Golgi Phosphoprotein 3 mediates the association between β3GalT-IV and ST3Gal-II glycosyltransferases

MARTÍNEZ-KOTESKI, NATALIA; RUGGIERO, FERNANDO M.; CAVIERES, VIVIANA A ; MARDONES, GONZALO ; FIDELIO, GERARDO D.; VILCAES, A. ALEJANDRO

Congreso; SAIB 2022; 2022

SAIB

Glycosphingolipids (GSL) are sialylated glycolipids located on the surface of eukaryotic cells. There they modulate the activities of membrane proteins, regulating several physiological processes. The expression levels of GSLs are specific to the cell type and changes in the glycosylation of these glycolipids are a hallmark of tumors that contributes to their development and progression. Recently it has been proposed that the Golgi oncoprotein GOLPH3 could regulate the synthesis of GSL by modulating the retention of glycolipid´s glycosiltransferases in Golgi. A feature that might contribute to GOLPH3 oncogenic trait. In the present work we analyzed the effect of GOLPH3 downregulation in the metabolism of complex sialylglycolipids. We observed by high performance thin-layer chromatography, flow cytometry and immunofluorescence that GOLPH3 knockdown (KD) in a glioblastoma cell line, produces a downregulation of GD1a ganglioside and an increase of GM1 ganglioside. Similar results were also obtained in a breast cancer cell line, suggesting that GOLPH3 may have a general role in glycolipid metabolism in multiple tumor types. By immunofluorescence we noticed that GOLPH3 depletion did not affect the subcellular localization of β3GalT-IV-HA-YFP (GM1 synthase) and ST3Gal-II-mCherry (GD1a synthase). Also, low levels of GOLPH3 did not alter the molecular weight of N-glycosylated ST3Gal-II, meaning that GOLPH3 levels are not involved in N-glycosylation of ST3Gall-II. Hence, the down regulation of GD1a in GOLPH3 KD cells may noy be attributable to a lack of posttranslational modifications of ST3GalII. Previously, it has been shown that the formation of complexes between certain glycosyltransferases, determines the channeling of substrates, thus the product of one enzyme is preferentially used by the second one and not a competing glycosyltransferase. This prompted us to ask whether there is a physical association between β3GalT-IV and ST3Gal-II and if it is modulated by GOLPH3 levels. By immunoprecipitation and FRET assays on control and GOLPH3 KD cells we show for the first time that β3GalT-IV and ST3Gal-II form a complex that depends on GOLPH3 expression. This association would be responsible for enhancing the synthesis of GD1a from GM1. Our results suggest a novel mode of regulation of glycolipids synthesis, where GOLPH3 plays a crucial role