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

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 an...

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Bibliographic Details
Published in:	Glycobiology (Oxford) 2015-01, Vol.25 (1), p.55-65
Main Authors:	Kong, Yun, Joshi, Hiren J, Schjoldager, Katrine Ter-Borch Gram, Madsen, Thomas Daugbjerg, Gerken, Thomas A, Vester-Christensen, Malene B, Wandall, Hans H, Bennett, Eric Paul, Levery, Steven B, Vakhrushev, Sergey Y, Clausen, Henrik
Format:	Article
Language:	eng
Subjects:	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 Original Peptides - chemical synthesis Peptides - chemistry Polypeptide N-acetylgalactosaminyltransferase Polysaccharides - chemistry Polysaccharides - metabolism Proteomics Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Substrate Specificity
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Summary:	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.
ISSN:	0959-6658 1460-2423