The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86

The main surface glycoprotein, hemagglutinin (HA), was obtained by treatment of influenza virus B/Leningrad/179/86 with bromelain. Amino acid and monosaccharide compositions of HA and neuraminidase (NA, earlier isolated from the same virus) were determined, thus showing HA and NA to contain 8-10 and 2 carbohydrate chains, respectively. The carbohydrate fragments were cleaved off by the alkaline LiBH4 treatment, the oligosaccharides released were reduced with NaB3H4 and fractionated by two-step HPLC on Ultrasphere-C18 and Zorbax-NH2 columns. Some higher mannose and complex oligosaccharides were identified in both cases by comparison with nonlabelled oligosaccharides of the known structure. The data obtained show that surface glycoproteins of influenza virus A and B are rather similar with regard to structure and heterogeneity of their carbohydrate chains.     

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.     

The structure of oligosaccharide fragments of glycoproteins from influenza virus A/Krasnodar/101/59/(H2N2)--heavy and light chains of hemagglutinin and neuraminidase

The structure and heterogeneity of carbohydrate chains of hemagglutinin (HA) and neuraminidase (NA), the surface glycoproteins of influenza virus A/Krasnodar/101/59 (H2N2), were investigated. Hemagglutinin was reduced with beta-mercaptoethanol and its heavy (HA1) and light (HA2) chains were separated by gel chromatography. Amino acid and sugar composition of HA1, HA2 and NA was elucidated. The carbohydrate chains of the glycoproteins were cleaved off by the alkaline LiBH4 treatment and oligosaccharides were reduced with NaB[3H]4. They were fractionated by subsequent two-step HPLC on Ultrasphere-C8 and Zorbax-NH2 columns with simultaneous identification using nonlabelled oligosaccharides of known structures. Some of the major oligosaccharides isolated from HA1, HA2 and NA were thus identified as high mannose chains, containing 5-9 mannose residues, and complex chains, first of all biantennary chains having or not having bisecting N-acetylglucosamine and/or fucose residues. The approach which has been developed enables one to study the structure and heterogeneity of carbohydrate chains starting from one nmole of a desialylated N-glycoprotein.     

Structures of oligosaccharides on beta-galactosidase from Aspergillus oryzae

The carbohydrate portions of beta-galactosidase from Aspergillus oryzae were found to be composed of two types of sugar chains. They were released equally well with endo-beta-N-acetylglucosaminidase H, but were distinct in their chain length. The long sugar chains (fraction I), corresponding to 4% of the total carbohydrate chains, were composed of galactomannan-type oligosaccharides, which consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 30.0, 16.4, 1.4, and 2.1 per mol of aspartic acid, respectively. The short sugar chains (fraction II), corresponding to 96% of the total carbohydrate chains, consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 9.4, 0.6, 0.3, and 1.7 per mol of aspartic acid, respectively. Both types of sugar chains were fractionated into neutral and acidic subfractions. The neutral subfraction of fraction I (I-N), corresponding to 1% of the total carbohydrate chains, was very heterogeneous in length and was resistant to digestion with alpha-mannosidase and beta-galactosidase. The neutral subfraction of fraction II (II-N), corresponding to 91% of the total carbohydrate, was composed of a mixture of oligosaccharides with oligomanneoside chains (Mann GlcNAcol). The major components were similar to high mannose-type oligosaccharides of mammalian origin in their composition and size (n = 5-9). However, digestion of II-N with alpha 1,2-mannosidase produced considerable amounts of Man6GlcNAcol, an unusual product in the case of high mannose-type oligosaccharides of mammalian origin, in addition to the common one, Man5GlcNAcol.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

The distribution of carbohydrate side chains along the polypeptide chain of glucoamylase

Glucoamylase is a starch-hydrolyzing enzyme with a glycoprotein structure, used industrially for the conversion of starch to glucose, citric acid, corn syrups, and high-fructose sweeteners. This enzyme possesses an unusual type of structure in which many carbohydrate side chains are linked O-glycosidically to serine and threonine residues of the polypeptide chain. The carbohydrate side chains may be single monosaccharide residues or oligosaccharides of mannose, glucose, galactose, and in some cases N-acetylglucosamine. New data from experiments on the CNBr fragmentation of glucoamylase followed by chemical and immunological characterization of the fragments show that the carbohydrate side chains are distributed randomly along the polypeptide chain. Such a structure is appropriately termed a random model reprensentation for the glucoamylase molecule.     

Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)     

Heterogeneity of carbohydrate fragments in heavy and light chains of influenza virus A/Leningrad/385/80 (H3N2) hemagglutinin

Comparative analysis of carbohydrate chains variations in influenza virus A/Leningrad/385/80 (H3N2) hemagglutinin (HA) and its heavy (HA1) and light (HA2) chains has been carried out. The carbohydrate chains of these three glycoproteins were eliminated by reductive cleavage of N-glucosaminidic linkages under LiBH4 - tert-BuOH treatment. Fractionation of the oligosaccharides thus obtained by means of gel chromatography and HPLC resulted in isolation of 21 individual oligosaccharides from each glycoprotein. Their monosaccharide composition revealed almost identical pattern of high-mannose as well as complex chains in HA1 and HA2 in spite of different number (6-7 in HA1 and only 1 in HA2) of glycosilated sites. The possibility of a great number of both high-mannose and complex chains attached at the same site of glycoprotein is shown.     

Structural analysis of carbohydrate chains of normal and myeloma immunoglobulins G using HPLC

A comparative analysis of carbohydrate chains from human normal IgG and myeloma IgG (subclass 4) was carried out using HPLC. It is shown that both IgGs contain the same carbohydrate chains (biantennary and bisected) but the relative amount of bisected and incomplete chains (with fewer terminal galactose residues) is higher in myeloma IgG4. Two earlier unknown minor oligosaccharides were isolated from normal IgG and structurally studied by means of the partial hydrolysis and the Smith degradation.     

The glycoprotein nature and antigenicity of a fungal D-glucosyltransferase

D-Glucosyltransferase (EC 2.4.1.24) from Aspergillus niger has been prepared in pure form by chromatography on DEAE-cellulose. The enzyme transfers D-glucosyl units from maltose and other alpha-linked D-glucosyl oligosaccharides to glucosyl co-substrates resulting in the synthesis of new types of oligosaccharides. The glucosyltransferase has been found to be a glycoprotein containing 20% of carbohydrate consisting of mannose, glucose, and galactose. The carbohydrate residues are attached as either single units or as short oligosaccharide chains by O-glycosyl linkages to the serine and threonine residues of the protein. Antibodies directed against glucosyltransferase have been induced in animals by appropriate immunization regimes. These antibodies combine with the carbohydrate components of the enzyme and, therefore, the carbohydrate residues are the immunodeterminant groups of the glucosyltransferase.     

Structural studies of the carbohydrate moieties of rat kidney gamma-glutamyltranspeptidase. An extremely heterogeneous pattern enriched with nonreducing terminal N-acetylglucosamine residues

The carbohydrate moieties of gamma-glutamyltranspeptidase purified from rat kidney were released as oligosaccharides by hydrazinolysis. Fractionation of the oligosaccharide mixture by paper electrophoresis and Bi-Gel P-4 column chromatography and structural study of each component by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation have revealed that it is composed of 23 neutral oligosaccharides, monosialyl derivatives of 67 oligosaccharides, disialyl derivatives of 62 oligosaccharides, and trisialyl derivatives of 5 oligosaccharides. The neutral oligosaccharides are either high mannose type or biantennary complex type, and the acidic oligosaccharides are bi-, tri-, and tetranntennary complex type sugar chains. Most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the beta-mannosyl residue of their trimannosyl core. Another characteristic feature of these complex type sugar chains is that they are enriched with nonreducing terminal beta-N-acetylglucosamine residues.     

Purification and structures of oligosaccharide chains in swine trachea and Cowper's gland mucin glycoproteins

Mucin glycoproteins were purified from extracts of swine trachea mucosa and Cowper's gland. The gelatinous extracts were solubilized by reduction and carboxymethylation and then purified by chromatography on Sepharose CL-6B and DEAE-Sepharose. The structure of some of the carbohydrate units in these glycoproteins were determined and compared. Alkaline borohydride treatment indicated that more than 85% of the carbohydrate chains in these glycoproteins were linked to serine or threonine residues in the polypeptide chain through O-glycosidic bonds with N-acetylgalactosamine. Reduced oligosaccharides released by treatment with alkaline borohydride were isolated by gel filtration on Bio-Gel P-6 and chromatography on DEAE-cellulose and paper. The structures of the oligosaccharides were established by methylation analysis, gas chromatography, and sequential hydrolysis with specific exoglycosidases. The major oligosaccharides in Cowper's gland mucin glycoproteins were sialylated short chains: NeuAc alpha 2,6GalNAcol and NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAcol. In marked contrast, branched chains containing a Gal beta 1,3(GlcNAc beta 1,6)GalNAc core unit were the major components of trachea mucin glycoprotein. Ten of these chains had the following structures: (Formula: see text).     

The carbohydrate chains of the beta subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates.

The N-linked carbohydrate chains of the beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text]     

The mechanism of folding of pancreatic ribonucleases is independent of the presence of covalently linked carbohydrate

The role of asparagine-linked oligosaccharides for the mechanism of protein folding was investigated. We compared the stability and folding kinetics for two sets of pancreatic ribonucleases (RNases) with identical amino acid sequences and differences in glycosylation. First the folding of RNases A (carbohydrate free) and B (a single N-linked oligosaccharide) from bovine pancreas was investigated. The kinetics of refolding were identical under a wide range of conditions. The rate of unfolding by guanidinium chloride was decreased in RNase B. In further experiments the folding of porcine RNase (three carbohydrate chains at Asn-21, -34, and -76) was compared with the corresponding data for the deglycosylated protein. Even for this RNase with almost 40% carbohydrate content the mechanism of refolding is independent of glycosylation. Although the folding mechanism is conserved, the rates of individual steps in folding are decreased about 2-fold upon deglycosylation. We interpret this to originate from a slight destabilization of folding intermediates by carbohydrate depletion. In control experiments with nonglycosylated bovine RNase A it was ascertained that treatment with HF (as used for deglycosylation) did not affect the folding kinetics. The in vitro folding mechanism of glycosylated RNases apparently does not depend on the presence of N-linked oligosaccharide chains. The information for the folding of glycoproteins is contained exclusively in the protein moiety, i.e. in the amino acid sequence. Carbohydrate chains are attached at chain positions which remain solvent exposed. This ensures that the presence of oligosaccharides does not interfere with correct folding of the polypeptide chain.     

Oligosaccharides in lysosomal enzymes. Distribution of high-mannose and complex oligosaccharides in cathepsin D and beta-hexosaminidase

The distribution of the different types of oligosaccharides in cathepsin D and in beta-hexosaminidase synthesized in cultured human fibroblasts was studied by using endo-beta-N-acetylglucosaminidase H as a probe for high-mannose oligosaccharides. The enzymes were specifically labelled in the protein or the carbohydrate moiety. In both enzymes, resistant and cleavable oligosaccharides were found. The resistant oligosaccharides prevailed in the secreted enzymes. Precursor molecules of cathepsin D contained two oligosaccharide side chains. Multiple forms of the precursor are synthesized with both, one or none of two oligosaccharides sensitive to the action of the endo-beta-N-acetylglucosaminidase H. In fibroblasts unable to phosphorylate lysosomal enzymes (mucolipidosis II) the excessively secreted lysosomal enzymes contained predominantly oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H.     

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.     

Inhibition of processing of asparagine-linked carbohydrate chains on IgG2a by using swainsonine has no influence upon antibody effector functions in vitro

Removal of asparagine (Asn)-linked carbohydrate chains from IgG antibody molecules reduces their antibody effector functions such as C activation and FcR binding. We have prepared IgG2a mAb with modified structure of carbohydrate chains by treating the hybridoma cells with swainsonine, which inhibits the processing of Asn-linked carbohydrate chains at the site of action of mannosidase II. These antibodies have obtained the capacity to bind lentil lectin and have become sensitive to endoglycosidase H digestion, indicating the structural changes of oligosaccharides from complex type to hybrid type. They behaved in an identical manner to the normal IgG2a antibodies with regards to extracellular secretion, Ag-binding capacity, C-mediated hemolysis and FcR-mediated functions. Critical moieties of Asn-linked carbohydrate chains on IgG molecules to retain their antibody effector functions were discussed.     

Structural characterization of neutral oligosaccharides of human H+Leb+ gastric mucin

The structure of carbohydrate chains of human gastric mucin was investigated. The mucin, purified from pronase-degraded gastric aspirates of the secretors with blood group H+Leb, was subjected to alkaline borohydride treatment and a heterogeneous population of neutral (72.1%) and acidic (27.9%) oligosaccharide alditols was obtained. Ten oligosaccharides (I-X), representing 80.3% of the neutral oligosaccharide fraction, have been purified to homogeneity and their structures determined. These oligosaccharides ranged in length from 4 to 12 sugar units and contained mono-, bi-, and triantennary structures. Based on hemagglutination inhibition data in H-anti-H, Leb-anti-Leb, and I-anti-I systems, and the results of structural analyses, we proposed the following structures for these oligosaccharides: (formula, see text)     

Structure of carbohydrate chains of dimeric molecules and individual subunits of gonadotropin from the Russian sturgeon]

Carbohydrate chains of gonadotropin from the Russian sturgeon hypophysis, as well as of alpha- and beta-subunits of the hormone, were split off and fractionated by gel-chromatography and HPLC. More than ten oligosaccharides released from the male and female hormones gave almost identical patterns, whereas differences between alpha- and beta-subunits were more noticeable. Basing on the chromatographic properties and monosaccharide compositions of the oligosaccharides isolated and the known structures of N-linked carbohydrates of mammalian hormones, the common carbohydrate chain of sturgeon gonadotropin is as follows: [formula: see text] Some oligomannosidic and/or hybrid chains and small oligosaccharides of the pentasaccharide core type were also found. Carbohydrate chains of fish gonadotropin have fewer sialic acid residues and significantly fewer (if any) sulphate groups than the mammalian hormones.     

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.