Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.
Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.
Codogno
P
P
Unité de Recherche sur la Glycobiologie et la Reconnaissance Cellulaire, INSERM U 180, UFR Biomédicale des Saints Pères, Paris, France.
Bauvy
C
C
Sève
A P
AP
Hubert
M
M
Ogier-Denis
E
E
Aubery
M
M
Hubert
J
J
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism
D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D009844:Oligosaccharides; D005643:Fucose