O-glycosylation of the nuclear forms of Pax-6 products in quail neuroretina cells.
Many transcription factors are demonstrated as being glycosylated with O-N-acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O-GlcNAc modification of the p48/p46 Pax-6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax-6 proteins were found in the nucleus of the neuroretina cells. WGA-selected Pax-6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O-GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N-acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax-6 primary sequence allowed us to determine potential O-GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA-selected proteins experiment points in favor of a main localization on the paired-box domain.
Lefebvre
Tony
T
Unité Mixte de Recherche 8576 du CNRS, Laboratoire de Chimie Biologique, 59655 Villeneuve d'Ascq, France.
Planque
Nathalie
N
Leleu
Denis
D
Bailly
Manuella
M
Caillet-Boudin
Marie-Laure
ML
Saule
Simon
S
Michalski
Jean-Claude
JC
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005136:Eye Proteins; D005700:Galactosyltransferases / Q000378:metabolism; D006027:Glycosides / Q000032:analysis; D006031:Glycosylation; D018398:Homeodomain Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011784:Quail; D012097:Repressor Proteins; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D002250:Carbon Radioisotopes; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D006027:Glycosides; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D014909:Wheat Germ Agglutinins; D004247:DNA; D005700:Galactosyltransferases; D000117:Acetylglucosamine