Role of plasma membrane-bound sialidase NEU3 in clathrin-mediated endocytosis

RODRIGUEZ WALKER M.; VILCAES A.; DANIOTTI J.L.

Philadelphia

Congreso; Annual Meeting of The American Society for Cell Biology; 2014

The American Society for Cell Biology (USA)

Gangliosides (GS) are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. They have been implicated in many physiological and pathological processes, e.g. in cell growth, cell adhesion and endocytosis; including its capacity to function as receptors for several toxins, viruses and antibodies. GS are synthesized at the Golgi complex, but recently a number of enzymes of GS anabolism and catabolism have been shown to be associated with the plasma membrane. NEU3, a key plasma membrane-bound sialidase for GS hydrolysis, has been implicated in similar cellular processes as GS, and its up-regulation has been observed in various human cancer cells. In the case of clathrin-mediated endocytosis, although this has been widely studied, the specific role of GS and NEU3 in this cellular process has not yet been well established. Using biochemical assays and confocal microscopy imaging, we studied the role of GS and NEU3 in different endocytic processes. Initially, we observed an increase in the internalization of transferrin (Tf), the archetypical cargo for internalization through clathrin-mediated endocytosis, in cell lines expressing GS with high level of sialylation. The ectopic expression of NEU3 led to a drastic decrease in Tf endocytosis, suggesting a participation of GS in this process. However, expression of NEU3 in GS-depleted cells only slightly affected its inhibition on Tf endocytosis, indicating that NEU3 modifies the internalization of Tf independently of its action on GS. Additionally, the internalization of low-density lipoprotein, another typical ligand in clathrin-mediated endocytosis, was also decreased in NEU3-overexpressing cells. In contrast, internalization of cholera toxin β-subunit, which is endocytosed by both clathrin-dependent and clathrin-independent mechanisms, remained unaltered. Kinetic assays carried out on NEU3 expressing cells revealed a significant reduction in the sorting of Tf to early and recycling endosomes, without significant changes recorded in the expression level of the Tf receptor. We found that NEU3 expression altered the subcellular distribution of clathrin adaptor AP-2 σ2 subunit, but did not reveal any changes in the membrane distribution of clathrin or phosphatidylinositol (4, 5)-bisphosphate. Finally, NEU3 did not alter the distribution of caveolin-1. Overall, these results suggest a specific and novel role of NEU3 in clathrin-mediated endocytosis.