Evidence for a lactose-mediated association between two nuclear carbohydrate-binding proteins

Abstract

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.

CBP70, a glycosylated nuclear lectin

“…The nuclear protein solution or the cytoplasmic extract, unlabeled or radiolabeled with [2-3 H]mannose, was adjusted to 0.7 mM CaCl 2 and 0.5 mM MgCl 2 (PBS 1 ) and affinity chromatography was performed as previously described [Sève et al, 1993]. To obtain purified nuclear or cytoplasmic CBP70, the proteins, eluted from GlcNAc columns with 0.2 M GlcNAc, were dialyzed overnight against PBS 1 at 4°C and then incubated with glucose columns and eluted with 0.2 M glucose.…”

“…The nuclear proteins or cytoplasmic extracts were incubated with 1 ml of immobilized lectins overnight at 4°C under batch conditions. After packing in columns, the same procedure as that described above [Sève et al, 1993] was applied. The proteins specifically retained on WGA were eluted with 0.2 M GlcNAc, those retained to RCA-I were eluted with 0.2 M galactose, those bound to PNA were eluted with 0.2 M lactose, those linked to GNA were eluted with 0.3 M mannose, and then all the fractions were concentrated on Centricon-10 filters.…”

“…Six nuclear carbohydrate-binding proteins (CBP) have since been isolated: CBP35 [Roff and Wang, 1983], recently named galectin-3 [Barondes et al, 1994] and CBP14 [Cuperlovic et al, 1995], which recognize lactose; CBP67 [Schröder et al, 1992], CBP70 [Sève et al, 1993], and CBP33 [Lauc et al, 1994], which bind to glucose (Glc). CBP70 was also described as an N-acetylglucosamine (GlcNAc)-binding protein, as was CBP22 .…”

Cell Cycle Regulation by Galectin-12, a New Member of the Galectin Superfamily

“…Galectin-7, on the other hand, has been demonstrated to promote apoptosis in transfected cells (22) and may be responsible for the proapoptotic function of p53 (23). Relevant to the intracellular functions is the fact that galectins have been shown to recognize intracellular proteins, including Bcl-2 (19), nuclear lectins (24), and cytokeratins (25).…”

Galectin-3 and Regulation of Cell Function

“…Another question is the dependence of intracellular functions of galectin-3 on carbohydrate-recognition properties. There is scanty evidence for intracellular recognition of glycoconjugates by galectins with the exceptions of cytokeratin [147] and nuclear CBP70 [148]. Saccharides, particularly O-linked varieties exist in the cytoplasm and may potentially be ligands of cytosolic galectins.…”