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The discovery that certain cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain, which is localized opposite to the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase involved in receptor phosphorylation and in specific signal transduction. It is expected that blood accumulation of free or membrane-bound glycan ligands of cytokines may dramatically perturb their endogenous function inducing specific immunodeficiencies.

The N-glycosidic carbohydrate chains of hen -ovomucoid were released from the protein by hydrazinolysis, and separated by HPLC. Primary structural analysis of 3 major fractions was conducted by applying 500-MHz 1H-NMR spectroscopy in combination with methylation analysis. One of the fractions investigated appeared to consist of an intersected penta-antennary structure extended with one Gal residue. The location of the latter in a certain branch could be established unambiguously by NMR. This structure is a novel member of the family of N-glycosidic carbohydrates of glycoproteins.

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-β-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from β-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to α-N-acetylglucosamine residues linked to mannose 6phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Manα1,6- (Manαl,3)Manαl,6(Manαl,3)ManβαGlcNAc and either one or two αl,2-linked mannose residues. peerReviewed

New born meconium contains at least a hundred oTigosaccharides. In this study the isolation and characterization of the major constituents is described. The structure elucidation of 15 neutral and acidic oligosaccharides was carried out by methylation analysis, mass spectrometry and 360-MHz 1H-NMR spectroscopy. The results show that the oligosaccharides accumulating in human meconium are probably products of the catabolism, of the O- and N-linked carbohydrate chains of glycoproteins. It is proposed that endo-N-acetyl-alpha-d-galactosaminidase, endo-beta-d-galactosidase and endo-N-acetyl-beta-d-glucosaminidase are involved in the production of these compounds.

13C-NMR spectroscopic studies were carried out on Manalpha(1->6)Man beta(1->4)GlcNAc beta(1->4)GlcNAc beta(1->N)-Asn and Man alpha(1->6)Man beta(1->4)GlcNAc beta(1->4)[Fuc alpha(1->6)]GlcNAc beta(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.

The 360-MHz proton magnetic resonance spectra of 2-acetamido-1-N-(4-l-aspartyl)- 2-deoxy-beta-d-glucopyranosylamine (GlcNAc beta1-> Asn) and 2-acetamido-6-O-(alpha-l-fucopyranosyl)-1-N -(4-l-aspartyl)-2-deoxy-beta-d-glucopyranosylamine (Fuc1-> 6GlcNAc1 Asn) in deuterium oxide were completely interpreted. The chemical shifts and coupling constants were refined by simulation of the spectra. By means of an adapted Karplus equation the pyranose ring conformation of the sugars was calculated. The change of the geminal coupling constant J6a,6b in the N-acetylglucosamine residue of Fuc alpha1-> 6GlcNAc beta1-> Asn with respect to GlcNAc beta1-> Asn is proposed to be characteristic for the (1-> 6) glycosidic linkage.

360-MHz proton nuclear magnetic resonance spectra were recorded of 10 sialyl-oligosaccharides isolated form urine of sialidosis patients. Their structures are related to the complex aspareagine-linked glydan chains of glycoproteins. By correlation of these spectra and comparison with spectra of reference glycopeptides and sialy-lactose isomers it was possible to assign all signals belinging to anomeric, mannose H-2, sialic acid H-3 and N-acetyl protons. The number of the constituting monosaccharide residues of the oligomers can be obtained by integration of the above-mentioned singnals. The chemical shifts of the anometic and mannose H-2 proteins give information about the type of glydan structure (mono-, bi-, triantennary) and the presence of terminal sialic acid at eachof the antennas. The chemical shfts of sialic acid H-3 protons are typical for sialic acid residues in 23 or 26 linkage ot galactose.

The mucus glycoproteins, the so-called nest-cementing substance, from the salivary gland of Chinese swiftlets (genus Collocalia) are mainly constituted of sialic acid-rich O-glycosylproteins. Alkaline reductive treatment of the crude material led to the release of some neutral and numerous monosialyl and disialyl oligosaccharides. These were fractionated by gel filtration, anion-exchange chromatography, and high- performance liquid chromatography. The structures of the monosialyl oligosaccharides were established by combination of sugar and methylation analysis, fast atom bombardment-mass spectrometry, and electron impact-mass spectrometry after permethylation and 1H NMR spectroscopy (at 500 MHz). Typically, some of the monosialyl oligosaccharides appeared to possess the core structure Gal β(1→3)[GlcNAcβ(1→6)]GalNAcα(1→3)GalNAc-ol. Moreover, the (1→6)-linked branch consisted of an unusual di- or trigalactosyl sequence, [Galα(1→4)0-1Galβ(1→4)Galβ(1→4)GlcNAcβ(1→6). Thus, the most complex representatives of the monosialyl fraction from Collocalia mucin were found to be: (Formula: see text) The other compounds identified are partial structures thereof.

Acetylneuraminic acid preparations from the urine of a patient with sialuria contain 12% of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. This new human sialic acid was isolated by ion-exchange and partition chromatography. The structure has been elucidated by mass spectrometry and confirmed by comparison with the synthetic compound. The properties of this unsaturated sialic acid in the orcinol/Fe3+ /HCl and the periodic acid/thiobarbituric acid tests as well as in thin-layer and gas-liquid chromatography are described. It does not react with acylneuraminate pyruvate-lyase. The origin of this new human sialic acid is discussed.

The conformation is described of the sialylalpha(2-> 3/6)N-acetyllactosamine structural element, frequently occurring in glycoproteins. NOE spectroscopy of NeuAcalpah(2-> 3) Galbeta(1-> 4)GlcNAcbeta(1-> N)Asn and NeuAcalpha-(2-> 6)Galbeta(1-> 4)GlcNAcbeta(1-> N)Asn is presented and for each glycosidic linkage, except for the alpha(2->6)-linkage, a number of interglycosidic NOEs are measured. The analysis of these effects is performed using a full relaxation matrix. Analysis of intraresidue NOEs provides a calibration of the calculation method. Hard-sphere exo-anomeric (HSEA) energy calculations indicate a single conformation for the beta(1-> 4)-linkage in both compounds, both being consistent with the NOE data. HSEA and molecular-mechanics force-field with hydrogen-bonding potential energy calculations both indicate the existence of three preferred conformations for the alpha(2-> 3)-linkage. The analysis of the NOE spectra are consistent with a distribution over two or three of these conformations; by combination with the energy diagram for this linkage the existence of onyl a single conformation can be excluded. The NOE spectrum of the compound with the alpha(2-> 6)-linkage indicates a gt orientation for the Gal C-6 hydroxymethyl group. On this basis, the HSEA energy calculations for the alpha(2->6)-linkage indicate an extended low-energy surface with a number of preferred conformations. The absence of NOEs across this linkage is interpreted in terms of a non-rigid, but overall folded conformation of the NeuAcalpha(2->6)Galbeta(1->4)GlcNAcbeta structural element. This provides an explanation for the shift effects induced by alpha(2->6) attachment of NeuAc to the N-acetyllactosamine unit.

The present study was initiated in order to determine the primary structures of the carbohydrate units of desialyzed human plasma al-acid glycoprotein. In an earlier investigation I6 glycopeptides which originated from four of the five glycosylation sites of this protein were prepared in the homogeneous state. Since the elucidation of the structures of such a large number of carbohydrate units by the conventional chemical and enzymatic procedures would require a very long period of time and considerable amounts of material, a new method-360-MHz H NMR spectroscopy-in combination with methylation analysis and partial acetolysis was introduced i n the present study. This highly sensitive method allowed the elucidation of the structures of the carbohydrate units of the mentioned glycopeptides to be carried out in a very short period of time. The present investigation revealed that the structures of the 16 heteroglycans can be grouped into five classes, and, except for those of two classes, the structures are new. Two classes designated A and B possess the known bi- and trimtennary structures, respectively. A third class designated C proved to be tetraantennary in its structure, whereas the other classes designated BF and CF are, in comparison with the structures of classes B and C, characterized by an additional fucose residue in a hitherto unknown position and linkage. The latter monosaccharide residue is attached in an a( 1-3) bond, vicinal to Gal@(1 +4), to the GlcNAc residue 7. The present study also revealed that each glycosylation site of pooled a]-acid glycoprotein possesses carbohydrate units with different structures.

The discovery that certain cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain, which is localized opposite to the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase involved in receptor phosphorylation and in specific signal transduction. It is expected that blood accumulation of free or membrane-bound glucan ligands of cytokines may dramatically perturb their endogenous function inducing specific immunodeficiencies.