Hyperfucosylation of alpha 1-acid glycoprotein during cirrhosis.
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Carbohydrate Sequence↗
Biomedical subjects
B Fournet
Publications and source records attributed to B Fournet.
Microheterogeneity and structures of neutral glycans present in quail ovomucoid.
Anionic chromatography of oligosaccharide-alditols obtained by alkaline hydrolysis of quail ovomucoid glycopeptides resulted in the isolation of a major neutral fraction (83%) which was separated into 14 fractions by high-performance liquid chromatography on an alkylamine silica column. The structures of nine oligosaccharide-alditols were determined. By the identification of mannobiosido-, and mannotriosido-di-N-acetylchitobiose mixed with N-acetyl-lactosaminic and oligomannosidic-types oligosaccharides, we demonstrate the high microheterogeneity of the glycans presents in the neutral fraction of this glycoprotein and suggest that ovomucoid glycosylation uses several pathways.
Animals↗
Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X.
Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?
Amino Acid Sequence↗
400-MHz 1H-NMR spectroscopy of fucosylated tetrasialyl oligosaccharides isolated from normal and cirrhotic alpha 1-acid glycoprotein.
The comparative study of fucosylated tetrasialyl-oligosaccharides isolated from normal and cirrhotic alpha 1-AGP was performed using permethylation and 400-MHz 1H-NMR spectroscopy. These results clearly show the tetraantennary structure of these two oligosaccharides with hyperfucosylation for the tetrasialylated fraction from cirrhotic alpha 1-AGP. In the latter oligosaccharide the simultaneous presence on two antennae (7 and 7') of the sialosyl Lewis X determinant NeuAc-(alpha 2-3) Gal(beta 1-4) [Fuc(alpha 1-3)] GlcNAc has been observed. Moreover the 5 and 5' antennae were alpha 2-6 sialylated but without fucose.
Ascitic Fluid↗
Purification and carbohydrate structure of natural murine interferon-beta.
Mouse interferon-beta (Mu-INF-beta) induced in C-243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation. DEAE-cellulose, monoclonal Mu-IFN-beta antibody affinity and Mono-S cation-exchange chromatographies. Specific activity of the purified Mu-IFN-beta ranged over 1.1-1.4 X 10(9) NIH units/mg protein. This preparation was submitted to pronase digestion and gel on Fractogel TSK HW-40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical-ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6-tetra-O-methylgalactose; 3,4,6-tri-O-methyl-mannose; 2,3,4- and 2,4,6-tri-O-methylgalactose; 2,4,di-O-methyl mannose and 3,6-di-O-methylglucosamine. These results when compared with data on N-glycans suggest the following structure for the carbohydrate moiety of Mu-INF-beta: (formula; see text).
Animals↗
Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector.
A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.
Amino Acid Sequence↗
Isolation of the major O-glycosidically linked oligosaccharides obtained by alkaline borohydride degradation of human meconium glycoproteins.
Neutral and acidic oligosaccharides derived from human meconium glycoproteins by alkaline borohydride degradation have been separated by high-performance liquid chromatography on a Micro-Pak anion-exchange column. In each class, oligosaccharides were purified by normal-phase (neutral and acidic oligosaccharides) and reversed-phase (neutral oligosaccharides) chromatography. Effective separations of neutral oligosaccharides and acidic oligosaccharides were achieved.
Borohydrides↗
Continuous-flow fast atom bombardment-mass spectrometry of permethylated oligosaccharides: a comparative study of direct mixture analysis with packed capillary column liquid chromatography-fast atom bombardment-mass spectrometry.
Conventional positive fast atom bombardment (FAB) and continuous-flow FAB analysis were carried out with permethylated lacto-N-tetraose. This latter method, a new approach, has been used to analyze a mixture of permethylated oligosaccharides by liquid chromatography-mass spectrometry (LC-MS) with a packed capillary fused-silica column and the continuous-flow probe as interface. Under these conditions, we found the continuous-flow probe to be superior to the conventional probe because its low matrix level increased the signal-to-noise ratio. The analysis of the mixture of permethylated oligosaccharide alditols obtained from hen ovomucoid by LC-MS using the continuous-flow probe as interface is described.
Animals↗
Acylgalactosylceramides in developing dysmyelinating mutant mice.
Acylgalactosylceramides (AGC) from forebrains of normal and dysmyelinating (quaking and shiverer) mice were purified by Florisil column chromatography and preparative TLC. These procedures resolved the AGC on the basis of their Rf values into two main fractions which comigrate with their homologs from rat forebrains. In control animals, AGC were detectable in mouse forebrains from the eighth postnatal day and reached maximal values within 20 days. The same developmental pattern was obtained in dysmyelinating shiverer mice but the AGC content was reduced to approximately 30% of control values. In quaking mutants, the AGC were hardly detected. They were also present in sciatic nerve of normal mice and to a lesser extent in trembler mice. Gas chromatography-mass spectrometry analysis of both ester- and amide-linked fatty acids isolated from AGC of normal and shiverer mice shows that the shiverer mutant AGC display a chemical structure similar to that of normal AGC. AGC constituents of control myelin are reduced by approximately 70% in shiverer myelin, indicating that these molecules can be considered as early markers of oligodendrocyte differentiation. The early arrest of myelinogenesis in the quaking animals and the near absence of AGC are in good agreement with this proposal. Moreover, the reduced amount of AGC in the trembler PNS indicates that AGC could also be early markers for differentiation of the Schwann cell.
Animals↗
Primary structure of the glycans of porcine pancreatic lipase.
The glycan primary structure of the main glycopeptide fraction obtained by pronase and carboxypeptidase A digestions of porcine pancreatic lipase has been investigated by 500-MHz 1H-NMR spectroscopy and methylation analysis. The results demonstrate that the glycopeptide fraction was a mixture containing the following structures: (formula; see text)
Animals↗
Primary structure of an N-glycosidic carbohydrate unit derived from Sophora japonica lectin.
The lectin isolated from Sophora japonica seeds is a glycoprotein which binds oligosaccharides with non-reducing terminal Gal beta(1----3/4)GlcNac beta 1----units. The carbohydrate moiety of the lectin is composed of fucose, xylose, mannose and N-acetylglucosamine. The major glycopeptide of the lectin, prepared by pronase digestion, was derivatized with fluorescein isothiocyanate, purified by PAGE and examined by exoglycosidase digestion as well as purified by gel filtration through Bio-Gel P6-DG and investigated by methylation analysis and 400-MHz 1H-NMR spectroscopy. The primary structure of the glycopeptide was established to be as follows. (Formula: see text). Structures similar to this containing a (beta 1-2)xylosyl substituent on the core beta-mannosyl residue and an inner core (alpha 1-3)fucosyl substituent seem to occur frequently in plant glycoproteins.
Carbohydrate Conformation↗
Alterations in the carbohydrate moiety of alpha-1-acid glycoprotein purified from human cirrhotic ascitic fluid.
The carbohydrate analysis of alpha 1-AGPc purified from cirrhotic ascitic fluid was performed by immunoaffinity chromatography. It showed a large increase in the fucosyl molar ratio and sugar content (47%). The molar ratio of the oligosaccharides which were released by hydrazinolysis and fractionated by high-performance liquid chromatography confirms the marked increase in fucosyl residues in each fraction. A shift towards fractions with a high degree of branching was also observed. Moreover, the studies of sugar molar ratios and methylation of the tetrasialylated fraction indicated the simultaneous presence of sialyl and fucosyl residues on one of the outer branches.
Ascitic Fluid↗
Primary structure of O- and N-glycosylic carbohydrate chains derived from murine submandibular mucin (MSM).
The carbohydrate moiety of mouse submandibular mucin (MSM) contains mainly D-mannose and 2-acetamido-2-deoxy-D-glucose together with sialic acid, D-galactose, and 2-acetamido-2-deoxy-D-galactose. O-Glycosylically bound saccharides, obtained by treatment of MSM with alkaline borohydride, were shown by methylation analysis to have the structure: alpha-NeuAc-(2----3)-beta-Gal-(1----3)-GalNAc-ol. N-Glycosylically bound saccharides obtained from MSM by hydrazinolysis, and analysed by 500-MHz 1H-n.m.r. spectroscopy, were shown to have the following comprehensive structures. (Formula: see text).
Animals↗
Purification of the lipopolysaccharide fraction from Klebsiella pneumoniae O1 K2 by high-performance liquid chromatography.
An high-performance liquid chromatography technique was applied to purify the lipopolysaccharide fraction from a lysate of Klebsiella pneumoniae O1 K2 (NCTC 5055). The separation of the lipopolysaccharide fraction from the proteins was carried out with a reversed-phase column. By this method the lipopolysaccharide fraction was obtained in a pure state, devoid of proteins but possessing the same biological properties as the lipopolysaccharide fraction prepared by the classical phenol-water technique.
Bacterial Proteins↗
Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance.
This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).
Amides↗
Identification of UDP-galactose: lactose (lactosylceramide) alpha-4 and beta-3 galactosyltransferases in human kidney.
Two galactosyltransferases were identified in human kidney microsomes which both transfer galactose from UDP Gal to lactose as well as to lactosylceramide. Using a solubilized and a partially purified enzyme preparation sufficient product could be obtained for detailed structural analysis. The trisaccharide products were isolated by gel permeation chromatography and separated by preparative high performance thin layer chromatography. The anomeric configuration of the transferred galactose was determined by specific glycosidase digestion and the linkage was identified by methylation and gas-liquid-chromatography. The glycolipid products were not separated but analyzed directly, before and after alpha or beta galactosidase digestion, by methylation, hydrolysis and thin layer chromatography. Into both acceptor substrates galactose was incorporated in alpha 1-4 (30%) and beta 1-3 (70%) linkages. The alpha 1-4 galactosyltransferase is responsible for the synthesis of the Pk antigen Gal alpha 1-4 Gal beta 1-4 Glc-ceramide in human kidney. The beta 1-3 galactosyltransferase has not previously been identified.
Antigens, CD↗
Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture.
Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.
Animals↗
Separation of sialyl-oligosaccharides by high-performance liquid chromatography. Application to the analysis of mono-, di-, tri- and tetrasialyl-oligosaccharides obtained by hydrazinolysis of alpha 1-acid glycoprotein.
High-performance liquid chromatography has been applied to the separation of isomers of mono-, di-, tri- and tetrasialylated oligosaccharides derived from alpha 1-acid glycoprotein by hydrazinolysis. The separation of the sialyl-oligosaccharides on the basis of their negative charges was carried out with quaternary amine-bonded silica. Within each class, the anionic oligosaccharides were fractionated on the basis of their net carbohydrate content on alkylamine-modified silica using a mobile phase consisting of a mixture of acetonitrile and potassium dihydrogen phosphate with 0.01% of 1,4-diaminobutane or 0.01% of tetraethylpentamine.
Chromatography, Gas↗