Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis
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Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis. / 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.
In: Glycobiology, Vol. 25, No. 1, 01.2015, p. 55-65.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis
AU - Kong, Yun
AU - Joshi, Hiren J
AU - Schjoldager, Katrine Ter-Borch Gram
AU - Madsen, Thomas Daugbjerg
AU - Gerken, Thomas A
AU - Vester-Christensen, Malene B
AU - Wandall, Hans H
AU - Bennett, Eric Paul
AU - Levery, Steven B
AU - Vakhrushev, Sergey Y
AU - Clausen, Henrik
N1 - © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
PY - 2015/1
Y1 - 2015/1
N2 - 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.
AB - 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.
U2 - 10.1093/glycob/cwu089
DO - 10.1093/glycob/cwu089
M3 - Journal article
C2 - 25155433
VL - 25
SP - 55
EP - 65
JO - Glycobiology
JF - Glycobiology
SN - 0959-6658
IS - 1
ER -
ID: 129784266