Henri Debray

Affinity of four immobilized Erythrina lectins toward various n-linked glycopeptides and related oligosaccharides

The behavior of N-acetyllactosamine-type oligosaccharides and glycopeptides on columns of four different Erythrina agglutinins immobilized on Sepharose was examined. The sugar-binding specificity of the four lectins is very similar and is directed toward unmasked N-acetyllactosamine sequences, the main difference between the four lectins being the relative strength of interaction of the lectins with a given glycan. Substitution of the N-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose completely abolishes the affinity of the lectins for the saccharides. The presence of one or several alpha-Fuc-(1----3)-GlcNAc groups decreases or completely inhibits the interaction between the glycopeptides and the Erythrina lectins. Substitution of the beta-mannose residue by an additional bisecting beta-(1----4)-N-acetylglucosamine residue decreases the affinity of the lectins for these structures as compared to the unsubstituted ones. Surprisingly, the affinity of the lectins for the oligosaccharides tested is higher than for the corresponding glycopeptides. Our findings show that, after careful calibration with well-defined oligosaccharides and glycopeptides, the immobilized Erythrina agglutinin-Sepharose columns provide valuable tools for the fractionation of N-acetyllactosamine-containing oligosaccharides and glycopeptides.

On the presence of two types of subunit in soybean agglutinin

Soybean Agglutinin (SBA), a glycoprotein lectin (mol. wt. 120 000) is comprised of four subunits of 30 000 daltons each [ 11. The lectin interacts specifically withN-acetyl-D-galactosamine and D-galactose [2] and possesses two saccharide binding sites per 120 000 daltons [ 11. Although the number of binding sites is half the number of subunits, no differences could be previously detected between the subunits [ 11. Recently it has been demonstrated that discontinuous polyacrylamide gel electrophoresis at alkaline pH, in the presence of urea or sodium dodecyl sulfate (SDS), has a high resolving power in separating similar polypeptide chains. With this technique, differences have been found between the subunits of tubulin [3,4] and of the lectin from Dolichos biflorus [5,6], proteins that were believed to be comprised of identical subunits. We have now reinvestigated the subunit structure of SBA using the above technique and found that the lectin is composed of two types of subunit. These were separated by ion exchange chromatography in the presence of urea and were found to differ in their charge.

Rat alpha-fetoprotein heterogeneity: Affinity chromatography on Ricinus communis Sepharose column

The interaction between Ricinus communis agglutinin (RCA) with whole alpha-fetoprotein (AFP) and with its electrophoretic variants by affinity chromatography using Sepharose coupled lectin has been examined. AFP was isolated from amniotic fluid fetal newborn sera and hepatoma bearing rat serum and was purified by affinity chromatography on anti AFP-Sepharose column. The data demonstrate that RCA1-reactive AFPA2 had a lower content of sialic acid (4 residues/mole) as compared to the RCA1 nonreactive AFPA1 (6 residues/mole). It is postulated that the lack of 2 N-acetylneuraminic acid residues exposes 2 D-galactose terminal sites in the reactive form allowing it to react with the Ricinus lectin. This observation was confirmed by enzymic desialation of AFPA which unmasks all D-galactopyranosyl sites and allows the quantitative binding of the desialated variant. AFPB glycans appeared to be less accessible as shown by their incomplete in vitro enzymic desialation and their nonreactivity with the Ricinus lectin. AFP1 is invariably found in fresh sera or adult hepatoma sera and is not an artefact due to the method of isolation. Dosage of these proteins in the serum of rats from birth until Day 24 revealed that AFPB and AFPA2 disappear faster than the AFPA1 which may signify either that they become preferentially localized in certain organs or that they are more rapidly catabolized in the sera.

Specificity of the isolectins from the plant cactus Machaerocereus eruca for oligosaccharides from porcine stomach mucin.

Sugar specificity of theMachaerocereus eruca isolectins, MeAI and MeAII, has been determined by comparing the capacity of glycans with well defined structures to inhibit their haemagglutinating activity. Both are galactose-specific isolectins with high affinity for O-glycans. However, the twoM. eruca isolectins recognize different oligosaccharidic sequences belonging to O-glycosidically linked glycans from porcine stomach mucin. The minimal structure recognized by MeAI on the porcine mucin glycans is the O-glycan core Galβ1, 3GalNAc-ol, whereas MeAII has a more extended site and interacts with a biantennary O-glycan possessing the terminal trisaccharide Fucα1,2 (GalNAcα1,3) Galβ1,4.

A comparative study on the purification of the Amaranthus leucocarpus syn. hypocondriacus lectin.

Amaranthus leucocarpus lectin is a homodimeric glycoprotein of 35 kDa per sub-unit, which interacts specifically with N-acetyl-galactosamine. In this work, we compared different glycoproteins that contain Galbeta1-3 GalNAcalpha1-3 Ser/Thr or GalNAcalpha1-3 Ser/Thr in their structure as ligands to purify the A. leucocarpus lectin. From the glycoproteins tested, fetuin was the most potent inhibitor of the hemagglutinating activity and the better ligand for lectin purification; however, the use of desialylated stroma from erythrocytes represented the cheapest method to purify this lectin. O-linked glycans released from the glycoproteins used as affinity matrix and those from different erythrocytes were less inhibitory than parental glycoproteins. The NH2-terminal of the lectin is blocked; moreover, this is the only example of a lectin isolated from this genus to be a glycoprotein. Analysis of the glycoprotein sequences with inhibitory activity for the lectin, showed a different pattern in the O-glycosylation, which confirms that A. leucocarpus lectin recognizes conformation and, probably, distances among O-linked glycans moieties.

Isolation and Characterization of Surface Glycopeptides from Adult Rat Hepatocytes in an Established Line

Publisher Summary nThe chapter discusses a micro method for the isolation and characterization of surface glycopeptides from adult rat liver cells in an established line and the first results obtained with this method. D–[U–14C] glucose was used as radioactive precursor, which allowed for the characterization of labeled surface glycopeptides liberated by mild tryptic digestion. The trypsinates were purified by gel filtration. Hydrolysis and analysis of the carbohydrate part of the excluded glycopeptides showed that the 14C label is found in each of the monosaccharide components. Efforts are being made to confirm the proposed structures for glycopeptides of the surface of rat liver cells, transformed because of viral infection or by chemical carcinogens. These include chemical (acetolysis and hydrazinolysis- nitrous deamination) and enzymic (exo- and endo-glycosidases) cleavage of polysaccharide chains.