Neutral oligosaccharides in the urine of a patient with glycogen storage disease type II

Neutral oligosaccharides were isolated from urine of an adult patient with glycogen storage disease type II, a deficiency of lysosomal acid alpha-glucosidase, by chromatography on columns of activated charcoal, Dowex 50 X 2 and Dowex 1 X 2. Total neutral oligosaccharides in the urine of the patient were increased about 5-fold as compared with those in normal controls. The most accumulated oligosaccharide was separated by Bio-Gel P-2 column chromatography, and finally purified by paper chromatography. Based on various studies, including carbohydrate analysis, chemical ionization mass spectrometry, fast atom bombardment mass spectrometry, degradation by glucoamylase and isopullulanase, and methylation analysis, the structure of this oligosaccharide was deduced to be Glc alpha 1----6Glc alpha 1----4Glc alpha 1----4Glc. This oligosaccharide appears to be accumulated in urine of the patient with acid alpha-glucosidase deficiency as an end product of the hydrolysis of glycogen.     

Purification and characterization of alpha-L-fucosidases from Streptomyces sp. OH11242

alpha-L-Fucosidases were found in the culture fluid of Streptomyces sp. OH11242 grown with porcine gastric mucin (PGM) as the sole carbon source. The alpha-L-fucosidases were purified by ammonium sulfate precipitation followed by chromatography on Sepharose CL-4B, hydroxyapatite, Resource Q and Mono Q. Two enzyme fractions, termed Fase-I and Fase-II, were obtained, each bearing different substrate specificity. Fase-I hydrolyzed fucose residues from fucose-containing oligosaccharide chains on PGM, but not p-nitrophenyl alpha-L-fucoside (Fucalpha-O-PNP). In contrast, Fase-II cleaved fucose from Fucalpha-O-PNP, but not fucose-containing oligosaccharides on PGM. Fase-I also hydrolyzed the alpha1-2 fucosidic linkage in various oligosaccharides, but not alpha1-3 and alpha1-4 fucosidic linkages. Fase-II was separated into two fractions, Fase-IIa and -IIb by Mono Q chromatography, Fase-IIb hydrolyzed alpha1-3 and alpha1-4 fucosidic linkages, but not alpha1-2 fucosidic linkages, while Fase-IIa hydrolyzed none of them. Fase-I was purified to homogeneity by SDS-polyacrylamide gel electrophoresis, the molecular mass was estimated to be approximately 59000 and 76000 Da by SDS-PAGE and gel-permeation chromatography, respectively. The optimum pH for Fase-I activity was 5.5-6.0. These fucosidases with different substrate specificities might be useful to reveal the physiological role of fucose-containing oligosaccharides in the gastric mucins.     

Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Fish egg polysialoglycoproteins: structures of new sialooligosaccharide chains isolated from the eggs of Oncorhynchus keta (Walbaum). Fucose-containing units with oligosialyl groups

A series of acidic oligosaccharide alditols having different neutral core oligosaccharides were isolated from salmon egg polysialoglycoproteins by alkali-borohydride treatment followed by anion-exchange chromatography and Iatrobead chromatography. Their structures were determined by methylation analysis, molecular secondary ion mass spectrometry of underivatized oligosaccharides, and enzymatic desialylation. The molecular secondary ion mass spectra of intact sialooligosaccharides exhibit pronounced quasi-molecular-ion peaks, (M + H)+, (M + Na)+, (M + 2Na - H)+, and/or (M + K)+, as well as some diagnostic sequence ion peaks. Of a number of oligosaccharide alditols, the following are novel: Fuc alpha 1 leads to 3GalNAc beta l1 leads to 3Gal beta 1 leads to 4Gal beta 1 leads to 3[(leads to 8NeuGc alpha 2)n leads to 6]GalNAcol (n = 1-6). The proton nuclear magnetic resonance spectra of these oligosaccharides are also reported and discussed.     

A urinary pentasaccharide in bovine mannosidosis.

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: alpha-D-Manp-(1 leads to 6)-beta-D-Manp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis.     

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Characterization of a novel type of chain-terminator Gal beta 1-6Gal beta 1-4)GlcNAc in an oligosaccharide related to N-glycosylated protein glycans isolated from GM1 the urine of patients with gangliosidosis

Two new oligosaccharides were isolated from the urine of a patient with GM1 gangliosidosis. Final purification of the oligosaccharides was accomplished by capillary supercritical fluid chromatography. Structural analysis was by chemical analysis, chemical-ionization mass spectrometry and 400-MHz 1H-NMR spectroscopy, leading to two primary structures. The first is derived from a classical triantennary N-acetyllactosamine-type glycan: Gal beta 1-4GlcNAc beta 1-4(Gal beta 1-4GlcNAc beta 1-2)Man alpha 1-3Man beta 1-4GlcNAc. The second is unusual with a terminal disaccharide Gal beta 1-6Gal, which had not yet been described for glycans of the N-acetyllactosamine type: Gal beta 1-6Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6Man beta 1-4GlcNAc.     

The Asn-linked carbohydrate chains of human Tamm-Horsfall glycoprotein of one male. Novel sulfated and novel N-acetylgalactosamine-containing N-linked carbohydrate chains.

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-beta-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, possessing the following terminal structural elements: [formula: see text]     

Structural analysis of O-glycosidic type of sialyloligosaccharide-alditols derived from urinary glycopeptides of a sialidosis patient

Sialidosis urine was fractionated by gel filtration on Bio-Gel P-6. All pooled fractions containing carbohydrates showed the presence of small amounts of GalNAc in non-reducing position, besides free N-acetyllactosamine type of oligosaccharides as major constituents. The fractions were subjected to reductive alkaline borohydride degradation, after which the major part of GalNAc was recovered as N-acetyl-D-galactosaminitol (GalNAc-ol). The GalNAc-ol-containing material was separated from the N-glycosidic oligosaccharides by a second gel-filtration step on AcA 202. Subsequently, the O-glycosidic sialyloligosaccharide-alditols were subfractionated by anion-exchange chromatography on Mono Q. Structural analysis by 500-MHz 1H-NMR spectroscopy revealed two major components in all fractions, namely: NeuAc alpha 2-3Gal beta 1-3GalNAc-ol and NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc-ol. Furthermore, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-6]GalNAc-ol was found as a minor component in some of the fractions. The presence of these carbohydrate chains in Bio-Gel fractions differing in molecular mass suggested that they are derived from glycopeptides which are heterogeneous in their peptide part.     

Structural characterization of novel complex oligosaccharides accumulated in the caprine beta-mannosidosis kidney. Occurrence of tetra- and pentasaccharides containing a beta-linked mannose residue at the nonreducing terminus

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.     

Polar glycosphingolipids in insect: chemical structures of glycosphingolipid series containing 2'-aminoethylphosphoryl-(----6)-N-acetylglucosamine as a polar group from larvae of the green-bottle fly, Lucilia caesar.

A series of glycosphingolipids containing 2'-aminoethylphosphoryl(----6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl----6GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, GalNAc alpha 1-4GalNAc beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, Gal beta 1-3GalNAc alpha 1-4GalNAc-beta 1-4(2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc beta 1-Cer, and GlcNAc beta 1-3Gal-beta 1-3GalNAc alpha 1-4GalNAc beta 1-4 (2'-aminoethylphosphoryl----6)GlcNAc beta 1-3Man beta 1-4Glc-beta 1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.     

Structural studies of the sugar chains of human parotid alpha-amylase

Human parotid amylase can be separated into three families of isoenzymes (A', A, and B) by Sephadex G-75 column chromatography. Isoenzymes in family B were free from carbohydrate, while those in family A were all glycoproteins. The carbohydrate moieties of family A isoenzymes were released from their polypeptide portions by hydrazinolysis and labeled by reduction with NaB[3H]4. The yield of total radioactive oligosaccharides indicated that family A isoenzymes all contain single asparagine-linked sugar chains in one molecule. The radioactive oligosaccharides were fractionated into one acidic and two neutral oligosaccharide fractions by paper electrophoresis and paper chromatography. By sequential exoglycosidase digestion in combination with a methylation study, their structures were determined to be: Gal beta 1 leads to 4 (Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 and 3[Gal beta 1 leads to 4 GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 (NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc.     

Structure of a major glycophosphoceramide from tobacco leaves, PSL-I: 2-deoxy-2-acetamido-D-glucopyranosyl(alpha1 leads to 4)-D-glucuronopyranosyl(alpha1 leads to 2)myoinositol-1-O-phosphoceramide.

The chemical structure of a major glycophosphoceramide from tobacco leaves, called PSL-I [K. Kaul and R. L. Lester (1975), Plant Physiol. 55, 120], has now been characterized as 2-deoxy-2-acetamido-D-glucopyranosyl(alpha1 leads to 4)D-glucuronopyranosyl(alpha1 leads to 2)myoinositol-1-O-phosphoceramide. Sites of glycoside linkage were determined by (1) methylation analysis on a trisaccharide isolated by degradation of carboxyl-reduced PSL-I and (2) periodate oxidation experiments on PSL-I. The resulting products were identified with gas chromatography/mass spectrometry. Anomeric configurations were determined by resistance of the sugars in the peracetylated trisaccharide to chromium trioxide treatment.     

Oligosaccharide structures of isolated human colonic mucin species

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.     

Structure of the carbohydrate moieties of bovine rhodopsin.

The sugar chains of bovine rhodopsin were released from the polypeptide moiety by hydrazinolysis and reduced with NaB[3H]4 after N-acetylation. The radioactive oligosaccharides thus obtained were fractionated into three components by paper chromatography. The structures of these components were elucidated as GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 and 6 Man alpha 1 leads to 6)Man beta leads to 4GlcNAc beta 1 leads to 4GlcNAc, and GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(Man alpha 1 leads to 3 (Man alpha 1 leads to 6)Man alpha 1 leads to 6)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, by sequential exoglycosidase digestion, methylation analysis, and endo-beta-N-acetylglucosaminidase D digestion. The unusual features of the sugar chains of rhodopsin molecule seem to support the proposed processing pathway for the biosynthesis of asparagine-linked sugar chains of glycoproteins.     

Isolation and characterization of novel phosphate-containing sialyloligosaccharides from normal human urine

Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(alpha)-P; NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc(alpha)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates.     

Characterisation by mass spectrometry and 500-MHz proton nuclear magnetic resonance spectroscopy of penta- and hexasaccharide chains of human foetal gastrointestinal mucins (meconium glycoproteins)

Structural studies using liquid secondary ion mass spectrometry, gas liquid chromatography/mass spectrometry and 500-MHz 1H NMR are described of the major penta- and hexasaccharides of a fraction of human foetal gastrointestinal mucins. Glycoproteins from a blood group H active meconium pool were studied after depletion of Ii antigenic activities by immunoaffinity chromatography and treatment with mild acid hydrolysis to reduce the chain heterogeneity. Oligosaccharides were released by mild alkali/borohydride degradation and purified by Bio-Gel P4 chromatography and HPLC. Eleven penta- and hexasaccharides have been fully characterised as a result of this study and one previous report [Hounsell et al. (1988) Biochem. J. 256, 397-401] and information obtained on additional oligosaccharides present in small amounts. These oligosaccharides show the following features: (table; see text) Sequences in these oligosaccharides not commonly found in mucins so far studied are chain-terminating GlcNAc alpha 1-4Gal, repeating-type-I (Gal beta 1-3GlcNAc) backbones, the backbone branch GlcNAc beta 1-6(GlcNAc beta 1-3)Gal and the backbone sequence GlcNAc beta 1-6Gal beta 1- in the absence of a substituent at C3 of galactose.     

Isolation and structural characterization of twenty-one sialyloligosaccharides from galactosialidosis urine. An intact N,N'-diacetylchitobiose unit at the reducing end of a diantennary structure

Galactosialidosis urine was fractionated by gel-permeation chromatography on Bio-Gel P-6. The obtained sialic acid-containing carbohydrate fractions were purified by reversed-phase chromatography and separated according to charge by medium-pressure anion-exchange chromatography on Mono Q. The Mono Q fractions, being mixtures of sialyloligosaccharides differing mainly in sialic acid-linkage type (alpha 2-3/alpha 2-6), were subfractionated by high-performance liquid chromatography on Lichrosorb-NH2. The purified compounds were analysed by 500-MHz 1H-NMR spectroscopy. Twenty-one fully and partially sialylated N-acetyllactosamine-type compounds include mono-, di-, tri- and tetra-antennary structures. All structures have the sequence Man beta 1-4Glc-NAc at the reducing terminus in common, except one diantennary structure bearing an intact N,N'-diacetylchitobiose unit at the reducing end, which is a new feature in human glycoproteinosis urine.