Sève A P, Hadj-Sahraoui Y, Felin M, Doyennette-Moyne M A, Aubery M, Hubert J
Laboratoire de Glycobiologie et de Reconnaissance Cellulaire, INSERM U180, UFR Biomédicale des Saints-Pères, (Université René Descartes, Paris V, France.
Exp Cell Res. 1994 Jul;213(1):191-7. doi: 10.1006/excr.1994.1190.
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.
我们之前报道过，在适当的亲和层析条件下，两种碳水化合物结合蛋白（CBP35和CBP70）可作为复合物从HL60细胞核提取物中分离出来。此外，我们还证明，在亲和层析过程中，乳糖与CBP35的结合可改变CBP70 - CBP35的结合。为了确定乳糖与CBP35结合后，CBP70 - CBP35的结合在细胞核中是否会被破坏，我们分析了在有无乳糖孵育的HL60细胞膜去除细胞核中CBP70和CBP35的行为。本研究使用了与CBP35和CBP70交叉反应的抗血清、专门针对CBP70产生的抗血清以及对CBP35有反应的单克隆抗体（Mac 2）。综合间接免疫荧光染色、免疫印迹实验和定量流式细胞荧光分析的结果表明：（i）在无乳糖孵育的细胞核中存在CBP70和CBP35且它们共定位；（ii）在有乳糖存在的情况下孵育时，所有CBP70分子离开细胞核，而可能与RNA结合的CBP35分子仍留在细胞核内；（iii）葡萄糖没有与乳糖相同的作用。这些结果强烈表明，在膜去除的细胞核中，乳糖与其碳水化合物识别结构域的结合所诱导的CBP35构象变化可改变CBP35和CBP70的相互作用。
