The Plasma Membrane Ganglioside Sialidase Cofractionates with Markers of Lipid Rafts

Kalka, Dorothee; Reitzenstein, Carolina; Kopitz, Jürgen; Cantz, Michael

doi:10.1006/bbrc.2001.4864

Cited by 82 publications

(46 citation statements)

References 35 publications

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“…Furthermore, recent results show that the enzyme is localized in the Triton X-100-resistant microdomains (i.e. 'classical' GEM) where it interacts with caveolin [40,41]. Then how can one explain the effect of Neu3 on the interactions of the protein, which is excluded from the classical lipid rafts?…”

Section: Discussionmentioning

confidence: 99%

Gangliosides play an important role in the organization of CD82-enriched microdomains

Odintsova

Butters

Monti

et al. 2006

Biochemical Journal

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Four-transmembrane-domain proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organization of TERM are poorly understood. In the present study, we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase [NB-DGJ (N-butyldeoxygalactonojirimycin) and PPMP (D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol · HCl)] resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasmamembrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased, but the association with EGFR [EGF (epidermal growth factor) receptor] was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together, our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these results demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

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Section: Discussionmentioning

confidence: 99%

Gangliosides play an important role in the organization of CD82-enriched microdomains

Odintsova

Butters

Monti

et al. 2006

Biochemical Journal

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“…The plasma membrane sialidase NEU3 3 is characterized by high substrate specificity for ganglioside substrates in the acidic range of pH (26). In addition, MmNEU3 has been demonstrated to be associated with SED in the neuroblastoma cell line SK-N-MC (27) as well as in HeLa and COS-1 cells, where it is closely associated with caveolin (28).…”

Section: Glycosphingolipids (Gsls)mentioning

confidence: 99%

The Plasma Membrane-associated Sialidase MmNEU3 Modifies the Ganglioside Pattern of Adjacent Cells Supporting Its Involvement in Cell-to-Cell Interactions

Papini

Anastasia

Tringali

et al. 2004

Journal of Biological Chemistry

131

142

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We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-3 H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-3 H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-3 H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions. Glycosphingolipids (GSLs)1 expressed at the cell surface are well known as modulators of several aspects of signal transduction processes involved in the control of cell proliferation, survival, and differentiation (1). GSLs in the plasma membrane are able to interact laterally with other membrane molecules modulating their properties (cis-interactions). Lipid rafts or membrane microdomains result from dynamic clustering of sphingolipids and cholesterol to form the so-called sphingolipid-enriched domain (SED) or lipid rafts (2). These structures move within the fluid bilayer and function as platforms for the attachment of proteins when membranes are moved around the cell and during signal transduction (3, 4). In addition, the expression pattern of GSLs in several cells and tissues undergoes deep changes during development and neoplastic transformation (5). These events are usually characterized by dramatic changes in cell recognition, suggesting that GSLs as cell surface antigens could play relevant roles as receptor sites in cell-cell recognition (trans-interaction). The receptor role of GSLs has been hypothesized in the case of microbial infections based on their ability to interact with bacterial toxins and microbial lectins (6, 7). Conformational analysis confirms that the orientation of the oligosaccharide chains of glycolipids at the cell surface complies wit...

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“…We now present evidence that Neu3 is closely associated with caveolin-1 within caveolae microdomains and probably acts as a transducer molecule. In this context it is of interest that in the course of the present study, a study (20) was published that described gan-glioside sialidase activity co-fractionating with rafts from a neuroblastoma cell line.…”

mentioning

confidence: 95%

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

Wang¹,

Yamaguchi²,

Wada³

et al. 2002

Journal of Biological Chemistry

114

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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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The Plasma Membrane Ganglioside Sialidase Cofractionates with Markers of Lipid Rafts

Cited by 82 publications

References 35 publications

Gangliosides play an important role in the organization of CD82-enriched microdomains

Gangliosides play an important role in the organization of CD82-enriched microdomains

The Plasma Membrane-associated Sialidase MmNEU3 Modifies the Ganglioside Pattern of Adjacent Cells Supporting Its Involvement in Cell-to-Cell Interactions

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

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