Showing papers on "Galectin published in 1994"

Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides.

Abstract: Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.

Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion.

Abstract: Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29–35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissuesin vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cellsin vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.

Concomitant Increases in Galectin-1 and Its Glycoconjugate Ligands (Carcinoembryonic Antigen, Lamp-1, and Lamp-2) in Cultured Human Colon Carcinoma Cells by Sodium Butyrate

Abstract: Galactoside-binding lectins (galectins) with molecular masses of about 14.5 kilodaltons (galectin-1) and 31 kilodaltons (galectin-3) have been found in a variety of normal and malignant cells and have been implicated in the regulation of cell growth, cell adhesion, and metastasis. The KM12 human colon carcinoma cell line was found to express only galectin-3. Because the levels of both galectins are developmentally regulated and can be modulated during the differentiation of several cultured tumor cell lines, we studied the ability of 11 differentiation-inducing agents to induce galectin-1 expression in the KM12 cells. Treatment of these cells with sodium butyrate, an established differentiation-inducing agent for colon carcinoma cells, resulted in the induction of galectin-1, which was detected by immunoblotting as well as by affinity chromatography. This effect was not seen with any of the 10 other differentiating agents: hexamethylene bisacetamide, dimethyl sulfoxide, dimethyl formamide, herbimycin A, mycophenolic acid, retinoic acid, difluoromethyl ornithine, dibutyryl cAMP, 8-chloro cAMP, and transforming growth factor beta 1. Galectin-1 induction by butyrate was observed in seven other human colon carcinoma cell lines. Further studies with the KM12 cells revealed that butyrate caused cell flattening, suppressed cell proliferation and colony formation in agarose, and increased the level of carcinoembryonic antigen, a marker of human colon carcinoma cell differentiation, within 48 h of treatment. The increase in galectin-1 level was dependent linearly on butyrate concentration (range, 1-4 mM). Galectin-1 mRNA expression was detected by Northern blotting as early as 6 h, and the protein was detected after 24 h of treatment initiation. The level of the constitutively expressed galectin-3 was also increased by butyrate but to a lesser extent than the level of galectin-1. Butyrate-induced galectin-1 was detected on the cell surface by immunoprecipitation from radioiodinated cell surface proteins as well as by indirect immunofluorescence labeling. Affinity-purified human galectin-1 was found to bind to purified polylactosamine-containing glycoproteins and to detergent-solubilized cellular proteins electroblotted onto nitrocellulose membranes. Affinity chromatography of [3H]glucosamine-labeled KM12 cell extracts on immobilized galectin-1 followed by immunoprecipitation from the lactose-eluted material demonstrated that lysosome-associated membrane glycoprotein-1, carcinoembryonic antigen, and nonspecific cross-reacting antigen are the major galectin-1-binding proteins in these cells. These results indicate that galectin-1 expression may be associated with the differentiation of KM12 cells and that several glycoproteins shown to be important in colon carcinoma adhesion and metastasis are capable of functioning as its endogenous ligands.

Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1

Abstract: To identify critical amino acid residues for carbohydrate binding of galectins (soluble beta-galactoside-binding lectins found in the animal kingdom). site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992) J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.

(Carcinoembryonic Antigen, Lamp-i, and Lamp-2) in Cultured Human Colon Carcinoma Cells by Sodium Butyratet

Abstract: Galactoside-binding lectins (galectins) with molecalar masses of about 14.5 kilodaltons (galectin-1) and 31 kilodaltons (galectin-3) have been found in a variety ofnormal and madignnntcells and have been implicated In the regulation of cell growth, cell adhesion, and metastasis. The KM12 human colon carcinoma cell line was found to express only galecdn-3@ Becausethe levelsofboth galectins are developmentally regulated and can be modulated during the differentiation of several cultured tumor cell lines, we Studied the ability of 11 dIfferentiation-Inducing agents to induce galectln-1 expression in the KM12 cells. Treatment of these cells with sodium butyrate, an established differentiation-inducing agent for colon carcinoma cells, resulted In the Induction ofgalectin-1, which was detected by hnmunoblottingas well as by affinitychromatography.This effect was not seen with any of the 10 other differendating agents: hexamethylene bisacetamide, dimethyl sulfoxide, dimethyl formamide, herbimycin A, mycophenolic acid, retinoic acid, difluoromethyl ornithine, dibutyryl cAMP, 8-chloro cAMP, and transforming growth factor fll. Galectin-1 Induction by butyrate was observed In seven other human colon card noma cell lines. Further studies with the KM12 cells revealed that butyr ate caused cell flattening, suppressed cell proliferation and colony forma tion In agat-ose, and Increased the level of cardnoembryonic antigen, a marker of human colon carcinoma cell differentiation, within 48 h of treatment. The increase in galectln-1 level was dependent linearly on butyrate concentration (range, 1.-4 mM). Galectin-1 mRNA expression was detected by Northern blotting as early as 6 h, and the protein was detected after 24 h of treatment Initiation. The level of the constitutively expressed galectin-3 was also increased by butyrate but to a lesser extent than the level of galectin-1.Butyrate-Inducedgalectln-1was detected on the cell surface by immunoprecipitatlonfrom radiolodinatedcell surface proteins as well as by indirect Immunofluorescence labeling. Affinity purified human galectin-1 was found to bind to purified polylactosamine containing glycoproteins and to detergent-solubilized cellular proteins electroblotted onto nitrocellulose membranes. Affinity chromatography of [3H]glucosamine-labeled KM12 cell extracts on Immobilized galectin-1 followed by Immunoprecipitatlon from the lactose-elutedmaterial dam onstrated that lysosome-associated membrane glycoproteln-1, cardnoem bryonic antigen, and nonspecific cross-reacting antigen are the major galectln-1-bindlng proteins in these celia. These resnits indicate that ga lectin-1 expression may be associated with the differentiation of KM12 cells and that several glycoproteins shown to be Important in colon carcinoma adhesion and metastasis are capable of functioning as its endogenous ligands.