Carbohydrate-binding Protein 35 Research Articles

Rev protein suppression of complex formation between nuclear proteins and rev-responsive element-containing RNA of human immunodeficiency virus-1

The Rev protein from human immunodeficiency virus type 1 (HIV-1) is known to bind Rev responsive element (RRE) sequence of HIV-1 mRNA. This interaction is thought to enhance expression of viral structural proteins but the mechanism for this effect is uncertain. The aim of this study was to investigate (i) whether other cellular proteins also bind to the RRE sequence and (ii) whether binding of cellular proteins to RRE RNA is influenced by Rev protein. Our results revealed that a variety of RNA-protein complexes are formed when in vitro transcribed RRE-containing RNA is incubated with proteins present in HeLa nuclear extracts. The molecular masses of the most prominent bands in RNase protection assays were ∼90, 82, 67, 50, and 35–45 kDa. Addition of recombinant Rev protein suppressed the complex formation between RRE RNA and nuclear proteins. The strongest effect was observed when the RNA was preincubated with Rev before addition of HeLa proteins. Rev protein bound to the RRE-containing RNA under formation of oligomeric complexes with a Svedberg coefficient of 4.0–4.5 S. The β-galactosidespecific nuclear carbohydrate-binding protein 35 (CBP35), which is present in nuclear ribonucleoprotein (RNP) particles, was identified in the complexes formed between RRE RNA and HeLa nuclear protein. Binding of these complexes to a galactose affinity matrix was abolished by addition of Rev protein. We propose that binding of Rev protein to RRE-containing HIV transcripts prevents RNP complex formation between these transcripts and cellular proteins; this may inhibit spliceosome assembly allowing the transport of the unspliced or incompletely spliced HIV-1 mRNA into the cytoplasm. Such an effect could be expected to promote expression of viral structural proteins.

Intranuclear Distribution of Galectin-3 in Mouse 3T3 Fibroblasts: Comparative Analyses by Immunofluorescence and Immunoelectron Microscopy

The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli.

Expression of Nuclear Lectin Carbohydrate-Binding Protein 35 in Human Immunodeficiency Virus Type 1-Infected Molt-3 Cells

The nuclear carbohydrate-binding protein 35 (CBP35), a beta-galactoside-specific lectin with an M(r) of 35,000, has been identified in nuclear ribonucleoprotein complexes (RNPs) from a variety of mammalian tissues and cells. Here we determined that the expression of CBP35 mRNA greatly increases after infection of Molt-3 cells with human immunodeficiency virus type 1 (HIV-1), concomitantly with the onset of expression of the viral regulatory gene tat, and then declines. The increase in CBP35 mRNA level results in an enhanced synthesis of CBP35, as evidenced in nitrocellulose filter binding assay using radiolabeled, sugar-specific neoglycoprotein. Immunoblotting experiments showed that CBP35 is present in the 40S heterogeneous nuclear RNP complex from HIV-1-infected Molt-3 cells. CBP35 could also be detected using a novel photoreactive alpha-D-galactose probe designed for the specific detection of CBP.

Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains

The cDNA clone for carbohydrate-binding protein 35 (CBP35) was engineered into the bacterial expression vector pIN III ompA2, which directs the secretion of the expressed protein into the periplasmic space. Recombinant CBP35 was purified from this system, at a level of approximately 50 mg/liter of bacterial culture. Digestion of recombinant CBP35 with collagenase D, followed by purification using saccharide-specific affinity chromatography yielded a M(r) approximately 16,000 polypeptide, corresponding to the COOH-terminal domain (residues 118-264) of the CBP35 polypeptide. This indicates that the COOH-terminal half of CBP35 contains the carbohydrate recognition domain, consistent with its sequence homology to other S-type lectins. The NH2-terminal domain (residues 1-137) was derived by site-directed mutagenesis of the cDNA, in which stop codons are inserted in place of Gly138 and Gly139, and expression of the mutant cDNA in the same pIN III ompA2 system. The purified NH2-terminal domain failed to bind to saccharide-specific affinity resins. Differential scanning calorimetry of rCBP35 and its individual domains yielded transition temperatures of approximately 39 and approximately 56 degrees C for the NH2- and COOH-terminal domains, respectively. Lactose binding by the COOH-terminal domain shifted the transition temperature to 65 degrees C, whereas sucrose failed to yield the same effect. These results suggest that the individual domains of the CBP35 polypeptide are folded independently.

Carbohydrate-Binding Protein 35 Is the Major Cell-Surface Laminin-Binding Protein in Colon Carcinoma

The interaction of tumor cells with laminin is thought to be critical in invasion and metastasis. We found that an endogenous lectin, carbohydrate-binding protein 35 (CBP-35), is the major laminin-binding protein on human colon carcinoma cells and that its surface expression suggests involvement in metastasis. We identified CBP-35 by laminin-affinity chromatography and immunoblotting. Surface expression of CBP-35 on eight human colon carcinoma cell lines was compared by flow cytometry. Poorly differentiated cell lines and DLD-2, a signet-ring carcinoma cell line, expressed more surface CBP-35 than well-differentiated cell lines. Poorly differentiated cell lines and DLD-2 are characterized as aggressive cell lines because they adhere to and invade through reconstituted basement membrane significantly better than well-differentiated cell lines. These data suggest that CBP-35 is involved in tumor cell-basement membrane interactions and that an increase in CBP-35 surface expression may facilitate metastatic potential of colon carcinoma cells.

Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186.

Carbohydrate-binding protein 35 (CBP35), also known as the macrophage surface antigen Mac-2, is a lactosamine-specific lectin whose extracellular properties include the ability to agglutinate cells and to bind avidly to the basement membrane glycoprotein laminin. Although these and other properties would be facilitated by dimerization of this lectin, previous studies have argued against multimeric forms of this protein. We report here that macrophage CBP35, purified by laminin affinity chromatography, exists as several distinct species (Mr 35,000, 67,000, and 80,000) when analyzed under non-reducing conditions. This unexpected finding prompted us to study the biochemistry of multimerization using recombinant CBP35 (rCBP35). rCBP35 expressed in Escherichia coli forms disulfide-linked homodimers (Mr 67,000). The dimeric form of CBP35 binds to laminin with higher affinity than does monomer and by a lactosamine-dependent mechanism. Site-directed mutagenesis indicated that cysteine 186, the single cysteine residue in CBP35, is required for dimerization. These results raise the possibility that homo- and heterodimeric forms of CBP35 contribute to its postulated functions in cell-matrix interactions and growth regulation.

Expression of carbohydrate binding protein 35 in human fibroblasts: Variations in the levels of mRNA, protein, and isoelectric species as a function of replicative competence

We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to ~18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (p I 8.7) protein, as well as the phosphorylated (p I 8.2) derivative. In contrast, older (passage 27–35, 55–68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (p I 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.

Carbohydrate-binding protein 35. Isoelectric points of the polypeptide and a phosphorylated derivative.

Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.

Carbohydrate-binding Protein 35: Levels of transcription and mRNA accumulation in quiescent and proliferating cells

In previous studies, we observed proliferation-dependent expression and nuclear localization of the lectin, designated carbohydrate-binding protein 35 (CBP35), in 3T3 fibroblasts at the polypeptide level by Western blot and immunofluorescence analysis. In the present study, we have compared the expression of the CBP35 gene in quiescent and proliferative 3T3 cells at the levels of (a) accumulated mRNA by Northern blotting and (b) nuclear transcription by run-off assays. When serum-starved, quiescent cultures of 3T3 cells were stimulated by the addition of serum, there was an increase in the nuclear transcription of the CBP35 gene and in the accumulation of its mRNA early (1–3 h) in the activation process, well before the first wave of DNA synthesis. These increases were not dependent on de novo protein synthesis inasmuch as they occurred even in the presence of cycloheximide. There was also an elevated transcription rate and mRNA level in transformed cells when compared to their normal counterparts. Finally, the expression of CBP35 was compared between sparse, proliferating cultures of 3T3 cells and density-inhibited confluent monolayers of the same cells. Although the rate of transcription of the CBP35 gene was approximately the same in the two cultures, there was a higher level of CBP35 mRNA in the dense cells. Thus, it appears that posttranscriptional mechanisms may be involved in the accumulation of mRNA.

Expression of carbohydrate binding protein 35 in human fibroblasts: Comparisons between cells with different proliferative capacities

Carbohydrate Binding Protein 35 (CBP35) is a galactose-specific lectin found in the nucleus and cytoplasm of mouse 3T3 fibroblasts. In these cultures, the level of expression and nuclear localization of CBP35 was correlated with the proliferative state of the cells. CBP35 is also found in human fibroblasts. We have compared the expression and localization of CBP35 in human fibroblasts of different replicative capacities: young (passage 11), intermediate (passage 19), and old (passage 33) SL66 cells, and fibroblasts derived from a patient with Werner's syndrome. The results indicate that the expression of CBP35 in cells with either age-acquired or congenital replicative deficiencies was unresponsive to serum stimulation, in contrast with that found in young normal human fibroblasts and in 3T3 cells.

Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex

In previous studies, a lectin designated as carbohydrate binding protein 35 (CBP35) was identified in the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts. In the present study, we observed that treatment of Triton X-100 permeabilized 3T3 cells with ribonuclease A released CBP35 from the nuclei, while parallel treatment with deoxyribonuclease I failed to do so. This conclusion was based on (a) immunofluorescence analysis of the nuclear residue after detergent and enzymatic treatments and (b) immunoblotting analysis of the supernatant fraction produced by these treatments. These results indicate that CBP35 may be associated with the ribonucleoprotein elements of the 3T3 cell nuclei. In corroboration with this conclusion, fractionation of the nucleoplasm derived from 3T3 cells on a cesium sulfate gradient (1.25-1.75 g/mL) localized CBP35 in fractions with densities of 1.30-1.32 g/mL, corresponding to the range of densities reported for heterogeneous nuclear ribonucleoprotein complex (hnRNP). Conversely, when nucleoplasm was fractionated on an affinity column of Sepharose derivatized with N-(epsilon-aminocaproyl)-D-galactosamine, the bound and eluted fraction contained RNA, as well as a set of polypeptides whose molecular weights matched those reported for the core particle of hnRNP. One of these polypeptides was identified as CBP35. These results suggest that CBP35 is a component of hnRNP.

Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.

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 of serum-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.

Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a λgt11 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 β-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 affinity 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 β-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts.

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, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.