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PubMed1982

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors.

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.

Animals↗
PubMed1984

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1.

Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.

Carbohydrate Sequence↗
PubMed1983

Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma.

The H-2Kk glycoprotein has been isolated by monoclonal antibody affinity chromatography, and an analysis of the asparagine-linked oligosaccharides present at the two major glycosylation sites has been performed. Antigen obtained from the AKTB-1b B-cell lymphoma that had been labeled with [2,6-3H]mannose for 5 or 21 h or for 5 h followed by a 5-h chase was digested exhaustively with trypsin. Each glycosylation site was then isolated by reverse phase high performance liquid chromatography using a C18 column. After removal from the peptide backbone by the almond emulsin peptide: N-glycosidase, the oligosaccharides from each isolated site were analyzed by gel filtration, ion exchange chromatography, concanavalin A affinity chromatography, and glycosidase treatment to assess the contribution of sialic acid and branching patterns of the oligosaccharide backbones to the overall microheterogeneity. The glycosylation of the H-2Kk antigen derived from several different AKTB-1b tumor preparations was examined during a period covering 1 year, during which time the tumor was passaged continuously in vivo in 2-week cycles. Our results conclusively demonstrate that the pattern of oligosaccharide microheterogeneity at the two glycosylation sites of the H-2Kk antigen derived from AKTB-1b cells is stable and that each site differs as to the specific array of oligosaccharide types found on the fully processed glycoprotein. In addition, this report describes an analytical scheme employing reverse phase high performance liquid chromatography to follow oligosaccharide processing and hydrolysis of the N-glycosidic bond by the peptide: N-glycosidase.

Animals↗
PubMed1983

Structures of the O-glycosidically linked oligosaccharides of human IgD.

In the previous communication (Mellis, S. J., and Baenziger, J. U. (1983) J. Biol. Chem. 258, 11546-11556), the structures of the oligosaccharides present at the 3 asparagine glycosylation sites of a human IgD myeloma protein were defined. In this communication, we present the structures of the O-glycosidically linked oligosaccharides located in the hinge region of IgD:WAH. Three or four threonine residues and one serine residue in the region bear O-glycosidically linked oligosaccharides. Approximately 50% of these molecules have the structure Gal beta 1 leads to 3 GalNAc which is identical with the structure of the predominant oligosaccharide in the hinge region of human IgA1 myeloma proteins (Baenziger, J. U., and Kornfeld, S. (1974) J. Biol. Chem. 249, 7270-7281). The remainder of the oligosaccharides contain 1 or 2 residues of N-acetylneuraminic acid and have the structures NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc (30%), Gal beta 1 leads to (NeuAc alpha 2 leads to 6)GalNAc (12%), and NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc (8%). The sialylated molecules have not been encountered previously on other human immunoglobulin heavy chains. These structures, however, have been described on a number of secreted and membrane glycoproteins. Examination of oligosaccharides isolated from different subregions of the IgD hinge indicated that a specific distribution of the sialylated structures among the glycosylated amino acids of the hinge region is not likely.

Amino Acid Sequence↗
PubMed1983

Biosynthesis of O-linked oligosaccharides on proteoglycans by chondrocytes from the swarm rat chondrosarcoma.

The core protein of proteoglycans from cartilage is substituted with glycosaminoglycans as well as N- and O-glycosidically linked oligosaccharides. We have taken advantage of the long intracellular half-life of the core protein precursor to the rat chondrosarcoma proteoglycan to study the temporal relationship between the addition of the chondroitin sulfate chains and the O-linked oligosaccharides onto the core protein during the formation of the completed proteoglycan molecule. Chondrocyte cultures were pulsed on day 2 with [6-3H]glucosamine for times ranging from 30-420 min. Media and corresponding 4% zwittergent, 4 M guanidine HCl extracts were then pooled and subjected to dissociative density gradient ultracentrifugation to yield purified proteoglycan monomers which were then subjected to alkaline borohydride treatment. The released chondroitin sulfate chains were then purified by precipitation with 50% (v/v) ethanol. The O-linked oligosaccharide-alditols in the supernatant fractions were purified by molecular sieve chromatography on Bio-Gel P-6, and analyzed after digestion with alpha-neuraminidase and subsequent chromatography on Bio-Gel P-2. The different O-linked oligosaccharide-alditols were identified from their hexosamine and hexosaminitol contents. The kinetics of entry of 3H label into N-acetylgalactosamine of chondroitin sulfate was indistinguishable from that into either N-acetylglucosamine or N-acetylgalactosaminitol residues of the oligosaccharide-alditols, with half-times to linear incorporation of 10-17 min. These results show that initiation as well as completion of the O-linked oligosaccharides on the core protein occurs essentially at the same time that chondroitin sulfate chains are added. The results suggest that these biosynthetic processes occur in the Golgi apparatus during the last few minutes of the total intracellular dwell time (half-time of about 90 min) of the core protein acceptor.

Animals↗
PubMed1982

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).

Carbohydrate Conformation↗
PubMed1981

Purification of human midcycle cervical mucin and characterization of its oligosaccharides with respect to size, composition, and microheterogeneity.

Human cervical mucin was solubilized from the gel phase of pooled midcycle cervical mucus using 6 M guanidine hydrochloride and 10 mM dithiothreitol and was then alkylated with iodoacetamide. Mucin was then purified by gel filtration on Bio-Gel A-50m resin in buffer containing 0.1% sodium dodecyl sulfate. The purified mucin gave a single band upon electrophoresis in either 5% acrylamide or 1% agarose gels. Protein comprised 21% of the glycoprotein by weight and amino acid analysis revealed a high content of Ser and Thr. Saccharide analysis yielded approximate molar ratios of Fuc:Gal:GlcNAc:GalNAc:NeuAc = 1:2:1:1:0.5. Inorganic sulfate, 1% by weight, was detected, but mannose was absent. Reductive alkali treatment of mucin resulted in release of oligosaccharides with concomitant conversion of 77% of GalNAc to its reduced derivative N-acetylgalactosaminitol (GalNAcol) thus demonstrating O-glycosidic linkage of GalNAc to protein. Reduced oligosaccharides were purified by ion exchange chromatography on DEAE-cellulose, paper chromatography, and high resolution gel filtration on Bio-Gel P-2 resin. A total of 16 reduced oligosaccharides were identified by thin layer chromatography. These included neutral, sialylated, and sulfated oligosaccharides and they varied in size from a disaccharide to a nonasaccharide. The major neutral oligosaccharide isolated (21% of recovered GalNAcol) was a tetrasaccharide, Gal:GlcNAc:GalNAcol = 2:1:1, and the major acidic oligosaccharide isolated (11% of recovered GalNAcol) was a trisaccharide, Gal:GalNAcol:NeuAc = 1:1:1.

Amino Acids↗
PubMed1982

Effects of mannoprotein mutations on Saccharomyces cerevisiae core oligosaccharide structure.

By the combined actions of an endo-alpha-1 leads to 6-mannanase and an endo-beta-N-acetylglucosaminidase, the core oligosaccharides can be released from Saccharomyces cerevisiae X2180 mnn2 mannoproteins. The effects of various mannoprotein mutations were evaluated by structural comparison of these core oligosaccharides with those prepared from double mutant strains with the genotypes mnn1 mnn2, mnn2 mnn3, mnn2 mnn4, and mnn2 mnn5. The results indicate that only the mnn1 lesion has a major effect on the mannoprotein core structure. Whereas the mnn2 mannoprotein yields a core composed of 6 fragments that differ in size from each other by single mannose units, only the two smallest species predominate in the mnn1 mnn2 preparation. This change is correlated with a loss of terminal alpha 1 leads to 3-mannosyl residues, an effect on the mnn1 lesion that is found also in the polysaccharide outer chain and hydroxyamino acid-linked mannooligosaccharides. The mnn3 and mnn5 mutations also had slight effects on the core size, but clear differences in linkage composition were not apparent. The results suggest that core oligosaccharides have an average composition of Man11GlcNAc, whereas Man9GlcNAc is the major oligosaccharide in strains containing the mnn1 defect. These values are 2 to 3 sugars less than those estimated previously (Nakajima, T., and Ballou, C. E. (1975) Biochem. Biophys. Res. Commun. 66, 870-879). Detailed analysis of the major core oligosaccharide from the mnn1 mnn2 mutant revealed that the two mannoses in alpha 1 leads to 3 linkage to the backbone were adjacent to each other and that the oligosacccharide is nearly identical with one isolated from chinese hamster ovary cell membranes (Li, E., and Kornfeld, S. (1979) J. Biol. Chem. 254, 1600-1605). This finding provides strong evidence for the evolutionary conservation of this structural feature of the high mannose core oligosaccharides.

Acetylglucosaminidase↗
PubMed1982

Structural studies of the phosphorylated high mannose-type oligosaccharides on human beta-glucuronidase.

Phosphomannosyl residues present on numerous acid hydrolases serve a critical role in mediating the endocytosis and intracellular transport of these glycoproteins. Previous work established that the mannose 6-phosphate on lysosomal enzymes is present on endoglycosidase H releasable oligosaccharides and that much of the phosphate is in diester linkage. In order to determine the number and location of the phosphates as well as the precise arrangement of the neutral sugar residues, we examined the structures of the phosphorylated oligosaccharides from a single acid hydrolase, human beta-glucuronidase isolated from spleen. The beta-glucuronidase-derived phosphorylated oligosaccharides are all high mannose-type oligosaccharides whose linkages correspond to previously described prototypical high mannose structures. They contain 1 or 2 moieties of mannose 6-phosphate/oligosaccharide. The major species contains 1 phosphate in diester linkage and represents approximately 63% of the phosphorylated oligosaccharides. Only 15% of the phosphorylated oligosaccharides have their phosphate exclusively in monoester linkage. The phosphate(s) present on these molecules is heterogeneous in location, but all of the phosphate present on the branch linked to the 3-carbon of the beta-linked mannose is found on its innermost alpha-1,2-linked mannose. Analysis of the phosphate-covering moiety showed it to be alpha-linked N-acetylglucosamine in most, if not all, cases.

Alkaline Phosphatase↗
PubMed1982

Structures of N-linked and O-linked oligosaccharides on proteoglycan monomer isolated from the Swarm rat chondrosarcoma.

Monomer proteoglycans isolated from the Swarm rat chondrosarcoma contain O- and N-glycosidically linked oligosaccharides. The O-glycosidically linked ones were released from the protein core by alkaline borohydride treatment. After removal of sialic acid, the resulting oligosaccharide alditols were converted to N-trifluoroacetyl derivatives by trifluoroacetolysis. The N-trifluoroacetylated oligosaccharide alditols show shorter retention time on gas-liquid chromatography, as permethylated derivatives, than the corresponding N-acetyl derivatives. The mass spectra of these new derivatives also give more structural information and provide definitive structures. Alkaline borohydride treatment of the proteoglycans also released N-glycosidically linked oligosaccharide-peptides. These were purified and analyzed by Smith degradation, trifluoroacetolysis, and chromium trioxide oxidation. The results indicate that more than 70% of the N-glycosidically linked oligosaccharides have the following structure: (formula, see text) with the Fuc present on only about 60% of the oligosaccharides.

Animals↗
PubMed1980

N-Linked glycoprotein assembly. Evidence that oligosaccharide attachment occurs within the lumen of the endoplasmic reticulum.

The transbilayer orientation of the oligosaccharide chain transferred from oligosaccharide-lipid to endogenous protein acceptors in sealed hen oviduct microsomes has been examined using endo-beta-N-acetylglucosaminidase H as a topological probe. The oligosaccharide moiety of these acceptors was released by the enzyme only under conditions where the microsomes were made permeable to macromolecules. The release of the oligosaccharide chain by endo-beta-N-acetylglucosaminidase H was not increased by removal of ribosomes or by mild trypsinization of the sealed microsomes. The endogenous acceptors were shown to be membrane-associated proteins that are not a part of the mRNA . ribosome . tRNA . nascent chain complex. From these results we conclude that the transfer of oligosaccharide from oligosaccharide-lipid occurs at the luminal face of the rough endoplasmic reticulum.

Acetylglucosaminidase↗
PubMed1980

Asparagine-linked sugar chains of glycoproteins in calf thymocyte plasma membrane. Isolation and fractionation of oligosaccharides liberated by hydrazinolysis.

The plasma membrane glycoproteins of calf thymocytes were converted to glycopeptides by exhaustive pronase digestion. Glycopeptides with asparagine-linked sugar chains were separated from those with mucine-type sugar chains by Bio-Gel P-10 column chromatography. The asparagine-linked sugar chains were released as oligosaccharides from the peptide moiety by hydrazinolysis and labeled by reduction with NaB[3H]4. The radioactive oligosaccharides were fractionated into fifteen acidic components and ten neutral components by combination of paper electrophoresis and Bio-Gel P-4 column chromatography. The acidic nature of all fifteen acidic components can be ascribed to their N-acetylneuraminic acid residues. The Bio-Gel P-4 column chromatographic patterns of the neutral oligosaccharide fraction and of the neutral fraction obtained on sialidase treatment of the pooled acidic oligosaccharide fraction were totally different, indicating that the acidic oligosaccharides are not simple sialyl derivatives of the neutral oligosaccharides.

Animals↗
PubMed1981

Observation by 13C NMR of interactions between cholera toxin and the oligosaccharide of ganglioside GM1.

The initial event in the action of cholera toxin on intact cells is its recognition of cell-surface receptors, molecules of ganglioside GM1. We have studied details of this interaction by 13C NMR, which enables us to examine simultaneously both the protein and the ganglioside or, as in the present instance, its oligosaccharide portion. 13C NMR spectra of the toxin are consistent with the long correlation times expected for this 84,000-dalton protein. They show, however, some resolved resonances (including one tentatively assigned to the epsilon 2 carbon of tryptophan, 138.3 ppm downfield from tetramethylsilane). When oligosaccharide is added to the toxin this resonance broadens or moves further upfield to reside under phenylalanine resonances at 136.7 ppm. Of the seven tryptophan residues in cholera toxin, five are in the B subunits which bind GM1, so that the data are consistent with a resonance shift for the epsilon 2 carbons of these residues on binding the oligosaccharide. Resonances arising from the anomeric and methylene carbons of the sialic acid moiety of the oligosaccharide are also shifted. Comparison with corresponding chemical shifts in a series of model compounds suggest that the latter effects may originate in a toxin-induced conformational change in the oligosaccharide. At high resolution, anomeric carbon resonances of terminal galactose residues in free and bound oligosaccharide are also resolved.

Carbohydrate Conformation↗
PubMed1993

Purification and characterization of an enzyme releasing lacto-N-biose from oligosaccharides with type 1 chain.

An enzyme specific for oligosaccharides with type 1 chain was purified 7,000-fold from the culture broth of Streptomyces sp. 142. The enzyme, lacto-N-biosidase, was induced and secreted into culture medium when the strain was cultured in the presence of porcine stomach mucin. The enzyme was purified by anion-exchange chromatography on Q Sepharose, cation-exchange chromatography on S Sepharose, fast protein liquid chromatography on a Mono S column, and gel filtration chromatography on TSK gel HW55S. To remove contaminating alpha-1,3/4-fucosidase and beta-N-acetylglucosaminidase, final purification was done by fast protein liquid chromatography on a Mono S column and affinity chromatography on N-acetylglucosamine agarose. The purified enzyme gave only one major protein band with an apparent M(r) of 60,000 on sodium dodesyl sulfate-polyacrylamide gel electrophoresis. The enzyme had maximum activity at pH 5.5 and was stable at the pH range of 4.0-10.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine showed that the enzyme specifically hydrolyzed lacto-N-tetraose and the N-acetyllactosamine type of triantennary sugar chain with the type 1 chain, but did not hydrolyze type 2 chain oligosaccharides or the type 1 chain oligosaccharides with fucose or sialic acid including lacto-N-fucopentaose I and II and alpha-2,3-sialyl lacto-N-tetraose. The enzyme released lacto-N-biose from asialofetuin, and almost all oligosaccharides in asialofetuin were found to have only type 2 chains. Sequential digestion of extended type 1 chain oligosaccharides with alpha-1,3/4-fucosidase and lacto-N-biosidase was possible.

Asialoglycoproteins↗
PubMed1994

Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.

Lipoprotein lipase (LpL) catalyzes the hydrolysis of triglycerides in plasma lipoproteins at the luminal surface of the vascular endothelium. This enzyme is bound via electrostatic interactions to heparan sulfate (HS). The specific endothelial cell surface HS oligosaccharide sequences that are necessary for binding of LpL to HS have not been characterized. To identify this LpL-binding oligosaccharide sequence, oligosaccharides were isolated from bovine aortic endothelial cell-derived HS and assessed for LpL binding properties. Endothelial HS chains that were isolated from endothelial total cell-associated proteoglycans were deacetylated by complete hydrazinolysis, cleaved with nitrous acid (pH 4.5), and reduced with [3H]NaBH4. The resulting fragments composed of N-sulfated glucosamine-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) were chromatographed on a LpL-Sepharose column. A high affinity decasaccharide was isolated and characterized. Disaccharide analysis of this decasaccharide indicated that it yielded only the disaccharide IdceA(2-SO4)-->AManR(6-SO4) on treatment with nitrous acid at low pH. Therefore, the sequence of the LpL-binding decasaccharide is [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4)) alpha 1-4]4-IdceA(2-SO4) alpha 1-4AManR(6-SO4) and is distinct from those that bind antithrombin and basic fibroblast growth factor. Partial depolymerization of endothelial HS chains with hydrazine/high pH nitrous acid treatment gave rise to lipase-binding oligosaccharides larger than decasaccharide. However, further complete depolymerization of these oligosaccharides resulted in only a high affinity decasaccharide composed of repeating disaccharide units of [IdceA(2-SO4) alpha 1-4GlcNSO4(6-S0(4))]. These results indicate that the decasaccharide is the active fragment that binds to LpL with high affinity. Molecular modeling studies of the decasaccharide indicate that it presents a linear array of negatively charged sulfate groups that may adopt a favorable disposition to bind to peptide region(s) comprised of basic amino acid residues of LpL with high affinity.

Animals↗
PubMed1993

Development of neoglycoproteins conjugated with natural oligosaccharides through carboxyl residues of proteins and its application to recombinant human interleukin 1.

In order to develop glycosylated cytokines, neoglycoproteins were synthesized utilizing naturally occurring oligosaccharides. High mannose type oligosaccharide-asparagine (Asn)s, containing Man8GN2-Asn, Man7GN2-Asn and Man6GN2-Asn, were obtained from quail ovalbumin and were coupled to bovine serum albumin (BSA) by carbodiimide-mediated coupling. Major reaction occurred between amino residues of oligosaccharide-Asns and carboxyl residues of BSA. Approximately one molecules of oligosaccharides-Asns were coupled to per molecule of BSA with 50% yield of glycosylation. Among the oligosaccharides, Man6GN2-Asn appeared to be conjugated predominantly. While this strategy was applied to recombinant human interleukin 1 alpha (IL-1 alpha), three molecules of oligosaccharide-Asns were introduced into per molecule of IL-1 with 10% yield of glycosylation.

Animals↗
PubMed1993

Biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked glycans labeled by UDP-[6-3H]galactose, CMP-[9-3H]N-acetylneuraminic acid, and [acetyl-3H]acetyl-coenzyme A.

When a rat liver Golgi apparatus-enriched subcellular fraction is incubated with UDP-[3H]Gal, CMP-[3H] Neu5Ac, or [acetyl-3H]acetyl (Ac)-CoA, label is efficiently transferred to endogenous acceptors, which are resistant to added proteases, unless detergent is added at a sufficiently high concentration. Thus, the acceptors are within the lumen of intact compartments of correct topological orientation, which are likely to be similar to those of the Golgi apparatus in the intact cell. In each case, approximately 90% of the macromolecular radioactivity is specifically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase digestion, as labeled N-linked oligosaccharides. Label from UDP-[3H]Gal is transferred to several distinct N-linked oligosaccharides, and many of these carry sialic acid (Sia) residues. This amount increases if the transfer reaction is chased with CMP-Neu5Ac. A major fraction of the [3H]Gal is directly "covered" with Sia residues, indicating that at least a portion of the beta-galactosyltransferase(s) are co-localized with one or more sialyltransferases. The majority of the [3H]Gal is found in a beta 1,3-linkage, rather than the more common beta 1,4-linkage. The N-linked oligosaccharides labeled by CMP-[3H] Neu5Ac carry labeled Sia residues in either alpha 2,3 or alpha 2,6 linkage, and showed a range of charge distribution. The transferred [3H]Neu5Ac is not O-acetylated even when Ac-CoA is added at saturating concentrations, implying that the sialyltransferases and the O-acetyltransferase(s) are not functionally co-localized. However, approximately 20% of label released from N-linked oligosaccharides by sialidase does not co-migrate with authentic Neu5Ac in high performance liquid chromatography analysis, indicating that transferred [3H] Neu5Ac is modified by unknown enzymes in the Golgi. Most of the [3H]acetate transferred from [acetyl-3H] Ac-CoA to N-linked oligosaccharides is on Sia residues that are exclusively alpha 2,6-linked, and is enriched on tri- and tetra-antennary chains that do not appear to carry any 2,3-linked Sia residues. These data indicate a restricted substrate preference of the O-acetyltransferase(s). About one-quarter of the [3H]acetate transferred is sialidase-resistant, indicating either transfer to monosaccharides other than sialic acid, or to sialidase-resistant sialic acids. While most of these sialidase-resistant oligosaccharides remain negatively charged, about 10% are neutralized by sialidase, confirming transfer of [3H]acetate to monosaccharides other than sialic acid.

Acetyl Coenzyme A↗
PubMed1977

Structural analysis of oligosaccharides isolated from the urine of a blood group A, secretor, woman during pregnancy and lactation.

Twenty different oligosaccharides have been isolated from urine collected from an A, Le(a- b+), secretor woman during her pregnancy and subsequent lactation. Nine of these have been found previously in the milk of Le (a- b+) individuals. Three new fucose-containing oligosaccharides denoted lacto-N-neotrifucoheptaose II (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc), lacto-N-neodfucohexaose I (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 4)-[alpha-L-Fuc-(1 leads to 3)]beta-D-GlcNAc-(1 leads to 3)-beta-D-Gal-(1 leads to 4)-D-Glc), and lacto-N-difucohexaose IV (alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 3)-beta-D-GlcNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 4)-D-Glc) are described. Three new myo-inositol-containing oligosaccharides which are characteristic for pregnancy have also been isolated. Their structures are: alpha-D-GalNAc-(1 leads to 3)-[alpha-L-Fuc-(1 leads to 2)]-beta-D-Gal-(1 leads to 0)-[alpha-L-Fuc-(1 leads to 0)]-myo-inositol; alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-[alpha-D-Fuc-(1 leads to 0)]myo-inositol; and alpha-L-Fuc-(1 leads to 2)-beta-D-Gal-(1 leads to 0)-myo-inositol, respectively. Three oligosaccharides containing only glucose were found and partially characterized. In addition, two oligosaccharides described previously (a glucose tetrasaccharide (Hallgren, P., Hansson, G., Henriksson, K.-G., Häger, A., Lundblad, A., and Svensson, S. (1974) Eur. j. clin. Invest. 4, 429-433) and a urine A pentasaccharide (Lundblad, A., and Svensson, S. (1973) Carbohyd. Res. 30, 187-189) were also isolated and characterized. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, and preparative paper chromatography, Structural determination involved sugar and methylation analyses. Tri- and tetrasaccharides were investigated by combined gas-liquid chromatography-mass spectrometry. Sequence analyses of larger oligosaccharides were performed by combined gas-liquid chromatography-mass spectrometry of di- and trisaccharides obtained after partial acid hydrolysis of the parent compound. Anomeric configurations were deduced from optical rotations and by comparison with authentic samples.

ABO Blood-Group System↗