Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases

Erhard Bieberich, Sarah MacKinnon, Maria J Silva, Donna D. Li, Tewin Tencomnao, Louis Irwin, Dmitri Kapitonov, Robert K Yu

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)11479-11487
Number of pages9
JournalBiochemistry
Volume41
Issue number38
DOIs
StatePublished - Sep 24 2002

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Glycosyltransferases
Gangliosides
Biosynthesis
Enzymes
Transfection
Fusion reactions
N-Acetylgalactosaminyltransferases
Sialyltransferases
Thin layer chromatography
Fluorescence microscopy
Thin Layer Chromatography
Green Fluorescent Proteins
Neuroblastoma
Fluorescence Microscopy
Immunoprecipitation
Detergents
Assays
Proteins
Chemical analysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. / Bieberich, Erhard; MacKinnon, Sarah; Silva, Maria J; Li, Donna D.; Tencomnao, Tewin; Irwin, Louis; Kapitonov, Dmitri; Yu, Robert K.

In: Biochemistry, Vol. 41, No. 38, 24.09.2002, p. 11479-11487.

Research output: Contribution to journalArticle

Bieberich, E, MacKinnon, S, Silva, MJ, Li, DD, Tencomnao, T, Irwin, L, Kapitonov, D & Yu, RK 2002, 'Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases', Biochemistry, vol. 41, no. 38, pp. 11479-11487. https://doi.org/10.1021/bi0259958
Bieberich E, MacKinnon S, Silva MJ, Li DD, Tencomnao T, Irwin L et al. Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. Biochemistry. 2002 Sep 24;41(38):11479-11487. https://doi.org/10.1021/bi0259958
Bieberich, Erhard ; MacKinnon, Sarah ; Silva, Maria J ; Li, Donna D. ; Tencomnao, Tewin ; Irwin, Louis ; Kapitonov, Dmitri ; Yu, Robert K. / Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases. In: Biochemistry. 2002 ; Vol. 41, No. 38. pp. 11479-11487.
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