Alpha-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes.

An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.

[The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue]. / Rasshcheplenie glikoproteinov pod vozdeistviem borgidrida litiia. Vydelenie i analiz o-glikopeptidov s vosstanovlennoi C-kontsevoi aminokisloto i.

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.

Alpha-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM 701.

An alpha-galactosidase that inactivates the group specificity of B erythrocytes (group III) of human blood and does not affect A erythrocytes (group II) was isolated from the marine bacterium Pseudoalteromonas sp. KMM 701. The enzyme preparation did not contain lectin, hemolytic, sialidase, endoglycanase, or glycosidase activities. The enzyme is stable at 20 degreesC for 24 h, has pH optimum for catalysis within the range of 6.7-7.7, and is stable to high concentrations of NaCl. It is 4-fold more efficient than the alpha-galactosidase from green coffee beans. At pH 7.0 the Km for p-nitrophenyl-alpha-D-galactopyranoside is 0.29 mM. The molecular weight of the enzyme determined by gel-filtration is 195 +/- 5 kD. The alpha-galactosidase is denatured by urea and guanidine hydrochloride. Its activity does not depend on the presence of metal ions. It contains a sulfhydryl group essential for its catalytic activity.

Structure of an acidic polysaccharide present in the bacteriolytic complex lysoamidase.

The structure of an acidic polysaccharide component of a bacteriolytic complex (lysoamidase), isolated from a bacterium of the genus Xanthomonas, was studied. On the basis of sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopic study of the initial polysaccharide and its O-deacetylated and carboxyl-reduced derivatives, the following structure of the trisaccharide repeating unit of the polysaccharide was established [formula: see text] where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively.

[Structure of an acidic polysaccharide found in a bacteriolytic complex of lysoamidase]. / Struktura kislogo polisakharida, vkhodiashchego v sostav bakterioliticheskogo kompleksa lizoamidazy.

A structural study of an acidic polysaccharide, a component of the lysoamidase bacteriolytic complex has been carried out. Analysis of the monosaccharide composition of the original polysaccharide, of the product of carboxyl groups reduction and of the 13C-NMR and 1H-NMR spectra of the original and the O-deacetylated polysaccharides using two-dimensional NMR spectroscopy has made it possible to establish the structure of the repeating trisaccharide unit of the polysaccharide as follows: [formula: see text] where ManNAcA and GalNAcA are 2-acetamido-2-deoxymannuronic acid and 2-acetamido-2-deoxygalacturonic acid, respectively. Also small amount of a neutral polysaccharide containing of rhamnose is present in the lysoamidase preparation.

Structure of simusan, a new acidic exopolysaccharide from Arthrobacter sp.

Simusan, a major exopolysaccharide produced by an ethanol-utilizing Arthrobacter sp. strain CE-17, contains D-glucose, D-mannose, D-galactose, L-rhamnose, D-glucuronic acid, and pyruvic acid in the ratios approximately 3:2:1:1:1:1 as well as O-acyl groups (presumably (presumably residues of acetic and palmitic acid). On the basis of chemical modifications of the polysaccharide, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and an octa-saccharide fragment, Smith degradation, and 1H and 13C NMR analysis, the following structure of the repeating unit was established: [formula: see text] It is suggested that at least one of the glucose residues and the galactose residue are O-acetylated.

Structure of the major exopolysaccharide produced by Azotobacter beijerinckii B-1615.

A. beijerinckii strain B-1615 produced two acidic exopolysaccharides in the ratio approximately 9:1. The minor polysaccharide contained mannuronic and guluronic acids in the ratio 2.3:1 and is a bacterial alginate. The major polysaccharide consisted of D-galactose, L-rhamnose, and pyruvic acid in the ratios 2:1:1 and was acetylated. On the basis of methylation analysis, and 1H- and 13C-n.m.r. spectroscopy of the polysaccharide before and after removal of the pyruvic acid residues and O-deacetylation, it was concluded that the major polysaccharide had the structure [formula; see text] with up to 1.5 OAc groups per repeating unit.

[Structure of the carbohydrate chain of riboflavin-binding glycoprotein from hen egg white. Neutral oligosaccharides of the hybrid type]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteinabelka kurinogo iaitsa. Neitral'nye oligosakharidy gibrid tipa.

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.

[The structure of carbohydrate chains of riboflavin-binding glycoprotein from chicken egg protein. III. 1H-NMR spectroscopy (500 MHz) of neutral fucosylated oligosaccharides]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteina belka kurinogo iaitsa. III. Spektroskopiia 1H-IaMR (500 MHz) neitral'nykh fukozilirovannykh oligosakharidov.

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.

A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

[Synthesis of N-glycoproteins with a native type of carbohydrate-peptide bond]. / Sintez N-glikoproteinov s prirodnym tipom uglevod-peptidnoi sviazi.

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.

[Structure of carbohydrate chains of the riboflavin-binding glycoprotein of chicken egg protein. II. 1H-NMR (500 MHz) spectroscopy of the major neutral oligosaccharides]. / Struktura uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteina belka kurinogo iaitsa. II. Spektroskopiia 1H-YaMR (500 MGts) glavnykh neitral'nykh oligosakharidov.

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.

[The structure of chordin: characteristics of protein and carbohydrate components and their role in antigenicity]. / Struktura khordina: kharakteristika belkovogo i uglevodnogo komponentov i ikh rol' v antigennosti.

Using immobilized monoclonal antibodies, a tissue-specific antigen, chordin, was isolated from cell extracts of giant sturgeon (beluga) notochord. The antigen was further purified by gel filtration through SP-Sephadex (pH 2.1) and gel chromatography on TSK Toyopearl HW-60. Purified chordin preparations contained 40% of protein and 60% of carbohydrates. The predominant polar amino acids were threonine, serine, glycine, asparagine and glutamine (or aspartic and glutamic amino acids). The carbohydrate moiety comprised mannose, fucose, galactose, galactosamine and glucosamine. Treatment of chordin with three enroglycosidases specifically hydrolyzing the carbohydrate chains of proteoglycans did not affect the antigenic properties of chordin or its behaviour on gel filtration. These findings and the fact that 75% of galactosamine was converted to galactosaminite after treatment with alkaline NaBH4 permitted to relate chordin to glycoproteins carrying O-glycosidic carbohydrate-peptide bonds between the N-acetyl-galactosamine and beta-hydroxyamino acid residues. Besides, chordin seems to contain a N-glycosylamide carbohydrate-peptide bond as can be judged from glucosaminite formation after treatment of the antigen with alkaline LiHB4. The changes in the antigenic properties of chordin after its treatment with neuraminidase, pronase, sodium periodate, alkali, alkaline NaBH4 or LiBH4 suggest that the polypeptide moiety of the chordin molecule and, perhaps, the N-acetylgalactosamine within the composition of the carbohydrate-peptide bond are involved in the construction of its most immunogenic determinants (P-determinants).

[Fractionation of chicken egg glycoproteins and their purification using preparative HPLC]. / Fraktsionirovanie glikoproteinov kurinogo iaitsa i ikh ochistka s pomoshch'iu preparativnoi VEZhKh.

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.

The carbohydrate chains of influenza virus hemagglutinin.

The major surface antigen of influenza virus A/Leningrad/385/80 (H3N2), H3 hemagglutinin, as well as its heavy and light subunits were obtained by bromelain treatment, followed by gel chromatography. Carbohydrate chains were split off from both subunits by lithium borohydride-lithium hydroxide in aqueous 2-methyl-2-propanol, and individual oligosaccharides isolated. The main oligosaccharides, whose structure was determined by 1H-n.m.r. spectroscopy and chemical methods, are of the ordinary oligomannoside and complex types. It was found that, in spite of the great difference in number of glycosylation sites in heavy and light subunits, the amount and even relative abundance of variants of carbohydrate chains in both subunits are very similar.

[Isolation and characteristics of carbohydrate chains of riboflavin-binding glycoprotein from the chicken egg]. / Vydelenie i kharakteristika uglevodnykh tsepei riboflavinsviazyvaiushchego glikoproteina belka kurinogo iaitsa.

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.

[Heterogeneity of carbohydrate fragments in heavy and light chains of influenza virus A/Leningrad/385/80 (H3N2) hemagglutinin]. / Geterogennost' uglevodnykh fragmentov v tiazheloi i legkoi tsepiakh gemaggliutinina virusa grippa A/Leningrad/385/80 (H3N2).

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.

[Preparative method of isolating influenza virus glycoproteins]. / Preparativnyi metod vydeleniia glikoproteinov virusa grippa.

Two preparative methods for isolation of biologically active glycoproteins from influenza virus A - hemagglutinin and neuraminidase, were elaborated. A three-step procedure involves solubilization of glycoproteins with nonionic (Triton N-101, TN) or cationic (cetylpyridinium chloride, CPC) detergents, separation from degraded virions by centrifugation, and removal of detergents and lipids by precipitation of the glycoproteins with butanol (TN), or, alternatively, precipitation of CPC upon cooling. Using virion concentration of approximately 1 mg/ml and optimal weight ratio of detergent to virus (protein) of approximately 20:1 (for TN) and 1:1 (for CPC), the glycoproteins were obtained with the overall yield of 70-80%. The isolated glycoproteins exhibit the same immunological and enzymatic activities as intact virus A/Texas/77 and A/Leningrad/80.

Specific method for the fragmentation of the polypeptide chain of glycoproteins. Distribution of carbohydrate chains on the peptide core of blood-group-specific glycoprotein.

A method for specific fragmentation of the polypeptide backbone of glycoproteins at the glycosylated serine and threonine residues has been developed. The fragmentation includes beta-elimination of the carbohydrate chains, followed by bromination of the resulting enamine groups, and cleavage of the brominated amino acid residues by alkaline sodium borohydride. The method was employed for fragmentation of the peptide core of pig blood-group substance H. Essentially all the serine and threonine residues were shown to be O-glycosylated, and rather frequently either adjacent or separated by a single amino acid (mainly alanine). When they were separated by two or three amino acid residues, proline was preponderant.