Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum: In vivo studies on glycosylation of mucin MUC1 and MUC2 repeats

Eva Jung, Andrew A. Gooley, Nicolle H. Packer, Peter Karuso, Keith L. Williams

Research output: Contribution to journalArticleResearchpeer-review

Abstract

One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Set and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc:polypeptide N- acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in a configuration as determined by NMR studies.

LanguageEnglish
Pages517-524
Number of pages8
JournalEuropean Journal of Biochemistry
Volume253
Issue number2
Publication statusPublished - 15 Apr 1998

Fingerprint

Glycosylation
Dictyostelium
Acetylglucosamine
Mucins
Peptides
Proteins
Electrospray ionization
Uridine Diphosphate
Electrospray Ionization Mass Spectrometry
Glycopeptides
Monosaccharides
Protein Sequence Analysis
Glutathione Transferase
Eukaryota
Mass spectrometry
Membrane Proteins
Fusion reactions
Nuclear magnetic resonance
Tissue
Membranes

Keywords

  • Acceptor site
  • Dictyostelium discoideum
  • O-glycosylation
  • O-linked N- acetylglucosamine

Cite this

@article{76858caeaad04b8aaaa50bc154663400,
title = "Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum: In vivo studies on glycosylation of mucin MUC1 and MUC2 repeats",
abstract = "One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Set and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc:polypeptide N- acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in a configuration as determined by NMR studies.",
keywords = "Acceptor site, Dictyostelium discoideum, O-glycosylation, O-linked N- acetylglucosamine",
author = "Eva Jung and Gooley, {Andrew A.} and Packer, {Nicolle H.} and Peter Karuso and Williams, {Keith L.}",
year = "1998",
month = "4",
day = "15",
language = "English",
volume = "253",
pages = "517--524",
journal = "FEBS Journal",
issn = "1742-4658",
publisher = "Wiley-Blackwell, Wiley",
number = "2",

}

Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum : In vivo studies on glycosylation of mucin MUC1 and MUC2 repeats. / Jung, Eva; Gooley, Andrew A.; Packer, Nicolle H.; Karuso, Peter; Williams, Keith L.

In: European Journal of Biochemistry, Vol. 253, No. 2, 15.04.1998, p. 517-524.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum

T2 - FEBS Journal

AU - Jung, Eva

AU - Gooley, Andrew A.

AU - Packer, Nicolle H.

AU - Karuso, Peter

AU - Williams, Keith L.

PY - 1998/4/15

Y1 - 1998/4/15

N2 - One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Set and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc:polypeptide N- acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in a configuration as determined by NMR studies.

AB - One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Set and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc:polypeptide N- acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in a configuration as determined by NMR studies.

KW - Acceptor site

KW - Dictyostelium discoideum

KW - O-glycosylation

KW - O-linked N- acetylglucosamine

UR - http://www.scopus.com/inward/record.url?scp=0032522587&partnerID=8YFLogxK

M3 - Article

VL - 253

SP - 517

EP - 524

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-4658

IS - 2

ER -