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In the past decades, a gradual increase in the resistance to antibiotics has been observed, leading to a serious thread for successful treatment of bacterial infections. This feature in addition to difficulties in developing adequate drugs against (tropical) diseases caused by parasites has stimulated the interest in vaccines to prevent infections. In principle, various types of cell surface epitopes, characteristic for the invading organism or related to aberrant growth of cells, can be applied to develop vaccines. The progress in establishing the structure of carbohydrate immuno-determinants in conjunction with improvements in carbohydrate synthesis has rendered it feasible to develop new generations of carbohydrate-based vaccines.

The structural analysis of protein-carbohydrate interactions is essential for the long-range aim to sort out entropic/ enthalpic factors in the binding process. Of conspicuous clinical interest, this work can also offer the perspective to devise new classes of therapeuticals which interfere with disease-related glycan recognition. We have shown that it is possible to use exchangeable hydroxyl protons of carbohydrate ligands as conformational sensors for defining their bound-state topology by measurements in dimethyl sulfoxide(d6) (Siebert et al. (2000) ChemBioChem, 1, 181-195). However, the proteins are required to maintain binding capacity in the aprotic solvent. To define conditions to limit its harmful effect on sensitive protein structures while still being able to pick up solvent-exchangeable hydroxyl signals we systematically tested binary solvent mixtures of dimethyl sulfoxide and acetone with water. These solvent mixtures did not preclude to monitor hydroxyl protons of carbohydrate ligands even at temperatures well above 0 degrees C. Notably, hydrogen bonding of the two tested disaccharides (Galbeta1-4Glcalpha/beta and Galalpha1-3Galalpha/beta or Galalpha1-3Galbeta1-OCH(3)), which are common lectin ligands, resembled the situation under physiological conditions. Also, a refined topological description for hydroxyl positioning could be achieved for Galalpha1-3Gal. At least equally important, this approach worked for elucidation of the mistletoe-lectin-bound topology of lactose in its syn-conformation with indication for formation of a characteristic interresidual hydrogen bond. These measurements were performed in a binary dimethyl sulfoxide(d6):water mixture (6:4 ratio, v/v) at -12 degrees C and encourage to pursue this line of investigation by monitoring in the course of stepwise temperature increases. Our experiments reveal that binary mixtures have favorable properties for the conformational analysis of the free- and bound-state topologies of bioactive ligands.

13C-NMR spectroscopic studies were carried out on Manα(1↵6)Manβ(1↵4)GlcNAcβ(1↵4)GlcNAcβ (1↵N)Asn and Manα(1↵6)Manβ(1↵4)GlcNAcβ(1↵4)[Fucα(1↵6)]GlcNAcβ(1↵N)Asn, which are part of the invariant core of N-glycosidically linked carbohydrates of glycoproteins. All resonances of proton-bearing carbon atoms were unambiguously assigned. Where possible, the assignments were confirmed by selective proton-decoupling experiments. [ABSTRACT FROM AUTHOR]

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-β- glucosaminyl)asparagine amidase F and subsequently separated from the O- glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O- linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel- permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two- dimensional ¹H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal β 1- 4GlcNAc β 1-3Gal β 1-3GalNAc-ol, Gal β 1-4(6SO4 )GlcNAc, and α 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac α2-3]0-1[Gal β 1-4(6SO4-)GlcNAc β1-3]nGal β 1-4GlcNAc β 1-3Gal β 1- 3GalNAc-ol. In addition, smaller compounds were identified in which the Gal β 1-3GalNAc-ol core is substituted by Neu5Gc/Ac α 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac α 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O- sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc β 1-3GalNAc-ol core structure. Analysis of the endo-β- galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N- acetyllactosamine) chains. [ABSTRACT FROM AUTHOR]

Human Tamm-Horsfall glycoprotein has been purified from the urine of one male. The Asn-linked carbohydrate chains were enzymically released by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F, and separated from the remaining protein by gel-permeation chromatography on Bio- Gel P-100. Fractionation of the intact (sulfated) sialylated carbohydrate chains was achieved by a combination of three liquid-chromatographic techniques, namely, anion-exchange FPLC on Q- Sepharose, amine-adsorption HPLC on Lichrospher-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. In total, more than 150 carbohydrate-containing fractions were obtained. some of which still contained mixtures of oligosaccharides. The primary structure of 30 N-glycans, including 10 novel oligosaccharides, were determined by one- and two-dimensional 1H-NMR spectroscopy at 500 MHz or 600 MHz. The types of compounds identified range from non-fucosylated, monosialylated, diantennary to fucosylated, tetrasialylated, tetraantennary carbohydrate chains, pos- sessing the following terminal structural elements: [This symbol cannot be presented in ASCII format] [ABSTRACT FROM AUTHOR]

From the carbohydrate-protein linkage region of whale cartilage proteoglycans, which bear predominantly chondroitin 4-sulfate, one nonsulfated, two monosulfated and one disulfated hexasaccharide alditols were isolated after exhaustive digestions with Actinase E and chondroitinase ABC, and subsequent β-elimination. Their structures were analyzed by chondroitinase ACII digestion in conjunction with HPLC and by 500-MHz ¹H-NMR spectroscopy. The nonsulfated compound (A) had the following conventional structure: ΔGlcA(β1–3)-GalNAc(β1–4)GlcA(β1–3)Gal(β1–3)Gal(β1–4)Xylol, where GlcA, ΔGlcA and GalNAc are glucuronic acid; 4,5-unsaturated glucuronic acid and 2-deoxy-2-N-acetylamino-D-galactose, respectively. The other compounds were sulfated derivatives of compound A. Two monosulfated compounds (B and C) had an ester sulfate on C4 or C6 of the GalNAc residue, respectively and the disulfated compound (D) had two ester sulfate groups, namely, one on C4 of the GalNAc and the other on C4 of the Gal residue substituted by GlcA. The molar ratio of A/B/C/D was 0.21:0.16:0.36:0.27. The compound containing Gal-4-O-sulfate was previously isolated by us in the form of a sulfated glycoserine [ΔGlcA(β1–3)GalNAc(4-O-sulfate)(β1–4)GlcA(β1–3)Gal(4-O-sulfate)(β1–3)Gal(β1–4)Xyl/β1-O-Ser] from the carbohydrate-protein linkage region of rat chondrosarcoma chondroitin-4-sulfate proteoglycans [Sugahara K., Yamashina, I., DeWaard, P., Van Halbeek, H. & Vliegenthart, J. F. G. (1988) J. Biol. Chem. 263, 10168–10174]. The discovery of this structure in the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans from nontumorous cartilage indicates that it is not a tumor-associated product but rather a physiological biosynthetic product since it represents a significant proportion. The biological significance of this structure is discussed in relation to glycosaminoglycan biosynthesis. [ABSTRACT FROM AUTHOR]

The carbohydrate moieties of equine chorionic gonadotropin α and β subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the β subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly α1–6 fucosylated at the Asn-bound GlcNAc residue and having α2–6 and α2–3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the α subunit. The major part of the O-linked carbohydrate chains, occurring solely in the β subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the β subunit. [ABSTRACT FROM AUTHOR]

Prostaglandin endoperoxide synthase was isolated froth sheep seminal vesicles. Sugar analysis of the glycoprotein revealed the presence of mannose and N-acetyiglucosamine only. The carbohydrate moiety was released from the polypeptide backbone by hydrazinolysis. After re-N-acetylation and reduction, the resulting mixture of oligosaccharide-alditols was fractionated on Bio-Gel P-4 and their structures were investigated by 500-MHz 1H-NMR spectroscopy. The carbohydrate chains turned out to be of the oligomannoside type containing six to nine mannose residues. The largest and most abundant compound was established to be: [This symbol cannot be presented in ASCII format] For the smaller structures heterogeneity occurs with respect to the outer α(1→2)-linked mannose residues. Furthermore, a small amount of Man6GlcNAc-ol (artefact of the hydrazinolysis procedure) was detected by 1H-NMR spectroscopy and fast atom bombardment mass spectrometry. [ABSTRACT FROM AUTHOR]

In blood-group H substance from pig stomach cell linings, carbohydrate structures are known to occur which are O-glycosidically linked via GalNAc to Ser or Thr of the polypeptide backbone. They have in common the Gal(β1→3)GalNAc core unit, which bears one or more N-acetyllactosamine branches. The latter are terminated either by Fuc in (α1→2) linkage or by GlcNAc in (α1→4) linkage to Gal. Both terminal sequences are considered to be porcine blood-group H antigenic determinants. Employing 500-MHz 1H-NMR spectroscopy, the spectral features of the oligosaccharide-alditols corresponding to these chains were established. Insight could be gained into the microheterogeneity displayed by some of the alditol fractions as to the distribution of the terminating Fuc and GlcNAc residues over the various branches. Such information can hardly be obtained using other approaches. [ABSTRACT FROM AUTHOR]

The proton-magnetic-resonance spectra of three partial structures of the carbohydrate-protein linkage region that frequently occurs in proteoglycans, namely, β-D-Galp-(l→3)- β-D-Galp-(1→4)- β-D-Xylp-(1→O)-L-Ser, were recorded in ²H2O at 500 MHz; they could be completely interpreted, both for the glyco-serines and for the corresponding glyco-xylitols. The chemical shifts and the coupling constants were refined by computer simulation of the spectra. The change in the chemical shift of H-4 of a D-galactopyranosyl residue upon substitution at C-3 by β-D-galactopyranosyl group is proposed to be characteristic for this particular attachment, making H-4 of galactose a structural-reporter group. The three constituting monosaccharides adopt the 4C1 (D) ring conformation. The terminal galactopyranosyl group and the internal galactopyranosyl residue differ as to the population of rotamers around the C-5/C-6 axis. Concomitantly, the flexibility of their glycosidic linkages is distinct. [ABSTRACT FROM AUTHOR]