Differential Expression of Three Sialidase Genes in Rat Development

Hasegawa, Takafumi; Carnero, Carmen Feijoo; Itoyama, Yasuto; Miyagi, Taeko

doi:10.1006/bbrc.2000.4186

Cited by 34 publications

(24 citation statements)

References 32 publications

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“…Sialidase Activity Assays-Crude homogenates from tissues and cells were used for sialidase activity assays with mixed gangliosides (Sigma) as substrates in the presence of Triton X-100 as described previously (11,12). One unit of sialidase was defined as the amount of enzyme catalyzing the release of 1 nmol of sialic acid/h.…”

Section: Methodsmentioning

confidence: 99%

“…The fact that plasma membrane sialidase is unique in specifically hydrolyzing gangliosides (4 -10) and in its subcellular localization in plasma membranes, where levels of other glycosidases are very low, suggests participation in cell surface events through modulation of gangliosides. To obtain evidence of this, we previously cloned sialidase cDNAs of mammalian origin (8,9,11,12), and we have recently investigated effects of overexpression in transgenic mice by using the human ortholog NEU3 cDNA. Unexpectedly, we have found that they develop insulin-resistant diabetes mellitus.…”

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confidence: 99%

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Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Sasaki

Hata

Suzuki

et al. 2003

Journal of Biological Chemistry

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108

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Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18 -22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased ␤-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.Gangliosides are a family of sialic acid-containing glycosphingolipids present in the cell surface membranes. Several lines of evidence suggest their important functional roles in regulating a wide range of biological processes including cell growth, cell differentiation, and transmembrane signaling (1-3). Most of the observations on ganglioside function, however, have been performed so far by using bacterial sialidases and exogenous gangliosides to mimic in vivo expression. To obtain further insights into their physiological significance and regulation mechanism, it is necessary to focus on endogenous sialidases responsible for ganglioside hydrolysis inside the cells. Three types of mammalian sialidase have been cloned and established to have low identity with each other in their primary structure. They differ in enzymatic properties and subcellular localization, being mainly found in lysosomes (Neu1), cytosol (Neu2), and plasma membranes (Neu3). Other than the contribution of lysosomal sialidase to glycoconjugate catabolism in lysosomes, their cellular roles are not well understood. The fact that plasma membrane sialidase is unique in specifically hydrolyzing gangliosides (4 -10) and in its subcellular localization in plasma membranes, where levels of other glycosidases are very low, suggests participation in cell surface events through modulation of gangliosides. To obtain evidence of this, we previously cloned sialidase cDNAs of mammalian origin (8, 9, 11, 12), and we have recently investigated effects of overexpression in transgenic...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Sasaki

Hata

Suzuki

et al. 2003

Journal of Biological Chemistry

Self Cite

108

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show abstract

“…The reaction mixture contained 50 nmol of substrate as bound sialic acid, 0.2 mg of bovine serum albumin, 10 mol of sodium acetate (pH 4.6), and 0.2 mg of Triton X-100 in 200 l. After incubation at 37°C for 10 -60 min, released sialic acid was determined by the thiobarbituric acid method after passing through an AG1X-2 mini-column as described elsewhere (7). Protein was determined by dye-binding assay (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

A Close Association of the Ganglioside-specific Sialidase Neu3 with Caveolin in Membrane Microdomains

Wang¹,

Yamaguchi²,

Wada³

et al. 2002

Journal of Biological Chemistry

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115

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

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“…Recently, we demonstrated that its overexpression induces a marked decrement of proliferation and spontaneous differentiation of myoblast C2C12 cells [16]. Although the sialidase Neu2 is expressed in other tissues such as liver [17], thymus [18] and brain [19], its enzymatic activity is often hardly detectable, thus making its study difficult.…”

Section: Discussionmentioning

confidence: 99%

“…Despite its relative abundance in skeletal muscle, cytosolic sialidase was found also in other tissues, such as liver [17], thymus [18] and, although at low levels, brain [19].…”

Section: Introductionmentioning

confidence: 99%

Cytosolic sialidase Neu2 upregulation during PC12 cells differentiation

FANZANI

2004

FEBS Letters

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The cytosolic sialidase Neu2 is known to be involved in myoblast differentiation. Here, we observed a Neu2 transcriptional induction during nerve growth factor, fibroblast growth factor 2 and epidermal growth factor treatments of PC12 cells, a favored model to study neuronal differentiation. The expression analysis of Neu2 deleted promoter revealed a remarkable increase of luciferase activity in treated PC12 cells, suggesting that in this cell line the Neu2 transcriptional levels are highly regulated.The enzymatic activity of cytosolic sialidase Neu2 was found to increase transiently only during differentiation, whereas was undetectable in untreated PC12 cells. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells.

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Differential Expression of Three Sialidase Genes in Rat Development

Cited by 34 publications

References 32 publications

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

Overexpression of Plasma Membrane-associated Sialidase Attenuates Insulin Signaling in Transgenic Mice

A Close Association of the Ganglioside-specific Sialidase Neu3 with Caveolin in Membrane Microdomains

Cytosolic sialidase Neu2 upregulation during PC12 cells differentiation

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