H. van Halbeek

High-resolution, 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins

Publisher Summary This chapter discusses the application of high-resolution, 1H-nuclear magnetic resonance (NMR) spectroscopy to the structural analysis of carbohydrates related to glycoproteins. Glycoproteins are biopolymers consisting of a polypeptide backbone bearing one or more covalently linked carbohydrate chains. The carbohydrate chains of glycoproteins may be classified according to the type of linkage to the polypeptide backbone. N-Glycosylic chains are attached to the amide group of asparagine (Asn), whereas the O-glycosylic chains are linked to the hydroxyl group of amino acid residues such as serine (Ser), threonine (Thr), and hydroxylysine (Hyl). The high-resolution, 1H-NMR spectroscopy technique, in conjunction with methylation analysis, is extremely suitable for the structural studies of N-, as well as on O-, glycosylic glycans. For the interpretation of the 1H-NMR spectrum of a carbohydrate chain in terms of primary structural assignments, the concept of “structural reporter groups” was developed. This means that the chemical shifts of protons resonating at clearly distinguishable positions in the spectrum, together with their coupling constants and the line widths of their signals, bear the information essential to permit the assigning of the primary structure. This chapter presents literature data on the high-resolution, 1H-NMR spectroscopy of carbohydrates derived from glycoconjugates. It also discusses the results for carbohydrates related to the glycoproteins of N-glycosylic type.

A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow κ-casein

Abstract 360-MHz 1 H-NMR spectra were recorded of NeuAcα(2 → 3)Galβ (1 → 3)GalNAc-ol (I), Galβ(1 → 3)[NeuAcα(2 → 6)] GalNAc-ol (II) and NeuAcα(2 → 3)-Galβ(1 → 3) [NeuAcα(2 → 6)]GalNAc-ol (III). The chemical shifts and coupling constants of the anomeric protons, the H-3ax and H-3eq of NeuAc, the GalNAc-ol skeleton protons, the H-3 of Gal and the N -acetyl protons of GalNAc-ol and NeuAc provide conclusive evidence for the identification of the primary structures. Compound II represents a novel carbohydrate chain of κ-casein.

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Abstract Partial in vitro sialylation of biantennary and triantennary glycopeptides of α1-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution 1H-NMR spectroscopic analysis of the isolated products enabled the assignment of the Galβ(1→4)GlcNAcβ(1→2)Man α(1→3) Man branch as the most preferred substrate site for sialic acid attachment. The Galβ(1→4)GlcNAcβ(1→2)Man α(1→6) Man branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal N -acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.

G. NMR spectroscopy of sialic acids

High-resolution NMR spectroscopy has become an invaluable technique in the study of biopolymers and of their constituents. Using 1H or 13C nuclei as probes information can be obtained about primary structures, conformations and intermolecular interactions of biomolecules in solution. In particular, the possibility to record spectra of underivatized compounds in aqueous solutions allows to afford further insight into the way of action of biomolecules under physiological conditions. For general reviews of high-resolution NMR spectroscopy in the study of biological systems the reader is referred to the recent books of Berliner and Reuben (1978, 1980), Jardetzky and Roberts (1981), and Shulman (1979).

A 500 MHz 1H NMR study of urinary oligosaccharides from patients with mannosidosis

H. VAN HALBEEK, L. DORLAND, G. A. VELDINK, J. F. G. VLIEGENTHART, G. STRECKER+, J.-C. MICHALSKI+, J. MONTREUIL+ and W. E. HULL* Department of Bio-Organic Chemistry, University of Utrech t, Croesestraat 79, 3522 AD Utrecht, The Netherlands, +Laboratoire de Chimie Biologique, Universitt? des Sciences et Techniques de Lille 1, Villeneuve d’Ascq, France and *Bruker Analytische Messtechnik, GmbH, D-7512 Rheinstetten-Fo., FRG

STRUCTURAL ANALYSIS OF THE CARBOHYDRATE MOIETIES OF HUMAN TAMM-HORSFALL GLYCOPROTEIN

Glycopeptides present in a pronase digest of human Tamm-Horsfall glycoprotein were fractionated by chromatography on DEAE-Sephadex A25 in 0.1M acetic acid. The separated glycopeptides were characterised by 500-MHz 1H-n.m.r. spectroscopy, in conjunction with sugar and amino acid analysis, and they were shown to be of the N-glycosylic, N-acetyllactosamine type. Each fraction consisted mainly of a tetra-antennary entity having various degrees of complexity, with lesser amounts of the triantennary structure, and even smaller amounts of the diantennary type. There was extensive heterogeneity in non-reducing terminal groups in each of the glycopeptides, whereas the peptide portions were similar. The extent to which any one of the galactose residues in the N-acetyllactosamine units was substituted, and the type of substitution, differed. The substituents were alpha-NeuAc-(2----6), alpha-NeuAc-(2----3), and alpha-NeuAc-(2----3)[beta-GalNAc-(1----4)]. The carbohydrate moieties of the glycoprotein were heterogeneous also because of an uneven distribution of the fucose residues, which were attached to GlcNAc residues, both that linked to asparagine and one or more of those present in the N-acetyllactosamine units. The alpha-NeuAc-(2----3)[beta-GalNAc-(1----4)]-beta-Gal-(1---- sequence forms, at least in part, the Sda immunodeterminant. The pKa of the carboxyl group of the sialic acid residue in this entity is lower than that for molecules lacking Gal-NAc in this position. Thus, the difference in the number of Sda determinants carried by the glycopeptides enabled the latter to be fractionated on DEAE-Sephadex.

A 500-MHz 1H-NMR study on the N-linked carbohydrate chain of bromelain. 1H-NMR Structural-reporter-groups of fucose α(1-3)-linked to asparagine-bound N-acetylglucosamine

The 500-MHz1H-NMR characteristics of theN-linked carbohydrate chain Manα1-6[Xylβ1-2]Manβ1-4GlcNAcβ1-4[Fucα1-3]GlcNAcβ1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The1H-NMR structural-reporter-groups of the α(1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.The 500-MHz1H-NMR characteristics of theN-linked carbohydrate chain Manα1-6[Xylβ1-2]Manβ1-4GlcNAcβ1-4[Fucα1-3]GlcNAcβ1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The1H-NMR structural-reporter-groups of the α(1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.

Structural characterization of a novel acidic oligosaccharide unit derived from cow colostrum κ-casein: A 500 MHz 1H-NMR study

The glycan part of the cow milk glycoprotein K-casein is constituted of only three different monosaccharides: galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylneuraminic acid (NeuAc). The carbohydrate chains exhibit heterogeneity in their primary structures. So far, as products after alkaline borohydride reductive cleavage, the bisialo tetrasaccharide-alditol NeuAccu(2 + 3)Gal/3( 1 -+ 3) [NeuAccw(2 + 6)] GalNAc-ol as well as two monosialo trisaccharide-alditols, NeuAccw(2 + 3)Ga@( 1 + 3)GalNAc-ol and Ga@( 1 + 3) [NeuAcol(2 + 6)] GalNAc-ol which can be conceived as partial structures of the tetrasaccharide-alditol, have been identified [l-4]. allows the deduction of primary structures of carbohydrates derived from glycoproteins, even if low quantities are available in mixtures of closely related compounds [8-l 11.

Primary structure of a novel N-glycosidic carbohydrate unit, derived from hen ovomucoid

This structure is a novel member of the family of N‐glycosidic carbohydrates of glycoproteins.

Structure elucidation of oligomannoside-type asparagine-bound carbohydrate chains of glycoproteins by 500 MHz 1H NMR spectroscopy

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