Evidence that lactose binding to CBP35 disrupts its interaction with CBP70 in isolated HL60 cell nuclei.
We have previously reported that two carbohydrate-binding proteins (CBP35 and CBP70) can, under appropriate conditions of affinity chromatography, be isolated from HL60 cell nuclear extracts as a complex. Moreover, we have demonstrated that, during affinity chromatography, the CBP70-CBP35 association can be modified by the binding of lactose to CBP35. To determine whether the CBP70-CBP35 association could be disrupted in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, incubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that was specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immunofluorescent staining, immunoblotting experiments, and quantitative flow-cytofluorometric analysis show that (i) CBP70 and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuclei, and (iii) glucose failed to have the same effect as lactose. These results strongly suggest that, in membrane-depleted nuclei, CBP35 and CBP70 interactions can be altered by a conformational change of CBP35 induced by the binding of lactose to its carbohydrate-recognition domain.
Sève
A P
AP
Laboratoire de Glycobiologie et de Reconnaissance Cellulaire, INSERM U180, UFR Biomédicale des Saints-Pères, (Université René Descartes, Paris V, France.
Hadj-Sahraoui
Y
Y
Felin
M
M
Doyennette-Moyne
M A
MA
Aubery
M
M
Hubert
J
J
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000943:Antigens, Differentiation / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D004273:DNA, Neoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D037502:Galectin 3; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007785:Lactose / Q000378:metabolism*; D015473:Leukemia, Promyelocytic, Acute; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D037502:Galectin 3; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; D007785:Lactose