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

Abstract

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...

Activity of plasma membrane β‐galactosidase and β‐glucosidase

“…An important point is to demonstrate that the plasma membrane associated activities were conserved in living cells. For Neu3 sialidase we have already demonstrated its activity on glycoconjugates located at the cell surface of neighboring cells [13]. To look for b-galactosidase and b-glucosidase activities in living cells, we used both artificial and natural substrates as described in Section 2.…”

Neobiosynthesis of Glycosphingolipids by Plasma Membrane-associated Glycosyltransferases*

“…However, an additional level of regulation of glycosphingolipid expression has been recently suggested to occur at the plasma membrane level, with the existence being reported of a plasma membrane-associated sialidase, termed Neu3, which is able to trigger selective ganglioside desialylation in different cell types (17)(18)(19). Moreover, ␤-hexosaminidase (20), ␤-glucosidase, and ␤-galactosidase (21) activities have also been demonstrated at the cell surface, suggesting their role in the remodeling of the plasma membrane glycosphingolipids, thus contributing to modulate lipid composition and membrane organization, and consequently, different signaling processes (21,22).…”

Identification and characterization of mature β-hexosaminidases associated with human placenta lysosomal membrane

“…These lipids play key role as signaling molecules in both physiological and pathological processes regulating cell-to-cell and/or cell-environment interactions [3,38]. Several recent studies revealed the presence of glycohydrolases not only in lysosomes but also at cell surface [5,18,39,40]. They stress the potential role of a remodelling of glycosphingolipids during cellular processes [6,7].…”

“…Nevertheless, new studies show the presence of glycohydrolases at the plasma membrane (PM) and point to their potential role in modifying glycosphingolipids during signaling and cellular communication [3,4]. For instance, evidence for PM sialidase Neu3 activity towards gangliosides exposed to the surface of adjacent cells has been recently demonstrated by co-culture experiments [5,6]. Moreover, the presence of the lysosomal sialidase Neu1 anchored to both lysosomal and plasma membranes has been established and an important immunoregulatory role for this enzyme has been suggested [7,8].…”