Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis

Glycobiology. 2015 Jan;25(1):55-65. doi: 10.1093/glycob/cwu089. Epub 2014 Aug 25.

Abstract

N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

Keywords: GALNT; ISOGlyP; MALDI-TOF; NetOGlyc4.0; SimpleCell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbohydrate Sequence
  • Enzyme Assays
  • Gene Expression Regulation
  • Glycomics
  • Glycosylation
  • Golgi Apparatus / chemistry
  • Golgi Apparatus / metabolism
  • Humans
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Molecular Sequence Data
  • N-Acetylgalactosaminyltransferases / chemistry*
  • N-Acetylgalactosaminyltransferases / genetics
  • N-Acetylgalactosaminyltransferases / metabolism
  • Peptides / chemical synthesis
  • Peptides / chemistry*
  • Polysaccharides / chemistry*
  • Polysaccharides / metabolism
  • Proteomics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Isoenzymes
  • Peptides
  • Polysaccharides
  • Recombinant Proteins
  • N-Acetylgalactosaminyltransferases
  • polypeptide N-acetylgalactosaminyltransferase