Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span

Abstract

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

A cytometric study of the red blood cells in Gaucher disease reveals their abnormal shape that may be involved in increased erythrophagocytosis

“…According to the protocol described by Bratosin et al (27), a solution (50 lL) of FITC-labeled lectins in phosphate buffered saline (PBS)-phenylmethysulfonyl fluoride (PMSF) buffer, pH 7.4 (10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 140 mM NaCl, 2.7 mM KCl, 0.2 mM PMSF), was added to 50 lL suspension of RBCs in the same buffer (corresponding to 2 Â 10 6 red cells). After 1 h incubation at 4 C in the dark, 10,000 cells were analyzed directly using a Becton-Dickinson FACScan cytometer (San Jose, CA).…”

Nuclear Localization Signal of Murine CMP-Neu5Ac Synthetase Includes Residues Required for Both Nuclear Targeting and Enzymatic Activity

“…5 and 6; Ref. 7), biological aging (8), as well as development (6,9) and progression of malignancies (10 -12) are accompanied by alterations in the cellular sialylation pattern. In bacteria sialic acids are found as components of capsules and lipooligosaccharides and often are important virulence factors, mediating resistance to host defense mechanisms (reviewed in Refs.…”

Lectinomics I. Relevance of exogenous plant lectins in biomedical diagnostics

“…The determination of sialic acids in order to monitor control of life span of human erythrocytes (Bratosin et al 1995;Marikovsky & Marikovsky 2002), or detection of multidrug resistance in cancer cells (Greer & Ivey 2007), accompanied by the formation of P-glycoprotein illustrate these trends. Plant lectins-related drug delivery strategies compose another field of growing interest.…”

“…Molecular mechanisms of erythrophagocytosis is based on desialylation of erythrocyte membrane glycoconjugates, which acts as a signal for red blood cell (RBC) capture by macrophages (Bratosin et al 1995). Flow cytometry using FITC labeled lectins was used to determine that aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes (Bratosin et al 1995). An immuno-electron microscopic method was used to evaluate the binding of WGA-gold conjugates to young and old human erythrocytes (Marikovsky & Marikovsky 2002).…”