Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts (original) (raw)
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Proceedings of the National Academy of Sciences, 1987
Proliferating 3T3 mouse fibroblasts contain higher levels of the lectin carbohydrate-binding protein 35 (CBP35) than do quiescent cultures of the same cells. An immunofluorescence study was carried out with a rabbit antiserum directed against CBP35 to map the cellular fluorescence distribution in a large population of cells under different growth conditions. This cytometric analysis showed that the lectin is predominantly localized in the nucleus of the proliferating cells. In quiescent 3T3 cultures, the majority of the cells lost their nuclear staining and underwent a general decrease in the overall fluorescence intensity. Stimulation ofserum-starved quiescent 3T3 cells by the addition of serum resulted in an increase in the level of CBP35. The percentage of cells showing distinct punctate intranuclear staining reached a maximum at about the same time as the onset of the first S-phase of the cell cycle. All of these results suggest that CBP35 may be a protein whose presence in the nucleus, in discrete punctate distribution, is coordinated with the proliferation state of the cell.
Biochimica et Biophysica Acta (BBA) - General Subjects, 2004
This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membranemembrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase a. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations. D
Gene, 1987
antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a 2gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing /I-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by afftnity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against /I-galactosidase.
Glycoconjugate Journal, 1987
The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cells in vitro during three to seven days of cultivation in thepresence of dimethylsulfoxide (DMSO). it was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation. The binding of wheat germ agglutinin (WGA), concanavalin A (Con A), Ricinus communis agglutinin-I (RCA-I) and Ulex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell. Cell-cell recognition employs specific reciprocal protein-carbohydrate interactions, as well as non-specific hydrophobic and ionic forces. The recognition is reciprocal in the sense that lectin-like su bstances and corresponding carbohydrate receptors may occur either on bacterial I1-3] or on mammalian [4, 5] cell membranes; either way, cell-cell contact is promoted [5, 6 I. Different glycoconjugates have come into focus as receptor molecules for bacterial timbriae (pili) which bind specifically to different short oligosaccharides [2]. The role of mammalian lectins is particularly well-studied in different liver cells which show affinity for molecules and particles displaying mannose, galactose or fucose residues [7-9]. Lectin-like activity has also been identified as important recognition markers of malignant transformation I5,
Lectin-binding sites are found in rat liver cell plasma membrane only on its extracellular surface
Journal of Cell Science, 1978
In the present study ultrastructural localization of binding sites for 5 lectins was studied in rat liver cell surface membrane fractions. For this purpose ferritin-coupled Concanavalin A, wheat germ agglutinin, soybean agglutinin, Ricinus communis agglutinin 120 and Lotus tetragonolobus agglutinin I were used as probes for mannose, N-acetyl glucosamine, N-acetyl galactosamine, galactose and fucose moieties in glycoproteins and glycolipids. Although recent reports suggest presence of glycogroups on the cytoplasmic surface of cellular membranes ultrastructural identification of membrane surfaces in the present study indicated an asymmetric localization of lectin-binding sites exclusively on the extracellular side of the membranes.
Cell, 1979
Three cell surface components of mouse embryonal carcinoma (EC) cells, F9 antigens and the receptors to the lectins FBP and PNA, have been isolated from radiolabeled EC cells by indirect immunoprecipitation. All three were efficiently labeled with fucose, galactose and glucosamine, but scarcely at all with mannose. The high molecular weight glycopeptides characteristic of early embryonic cells were released as the major glycopeptides upon pronase digestion of the three markers. The binding sites to the two lectins are present in the high molecular weight glycopeptides. Furthermore, a close correlation exists between the disappearance of the high molecular weight glycopeptides from differentiating EC cells and the disappearance of the three markers from the surface of these cells. The large glycopeptides from the three markers have the following properties in common. First, they are not mucin-type glycopeptides with short oligosaccharides, glycolipids and acidic mucopolysaccharides, nor are they products of incomplete pronase digestion of conventional complex-type glycopeptides. Second, they do not contain appreciable amounts of Fuclul+PGal or Fucal-+GGlcNAc linkages. Third, a significant fraction of the glycopeptides have the GlcNAc/&Gal sequence in their core structure. We propose that the cell surface markers of EC cells have a class of large carbohydrate chains not found in typical surface markers of adult cells such as H-2, la and LETS.