Carbohydrate-binding proteins of tumor lines with different growth properties

Abstract

Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca2+ -dependent and Ca2+ -independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.

Analysis of MonoPEGylated Human Galectin-2 by Small-Angle X-ray and Neutron Scattering: Concentration Dependence of PEG Conformation in the Conjugate

“…The production of the C57M mutant of hGal-2 and its site-specific PEGylation have been described previously . In brief, recombinant production under optimal conditions was followed by affinity chromatography on lactosylated Sepharose 4B as the crucial step and quality controls/activity assays to ensure purity and functionality as lectin . PEGylation was then carried out via selective reaction between the maleimide group of activated PEG and the thiol group of the single cysteine residue at position 75 of hGal-2, followed by a purification step using ion exchange chromatography.…”

Binding studies of adhesion/growth-regulatory galectins with glycoconjugates monitored by surface plasmon resonance and NMR spectroscopy

“…28-30 Following affinity chromatography on lactose-bearing Sepharose 4B, obtained by resin activation with divinyl sulfone, the proteins were checked for purity by one-and two-dimensional gel electrophoresis and nanoESI mass spectrometry, for quaternary structure by gel filtration and for activity by haemagglutination and cell-binding assays. [31][32][33][34] Proteolytic truncation of murine galectin-3 by digestion with commercial collagenase D was carried out as described followed by rechromatography and product analysis by mass spectrometry. 29, 35 The obtained C-terminal section constitutes the carbohydrate recognition domain (CRD) of galectin-3.…”

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

“…[22][23][24] Lectin activity was measured by haemagglutination using trypsin-treated and glutaraldehyde-fixed rabbit erythrocytes and cell binding/growth inhibition as well as solid-phase assays using the glycoprotein asialofetuin as ligand as described. 21,[25][26][27][28] Loss of activity owing to SH-group oxidation was minimized by exposing the lectin to a cysteine-modifying reagent (iodoacetamide) during elution from the affinity resin. 29,30 Controls by isothermal titration calorimetry ensured the presence of one active binding site for lactose per subunit of the hGal-1 homodimer.…”