Structures of the asparagine-linked sugar chains of laminin

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.     

Glycosylated major urinary protein of the house mouse: characterization of its N-linked oligosaccharides

A minor component of the major urinary protein complex of the house mouse was chromatographically isolated and ascertained to be a previously suspected glycoprotein. Using highly sensitive mass-spectrometric techniques for sequencing and linkage analysis, the N-linked oligosaccharides of this glycoprotein were characterized. They were determined to be of the complex type with a wide heterogeneity. The heterogeneity was due to both the degree of sialylation and the presence of galactose residues in either beta(1-3) or beta(1-4) linkages. The biantennary structures were the most pronounced glycans, while tri- and tetraantennary entities were minor.     

Structural studies on sialylated and sulphated O-glycosidic mannose-linked oligosaccharides in the chondroitin sulphate proteoglycan of brain.

We have previously described the structures of neutral and sialylated O-glycosidic mannose-linked tetrasaccharides and keratan sulphate polysaccharide chains in the chondroitin sulphate proteoglycan of brain. The present paper provides information on a series of related sialylated and/or sulphated tri- to penta-saccharides released by alkaline-borohydride treatment of the proteoglycan glycopeptides. The oligosaccharides were fractionated by ion-exchange chromatography and gel filtration, and their structural properties were studied by methylation analysis and fast-atom-bombardment mass spectrometry. Five fractions containing [35S]sulphate-labelled oligosaccharides were obtained by ion-exchange chromatography, each of which was eluted from Sephadex G-50 as two well-separated peaks. The apparent Mr values of both the large- and small-molecular-size fractions increased with increasing acidity (and sulphate labelling) of the oligosaccharides. The larger-molecular-size fractions contained short mannose-linked keratan sulphate chains of Mr 3000-4500, together with some asparagine-linked oligosaccharides. The smaller tri- to penta-saccharides, of Mr 800-1400, appear to have a common GlcNac(beta 1-3)Manol core, and to contain one to two residues of sialic acid and/or sulphate.     

The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides

The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

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.     

Structural Features of the Nerve Growth Factor Inducible Large External Glycoprotein of PC12 Pheochromocytoma Cells and Brain

Abstract: We have examined the oligosaccharide structure of a major M_r= 230,000 cell surface glycoprotein from rat PC12 pheochromocytoma cells, and of the immunochemically cross-reactive species present in brain. In response to nerve growth factor (NGF) the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. These morphological changes are accompanied by a 3- to 5-fold increase in the concentration and labeling of this cell surface glycoprotein, which has previously been named the NGF-inducible large external, or NILE, glycoprotein. Tri- and tetraantennary complex oligosaccharides are the predominant carbohydrate units present in the NILE glycoprotein from both brain and PC12 cells, where they represent 77–90% of the biosynthetically labeled oligosaccharides. Most of these are not substituted by fucose on the core N -acetylglucosamine which is linked to asparagine, and are accompanied by smaller proportions of biantennary and high-mannose oligosaccharides. Sequential lectin-agarose affinity chromatography employing concanavalin A, lentil lectin, and the leukoagglutinating lectin of Phaseolus vulgaris , together with neuraminidase treatment of the fractionated glycopeptides, demonstrated a moderate degree of microheterogeneity among the predominant tri- and tetraantennary oligosaccharide units with respect to the presence of core fucose, outer galactose and sialic acid residues, and the substitution positions on the α-linked mannose residues. NGF treatment of the PC12 cells had no significant effect on the oligosaccharide structure of the NILE glycoprotein. The greater molecular size of the PC12 cell NILE glycoprotein as compared to the immunochemically cross-reactive species present in brain (M_r= 205,000) is apparently due to the greater size of the PC12 cell tri- and tetraantennary complex oligosaccharides.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

Structural analysis of the oligosaccharides of DR1 and DQw1 molecules

The major glycopeptide fractions of the alpha- and beta-chains of HLA-DR1 and DQw1 molecules were isolated on columns of immobilized concanavalin A (Con A), Lens culinaris (Lens), Ricinus communis agglutinin Type I (RCA), and leuko-phytohemagglutinin. Oligosaccharides were prepared from these fractions by enzymatic digestion with Endoglycosidases H or F and were analyzed on Bio-Gel P-6. The glycopeptides tightly bound to Con A (ConA III) were mostly associated with alpha-chains and were resolved as a single oligosaccharide peak (Kd = 0.72) on Bio-Gel P-6 after Endo H digestion. Man-5 is the minimal polymannosyl structure which can be deduced for the ConA III fractions of either DQw1 or DR1 oligosaccharides. The major component of the glycopeptides of the alpha-chains of either DR1 or DQw1 molecules which were weakly bound to Con A (ConA II fraction) did not interact with RCA before or after mild acid hydrolysis or neuraminidase treatment. This component represents a biantennary complex with neither terminal galactose nor sialic acid residues with a minimal structure terminating in N-acetyl glucosamine on the Mannose alpha 1----6 arm, referred to as GnM. The ConA II fractions, which constitute 10% of the total glycopeptides of beta-chains, are associated primarily with fucosylated, sialylated biantennary oligosaccharides not seen on the alpha-chains. The ConA I unbound fractions of either alpha- or beta-chains were mostly bound to RCA after mild acid hydrolysis, suggesting that the minimal structure was a sialylated triantennary structure. The major component associated with the beta-chains was bound to Lens such that a more definite structural assignment can be made, i.e., a triantennary structure with the Mannose on the alpha 1----6 arm substituted at C-2 and C-6. The oligosaccharides of alpha- and beta-chains were resolved as broad peaks on Bio-Gel P-6, suggesting that a mixture of tri- and tetraantennary structures with variable degrees of sialylation and galactosylation were present. The structural differences reported here between oligosaccharides of alpha- and beta-chains of DQw1 and of the two subsets of DR1 molecules could be responsible in part for the differential recognition properties expected of human class II molecules encoded by distinct loci.     

Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH2. The structures of the carbohydrate chains were determined by 500- or 600-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (10%), disialylated diantennary (43%), disialylated tri-antennary (5%), trisialylated tri-antennary (13%), trisialylated tri'-antennary (8%), and tetrasialylated tetraantennary (12%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively alpha 2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing alpha 2-3- as well as alpha 2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an alpha 1-6-linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri'- and tetra-antennary oligosaccharide fractions small amounts (less than 5%) of compounds were detected having one or more additional N-acetyllactosamine units.     

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.     

Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin

The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.     

Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

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.     

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13 % of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2 %), bi-(23.9 %), tri-(9.0 %), tetra-(2.7 %), and penta-(2.8 %) antennary oligosaccharides. However, OM contained mostly tri-(33.5 %) and penta-(31.2 %) antennary oligosaccharides. The N-glycan–containing bisecting N-acetylglucosamine comprised 43.4 % and 79.8 % of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Major oligosaccharides in the glycoprotein of Friend murine leukemia virus: structure elucidation by one- and two-dimensional proton nuclear magnetic resonance and methylation analysis

The highly microheterogeneous, N-glycosidically linked oligosaccharides in the glycoproteins of Friend murine leukemia virus (as produced by Eveline cells) were liberated with endo-beta-N-acetylglucosaminidase H and by alkaline hydrolysis. They were fractionated (as desialylated oligosaccharitols) by gel filtration and by concanavalin A affinity chromatography, and the major fractions were analyzed by methylation-gas chromatography-mass spectrometry, by digestion with exoglycosidases, and, especially, by one- and two-dimensional proton nuclear magnetic resonance spectroscopy. Guidelines for qualitative and quantitative analysis of complex oligosaccharide mixtures by NMR were worked out and the results compared with those obtained by methylation analysis. It was found that these major fractions consist of bi-, tri-, and tetraantennary oligosaccharitols of the "complex" type (comprising a minority of species with N-acetyllactosamine repeating units), which are, in part, substituted by nonreducing terminal Gal alpha (1----3) and/or bisecting GlcNAc beta (1----4) residues.     

Structures of the N-linked oligosaccharides of Gp63, the major surface glycoprotein, from Leishmania mexicana amazonensis

The extent of protein N-glycosylation in Leishmania mexicana amazonensis has been proposed to be a factor in the virulence of the parasite. The N-linked oligosaccharides of gp63, the major surface glycoprotein of L. mexicana amazonensis, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P-4 yielded four fractions, and the oligosaccharides present were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis, and methylation analysis. Four major structures were found and were biantennary oligomannose type with compositions of Glc1Man6GlcNAc2 (La), Man6GlcNAc2 (Lb), Man5GlcNAc2 (Lc), and Man4GlcNAc2 (Ld). The largest oligosaccharide (La) was shown to contain a terminal glucopyranosyl residue on the alpha (1----3) arm. The biantennary oligomannose structures (Lb and Lc) and the glucosylated structure Glc1Man6GlcNAc2 (La) have not previously been reported as a component of a mature glycoprotein from any source.     

Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones

We have elucidated the structures of the anionic asparagine-linked oligosaccharides present on the glycoprotein hormones lutropin (luteinizing hormone), follitropin (follicle-stimulating hormone), and thyrotropin (thyroid-stimulating hormone). Purified hormones, isolated from bovine, ovine, and human pituitaries, were digested with N-glycanase, and the released oligosaccharides were reduced with NaB[3H]4. The 3H-labeled oligosaccharides from each hormone were then fractionated by anion-exchange high performance liquid chromatography (HPLC) into populations differing in the number of sulfate and/or sialic acid moieties. The anionic oligosaccharides were further purified as well as structurally characterized using a variety of preparative and analytical techniques, including HPLC, endo- and exoglycosidase digestions, and lectin affinity chromatography. The sulfated, sialylated, and sulfated/sialylated structures, which together comprised 67-90% of the asparagine-linked oligosaccharides on the pituitary glycoprotein hormones, were highly heterogeneous and displayed hormone- as well as animal species-specific features. The sulfated oligosaccharides consisted of hybrid and complex type oligosaccharides with one or two branches terminating in SO4-4GalNAc beta 1,4. In contrast, the sialylated oligosaccharides consisted of a wide array of differing structures containing two or three peripheral branches as well as one, two, or three sialic acid moieties. A previously uncharacterized dibranched oligosaccharide, bearing one residue each of sulfate and sialic acid, was found on all of the hormones except bovine lutropin. In this study, we describe the purification and detailed structural characterizations of the sulfated, sialylated, and sulfated/sialylated oligosaccharides found on lutropin, follitropin, and thyrotropin from several animal species. In the accompanying paper (Green, E.D., and Baenziger, J.U.(1987) J. Biol. Chem. 262, 36-44) we demonstrate the marked quantitative differences among the pituitary glycoprotein hormones in terms of sulfation, sialylation, and underlying oligosaccharide structures, as well as provide evidence for site-specific synthesis of oligosaccharides on individual hormones.     

Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule.