title: Trans-activity of plasma membrane-associated ganglioside sialyltransferase in mammalian cells.
abstract: Gangliosides are acidic glycosphingolipids that contain sialic acid residues and are expressed in nearly all vertebrate cells. They are synthesized at the Golgi complex by a combination of glycosyltransferase activities followed by vesicular delivery to the plasma membrane, where they participate in a variety of physiological as well as pathological processes. Recently, a number of enzymes of ganglioside anabolism and catabolism have been shown to be associated with the plasma membrane. In particular, it was observed that CMP-NeuAc:GM3 sialyltransferase (Sial-T2) is able to sialylate GM3 at the plasma membrane (cis-catalytic activity). In this work, we demonstrated that plasma membrane-integrated ecto-Sial-T2 also displays a trans-catalytic activity at the cell surface of epithelial and melanoma cells. By using a highly sensitive enzyme-linked immunosorbent assay combined with confocal fluorescence microscopy, we observed that ecto-Sial-T2 was able to sialylate hydrophobically or covalently immobilized GM3 onto a solid surface. More interestingly, we observed that ecto-Sial-T2 was able to sialylate GM3 exposed on the membrane of neighboring cells by using both the exogenous and endogenous donor substrate (CMP-N-acetylneuraminic acid) available at the extracellular milieu. In addition, the trans-activity of ecto-Sial-T2 was considerably reduced when the expression of the acceptor substrate was inhibited by using a specific inhibitor of biosynthesis of glycolipids, indicating the lipidic nature of the acceptor. Our findings provide the first direct evidence that an ecto-sialyltransferase is able to trans-sialylate substrates exposed in the plasma membrane from mammalian cells, which represents a novel insight into the molecular events that regulate the local glycosphingolipid composition.
authors.affiliation: Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
authors.forename: Aldo A
authors.initials: AA
authors.lastname: Vilcaes
authors.forename: Vanina Torres
authors.initials: VT
authors.lastname: Demichelis
authors.forename: Jose L
authors.initials: JL
authors.lastname: Daniotti
mesh_terms: D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002645:Chickens; D004285:Dogs; D005679:G(M3) Ganglioside / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*
chemical_list: D005679:G(M3) Ganglioside; D005732:Gangliosides; D008565:Membrane Proteins; D012794:Sialic Acids; C026226:ganglioside, GD3; D012799:Sialyltransferases