Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases.
Three key regulatory enzymes in ganglioside biosynthesis, sialyltransferase I (ST1), sialyltransferase II (ST2), and N-acetylgalactosaminyltransferase I (GalNAcT), have been expressed as fusion proteins with green, yellow, or red fluorescent protein (GFP, YFP, or RFP) in F-11A cells. F-11A cells are a substrain of murine neuroblastoma F-11 cells that contain only low endogenous ST2 and GalNAcT activity. The subcellular localization of the fusion proteins has been determined by fluorescence microscopy, and the ganglioside composition of these cells was analyzed by high-performance thin-layer chromatography (HPTLC). ST2-GFP (85 kDa) shows a distinct Golgi localization, whereas ST1-YFP (85 kDa) and GalNAcT-RFP (115 kDa) are broadly distributed in ER and Golgi. Untransfected F-11A cells contain mainly GM3, whereas stable transfection with ST2 or GalNAcT results in the predominant expression of b-series complex gangliosides (BCGs). This result indicates that the expression of ST2 enhances the activity of endogenous GalNAcT and vice versa. The specificity of this reaction has been verified by in vitro activity assays with detergent-solubilized enzymes, suggesting the formation of an enzyme complex between ST2 and GalNAcT but not with ST1. Complex formation has also been verified by co-immunoprecipitation of ST2-GFP upon transient transfection with GalNAcT-HA-RFP and by GFP-to-RFP FRET signals that are confined to the Golgi. FRET analysis also suggests that ST2-GFP binds tightly to pyrene-labeled GM3 but not to ST1. We hypothesize that an ST2-GM3 complex is associated with GalNAcT, resulting in the enhanced conversion of GM3 to GD3 and BCGs in the Golgi. Taken together, our results support the concept that ganglioside biosynthesis is tightly regulated by the formation of glycosyltransferase complexes in the ER and/or Golgi.
Bieberich
Erhard
E
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA. ebieberich@mail.mcg.edu
MacKinnon
Sarah
S
Silva
Jeane
J
Li
Donna D
DD
Tencomnao
Tewin
T
Irwin
Louis
L
Kapitonov
Dmitri
D
Yu
Robert K
RK
D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D018920:Coculture Techniques; D017931:DNA Primers; D018076:DNA, Complementary; D005727:Ganglia, Spinal / Q000378:metabolism; D005732:Gangliosides / Q000096:biosynthesis*; D017930:Genes, Reporter; D016695:Glycosyltransferases / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006706:Homeostasis; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D009447:Neuroblastoma; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D014162:Transfection; D014312:Trisaccharides / Q000096:biosynthesis / Q000737:chemistry; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D005732:Gangliosides; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; D049452:Green Fluorescent Proteins; D016695:Glycosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase