Nuclear proteins were extracted in 2 M NaCl from membrane-depleted nuclei isolated from HL60 cells. Extracted proteins were submitted to affinity chromatography columns containing immobilized glucose, galactose or lactose. The polypeptides present in the different eluted fractions were resolved by SDS-PAGE and were either silver stained or analysed by immunoblotting with monoclonal or polyclonal antibodies, respectively, raised against the glucose-binding protein CBP67 and the galactose-binding proteins CBP35 and L14. The results presented here show that HL60 cell nuclei contain CBP35 and a glucose-binding lectin of 70 kDa (CBP70). These data account for the previously reported binding of neoglycoproteins containing glucosyl and galactosyl residues to HL60 cell nuclei. Furthermore, the present study provides the new information that CBP35 can associate with CBP70 by interactions dependent on the binding of CBP35 to lactose, and the results of some affinity chromatography experiments strongly suggest that CBP35 and CBP70 associate by protein-protein interactions. The potential function of this lactose-mediated interaction is discussed with respect to data recently reported by others showing that CBP35 is involved in in vitro mRNA splicing and that lactose inhibits the processing of the pre-RNA substrate.
Since nuclear lectins were first characterized several years ago, six lectins have been isolated. Furthermore, the existence of nuclear glycoproteins containing N-linked complex-oligosaccharide chains or O-linked GlcNAc residues was evidenced. These latter are abundant in the nucleus and are well-studied so far. The presence of both glycoprotein and lectin in the cell nucleus led us to postulate that these two proteins could interact and play a role in some nuclear activities such as the modulation of transcription and/or nuclear cytoplasmic exchanges or by the disruption of protein-protein interactions. In such context, the recent data concerning the GlcNAc-binding activity of CBP70 argued this postulate. However, to study the possible role of a glycoprotein-lectin complex, it was critical to isolate the two partners. Because CBP70 was also a cytoplasmic protein, the lectin was isolated in both cytoplasmic and nuclear compartments in order to investigate the putative ligand in the two cellular compartments. The results obtained with cross-linking experiments on isolated and membranedepleted nuclei incubated with the CBP70 bearing an iodinatable, cleavable, photoreactive cross-linking agent (sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3'-dithiopropionate) and immunoprecipitation experiments with polyclonal antibodies raised against CBP70, revealed that both nuclear and cytoplasmic CBP70 have the same 82 kDa nuclear ligand which is absent in the cytoplasmic fraction. In addition, this ligand is glycosylated, containing GlcNAc residues, and, therefore, the complex between CBP70 and the 82 kDa polypeptide could be due to a glycoprotein-lectin interaction. These results raised the possibility that nuclear glycoprotein-lectin interaction could be involved in nuclear activities.
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