An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum.
To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated.
Jung
E
E
MUCAB (Macquarie University Center for Analytical Biotechnology), School of Biological Sciences, Macquarie University, Sydney, Australia.
Gooley
A A
AA
Packer
N H
NH
Slade
M B
MB
Williams
K L
KL
Dittrich
W
W
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D004023:Dictyostelium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000378:metabolism; D005786:Gene Expression Regulation; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000032:analysis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis
D000911:Antibodies, Monoclonal; D009005:Monosaccharides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016695:Glycosyltransferases; D015951:Factor Xa; D000117:Acetylglucosamine