Modulation of epidermal growth factor receptor phosphorylation by endogenously expressed gangliosides.

The effect of changing the ganglioside composition of Chinese hamster ovary K1 cells on the function of the epidermal growth factor receptor (EGFr) was examined by studying the signalling pathway generated after the binding of epidermal growth factor (EGF) both in cells depleted of glycosphingolipids by inhibiting glucosylceramide synthase activity and in cell lines expressing different gangliosides as the result of stable transfection of appropriate ganglioside glycosyltransferases. After stimulation with EGF, cells depleted of glycolipids showed EGFr phosphorylation and extracellular signal-regulated protein kinase 2 (ERK2) activity as parental cells expressing GM3 [ganglioside nomenclature follows Svennerholm (1963) J. Neurochem. 10, 613-623] or as transfected cells expressing mostly GM2 and GD1a as the result of stable transfection of UDP-GalNAc:LacCer/GM3/GD3 N-acetylgalactosaminyltransferase. However, cells stably transfected with CMP-NeuAc:GM3 sialyltransferase and expressing GD3 at the cell surface showed both decreased EGFr phosphorylation and ERK2 activation after stimulation with EGF. Results suggest that changes in the ganglioside composition of cell membranes might be important in the regulation of the EGF signal transduction.

GM3 alpha2,8-sialyltransferase (GD3 synthase): protein characterization and sub-golgi location in CHO-K1 cells.

GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in concert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope-tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expressed protein was enzymatically active both in vitro and in vivo and showed a molecular mass of approximately 47 or approximately 95 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of, respectively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impaired glycosylation, indicating that it was formed in the ER. Confocal immunofluorescence microscopy showed Sial-T2 localized to the Golgi complex and, within the organelle, partially co-localizing with the mannose-6-phosphate receptor, a marker of the trans-Golgi network (TGN). In cells treated with brefeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overloading. In experiments of incorporation of sugars into endogenous acceptors of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-NeuAc were converted to GD2 upon incubation with UDP-GalNAc. These results indicate that Sial-T2 localizes mainly to the proximal Golgi, although a fraction is located in the TGN functionally coupled to GalNAc-T. Consistent with this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking approximately 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.

Mouse beta 1,3-galactosyltransferase (GA1/GM1/GD1b synthase): protein characterization, tissue expression, and developmental regulation in neural retina.

The composition of cell surface gangliosides is largely dependent on the relative activities of Golgi resident glycosyltransferases. In the brain of birds and mammals, complex gangliosides (GM2, GM1, GD1a, GD1b, GT1b) abound at late stages of development and in the adult, due to the relatively high activities of the UDP-GalNAc:LacCer/GM3/GD3 beta 1,4-N-acetylgalactosaminyltransferase (GalNAc-T) and the UDP-Gal: GA2/GM2/GD2 beta 1, 3-galactosyltransferase (Gal-T2) relative to that of CMP-NeuAc:GM3 alpha 2,8-sialyltransferase (Sial-T2). Unlike brain, the mature mammalian neural retina abundantly expresses the simple ganglioside GD3, in relation to complex gangliosides, due to the low activity of GalNAc-T and Gal-T2 relative to Sial-T2. Here we describe the isolation and characterization of a mouse Gal-T2 cDNA that drives the synthesis of an epitope-tagged protein of molecular mass 43 kDa, which was enzymatically active and localized to the Golgi complex in transfected cell lines. Using this cDNA as a probe, it was found that Gal-T2 is coded by a single gene located in chromosome 17, and that the coding sequence is contained in a single exon. The expression of the specific Gal-T2 mRNA (approximately 1.8 kb) was highest in testis, which also showed elevated Gal-T2 activity. In the postnatal neural retina, Gal-T2 mRNA increased after day 3, maintained high levels of expression by days 4-7, and then decreased to initial values by day 10. The developmental pattern of mRNA expression was temporally coincident with that of Gal-T2 activity expression, indicating that this enzyme is under transcriptional control in the neural retina.