Ganglioside glycosyltransferases are S-acylated at conserved cysteine residues involved in homodimerisation

Biochem J. 2017 Aug 7;474(16):2803-2816. doi: 10.1042/BCJ20170124.

Abstract

Ganglioside glycosyltransferases (GGTs) are type II membrane proteins bearing a short N-terminal cytoplasmic tail, a transmembrane domain (TMD), and a lumenal catalytic domain. The expression and activity of these enzymes largely determine the quality of the glycolipids that decorate mammalian cell membranes. Many glycosyltransferases (GTs) are themselves glycosylated, and this is important for their proper localisation, but few if any other post-translational modifications of these proteins have been reported. Here, we show that the GGTs, ST3Gal-V, ST8Sia-I, and β4GalNAcT-I are S-acylated at conserved cysteine residues located close to the cytoplasmic border of their TMDs. ST3Gal-II, a GT that sialylates glycolipids and glycoproteins, is also S-acylated at a conserved cysteine located in the N-terminal cytoplasmic tail. Many other GTs also possess cysteine residues in their cytoplasmic regions, suggesting that this modification occurs also on these GTs. S-acylation, commonly known as palmitoylation, is catalysed by a family of palmitoyltransferases (PATs) that are mostly localised at the Golgi complex but also at the endoplasmic reticulum (ER) and the plasma membrane. Using GT ER retention mutants, we found that S-acylation of β4GalNAcT-I and ST3Gal-II takes place at different compartments, suggesting that these enzymes are not substrates of the same PAT. Finally, we found that cysteines that are the target of S-acylation on β4GalNAcT-I and ST3Gal-II are involved in the formation of homodimers through disulphide bonds. We observed an increase in ST3Gal-II dimers in the presence of the PAT inhibitor 2-bromopalmitate, suggesting that GT homodimerisation may be regulating S-acylation.

Keywords: S-acylation; gangliosides; glycosyltransferases; palmitoylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cell Line
  • Conserved Sequence
  • Cricetulus
  • Cysteine / metabolism
  • Dimerization
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Mutation
  • N-Acetylgalactosaminyltransferases / chemistry
  • N-Acetylgalactosaminyltransferases / genetics
  • N-Acetylgalactosaminyltransferases / metabolism*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Phylogeny
  • Protein Interaction Domains and Motifs
  • Protein Processing, Post-Translational*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sialyltransferases / chemistry
  • Sialyltransferases / genetics
  • Sialyltransferases / metabolism*

Substances

  • Luminescent Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • N-Acetylgalactosaminyltransferases
  • beta-1,4-N-acetyl-galactosaminyl transferase 1, human
  • Sialyltransferases
  • beta-galactoside alpha-2,3-sialyltransferase
  • alpha-N-acetylneuraminate alpha-2,8-sialyltransferase
  • haematoside synthetase
  • Cysteine