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By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.

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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.

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Reductive cleavage of the riboflavin-binding glycoprotein from hen egg white with LiBH4/tert-BuOH followed by NaBH4 treatment gave rise to oligosaccharide alditols. After fractionation by HPLC two individual oligosaccharide alditols of a hybrid type were isolated. Their structures were proved by 1H NMR 500 MHz spectroscopy and methylation analysis. One of the oligosaccharides has earlier been found in ovalbumin, whereas the other is identified in glycoproteins for the first time.

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Using LiBH4/ButOH treatment, oligosaccharides were cleaved off the hen egg white riboflavin-binding glycoprotein. HPLC led to the isolation of four fucose-containing oligosaccharide alditols, whose structure was elucidated by means of 1H NMR 500 MHz spectroscopy. The main fucosylated oligosaccharide, also present in hen ovomucoid, was found to be a biantennary carbohydrate chain of N-acetyllactosamine type.

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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.

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Reaction of beta-oligoglycosylamines obtained from carbohydrate chains of N-glycoproteins (ovalbumin, ovomucoid, riboflavin-binding glycoprotein from hen egg white, and asialofetuin) with bovine serum albumin, lysozyme, and poly(L-Asp) in presence of water-soluble carbodiimide gave rise to a series of glycoconjugates, modelling natural N-glycoproteins. Carbohydrate-peptide bond was shown to be of N-glycosylamide type with participation of Asp and Glu residues. The method allows one to obtain synthetic N-glycoproteins from oligomannoside, complex and hybrid oligosaccharide chains, and may find application both in biochemistry and biotechnology for improvement of physico-chemical properties of unglycosylated proteins.

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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.

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Reductive cleavage of riboflavin-binding glycoprotein from hen egg white (RF-GPw) with LiBH4/tert-BuOH followed by NaBH4/NaOH treatment gave rise to oligosaccharide alditols, fractionated by a successive HPLC on muBondapak C18 and Zorbax NH2 columns. Seven main individual oligosaccharide alditols were isolated and their structure was investigated by 1H NMR 500-MHz spectroscopy. The structure and relative content of the main oligosaccharide chains were proved to be identical in RF-GPw and ovomucoid. Structure of polypeptide chains and their molecular weight, number of glycosylation sites and their structure had little or no effect on the glycosylation pattern in both glycoproteins. HPLC of the oligosaccharide alditols from another egg white glycoprotein, ovotransferrin, also revealed its high microheterogeneity and close resemblance to those of ovomucoid and RF-GPw.

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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.

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An earlier developed method of identification of oligosaccharides by HPLC was used for studying the carbohydrate chains of three hemagglutinins from various influenza virus strains. The structures of main oligosaccharides of the complex type were elucidated on the basis of their chromatographic characteristics and monosaccharide composition. Oligosaccharide patterns varied in the above hemagglutinin samples but in all cases the major complex chains were fucosylated and nonfucosylated biatennary chains; bisected and triantennary chains were also found.

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Current state of research on the mechanism of biosynthesis of carbohydrate chains of N- and O-glycoproteins is reviewed. Functional predetermination of a multistage mechanism of the carbohydrate components' biosynthesis in N-glycosylproteins is suggested. Origin and character of heterogeneity of the carbohydrate chains in these biopolymers are discussed.

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A new method of fractionation of hen egg glycoproteins has been developed. The procedure involves high-speed mass ion-exchange chromatography on ZetaPrep cartridges, differential precipitation, and ultrafiltration on "Minitan" tangential-flow system. Six fractions were obtained from egg white (ovomucin, avidin, riboflavin-binding glycoprotein RF-GPw, ovoinhibitor, ovalbumin-ovotransferrin, and ovomucoid fractions), and two fractions from egg yolk (riboflavin-binding glycoprotein RF-GPy and phosvitin fractions). Using ion-exchange HPLC on columns (150 X 21.5 mm) Protein PAK DEAE-5PW and SP-5PW, six homogenous glycoproteins (avidin, RF-GPw, ovalbumin, ovotransferrin, ovomucoid, and RF-GPy) were isolated in preparative quantities (0.1-1 g). Ion-exchange HPLC also resolves some glycoproteins' isoforms with different pI values.

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For comparative study of carbohydrate chains of N-glycoproteins, method of "oligosaccharide maps" has been developed. It consists in fractionation of reduced oligosaccharide fragments by gel-chromatography and HPLC on reverse phase and amino columns. Using two HPLC retention time values for each oligosaccharide, two-dimensional maps for both variants of H1 hemagglutinin were constructed. The monosaccharide composition of the majority of oligosaccharides isolated was also elucidated. The carbohydrate chain's patterns for the H1 hemagglutinin variants were found to be similar but to differ considerably from those for H3 hemagglutinin. The data obtained show that the glycosylation pattern depends on virus strain, i.e. on the structure of the polypeptide chain of hemagglutinin.

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Isolation and characterization of oligosaccharides of riboflavin binding glycoprotein from hen white is described. Reductive cleavage of the N-glycosylamide carbohydrate-peptide bond with LiBH4/tert-BuOH followed by NaBH4-NaOH treatment gave rise to alditols, which were fractionated by means of HPLC. Twelve alditols were isolated in quantities sufficient for the monosaccharide analysis. Possibility of an ovomucoid-type oligosaccharide structure for all the alditols is discussed.

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Cyanogen bromide and chymotryptic fragments both containing a carbohydrate chain were obtained from the glycosylated form of porcine prolactin. By means of the glycosidase hydrolysis and the Smith degradation the carbohydrate chain of the glycoprotein was shown to be of the "complex" type of N-bound oligosaccharides and identical to the chain of the luteinizing hormone.

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The structure of four oligosaccharides which are the main carbohydrate chains of hemagglutinin of influenza virus A/Leningrad/385/80 (H3N2) has been elucidated. It was shown by means of enzymatic and mild acid hydrolysis, Smith degradation and acetolysis that the oligosaccharides have very similar structures (noncomplete triantennary) and differ from each other only in the number (0, 1 or 2) and position of fucose residues. The peculiarities of glycosylation of H3 hemagglutinin from different strains of influenza virus were discussed.

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The structure of four main oligomannosidic carbohydrate chains isolated from influenza virus A/Leningrad/385/80 (H3N2) hemagglutinin has been elucidated using 1H NMR spectroscopy. The data obtained suggest that splitting off four alpha 1-2 linked mannose residues under alpha-mannosidase action is the limiting and selective stage of transformation of high mannose carbohydrate chain to complex chain during biosynthesis of glycoproteins.

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