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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]

The N-linked carbohydrate chains of the β subunit of human chorionic gonadotropin (hCG-β) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N&sup4;-(N-acetyl-β-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. ¹H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [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]

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3-deoxy-manno-2-octulosonic acid (42%). In addition two other 2-keto-sugar acids are present, namely, 3-deoxy-5-O-methyl-manno-2-octulosonic acid (7%), the first methylated derivative of 3-deoxy-manno-2octulosonic acid found in nature, and 3-deoxy-lyxo-2-heptulosaric acid (11%). The characterization of the three 2-keto-sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC-MS and 500-MHz ¹H-NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC-MS. Other monosaccharides occurring in the cell wall are D-galacturonic acid (14%), D-galactose (4%), D-gulose (2%), D-glucose (1%) and L-arabinose (1%). [ABSTRACT FROM AUTHOR]

Numerous studies have shown that glycosylation of the α-subunit of human chorionic gonadotropin (αhCG) is essential for the biological activity of this hormone. To obtain detailed insight into the function of N-glycosylation, the availability of site-specifically and fully deglycosylated α-subunits obtained under non-denaturing conditions is a prerequisite. NMR spectroscopy in combination with FAB-mapping demonstrates that only Asn52 of the α-subunit is accessible to digestion by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F under native conditions. Treatment of native αhCG with endo-β-N-acetylglucosaminidase B results in full deglycosylation yielding αhCG with one GlcNAc residue at both Asn52 and Asn78. [ABSTRACT FROM AUTHOR]

The N-linked carbohydrate chains of phospholipase A[sub2] from honeybee (Apis mellifera) were released from glycopeptides with peptide-N-glycanase A and reductively aminated with 2-aminopyridine. The fluorescent derivatives were separated by size-fractionation and reverse-phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500-MHz [sup1]H-NMR spectroscopy. Ten oligosaccharides consisted of mannose, N-acetylglucosamine and fucose α 1-6 and/or α 1-3 linked to the innermost N-acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with N-acetylgalactosamine. The structures of the 14 N-glycans from honeybee phospholipase A2 can be arranged into the following three series: (formula???) [ABSTRACT FROM AUTHOR]

Incubation of honeybee (Apis mellifica) venom-gland extracts with GDP-[14C]fucose and GlcNAcβ1 → 2Manα1 → 6(GlcNAcβ1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(Fucα1 → 6)GlcNAcβ1 → N-Asn-peptide(NAc) gave a labeled product in 40% yield. Analysis by 500-MHz 1H-NMR spectroscopy indicated the transferred fucose (Fuc) residue to be α1 → 3-1inked to the Asn-bound GlcNAc. Further proof was provided by one-dimensional and two-dimensional 1H-NMR analysis of the incubation mixture, after incubation with β-N-acetylhexosaminidase. The established carbohydrate structure [This equation cannot be converted into ASCII text] proves the existence of a novel α1 → 3-fucosyltransferase with the ability to effect difucosylation of the Asn-bound GlcNAc in N-glycans. [ABSTRACT FROM AUTHOR]

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H-ras were analyzed by 500-MHz ¹H-NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D-[³H]glucosamine or L-[³H]fucose and the glycopeptides included in Bio-Gel P-10 (Mr 5000-3500) were separated into neutral and charged fractions on DEAE-cellulose. The major portion (80%) of these [³H]fucose glycopeptides from the non-transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues. The structure of the predominant neutral glycopeptide from the non-transformed NIH 3T3 cells was determined by ¹H-NMR spectroscopy to be tetraantennary containing terminal Ga1α1→3. Multiple line equations cannot be represented in ASCII text. This structure was verified by binding to the immobilized u-Gal-specific lectin, Griffonia simplicifolia I and leukoagglutinating phytohemagglutinin from Phaseolus vulgaris (L-PHA), which binds certain trior tetraantennary glycopeptides. In contrast, the structure derived by NMR spectroscopy of one of the predominant charged glycopeptides from the transformed cells was triantennary containing terminal NeuNAcα2→3 in addition to Galα1→3. Multiple line equations cannot be represented in ASCII text. In attempting to verify this structure by lectin-binding properties it was found thai removal of NeuNAca2-*3 reduced the affinity to L-PHA-agarose. The other major glycopepfides of the transformed cells which were more charged also contained NeuNAca2-,3 but no NeuNAca2-*6 or Galed --,3. A tentative structure was proposed for the major glycopeptide of the first charged class from NIH 3T3 cells on the basis of lectin-binding properties and the NMR spectrum which showed, in addition to NeuNA@2-@3. the presence of NeuNAca2-*6 and Gal@l -*3. On the basis of the NMR spectrum and other results, it is concluded that the presence of tetraantennary oligosaccharides are not sufficient for the transformed oligosaccharide phenotype. Rather, the tri- or tetraantennae must be sialylated in @,2---3 linkage, on more than one antennae, when properties of transformation are expressed in NIH 3T3 cells. Prior to transformation the tetraantennary oligosaccharides of these cells are terminated in @,Gal residues, whereas after transformation s-Gal residues appear to be replaced by NeuNAca2 -* 3. Thus, in this case, transformation may be accompanied by a change in the glycosyltransferases responsible for termination of the branches rather than those conferring branch configurations. [ABSTRACT FROM AUTHOR]

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: [This symbol cannot be presented in ASCII format] [ABSTRACT FROM AUTHOR]