Jean Montreuil

Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin

In previous papers [l-4] we have described 2 glycopeptides (Carbohydrate -+ Asn-Lys and Ser-Asn c Carbohydrate) isolated from pronase digests of human asialo-serotransferrin and we have demonstrated that in both glycopeptides the glycans were bound through a 4-N-(2-acetamido-2-deoxy-/3-D-glucopyranosyl)L-asparagine linkage. Evidence that the 2 glycans proceed from two different parts of the polypeptide chain was demonstrated by determination of amino-acid sequences of tryptic and chymotryptic glycopeptides : glycan I is conjugated in the N-terminal part of the peptidic chain and corresponds to the dipeptide Asn-Lys whereas glycan II is conjugated in the C-terminal part and corresponds to the dipeptide Ser-Asn [5-81. In this paper we report the primary structure of glycans I and II determined by application of different methods: exhaustive methylation, mild hydrolysis, acetolysis, hydrazinolysis as well as use of specific glycosidases.

A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsulfinyl carbanion

Treatment of dimethyl sulfoxide with butyllithium leads to rapid formation of lithium methylsulfinyl carbanion. The reaction products tend to be significantly freer from impurities when lithium methylsulfinyl carbanion is used rather than sodium or potassium methylsulfinyl carbanion. This reagent gives less background in g.l.c. and thus may be used to methylate micro-quantities of glycoprotein glycans (down to 10 micrograms) without the necessity of identifying methyl ethers by mass spectrometry.

Gas-liquid chromatography and mass spectrometry of methylated and acetylated methyl glycosides. Application to the structural analysis of glycoprotein glycans☆

Abstract The mass spectra of 52 partially methylated and acetylated methyl glycosides of galactose, mannose, glucose, and N-acetylglucosamine have been determined. Each derivative was identified on the basis of its gas-liquid chromatography retention time and mass spectra. The analysis of methyl ethers obtained by methanolysis of fully methylated glycans of α1-acid glycoprotein is described as an application of the method.

Comparative study of the primary structures of sero-, lacto- and ovotransferrin glycans from different species.

In order to establish relationships between glycan structure and biological activity and to answer the question: Are glycans markers of evolution?, the authors undertook a comparative study of the glycan primary structures of different transferrins (sero-, lacto- and ovotransferrins) from several species. By associating permethylation--mass spectrometry and 1H NMR spectroscopy, the primary structure of the following transferrin glycans were determined: human, bovine, hen, horse, marsupial, mouse, rabbit, rat and sheep serotransferrins; human, mouse, bovine and goat lactotransferrins; hen and turkey ovotransferrins. The results obtained led to the conclusion that transferrin glycans are specific for each transferrin and, for a given transferrin, specific to the species. No relationship could be established a priori between primary structure and function of transferrin glycans.

The structure of the asialo‐carbohydrate units of human serotransferrin as proven by 360 MHz proton magnetic resonance spectroscopy

Human serotransferrin contains two identical carbohydrate chains [l-3] about the primary Structure of which controversy exsists. Jamieson et al. [3] suppose a branched structure, wherein the branching mannose, glycosidically linked to N-acetylglucosamine /3l+Asn, bears two chains: one consisting of a mannotriose, the other of an N-acetylglucosamine residue and both terminated by an N-acetylneuraminyliV-acetyllactosamine unit. Spik et al. [ 1,2] propose a more symmetrical structure, built up from a mannotriosido-di-N-acetylchitobiose core substituted by two N-acetylneuraminyl-N-acetyllactosamine units. This structure, presented in fig. 1 differs essentially from the foregoing structure since it has only 3 mannose residues instead of 4. In-this paper the investigation by high-resolution ‘H nuclear magnetic resonance spectroscopy of the structure of the asialoglycopeptide (asialo-glycanAsn) isolated from serotransferrin is described. In particular the signals of the anomeric protons, the mannose-H-2 protons and the N-acetyl methyl groups were analysed. For the assignment of these signals, spectra of the corresponding asialo-agalacto-glycanAsn-Lys, the glyco-amino acids GlcNAcpl+Asn [4] and Mancz(1+6)Man/3(1+4)G1cNAc/3(1+4) [Fu&l+6)] GlcNA@l+Asn [5] and the trisaccharide Mancu(l+3) Mar@(l+4) GlcNAc [6] , representing partial structures of the asialo-glycan-Asn have been used as reference compounds. The monosaccharide units in these substances are numbered in correspondence to the numbering in the serotransferrin glycopeptides (see fig.1 and table 1).

Novel Fluorescence Assay Using Calcein-AM for the Determination of Human Erythrocyte Viability and Aging

A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported.

Immunostimulatory activity of lactotransferrin and maturation of CD4− CD8− murine thymocytes

Human milk lactotransferrin at a concentration ranging from 1 to 10 micrograms/ml stimulated up to 5 times the humoral immune response to sheep red blood cells, expressed as the number of plaque-forming cells, when injected into mice 3 h before immunization. Further, lactotransferrin-treated thymocytes given intravenously into mice, enhanced the immune response to sheep red blood cells to the same extent as IL-1. In vitro, studies showed that CD4- CD8- thymocytes incubated with lactotransferrin and added to the splenocyte cultures, increased the immune response to sheep red blood cells. Flow cytometry analysis studies indicated that, after an overnight incubation with human lactotransferrin, CD4- CD8- thymocytes acquired the CD4 antigen characteristic for the helper cell phenotype. Taken together, these results suggest that lactotransferrin stimulates the immune response by a process which involves the promotion of T cell differentiation.

Aleuria aurantia agglutinin: a new isolation procedure and further study of its specificity towards various glycopeptides and oligosaccharides

A new procedure for isolating a L-fucose-specific lectin from the mushroom Aleuria aurantia is described. The fine specificity of the purified lectin was determined by inhibition of agglutination of human red blood cells by various glycopeptides and oligosaccharides, and by studying the affinity of the immobilized lectin towards glycopeptides and oligosaccharides. Results of inhibition of hemagglutination showed that the lectin presents the highest affinity towards alpha-(1----6)-linked L-fucosyl groups. Immobilized Aleuria aurantia agglutinin interacts strongly with all N-glycosylpeptides or related glycans possessing an alpha-L-fucopyranosyl group linked to O-6 of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl residue involved in the glycosylamine linkage. In addition, presence of alpha-(1----3)-linked L-fucosyl groups greatly enhances the affinity of the lectin for the alpha-(1----6)-L-fucosylated glycans. The immobilized Aleuria lectin is a powerful tool for the resolution of the microheterogeneity of L-fucosylated glycopeptides and glycans of the N-acetyl-lactosamine type.

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

Primary structure of two sialylated triantennary glycans from human serotransferrin

Transferrin NMR analysis Glycan structure Glycoprotein heterogeneity