Galectin- and C-type lectin-reactive asialofetuin induces an increase of cytoplasmic pH in implant-colonizing rat macrophages - an ex vivo study

摘要

The carbohydrate chains of glycoconjugates establish cell surface signatures which are related to recognitive processes. In addition to serving as docking points for receptors such as lectins, the protein-carbohydrate interaction may trigger biosignaling processes in cells, warranting the need to employ model systems for this study. Asialofetuin (ASF) which presents with defined N- and O-linked b-galactoside-terminating oligosaccharide chains with reactivity to C-type lectins and galectins can be used as a model ligand, cell binding and ensuing response(s) to the sugar-specific interaction can be monitored. Due to the clinical relevance of this model our ex vivo experiments were focused on the inflammatory macrophages colonizing the surface of implanted cellophane foil. In addition to the visualization of b-galactoside-binding sites by biotinylated asialofetuin, impact of sugar-dependent probe binding on the cytoplasmic pHi is assayed by fluorescence excitation ratio-imaging microscopy. The experiments revealed pronounced binding of ASF to mononuclear macrophages and foreignbody giant multinucleate cells. Cell surface association of ASF (20 μg/ml) increased the pHi of the cytoplasm of inflammatory macrophages colonizing the surface of the implant relative to human serum albumin used as a carbohydrate free control. Owing to a slight Ca2+-sensitivity of pHi upregulation by ASF, a C-type b-galactoside- recognizing lectin is likely to be involved in this reaction. Since the use of EDTA (an inhibitor of C-type lectins) caused strong lectin-independent effects on the internal pH-value, presence of a chelating agent does not demonstrate unquestioned evidence suggested in the above experiments.