral sialidase, whereas 3.6% octylglucoside also solubilized the acidic sialidase. The neutral enzyme affected not only the artificial substrate but also any sialoderivatives of a ganglioside, glycoprotein, and oligosaccharide nature; the acidic enzyme did not affect sialoglycoproteins. Erythrocyte endogenous gangliosides were hydrolyzed by both sialidases, whereas the endogenous sialoglycoproteins responded to only the neutral enzyme. It was definitely proved that the acidic sialidase is located on the outer erythrocyte membrane surface, so presumably the neutral enzyme has the same location. It could be that the newly discovered neutral sialidase has a physiologic role in the releasing of sialic acid from erythrocytes during the erythrocyte aging process, leading to eventual phagocytosis by macrophages.

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.

Exercise performed at a competitive level could deeply modify the immune system and the cytokine response of athletes. In this report, we demonstrated that young elite female artistic gymnasts (n = 16; age: 9-15 years) showed an increase of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) mRNA expression in blood mononuclear cells (PBMCs), in comparison to girls performing the same sport at a recreational level (n = 16; age: 10-15 years). The increase of IL-6 and TNF-α mRNAs appeared to be directly linked to the intensity and duration of the training. Moreover, in elite athletes engaged in artistic gymnastics or in synchronised swimming (n =34; age: 9-15 years), IL-6 gene expression appeared to be modulated by the levels of circulating oestrogens: pre-pubertal athletes (n = 20; age: 11 ± 1 years) revealed a higher increase in IL-6 than pubertal athletes (n = 14; age: 14 ± 1.6 years). In pre-pubertal athletes, body mass index (BMI) percentile was inversely correlated with the increase of both IL-6 and TNF-α. The consequence of these events was the shift of the cytokine profile towards a pro-inflammatory status. These modifications, induced by training performed at an elite level, might negatively affect the growth of female children athletes.

Human sialidase NEU4 long (N4L) is a membrane-associated enzyme that has been shown to be localized in the outer mitochondrial membrane. A role in different cellular processes has been suggested for this enzyme, such as apop-tosis, neuronal differentiation and tumorigenesis. However, the molecular bases for these roles, not found in any of the other highly similar human sialidases, are not understood. We have found that a proline-rich sequence of 81 amino acids, unique to NEU4 sequence, contains potential Akt and Erk1 kinase motifs. Molecular modeling, based on the experimentally determined three-dimensional structure of cytosolic human NEU2, showed that the proline-rich sequence is accommodated in a loop, thus preserving the typical beta-barrel structure of sialidases. In order to investigate the role of this loop in neuronal differentiation, we obtained SK-N-BE neuroblastoma cells stably overexpres-sing either human wild-type N4L or a deletion mutant lacking the proline-rich loop. Our results demonstrate that the proline-rich region can also enhance cell proliferation and retinoic acid (RA)-induced neuronal differentiation and it is also involved in NEU4 interaction with Akt, as well as in substrate recognition, modifying directly or through the interaction with other protein(s) the enzyme specificity toward sialylated glycoprotein(s). On the whole, our results suggest that N4L could be a downstream component of the PI3K/Akt signaling pathway required for RA-induced differentiation of neuroblastoma SK-N-BE cells. © The Author 2013.

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.

The family of mammalian sialidases is composed of four distinct versatile enzymes that remove negatively charged terminal sialic acid residues from gangliosides and glycoproteins in different subcellular areas and organelles, including lysosomes, cytosol, plasma membrane and mitochondria. In this review we summarize the growing body of data describing the important role of sialidases in skeletal muscle, a complex apparatus involved in numerous key functions and whose functional integrity can be affected by various conditions, such as aging, chronic diseases, cancer and neuromuscular disorders. In addition to supporting the proper catabolism of glycoconjugates, sialidases can affect different signaling pathways by desialylation of many receptors and modulation of ganglioside content in cell membranes, thus actively participating in myoblast proliferation, differentiation and hypertrophy, insulin responsiveness and skeletal muscle architecture. ispartof: Skeletal Muscle vol:2 issue:1 ispartof: location:England status: published

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. J. Cell. Biochem. 113: 3207–3217, 2012. © 2012 Wiley Periodicals, Inc.

The sialylation level of molecules, sialoglycoproteins and gangliosides, protruding from plasma membranes regulates multiple facets of erythrocyte function, from interaction with endothelium to cell lifespan. Our results demonstrate that: (a) Both sialidases NEU1 and NEU3 are present on erythrocyte plasma membrane; (b) NEU1 is kept on the plasma membrane in absence of the protective protein/cathepsin A (PPCA); (c) NEU1 and NEU3 are retained on the plasma membrane, as peripheral proteins, associated to the external leaflet and released by alkaline treatments; (d) NEU1 and NEU3 are segregated in Triton X-100 detergent-resistant membrane domains (DRMs); (e) NEU3 shows activity also at neutral pH; and (f) NEU1 and NEU3 are progressively lost during erythrocyte life. Interestingly, sialidase activity released from erythrocyte membranes after an alkaline treatment preserves its functionality and recognizes sialoglycoproteins and gangliosides. On the other hand, the weak anchorage of sialidases to the plasma membrane and their loss during erythrocyte life could be a tool to preserve the cellular sialic acid content in order to avoid the early ageing of erythrocyte and processes of cell aggregation in the capillaries.

Neuroblastoma (NB) is a frequently lethal tumor that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the deregulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch and Wnt/β-catenin (CTNNB1) signaling. In this article, we demonstrate that sialidase NEU4 long (NEU4L) influences the differentiation/proliferation behavior of NB SK-N-BE cells by determining hyperactivation of the Wnt/β-catenin signaling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, nonphosphorylated β-catenin content, β-catenin/TCF transcriptional activity and β-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2) and CDC25A. In turn, these molecular features strongly modified the behavior of NEU4L SK-N-BE overexpressing cells, promoting the following: (1) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; (2) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133 and NES (nestin); (3) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/β-catenin signaling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment.

Several factors affect the skeletal muscle differentiation process, in particular modifications of cell-cell contact, cell adhesion, and plasma membrane characteristics. In order to support the role of the plasma membrane-associated sialidase NEU3 in skeletal muscle differentiation and to analyse which events of the process are mainly affected by this sialidase, we decided to stably over-express MmNEU3 in C2C12 cells by a lentiviral vector and to investigate cell behavior during the differentiation process. Vitally stained C2C12 and NEU3 over-expressing cells were counted to reveal modifications in differentiation induction. We found that NEU3 over-expressing cells remained proliferative longer than control cells and delayed the onset of differentiation. Expression of p21, myogenic transcription factors, and myosin heavy chain (MHC), assessed by real time PCR, confirmed this behavior. In particular, no MHC-positive myotubes were present in NEU3 over-expressing cells as compared to wild type C2C12 cells at day 3 of differentiation. Moreover, NEU3 over-expressing cells completed the differentiation process very quickly and formed hypertrophic myotubes. Analysis of MAPK/ERK pathway activation showed an increased ERK 1/2 phosphorylation in NEU3 over-expressing cells at the beginning of differentiation. We postulate that sialidase NEU3, decreasing plasma membrane ganglioside GM3 content, affects the EGF receptor and the downstream signaling pathways, promoting proliferation and delaying differentiation. Furthermore NEU3 improves myoblast fusion probably via neural-cell adhesion molecule (NCAM) desialylation. Therefore, this work further supports the central role of NEU3 as a key modulator in skeletal muscle differentiation, particularly in the myoblast fusion step.

The aggregative properties of gangliosides in diluted aqueous solutions are discussed on the basis of simple and well-established thermodynamic concepts. Theoretical assumptions are compared with experimental data obtained, mainly by scattering techniques, on GM3, GM2, GM1, GD1a, GalNAc-GD1a, GD1b, GD1b lactone and GT1b gangliosides, all containing ceramide portions of similar composition, and on GM1 molecular species containing different well-defined ceramide structures. We also report on mixed aggregates with amphiphilic compounds and on the ganglioside aggregate-soluble protein interaction effects which give rise to very stable lipoproteic complexes of well-defined ganglioside-protein composition.

Membrane-bound sialidase NEU3 increase during skeletal muscle differentiation has been shown to protect myoblasts from apoptosis and drive the differentiation process [1]. Thus, the objective of this study was to assess whether up-regulation of NEU3 would enhance the ability of murine skeletal muscle cells to resist to hypoxia, ultimately opposing cell death. We found that C2C12 myoblasts overexpressing NEU3 (L-NEU3) became highly resistant to 1% oxygen or 200 mM deferoxamine induced hypoxia. Moreover, L-NEU3 myoblasts survived a seven-day treatment of combined hypoxia and low serum (2% horse serum used to induce myoblast differentiation), without any significant cell loss. On the contrary, wild type C2C12 could not resist to these culturing conditions and all died within 48h. Real Time PCR showed NEU3 expression increase during all hypoxic treatments both in C2C12 and L-NEU3 cells, suggesting an endogenous NEU3 activation under these conditions. Moreover, we found that NEU3 over-expression activated pro-survival signalling pathways through up-regulation and activation of EGF receptor. Overall, our data support the hypothesis that NEU3 may play a critical role in the response of skeletal myoblasts to hypoxia and the preservation of cell viability by activating pro-survival signalling pathways. [1] Anastasia L. et al. J.Biol.Chem. 2008, 283 (52): 36265–36271.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.

The aggregative properties of gangliosides in diluted aqueous solutions are discussed on the basis of simple and well-established thermodynamic concepts. Theoretical assumptions are compared with experimental data obtained, mainly by scattering techniques, on GM3, GM2, GM1, GD1a, GalNAc-GD1a, GD1b, GD1b lactone and GT1b gangliosides, all containing ceramide portions of similar composition, and on GM1 molecular species containing different well-defined ceramide structures. We also report on mixed aggregates with amphiphilic compounds and on the ganglioside aggregate-soluble protein interaction effects which give rise to very stable lipoproteic complexes of well-defined ganglioside-protein composition.

Glycoglycerolipid analogues, derived from 2-O-β- d -galactosylglycerol, have been synthesized on the base of the structure of natural glycoglycerolipids showing anti-tumor and anti-inflammatory efficacy. These compounds have been previously demonstrated to inhibit phorbol 12-myristate-13-acetate (PMA) induced tumor promotion in mouse skin, but their mechanism of action has never been elucidated. In this work, we studied the effects of glycoglycerolipid analogues on PKC activation induced by PMA and its downstream target molecules, in human fibroblasts. Our results proved that: a) the tested compounds were able to block PKC translocation to the plasma membrane, promoted by PMA, in a dose-dependent manner (IC 50 : 0.48 μM for the most active compound 2 ); b) the efficacy of these compounds was strongly connected to their acyl chain linked to galactose; in particular, the addition of hexanoyl and branched chains enhanced PKC inhibition, the presence of a cyclohexane ring and an excessive length of the acyl chain, or its lack, exerted a negative effect; c) the inhibition of PKC translocation blocked enzyme activation and downstream signaling pathways, MAPK and FAK, involved in proliferation and adhesion/migration control. In addition, the branched glycoglycerolipid (compound 2 ) was able to inhibit PKC translocation and activation in naturally highly PKC activating glioblastoma cells, U87MG. As consequence, U87MG cell proliferation and, especially, migration potential resulted to be markedly reduced (−30% and −84%, respectively). Thus, these results reveal the role of a PKC-dependent mechanism in glycoglycerolipid analogues mediated protective effects and highlight their possible employment in the field of prevention/treatment of cancer.

The possibility of reprogramming adult somatic cells into pluripotent stem cells (iPSCs) has generated a renewed interest into stem cell research and promises to overcome several key issues, including the ethical concerns of using human embryonic stem cells and the difficulty of obtaining large numbers of adult stem cells (Belmonte et al., Nat Rev Genet, 2009). This approach is also not free from challenges like the mechanism of the reprogramming process, which has yet to be elucidated, and the warranties for safety of generated pluripotent cells, especially in view of their possible therapeutic use. Very recently, several new reprogramming methods have surfaced, which seem to be more appropriate than genetic reprogramming. Particularly, chemically induced pluripotent cells (CiPSs), obtained with recombinant proteins or small synthetic molecules, may represent a valid approach, simpler and possibly safer than the other ones.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.

Membrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (-30 to 40%) and enzymatic activity toward ganglioside GD1a (-25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (-55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0-G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9-OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse.

Sialidases are widely distributed glycohydrolytic enzymes removing sialic acid residues from glycoconjugates. In mammals, several sialidases with different subcellular localizations and biochemical features have been described. NEU4, the most recently identified member of the human sialidase family, is found in two forms, NEU4 long and NEU4 short, differing in the presence of a 12-amino-acid sequence at the N-terminus. Contradictory data are present in the literature about the subcellular distribution of these enzymes, their membrane anchoring mechanism being still unclear. In this work, we investigate the human NEU4 long and NEU4 short membrane anchoring mechanism and their subcellular localization. Protein extraction with Triton X-114 and sodium carbonate and cross-linking experiments demonstrate that both forms of NEU4 are extrinsic membrane proteins, anchored via protein-protein interactions. Moreover, through confocal microscopy and subcellular fractionation, we show that the long form localizes in mitochondria, while the short form is also associated with the endoplasmic reticulum. Finally, mitochondria subfractionation experiments suggest that NEU4 long is bound to the outer mitochondrial membrane.

Light and heavy lysosomes of mouse forebrain were separated from each other by centrifugation on a Percoll gradient. Light lysosomes were then freed from mitochondria and membranes by sucrose density gradient centrifugation and further purified by floatation-centrifugation on a sucrose gradient. The final preparations of light and heavy lysosomes, fairly homogenous, carried sinlidase activity, assayed on MU-NeuAc. The optimal pH was 4.0 and 4.2, the apparent Km value 2.8 × 10−3 M and 4.2 × 10−3 M and the apparent Vmax value 0.11 and 0.47 mU mg−1 protein, for the light and heavy lysosome sialidase, respectively. From 4 days to adulthood the specific activity of the light and heavy lysosome sialidase increased 3-fold and 1.7-fold, respectively.

Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase", has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regards to GM3. Induced down-regulation of NEU3 in murine myoblasts C2C12, even when partial, totally inhibits their capability to differentiate by increasing GM3 level above a critical point, that causes EGFR inhibition (and ultimately its down-regulation), and an higher responsiveness of myoblasts to the apoptotic stimuli. ispartof: Journal of Biological Chemistry vol:283 issue:52 pages:36265-36271 ispartof: location:United States status: published

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.

Glycosphingolipids and glycoproteins play pivotal roles in the complex series of events governing cell adhesion and signal transduction. Aberrant glycosilation, typical of tumor cells, represents a key event in the induction of invasion and metastasis. Sialidases remove sialic acid residues from sialoconjugates and, in mammals, these enzymes have been proved to be involved in several cellular phenomena, including cell proliferation and differentiation, membrane function, and malignant transformation. Herein we show that only the lysosomal sialidase Neu1 and the plasma membrane-associated sialidase Neu3 are expressed in CFU-E erythroid precursors and K562 erythroleukemic cells. Tumour cells show much higher expression levels than CFU-E cells and, during differentiation, the content of the two enzymes progressively decreases. The sialoglycoconjugate pattern is different in the two cell types. In fact, the differentiating erythroid precursors show an increase of the typical erythrocyte sphingolipids, whereas K562 cells treated with butyrate show a marked increase of GD1a, GM2, PE, and ceramide. Finally, during differentiation the sialoglycoprotein content of erythroid cells shows a marked increase, and in K562 cells the process induces the synthesis of some sialoglycoprotein typical of the erythroid membrane. Overall, these results point out the great differences in sialoglycoconjugate and sialidase patterns exhibited by normal and tumour cells.

Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT–PCR (reverse transcription–PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

We studied some erythrocyte glycohydrolases, erythrocyte membrane fluidity, plasma hydroperoxides and total antioxidant defences in 23 Down syndrome (DS) individuals in comparison with healthy age-matched and elderly controls. With regard to erythrocyte plasma membrane fluidity, plasma hydroperoxides and total plasma oxidative defences, DS subjects resembled the age-matched controls more than the elderly ones. Membrane glycohydrolases in DS, however, presented a pattern partly similar to age-matched controls and partly to elderly controls. Concerning cytosol glycohydrolases, DS subjects had lower levels of hexosaminidase and N -acetyl-β- d -glucosaminidase, the latter specific for the hydrolysis of GlcNAc residues O-linked to proteins. In general, erythrocyte membrane and cytosol glycohydrolases decreased during erythrocyte ageing in DS subjects and in all controls. The increased levels of the same enzymes in DS plasma might be attributed to an alteration of their release-uptake mechanisms between the two different compartments, on account of the higher plasma hydroperoxide levels. These findings indicate that erythrocyte ageing in DS differs partially from that of age-matched and elderly controls. In any case, the accelerated ageing seen in DS is no fully comparable to physiological ageing.