The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

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 micros- copy analysis. In addition, administration of the radio- labeled 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 [1-3H]sphingosine allowed the character- ization 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 com- pared 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 gan- glioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those ob- served in the case of [1-3 H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside sub- strates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside sub- strates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.