Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts (original) (raw)
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The Journal of Cell Biology, 1986
In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet,...
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
Binding of lectins to mitotic chromosomes and interphase nuclear substructures
Cell biology international reports, 1980
We have investigated binding of the lectins wheat germ agglutin (WGA) and concanavalin A (Con-A) to mitotic chromosomes and interphase nuclear substructures using fluorescein and rhodamineconjugated lectins. Standard cytogenetic preparations of human lymphocyte chromosomes were incubated with WGA. Mitotic chromosomes and interphase nuclei are brightly fluorescent. Con-A did not detectably bind chromosomes prepared in this way. Mitotic chromosomes display a distinctive banding pattern with WGA similar to quinacrine banding. We also studied binding to interphase "nuclear scaffolds", which are prepared by treating isolated Hela cell nuclei with DNAse 1 and high salt. WGA binds nuclear scaffolds internally while Con-A binds to the periphery. The specific binding of lectins to chromosomal structures derived from these two different methods indicates that glycosides are true chromosomal components and that glycoproteins may have a role in chromosome organization.
Biochemical and Biophysical Research Communications, 1985
Immunofluorescenoe and Immunoblottlng experiments, using a monoclonal antibody to the 13 kDa mammalian .8-galactoside-binding lectin have shown that human lymphocytes contain nuclear and cytoplasmic proteins of apparent molecular masses of 130, 80, 65 and 13 kDa that are antlgonically related to the lectin and whose levels and patterns of expression change in association with transformation, or after stimulation with mitogens. These observations, together with the finding that the myelold cell line K562 is also rich in the ]30 kDa component, whereas the mature granulocytes of normal donors and of patients with chronic myeloid leukaemia are lacking in all of the Immunoreactlve forms, raise the possibility that this family of lectin-related proteins may be components of growth regulatory systems that are variously elicited in the transformed and stimulated cells.
Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores
The Journal of Cell Biology, 1987
Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin.
Journal of cell science, 1982
Changes in the cell and nuclear morphology of sparsely plated WI38 fibroblasts were followed as a function of time after increasing the serum concentration from 0.3% to 10%. Quantitative measurements were carried out in parallel on Feulgen-stained nuclei and Wright-stained cells using the Quantimet 720-D image analysis system. We report a rapid, significant change in nuclear morphology indicative of nuclear rounding taking place within 30 min after increasing the serum concentration. In contrast, cell morphology showed only a slight change within the first 30 min but showed a significant change, also indicative of cell rounding, between 30 min and 3 h after increasing the concentration. Thus our results indicate a coupling between cell and nuclear morphology, but one in which nuclear changes precede cellular changes. As variations in both cell and nuclear morphology have been linked to the control of cell growth and transformation we also discuss briefly the implications of our resu...
Journal of cell science, 1989
Undifferentiated F9 embryonal carcinoma (EC) cells bound fluorochrome-coupled Helix pomatia agglutinins (HPA) and peanut agglutinins (PNA) homogeneously, but were distinctly heterogeneous in their binding of Dolichos biflorus agglutinin (DBA) conjugates. Upon chemically induced differentiation the proportion of cells binding the DBA conjugates increased, but a distinct heterogeneity in the intensity of binding remained among the parietal endoderm (PE)-like F9 derivatives. These cells were heterogeneous in their binding of HPA conjugates as well, and many of them failed to bind PNA conjugates, apparently due to sialylation of the PNA-binding sites. Electrophoretic analysis of lectin-binding glycoproteins in the detergent-soluble fraction of the cells revealed the appearance of a doublet of polypeptides of Mr 300,000-400,000 upon differentiation induced by retinoic acid (RA). In addition, an Mr 220,000 polypeptide appeared upon differentiation induced by RA and dibutyryl cyclic AMP (d...
Biosynthesis of nuclear proteins after stimulation of quiescent Swiss mouse 3T3 cells
Journal of Cell Science, 1986
Stimulation of quiescent Swiss mouse 3T3 fibroblasts either by serum or by the low molecular weight hormones, prostaglandin F2 alpha and insulin, induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or these pure hormones there is an overall increase of two- to fourfold in the rate of biosynthesis of nuclear proteins. In addition, there is a relative decrease in some proteins (Mr = 200 X 10(3), pI 6.0-6.5), while others increase (e.g. actin). Two polypeptides show specific correlations with the exit from G0. The synthesis of p30 (Mr = 30 X 10(3), pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (Mr = 36 X 10(3), pI = 4.25) is first seen at 10–20 h after restimulation. Synthesis of p36 correlates well with the initiation of DNA synthesis. The synthesis of both proteins is stimulated by serum and by the hormones. Thus there are common biosynthetic responses to different stimuli indicating convergent pathways leading to...
Journal of Biological Chemistry, 1999
The Gal1-3GalNAc␣ (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein-and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 g/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 g/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.