Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells.

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.     

The major oligosaccharides in the large subunit of the hemagglutinin from fowl plague virus, strain Dutch. Structure elucidation by one-dimensional and two-dimensional 1H nuclear magnetic resonance and by methylation analysis

The N-glycosidically linked glycans in the large subunit (HA1) of the hemagglutinin from fowl plague virus, strain Dutch (containing about 15%, w/w, of carbohydrates), were liberated by alkaline hydrolysis, and were filtrated through Bio-Gel as the re-N-acetylated oligosaccharide alditols. One major fraction (90%, mol/mol) was obtained. It was subfractionated by concanavalin A affinity chromatography and was analyzed by methylation/capillary gas chromatography/mass fragmentography and especially by one-dimensional and two-dimensional 1H nuclear magnetic resonance. The major HA1 glycans, which are not sialylated, were thus found to comprise about 40%, 30% and 20% (mol/mol), respectively, of biantennary intersected, biantennary, and triantennary N-acetyllactosaminic ('complex') oligosaccharides. About two thirds of the internal GlcNAc residues in these glycans are substituted by Fuc(alpha 1----6), all the triantennary species carry the third Gal(beta 1----4)GlcNAc(beta 1----unit at the Man(alpha 1----6)-branch, and roughly one fourth of the N-acetyllactosamine units in the non-intersected biantennary oligosaccharides are incomplete.     

Detailed structural analysis of asparagine-linked oligosaccharides of the nicotinic acetylcholine receptor from Torpedo californica.

The structures of the major oligosaccharide moieties of the nicotinic acetylcholine receptor (AcChoR) protein from Torpedo californica have been reported [Nomoto, H., Takahashi, N., Nagaki, Y., Endo, S., Arata, Y. and Hayashi, K. (1986) Eur. J. Biochem. 157, 233-242] to be high-mannose types. Here we report detailed analyses of the structures of the remaining oligosaccharides in this receptor. The sialylated oligosaccharides released by glycopeptidase (almond) digestion were separated according to the number of sialic acid residues using high-performance anion-exchange chromatography with pulsed amperometric detection. After removal of sialic acid from each fraction, the resulting neutral oligosaccharides were separately pyridylaminated and were analyzed by a combination of sequential exoglycosidase digestion and HPLC, then identified on a two-dimensional sugar map. The structures of two desialylated pyridylamino-oligosaccharides were further analyzed by high-resolution proton NMR. Each oligosaccharide was composed of species containing varying numbers of sialic acids. The desialylated complex-type oligosaccharides of AcChoR consisted of ten, eight and one different biantennary, triantennary and tetraantennary oligosaccharide, respectively. The biantennary oligosaccharides were divided into two groups; oligosaccharides with fucose at the proximal N-acetylglucosamine (six varieties) and oligosaccharides without fucose (four varieties). Each group consisted of species differing in the number of terminal galactose residues. The major component of the biantennary oligosaccharides had two galactose residues at the non-reducing termini. The terminal alpha-galactose residue(s) linked to C3 of beta-galactose were found in the fucose-containing biantennary oligosaccharides (two varieties). The triantennary oligosaccharides were also divided into two groups; oligosaccharides with (four varieties) and without (four varieties) besecting N-acetylglucosamine. These groups were composed of species differing in the number of terminal galactose residues. The major component of the triantennary oligosaccharides was fully galactosylated with three galactose residues. An unusual group, Gal beta 1-3GlcNAc, was present in low levels in the triantennary oligosaccharides. In contrast, the tetraantennary oligosaccharide was composed of only one species, which is fully galactosylated with four galactose residues.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

In vivo ligand specificity of E-selectin binding to multivalent sialyl Lewisx N-linked oligosaccharides.

The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium.     

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of the N-linked oligosaccharides from murine glycophorin

Glycophorins, isolated from BALB/c mouse erythrocytes, were degraded under mild and strong reductive alkaline conditions and the N-linked oligosaccharides were isolated as alditols. The oligosaccharide alditols were fractionated and purified using gel filtration, concanavalin A-Sepharose affinity chromatography, and high-performance ion-exchange chromatography. Structural analysis was carried out by chemical analyses, periodate oxidation in combination with fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. The results revealed the presence of sialylated biantennary, triantennary, and tetraantennary complex type oligosaccharides, all fucosylated at the innermost N-acetylglucosamine residue. The tri- and tetraantennary oligosaccharide-containing fractions also contained species elongated by one and/or two N-acetyllactosamine (-3Gal beta 1-4GlcNAc beta 1-) sequences. The N-linked oligosaccharides were shown to be combined only with one (the low molecular weight) of the two mouse glycophorins.     

Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells

Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the     

Purification and characterization of GDP-L-fucose-N-acetyl beta-D-glucosaminide alpha 1----6fucosyltransferase from cultured human skin fibroblasts. Requirement of a specific biantennary oligosaccharide as substrate

GDP-L-fucose-N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase which catalyzes the transfer of fucose from GDP-L-fucose to the asparagine-linked N-acetyl-beta-D-glucosamine of N-linked glycoproteins has been purified 37,000-fold from cultured human skin fibroblasts. The Km values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2 microM, respectively. The Vmax was 1.4 mumols/mg/min. The key step in enzyme purification was affinity chromatography using the immobilized substrate asialoagalactotransferrin glycopeptide-CH-Sepharose. The affinity-purified enzyme had a minimum substrate requirement for a biantennary oligosaccharide with GlcNAc in terminal position, having a Km value of 55 microM. It was heretofore unexpected that the oligosaccharide would serve as substrate, since the site of enzyme activity is GlcNAc-1-linked to Asn. Although the presence of amino acids on this oligosaccharide enhanced the activity 3-fold, it is proposed that this may be the result of an alpha/beta anomeric mixture (2:1) of oligosaccharide used in these studies with only the beta anomer active as substrate. The implication is that the amino acid is required only to retain the beta anomeric position of the substrate. Removal of GlcNAc or addition of Gal to either the oligosaccharide or glycopeptide destroyed the ability to serve as substrates. In addition, di-N-acetylchitobiose, tri-N-acetylchitotriose and GlcNAc beta 1----Asn were nonpermissible substrates. This rigid substrate requirement is unique among fucosyltransferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Structural basis for the substrate specificity of endo-beta-N-acetylglucosaminidase F(3)

Endo-beta-N-acetylglucosaminidase F(3) cleaves the beta(1-4) link between the core GlcNAc's of asparagine-linked oligosaccharides, with specificity for biantennary and triantennary complex glycans. The crystal structures of Endo F(3) and the complex with its reaction product, the biantennary octasaccharide, Gal-beta(1-4)-GlcNAc-beta(1-2)-Man-alpha(1-3)[Gal-beta(1-4)-GlcNAc-be ta(1-2)-Man-alpha(1-6)]-Man-beta(1-4)-GlcNAc, have been determined to 1.8 and 2.1 A resolution, respectively. Comparison of the structure of Endo F(3) with that of Endo F(1), which is specific for high-mannose oligosaccharides, reveals highly distinct folds and amino acid compositions at the oligosaccharide recognition sites. Binding of the oligosaccharide to the protein does not affect the protein conformation. The conformation of the oligosaccharide is similar to that seen for other biantennary oligosaccharides, with the exception of two links: the Gal-beta(1-4)-GlcNAc link of the alpha(1-3) branch and the GlcNAc-beta(1-2)-Man link of the alpha(1-6) branch. Especially the latter link is highly distorted and energetically unfavorable. Only the reducing-end GlcNAc and two Man's of the trimannose core are in direct contact with the protein. This is in contrast with biochemical data for Endo F(1) that shows that activity depends on the presence and identity of sugar residues beyond the trimannose core. The substrate specificity of Endo F(3) is based on steric exclusion of incompatible oligosaccharides rather than on protein-carbohydrate interactions that are unique to complexes with biantennary or triantennary complex glycans.     

L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S_2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.     

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.     

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.     

Comparative structural study of the N-linked oligosaccharides of human normal and pathological immunoglobulin G

The structures of oligosaccharides of normal and pathological immunoglobulin G (IgG) are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide 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 reverse-phase high-performance liquid chromatography. It was possible to separate 15 out of the 16 kinds of oligosaccharides that have been suggested to exist in normal human IgG. High-resolution proton nuclear magnetic resonance spectroscopy was used along with chemical methods to determine the structures of the separated oligosaccharides. It has been shown that in normal IgG a biantennary complex-type oligosaccharide with a fucose residue (formula; see text) is predominant and four kinds of oligosaccharides, which are biantennary with bisecting N-acetylglucosamine and without fucose residues, exist only in a very small quantity. The results obtained for normal IgG were compared with those obtained for three myeloma IgG proteins. It has been found that the most abundant species that exist in the pathological proteins analyzed in the present work lack one or two galactose residues at the nonreducing terminal. We show that the fractions of fucose-containing oligosaccharides are markedly decreased in the heavy-chain disease protein Per. It is of particular interest that in this paraprotein the major component is a biantennary complex-type oligosaccharide that lacks a fucose residue and an oligosaccharide with the structure (Formula: see text) exists as one of the most abundant components.