Catherine Alonso

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

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 β-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 asErythrina cristagalli andErythrina corallodendron lectins specific for terminal β-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 α-2,6-sialyltransferase of fluorescent or radioactiveN-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 β-galactose specific macrophage lectin.(5)Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to thein vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages

Effect of okadaic acid on O-linked N-acetylglucosamine levels in a neuroblastoma cell line

O-Linked N-Acetylglucosamine (O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.

Characterization of Lex, Ley and A Ley antigen determinants in KDN‐containing O‐linked glycan chains from Pleurodeles waltlii jelly coat eggs

Novel acidic oligosaccharides were isolated in large amounts by reductive alkaline treatment of the jelly coat of Pleurodeles waltlii (Michah) eggs. The oligosaccharides were found to contain the newly described KDN as acidic monosaccharide and possess either the Lex, Ley and A Ley antigenic determinants. Occurrence of Lex and Ley determinants previously recognized as tumor‐associated antigen (TAA) demonstrates that mucins of lower animals may represent a rich and easily available source for preparing TAA. Moreover, it reinforces the hypothesis according to which TAA are evolution markers.