Bernard Fournet

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.

Structure of an exocellular polysaccharide produced by Streptococcus thermophilus.

Streptococcus thermophilus strains grown on skimmed milk produced a viscosifying, exocellular, and water-soluble polysaccharide which contains D-glucose, D-galactose, and N-acetyl-D-galactosamine in the ratio of 1:2:1. Methylation analysis identified the glycosidic linkages in the tetrasaccharidic repeating-unit, and Smith degradation and nitrous deamination after N-deacetylation gave the sequence of monosaccharides in the repeating-unit. The anomeric configurations of the sugar residues were determined by oxidation of the peracetylated polysaccharide with chromium trioxide and by 1H- and 13C-n.m.r. spectroscopy. The following structure was assigned to the repeating unit of the polysaccharide,----3)-beta-D-Galp-(1----3)-[alpha-D-Galp-(1----6)]-beta- D- Glcp-(1----3)-alpha-D-GalpNAc-(1----.

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).

Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: Influence of the collision energy and of the collision gas for the assignment of linkage position

Tandem mass spectrometry has been used to study the collision-induced decomposition of [M+Na]+ ions of permethylated oligosaccharides. It is shown that many linkage positions in one compound may be determined by the presence or absence, in a single spectrum, of specific fragment ions that arise from the cleavage of two ring bonds and that the yield of such ions depends strongly on the collision energy and nature of the collision gas. In contrast to the behavior of monolithiated native oligosaccharides, the collision-induced decomposition of the sodiated and permethylated oligosaccharide samples at low energy leads to preferential cleavage of glycosidic linkages. At high collision energies, the fragment ions formed by cleavage of more than one bond are greatly enhanced, especially when helium is replaced by argon as the collision gas. Furthermore, argon is the more efficient collision gas in inducing fragmentation of the precursor ions. As an example of the application of this method, the discrimination between the 1 → 3 and 1 → 6-linked mannose residues in the common core of N-glycans is described.

Structure of an exocellular β-d-glucan from Pediococcus sp., a wine lactic bacteria

Pediococcus sp. produces an exocellular slime containing exclusively D-glucose. The structure of the polysaccharide was determined by methylation analysis, Smith degradation, enzymic hydrolysis, and 13C-n.m.r. spectroscopy as having a trisaccharide repeating unit, ----3)-beta-D-Glcp-(1---- 3)-[beta-D-Glcp-(1----2)]-beta-D-Glcp-(1----.

Triterpenoid saponins from Argania spinosa

Five new oleanane saponins named arganine A, B, D, E and F and two known saponins: arganine C and mi-saponin A were isolated from the kernel of Argania spinosa. The structures of these saponins were elucidated by using 1H NMR, 1H-1H COSY NMR, 13C NMR, FAB mass spectrometry and chemical evidence.

The primary structure of the asialo-carbohydrate units of the first glycosylation site of human plasma α1-acid glycoprotein

The elucidation of the structures of the carbohydrate units linked to glycosylation site I of human plasma alpha 1-acid glycoprotein is described. These carbohydrate units can be grouped into compounds with bi- (class A) and triantennary (class B) structures and the triantennary structure with a fucose residue (class BF) (Fig. 1). The structural variability of the carbohydrate units of glycosylation site I and also of glycosylation sites II to V (Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206--5214) accounts largely for the microheterogeneity of alpha 1-acid glycoprotein.

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.