The Experience in Lectins Application to Assess Changes in the Carbohydrate Composition of Murine Thymocytes Glycocalyx in the Early and Late Apoptotic Stages

Abstract

The apoptotic process is an important step in the process of T-lymphocytes maturation and differentiation. This work is devoted to the analysis of changes in the composition of oligosaccharides of CBA mice thymocytes glycocalyx during the hydrocortisone induced apoptosis. A panel of 23 fluorescein isothiocyanate (FITC)-labeled lectins specific for mannose, mannose and glucose, galactose, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, fucose and N-acetylneuraminic acid residues was used. Flow cytometry was used for evaluation the binding of lectins to thymocytes of intact mice, as well as mice after administration of hydrocortisone. Based on the results of TMRM and 7AAD staining, the cells were divided into living thymocytes, cells in early and late apoptosis. It has been established that living cells carry carbohydrates on the surface of glycocalyx containing terminal residues of galactose and N-acetyl-D-galactosamine. With the transition of thymocytes to late apoptosis, the binding of all lectins increases, except for fucose-specific ones. The glycocalyx structures of living thymocytes are low in density and contain groups of oligosaccharides with N-acetyl-D-galactosamine and D-galactose in the terminal position. The membrane layer of glycocalyx is characterized by a high density and a wide variety of oligosaccharide structures that persist at the stage of late apoptosis of thymocytes. The results indicate nonuniform density and heterogeneity of oligosaccharides in glycocalyx, a significant part of which is lost in early apoptosis.