Plasma anti-α-galactoside antibody mediates lipoprotein(a) binding to macrophages.

Lipoprotein (a) [Lp(a)] is the dominant lipid in atherosclerotic plaques though it is much less numerous than LDL or HDL in circulation. Molecular mechanism of selective uptake of Lp(a) into macrophages is unclear. Lp(a) was reported to form circulating immune complexes with the IgG-dominated plasma anti-α-galactoside antibody (anti-Gal) using the serine- and threonine-rich peptide sequences ( STPS) on its apo(a) subunit as surrogate ligand but left the other binding site of antibody free. We examined if these monovalent immune complexes could bind to smaller STPS-containing molecules on macrophage surface. Using placental membrane O-glycosylated proteins (PMOP) isolated by lectin affinity chromatography as model it was shown that human cell surface glycoproteins were small enough to occupy both binding sites of anti-Gal since they increased the fluorescence of FITC label at Fc part of anti-Gal and inhibited binding of anti-Gal and Griffonia simplicifolia lectin of similar specificity to immobilized ligands. Pre-incubation with anti-Gal facilitated Lp(a) attachment to macrophages unless anti-Gal-specific sugar was present. Anti-Gal-mediated attachment of apo(a) to macrophages increased with the number of apo(a) subunits. Further, anti-Gal-mediated binding of the same sample of apo(a) increased with the specific activity of anti-Gal sample. Finally binding of anti-Gal and anti-Gal-apo(a) complex to PMOP and macrophages respectively was mostly inhibited by LDL suggesting STPS as major anti-Gal epitopes on the cell surface. Results indicated that circulating Lp(a)-anti-Gal immune complexes anchor on macrophages using STPS-bearing cell surface glycoproteins as ligands and offer a pathway for Lp(a) sequestration into macrophages.

Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors.

Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3'-deoxy-3,3'-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A-E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from µM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.

Expression of beta-galactosidase under the control of the human c-myc promoter in transgenic mice is inhibited by mithramycin.

In order to assess the functional contribution of the human c-myc promoter region in the expression of the c-myc gene, transgenic mouse lines containing a bacterial lac Z gene encoding beta-galactosidase under the control of the human c-myc protooncogene promoter were generated. Transgenic mouse embryos heterozygous for the human c-myc Z transgene demonstrate high amounts of beta-galactosidase activity as early as day 11 of embryogenesis by histochemical staining of whole embryos using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) as substrate, localizing specifically to early spinal cord tissue. beta-galactosidase activity can be demonstrated by histochemical staining in brain tissue of day 14 embryos, localizing mainly to the prefrontal cortex region, while relative amounts of beta-galactosidase in spinal cord tissue are reduced. Determination of specific activity of beta-galactosidase using resorufin-beta-galactopyranoside as substrate in homogenates of whole embryos heterozygous for the human c-myc/lac Z transgene demonstrates significantly elevated beta-galactosidase activity over control embryos in day 11 and day 14 embryos. Surprisingly, cell homogenates of brain tissue from adult G1 generation mice heterozygous for the human c-myc/lac Z transgene demonstrate greater than 10-fold higher specific activity of beta-galactosidase over normal control brain tissue. Specific inhibition of the c-myc/lac Z transgene was also demonstrated in developing embryos using mithramycin given at a dose of 150 micrograms kg-1 d-1 intraperitoneal to pregnant females on days 7-13 of gestation. Both histochemical staining of beta-galactosidase and specific activity assays of day 14 embryos demonstrated significantly lower levels of beta-galactosidase than untreated controls. These results are unique since we are able to detect expression of beta-galactosidase in developing embryonic central nervous system tissue along with adult brain tissue of animals carrying the human c-myc Z transgene and we are able to specifically inhibit expression of the transgene using mithramycin administered in utero.

[Comparative studies on the immunoactive action of galactoside-specific mistletoe lectin. Pure substance compared to the standardized extract]. / Vergleichende Untersuchungen zur immunaktiven Wirkung von Galaktosid-spezifischem Mistellektin. Reinsubstanz gegen standardisierten Extrakt.

Comparative Studies on the Immunoactive Potency of Galactoside-specific Lectin from Mistletoe/Pure substance against standardized extract. Cellular and humoral aspects of the immunomodulating activity of the galactoside-specific lectin from mistletoe (ML-1) were investigated in cancer patients suffering from mammary carcinoma and compared to the immunoactive potency of a proprietary mistletoe extract standardized for ML-1 (ML-1 stand., Eurixor). Regular subcutaneous injections of the optimal dose of ML-1 and ML-1 stand. (1 ng/kg body weight; twice a week; for 4 weeks) yielded statistically significant increases of certain lymphocyte subsets (helper T-lymphocytes, natural killer (NK)-cells) which are generally believed to be involved in antitumor activity. Moreover, administration of either ML-1 preparation resulted in enhanced expression of interleukin (Il)-2 receptors on lymphatic cells and significantly increased serum levels of defined acute phase reactants (c-reactive protein, haptoglobin, C3 complement) as indicator of cellular and humoral activity. In vitro, exposition of human lymphocytes to ML-1 and ML-1 stand. resulted in enhanced expression of Il-2 receptors, which substantiated the capacity of both ML-1 preparations to affect immunological parameters within the host defense system. The effects of ML-1 and ML-1 stand. were comparable.

Purification and characterization of a beta-galactoside-binding soluble lectin from rat and bovine brain.

A beta-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggregation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the beta-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30,000 for the bovine brain lectin and 32,000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15,000 and 16,000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for beta-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.