Gérard Strecker

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.

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.

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).

Fast-atom-bombardment mass-spectrometry for carbohydrate-structure determination

Abstract The potential of fast-atom-bombardment (f.a.b.) mass-spectrometry in the carbohydrate field was assessed with the aid of unmodified, permethylated, and peracetylated oligosaccharides and glycosphingolipids. F.a.b. spectra are presented for ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 → d -GlcNAc, permethylated ( d -Gal→ d -GlcNAc) 5 →( d -Man) 3 - d -[ 2 H]GlcNAcol, permethylated gangliotetraosylceramide, and reduced and peracetylated tetra- to hepta-saccharides from human milk. From unmodified oligosaccharides, pseudomolecular ions (M + Na + ) were obtained as major ions in the high-mass range. Permethylated oligosaccharides and glycosphingolipids gave pseudomolecular ions (M + H + ) of high intensity, together with fragment ions of high diagnostic importance. At ambient temperature, f.a.b. spectra could only be obtained for the lower homologs of per- O -acetylated oligosaccharides. Reduced and peracetylated penta- to hepta-saccharides from human milk gave f.a.b. spectra only after heating of the target.

Heterogeneity of bovine lactotransferrin glycans. Characterization of α-D-Galp-(1→3)-β-D-Gal- and α-NeuAc-(2→6)-β-D-GalpNAc-(1→4)-β-D-GlcNAc-substituted N-linked glycans

Abstract Lactotransferrin isolated from a pool of mature bovine milk has been shown to contain N-glycosidically-linked glycans possessing N-acetylneuraminic acid, galactose, mannose, fucose, N-acetylglucosamine and N-acetylgalactosamine. The glycopeptides obtained by Pronase digestion were fractionated by concanavalin A-Sepharose affinity chromatography into three fractions: slightly retained (A), retained (B), and strongly retained (C). The structure of the glycans of the three fractions has been determined by application of methanolysis, methylation analysis, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. Diantennary structures without GalNAc were present as partially sialylated and partially (1 → 6)-α- l -fucosylated structures in Fractions A and B. Sequences containing α- d -Galp-(1 → 3)-β- d -Gal on the α- d -Man-(1 → 6) antenna, and β- d -GalpNAc-(1 → 4)-β- d -GlcNAc and α-NeuAc-(2 → 6)-β- d -GalpNAc-(1 → 4)-β- d -GlcNAc on the α- d -Man-(1 → 3) antenna were characterized in the oligosaccharide-alditols obtained by reductive cleavage of Fraction B. A series of Man4 − 9-GlcNAc structures were identified in Fraction C after endo-N-acetyl-β- d -glucosaminidase digestion. These results show that the structures of bovine lactotransferrin glycans are more heterogeneous than those of previously characterized transferrin glycans.

The nematode Caenorhabditis elegans synthesizes unusual O-linked glycans: identification of glucose-substituted mucin-type O-glycans and short chondroitin-like oligosaccharides.

The free-living nematode Caenorhabditis elegans is a relevant model for studies on the role of glycoconjugates during development of multicellular organisms. Several genes coding for glycosyltransferases involved in the synthesis of N- and O-linked glycans have already been isolated, but, apart from repetitive dimers of glycosaminoglycans, no detailed structure of either type of component has been published so far. This study aimed to establish the structures of the major O-glycans synthesized by C. elegans to give an insight into the endogenous glycosyltransferase activities expressed in this organism. By the use of NMR and MS, we have resolved the sequence of seven of these components that present very unusual features. Most of them were characterized by the type-1 core substituted on Gal and/or GalNAc by (beta1-4)Glc and (beta1-6)Glc residues. Another compound exhibited the GalNAc(beta1-4)N-acetylglucosaminitol sequence in the terminal position, to which was attached a tetramer of beta-Gal substituted by both Fuc and 2-O-methyl-fucose residues. Our experimental procedure led also to the isolation of glycosaminoglycan-like components and oligomannosyl-type N-glycans. In particular, the data confirmed that C. elegans synthesizes the ubiquitous linker sequence GlcA(beta1-3)Gal(beta1-3)Gal(beta1-4)Xyl.

Description d'un nouveau type de méliturie: la sialurie jean montreuil, gérard biserte, gérard strecker, geneviève spik

Abstract The authors describe a new type of melituria: sialuria, discovered in a threeyear-old child and characterized by a daily elimination varying between 5.8 and 7.2 g of N-acetylneuraminic acid. This compound was isolated, crystallized and identified on the basis of physico-chemical and structural studies.The authors describe a new type of melituria: sialuria, discovered in a threeyear-old child and characterized by a daily elimination varying between 5.8 and 7.2 g of N-acetylneuraminic acid. This compound was isolated, crystallized and identified on the basis of physico-chemical and structural studies.

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.

The crystal and molecular structure of O-α-D-mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranose

Abstract The crystal structure of α- D -Man p -(1→3)-β- D -Man p -(1→4)-α- D -GlcNA cp has been determined by the direct method using the multi-solution, tangent formula, and “magic integer” procedures. The space group is P 2 2 , and 2 molecules are in the unit cell with a  9.894 (5), b  10.372 (6), c  11.816 (6) A, and β  95.03° (6). The structure was refined to R 0.059 for 2099 reflections measured with Mo Kα radiation. Difference synthesis showed all the hydrogen atoms, and indicated a partial (∼30%) substitution of the α-anomer molecules by the β-anomer molecules. The D -mannopyranose and the D -glucopyranose have the normal 4 C 1 conformation; an intramolecular hydrogen-bond O-3″-H.....O-5′ (2.703 A) stabilises the GlcNAc in relation to β- D -mannopyranose.

Carbohydrate structures of hen ovomucoid. A mass spectrometric analysis.

The apparently homogenous N‐glycosidically‐linked glycans 1, 7, 11 and 14 released by hydrazinolysis from hen ovomucoid were analysed by fast atom bombardment and electron‐impact mass spectrometry after reduction and permethylation. The spectra support the primary structures established independently [FEBS Letters (1983) 152, 145–152] using methylation analysis, partial acid hydrolysis and 500 MHz 1H NMR spectroscopy. In addition to the major constituents present in fractions 1, 7, 11 and 14, four minor components not detected by other methods could be characterized with the aid of the mass spectrometry data as: Man2GlcNAcGlcNAc‐ol, GlcNAc4Man3GlcNAc‐ol, GlcNAc6Man3GlcNAc‐ol and GalGlcNAc6Man3GlcNAc‐ol. Our results show that the physical techniques used provide valuable data on the structure of complex glycans. In addition they can be employed to ascertain the homogeneity of the compounds examined as well as to detect trace amounts of homologs that might not be noticed by other methods.