Oligosaccharide structures of human colonic mucin

Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

A computerized approach to the analysis of oligosaccharide structure by high-resolution proton n.m.r.

The 500 MHz proton-n.m.r. spectra of 21 oligosaccharides, predominantly of the lacto-N and lacto-N-neo series and their derivatives containing non-reducing terminal fucose, sialic acid or N-acetylgalactosamine and reduced-end hexitol or hexosaminitol, were examined with 2H2O as solvent. The chemical-shift data obtained from analysis of the spectra were collated with data from other laboratories who have used 250-500 MHz n.m.r. in the analysis of secreted and chemically synthesized oligosaccharides and of the O- and N-linked chains of glycoproteins. A referenced computer library was constructed that includes the chemical shifts of monosaccharides within oligosaccharide sequences that make up the majority of the carbohydrate structures found in mammalian glycoproteins. Examples are given of the computerized interrogation of this library for the assignment of possible structures of unknown oligosaccharides.     

Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin

The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)     

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group U (o 43) lipopolysaccharides

1. A series of oligosaccharides was isolated from Salmonella milwaukee lipopolysaccharide by partial acid hydrolysis. 2. Structural studies on these oligosaccharides indicated that the O-specific side chain of this lipopolysaccharide has a repeating pentasaccharide unit that is probably alpha-d-galactosyl-(1-->3)-beta-d-galactosyl- (1-->3)-N-acetylgalactosaminyl-(1-->3)-N-acetyl- d-glucosaminyl-(1-->4)-l-fucose. 3. Another oligosaccharide, which is not structurally related to the repeating pentasaccharide unit, has also been isolated and it is indistinguishable from an oligosaccharide obtained from Salmonella ;rough' (R) lipopolysaccharides. The isolation of this and similar core oligosaccharides from all chemotype VI lipopolysaccharides supports the view that Salmonella S-lipopolysaccharides have a common core that is probably identical with RII lipopolysaccharide.     

Structures of the asparagine-linked oligosaccharide chains of human von Willebrand factor. Occurrence of blood group A, B, and H(O) structures.

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the polypeptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophytohemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another noteworthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(O) structures. In the present study, this glycoprotein was shown to contain mono- (0.4% of the total oligosaccharides), bi-(78.2%), tri- (12.3%), and tetraantennary (2.3%) complex type oligosaccharides in addition to a series of high mannose type oligosaccharides, Man6-9GlcNAc2 (0.8%). Biantennary complex type oligosaccharide chains were those with (8.2%) and without (70.0%) a bisecting GlcNAc residue and approximately 13.2%, 2.2%, and 0.4% of these contained blood group H(O), A, and B structures, respectively. The tri- and tetraantennary complex type chains were those with and without N-acetyllactosamine repeats, and about 13.0% of the triantennary chains without the N-acetyllactosamine repeat contained the blood group H(O) structure. Occurrence of these asparagine-linked oligosaccharides with blood group A and B structures suggest that the repeated use of factor VIII/vWF pooled concentrate for the treatment of hemophiliacs could result in the production of antibodies against vWF with a different blood group from that of the patient, and this development may be pathogenic.     

The structure of carbohydrate unit B of porcine thyroglobulin.

The oligosaccharide fraction was obtained from porcine thyroglobulin by hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by successive chromatographies on columns of DEAE-cellulose and concanavalin A-Sepharose, and their structures were investigated by the combination of endo- and exo-glycosidase digestions, methylation analysis and Smith degradation. From the results of these studies, the structures of the unit B oligosaccharides were proposed to be as follows: see formula in text. Thus the glycoprotein was found to have triantennary and biantennary complex-type oligosaccharides as acidic sugar chains. Concerning the triantennary oligosaccharides, the following structural features were shown: (1) the sialic acid residues were not localized on certain specific branches but distributed on all three branches; (2) however, alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the terminal of the branch arising from C-4 of the branching alpha-mannose residue, whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of the other branches; (3) the outer branching alpha-mannose residue was attached to C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were observed to exist.     

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.     

Preparative, affinity chromatography of sheep gastric-mucins having blood-group Ii activity, and release of antigenically active oligosaccharides by alkaline-borohydride degradation

A large number of oligosaccharide fractions having blood-group Ii activity were obtained by degradation with alkaline borohydride of sheep gastric-glycoproteins which had been enriched for these blood-group activities by affinity chromatography on an anti-I adsorbent column. The approximate molecular weights of the oligosaccharides were estimated from their elution profiles on Bio-Gel P4 and from monosaccharide compositions. The fractions of smallest molecular weight with both I and i activities were mixtures of hexa- to octa-saccharides. From a comparison of the inhibitory activities of these with fractions of higher molecular weight and with synthetic oligosaccharides, it was concluded that the antigenic determinant recognised by anti-I Ma is a trisaccharide, whereas those recognised by other types of anti-I and by anti-i antibodies are longer than trisaccharides.     

The immunochemistry of Salmonella chemotype VI O-antigens. The structure of oligosaccharides from Salmonella group N (o 30) lipopolysaccharides

1. The lipopolysaccharides isolated from ;smooth' (S) strains of Salmonella godesberg and Salmonella urbana by the phenol-water method were purified in the ultracentrifuge. 2. These lipopolysaccharides have the same O-antigenic structure and on partial hydrolysis the same series of oligosaccharides was obtained in each instance. 3. The results of quantitative microanalysis, borohydride reduction, periodate oxidation, Morgan-Elson reactions and enzymic hydrolysis with alpha- and beta-glucosidases on the isolated oligosaccharides indicated that the O-specific side chains of these S-lipopolysaccharides have a repeating tetrasaccharide unit that is beta-d-glucosyl-(1-->3)-N-acetylgalactosaminyl-(1-->4)-l-fucose with a further glucose residue bound at the 4-position on the N-acetylgalactosamine. 4. Another oligosaccharide, a glucosylgalactose, has also been isolated and is indistinguishable from an oligosaccharide isolated from Salmonella R-lipopolysaccharides. These findings provide further evidence supporting the view that all Salmonella S-lipopolysaccharides have a core consisting of R-lipopolysaccharide.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin

Luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) from pituitary and chorionic gonadotropin (CG) from placenta are a family of closely related glycoproteins. Each hormone is a heterodimer, consisting of an alpha- and a beta-subunit. Within an animal species, the alpha-subunits of all four glyco-protein hormones have an identical amino acid sequence, whereas each beta-subunit is distinct and confers hormone-specific features to the heterodimer. LH and FSH are synthesized within the same cell, the gonadotroph of the anterior pituitary, but are predominantly stored in separate secretory granules. We have characterized the asparagine-linked oligosaccharides on bovine, ovine and human LH, FSH and TSH. The various pituitary hormones were found to contain unique sulfated oligosaccharides with the terminal sequence SO4-4GalNAc beta 1----4GlcNAc beta 1----2Man alpha, sialylated oligosaccharides with the terminal sequence SA alpha Gal beta GlcNAc beta Man alpha, or both sulfated and sialylated structures. Despite synthesis of LH and FSH in the same pituitary cell, sulfated oligosaccharides predominate on LH while sialylated oligosaccharides predominate on FSH for all three animal species. We have examined the reactions leading to synthesis of the sulfated oligosaccharides to determine which steps are hormone specific. The sulfotransferase is oligosaccharide specific, requiring only the sequence GalNAc beta 1----4GlcNAc beta 1----2Man alpha. In contrast, the GalNAc-transferase appears to be protein specific, accounting for the preferential addition of GalNAc to LH, TSH, and free (uncombined) alpha-subunits compared with FSH and other pituitary glycoproteins. The predominance of sulfated oligosaccharide structures on LH may account for sorting of LH and FSH into separate secretory granules. Differences in sulfation and sialylation of LH, FSH and TSH may also play a role in the regulation of hormone bioactivity.     

The catabolism of mammalian glycoproteins. Comparison of the storage products in bovine, feline and human mannosidosis.

Analysis of the neutral urinary oligosaccharides in bovine, feline and human mannosidosis by thin-layer and gel-permeation chromatography has shown that the patterns of stored oligosaccharides in the three species are different. In bovine and feline mannosidosis the most abundant urinary oligosaccharide is also the most abundant in the tissues of each species. The predominant oligosaccharides were purified by a combination of gel-filtration, ion-exchange and thin-layer chromatography and shown to contain only mannose and N-acetylglucosamine by g.l.c. and g.l.c.--mass spectrometry. The probable composition and size of each oligosaccharide were predicted from its chromatographic properties, sugar composition and the known structure of asparagine-linked oligosaccharides. The bovine and feline oligosaccharides belonged to a homologous series of general composition Mann (GlcNAc)2, whereas the human oligosaccharides belong to a different series, MannGlcNAc. These structures suggest that lysosomal endohexosaminidase is not present in bovine and feline tissues. The predominant feline storage product, Man3(GlcNAc)2, was the expected storage product from the catabolism of complex asparagine-linked glycans. In contrast, the predominant bovine oligosaccharide, Man2(GlcNAc)2, probably lacks one of the alpha-linked mannose residues in the core region. A similar situation occurs in human mannosidosis. It is predicted that in these species either that the residual mutant alpha-D-mannosidase retains activity towards one of the core alpha-linked mannose residues or that another form of lysosomal alpha-D-mannosidase that is unaffected in these disorders occurs. It is concluded that the differences in storage products are due to differences in the catabolic pathways of glycoproteins among the species.     

Comparison of the N-Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment and Relative Occurrence of Oligosaccharide Structures by Serial Lectin Affinity Chromatography of 14C-Glycopeptides

The N-linked oligosaccharide structures of human myelin-associated glycoprotein (MAG) and P0 have been characterized by serial lectin affinity chromatography (SLAC) of 14C-glycopeptides. 14C-Glycopeptides were prepared from purified MAG derivative and P0 by extensive proteolytic digestion and N-14C-acetylation. Assuming that all the 14C-glycopeptides were radiolabelled to the same specific radioactivity, the relative occurrence of the oligosaccharide structures was correlated to the amount of incorporated radioactivity. Sixteen and 15 fractions were generated by SLAC of MAG and P0 14C-glycopeptides, respectively. Despite this tremendous structural heterogeneity, the oligosaccharide "fingerprints" of MAG and P0 obtained by SLAC displayed similarities: (a) of the three types of N-linked oligosaccharides, the complex type accounted for 80.4% and 94.9% of MAG and P0 radioactivity, respectively; (b) biantennary complex oligosaccharides were the major structures present on MAG and P0; (c) approximately 60% of MAG and P0 oligosaccharides possessed a bisecting N-acetylglucosamine residue; and (d) large amounts of oligosaccharides with an alpha(1-6)fucose residue were found in both MAG and P0 and, noticeably, approximately 25% of the tri- and/or tetraantennary and approximately 90% of the bisected biantennary oligosaccharides of both glycoproteins contained alpha(1-6)fucose residues in the core. This study demonstrates that MAG and P0, both belonging to the immunoglobulin superfamily, display structural similarities in their N-linked oligosaccharide contents.     

Electrophoretic resolution and fluorescence detection of N-linked glycoprotein oligosaccharides after reductive amination with 8-aminonaphthalene-1,3,6-trisulphonic acid.

The relative mobilities of various N-linked oligosaccharides reductively aminated to the charged fluorophore 8-amino-naphthalene-1,3,6-trisulphonic acid (ANTS) were determined by electrophoresis on high-density polyacrylamide slab gels. Each ANTS-derivatized oligosaccharide was assigned a relative migration index (RMI) expressed in terms of glucose equivalents, which was conveniently estimated by reference to a homologous series of ANTS--maltooligosaccharides run on each gel as oligosaccharide size standards. High-mannose-, complex- and hybrid-type structures were generally well resolved and easily visualized at picomole levels by simple UV light excitation. Application of these methods for the qualitative analysis of the oligosaccharides released from bovine fetuin and bovine asialofetuin by peptide-N-glycosidase F illustrates the usefulness of these techniques as fast, simple, and inexpensive tools for the characterization of N-linked oligosaccharides attached to glycoproteins.     

Enzymatic transfer of mannose from mannosyl-phosphoryl-polyprenol to lipid-linked oligosaccharides by pig aorta.

A particulate enzyme preparation prepared from the intimal layer of pig aorta catalyzed the transfer of mannose from mannosyl-phosphoryl-polyprenol (MPP) into a series of oligosaccharides that were linked to lipid. The reaction required detergent with Triton X-100 and NP-40 being best at a concentration of 0.5%. Several other detergents were inactive or only slightly active. The pH optima for this activity was about 7 to 7.5 in Tris buffer and the apparent Km for MPP was about 2 x 10(-7) M. The reaction was not stimulated by the addition of divalent cation and, in fact, was inhibited by the high concentrations of cation. The addition of EDTA did not inhibit the transfer of mannose from MPP and was somewhat stimulatory. The transferase(s) activity was "solubilized" from the particles by treatment with Triton X-100. This solubilized enzyme still formed a series of lipid-linked oligosaccharides from either MPP or GDP-mannose. The oligosaccharides were released from the lipid by mild acid hydrolysis and were separated by paper chromatography. Some five or six radioactive oligosaccharides were formed from either MPP or from GDP-mannose and these oligosaccharides had similar mobilities upon paper chromatography. However, MPP was a better donor for the larger oligosaccharides (i.e. those containing 8, 9, or 10 sugar residues), whereas GDP-mannose was better for formation of the oligosaccharide containing 7 sugar residues. In the presence of EDTA and detergent no MPP was formed from GDP-mannose, but radioactivity was still incorporated into the lipid-linked oligosaccharides. Under these conditions essentially all of the radioactivity was in the oligosaccharide containing 7 sugar residues. Since much of this activity could be released as mannose by acetolysis, GDP-mannose may be the direct mannosyl donor for formation of 1 leads to 6 branches. Oligosaccharides 7, 8, 9, and 10 were isolated and partially characterized in terms of their molecular weights, sugar composition, susceptibility to alpha-mannosidase, and 14C products formed by acetolysis and periodate oxidation. The molecular weights ranged from 1310 for oligosaccharide 7 to 1750 for oligosaccharide 10. Hydrolysis of each oligosaccharide and reduction with NaB3H4 gave the expected ratio of [3H]hexitol to [3H]hexosaminitol based on the molecular weight of the oligosaccharide. However, the hexitol fraction contained [3H]mannitol and [3H]glucitol. Since the amount of radioactivity in glucitol was 2 to 4 times that in mannitol and since only glucosaminitol was found in the amino sugar peak, it seems likely that each 14C-oligosaccharide was contaminated with an unlabeled oligosaccharide of equal molecular weight containing glucose and GlcNAc. Acetolysis of the 14C-oligosaccharides gave rise to 14C peaks of mannose, mannobiose, and mannotriose. In the larger oligosaccharides, most of the radioactivity was in mannobiose whereas in oligosaccharide 7 most of the radioactivity was in mannose...     

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.     

ATP-Evoked Arachidonic Acid Mobilization in Astrocytes Is via a P2Y-Purinergic Receptor

The mouse monoclonal antibody HNK-1 and the human monoclonal IgM antibody present in patients with polyneuropathy both recognize carbohydrate epitope(s) on human myelin-associated glycoprotein and P0. In the present study, the oligosaccharide structures that bear the antibody epitope(s) were investigated. The extracellular derivative of myelin-associated glycoprotein (dMAG) was purified by immunoaffinity chromatography. P0 was electroeluted from gel slices. Western blot analysis of whole glycoproteins demonstrated that the epitopes for HNK-1 and the human monoclonal IgM antibody were different. The glycopeptides obtained by proteolysis of purified dMAG and P0 were separated and characterized by affinity chromatography on concanavalin A-Sepharose. Both dMAG and P0 displayed heterogeneity in their oligosaccharide structures, i.e., they both contained mainly tri- and tetraantennary oligosaccharides (approximately 80%), although biantennary (10%) and high-mannose and/or hybrid (10%) oligosaccharides were present. The human monoclonal IgM antibody epitope was present on all types of isolated oligosaccharide structures from either dMAG and P0. The HNK-1 epitope was present on all types of oligosaccharide structures of dMAG, whereas it was present only on tri- and tetraantennary structures of P0.     

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.     

Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation

The occurrence of N-linked oligosaccharides lacking galactose is significantly higher than normal in serum IgG of patients with rheumatoid arthritis (RA) in whom rheumatoid factor (RF), an autoantibody against autologous IgG, has been detected. In the present study, IgGs with and without RF activity (IgGRF and non-RF IgG, respectively) were prepared from sera of RA patients, and their oligosaccharide structures were characterized in order to investigate the relationship between RF activity and glycosylation. Three IgGRF fractions and a non-RF IgG fraction were obtained based on their ability to bind to an IgG-Sepharose column. The specific RF activity, as measured by immunoassays, was highest in the IgGRF fraction, which bound most avidly to the IgG-Sepharose. When the oligosaccharides were released by hydrazinolysis, and analyzed by MALDI-TOF mass spectrometry and HPLC, in combination with sequential exoglycosidase treatment, all the IgG samples were found to contain a series of biantennary complex-type oligosaccharides. The incidence of galactose-free oligosaccharides was significantly higher in both IgGRFs and non-RF IgG from RA patients compared with IgG from healthy individuals. In all IgGRFs, the levels of sialylation and galactosylation were lower than those in non-RF IgG from RA patients; the sialylation of non-RF IgG was the same as that of IgG from healthy individuals. In addition, the decreases in galactosylation and sialylation of oligosaccharides in IgGRF correlated well with the increase in RF activity. These findings could contribute to our understanding of the mechanisms of IgG-IgG complex formation and the pathogenicity of these complexes in RA patients.