Teichulosonic acid, an anionic polymer of a new class from the cell wall of Actinoplanes utahensis VKM Ac-674(T).

The cell wall of Actinoplanes utahensis VKM Ac-674(T) contains two anionic polymers: teichoic acid 1,3-poly(glycerol phosphate) that is widespread in cell walls of Gram-positive bacteria; and a unique teichulosonic acid belonging to a new class of bioglycans described only in microorganisms of the Actinomycetales order. The latter polymer contains residues of di-N-acyl derivative of sialic acid-like monosaccharide - 5,7-diamino-3,5,7,9-tetradeoxy-L-glycero-ß-L-manno-non-2-ulosonic or pseudaminic acid (Pse) which bears the N-(3,4-dihydroxybutanoyl) group (Dhb) at C7. This polymer has irregular structure and consists of fragments of two types, which differ in substitution of the Dhb residues at O4 either with ß-D-glucopyranose or with ß-Pse residues. Most of the ß-Pse residues (~80%) are glycosylated at position 4 with α-D-galactopyranose residues in both types of fragments. The glucose, galactose, and Dhb residues are partly O-acetylated. The structures of the polymers were established by chemical and NMR spectroscopy methods.

[Characteristics of carbohydrate chains of two forms of salmon gonadotropic hormone binding and not binding concanavalin A]. / Kharakteristika uglevodnykh tsepei sviazyvaiushcheisia i ne sviazyvaiushcheisia s konkanavalinom A form gonadotropnogo gormona gorbushi.

A comparative analysis of carbohydrate chains of two forms of hunchback salmon gonadotropin that bind or not with ConA-Sepharose was carried out. It was found that unbound, "protein", ConA(-)-form contains three times less carbohydrates and has slightly different amino acid composition as compared to the bound, normally glycosylated ConA(+)-form. HPLC fractionation showed the oligosaccharides released from both hormone forms to be mainly sialylated. The major oligosaccharides identified in ConA(+)-form are biantennary, fucosylated (approximately 20%) or nonfucosylated; minor bissected chains are also present. In ConA(-)-form the same oligosaccharides and also oligosaccharides with a higher degree of branching (tri- and tetraantennary) were detected. These two hormone forms isolated from pituitary glands of male or female fish differed neither in the type of oligosaccharides nor in their ratio. Essential structural differences in carbohydrate chains of hunchback salmon and sturgeon gonadotropins were distinctly demonstrated.

[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.

[Structure of major complex carbohydrate chains of influenza virus A/Leningrad/385/80 (H3N2) hemagglutinin]. / Stroenie glavnykh uglevodnykh tsepei kompleksnogo tipa gemaggliutinina virusa grippa A/Lenongrad/385/80 (H3N2).

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.

[The structure of carbohydrate chains of human IgG4-paraproteins differing in the ability to bind cell receptors]. / Struktura uglevodnykh tsepei IgG4-paraproteinov cheloveka, razlichaiushchikhsiia po sposobnosti sviazyvat'sia s kletochnymi retseptorami.

Comparative oligosaccharide analysis by HPLC revealed structural differences in the carbohydrate chains of human IgG4 paraproteins, varying in ability to induce the rhesus monkey's passive skin anaphylaxis. An atypical IgG4 paraprotein, which is inactive in this reaction and also does not bind the IgG4-subclass specific monoclonal antibody IH2, has a much higher proportion of the carbohydrate chains lacking terminal galactose residues than two typical IgG4 paraproteins. This structural feature may be one of the reasons for the atypical IgG4 not to bind by the mast cell Fc gamma receptor.

[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.

[Structure of major oligomannoside chains of influenza virus A/Leningrad/385/80 (H3 N2) hemagglutinin]. / Stroenie glavnykh oligomannozidnykh tsepei gemaggliutinina virusa grippa A/Leningrad/385/80 (H3 N2).

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.

[Comparative study of carbohydrate chains of H1 hemagglutinins of influenza viruses A/Kiev/59/79 and X/Leningrad/54/1 using oligosaccharide maps]. / Sravnitel'noe izuchenie uglevodnykh tsepei H1 gemaggliutinina virusov grippa A/Kiev/59/79 i X/Leningrad/54/1 s pomoshch'iu oligosakharidnykh kart.

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.

[Structure of the carbohydrate chain of glycosylated porcine prolactin]. / Struktura uglevodnoi chasti glikozilirovannogo svinogo prolaktina.

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.

[The structure of oligosaccharide fragments of glycoproteins from influenza virus A/Krasnodar/101/59/(H2N2)--heavy and light chains of hemagglutinin and neuraminidase]. / Struktura oligosakharidnykh fragmentov glikoproteinov virusa grippa A/Krasnodar/101/59(H2N2)--tiazheloi i legkoi tsepei gemaggliutinina i neiraminidazy.

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.

[Structural analysis of carbohydrate chains of normal and myeloma immunoglobulins G using HPLC]. / Strukturnyi analiz uglevodnykh tsepei normal'nogo i mielomnogo immunoglobulinov G s pomoshch'iu VEZhKh.

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 structure of complex carbohydrate chains of hemagglutinin from influenza viruses A/Kiev/59/79 (H1N1), A/Chile/1/83/25(H1N1) and X/79(H3N2)]. / Struktura uglevodnykh tsepei kompleksnogo tipa v gemaggliutinine virusov grippa A/Kiev/59/79(H1N1), A/Chili/1/83/25(H1N1) i X/79(H3N2).

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.

[Structure of carbohydrate chains of dimeric molecules and individual subunits of gonadotropin from the Russian sturgeon]. / Struktura uglevodnykh tsepei dimernoi molekuly i otdel'nykh sub''edinits gonadotropina russkogo osetra.

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.

[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.

[The structure of carbohydrate chains of hemagglutinin and neuraminidase of influenza virus B/Leningrad/179/86]. / Struktura uglevodnykh tsepei gemaggliutinina i neiraminidazy virusa grippa 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.

[Glycoproteins from Drosophila melanogaster cell culture contains O-bound carbohydrate chains of the Gal(beta1-3)GalNAc type]. / Glikoprotein iz kul'tury kletok Drosophila melanogaster soderzhit O-sviazannye uglevodnye tsepi tipa Gal(beta1-3)GalNAc.

The glycoprotein from cultured cells of D. melanogaster, also detected in various insect tissues as a component of the extracellular matrix, was characterized as a mucin-type glycoprotein not yet described in invertebrates. This glycoprotein with an apparent molecular mass of approximately 90 kDa contains about 40% of carbohydrates, largely represented mainly by GalNAc and Gal; its polypeptide moiety is enriched with Thr, Ser, Pro and Gly. An analysis of oligosaccharides liberated by treatment of the glycoprotein with alkaline NaBH4 or O-glycanase (endo-alpha-N-acetylgalactosaminidase) revealed Gal(beta 1-3)GalNAc as the major type of the sugar chains. About half of the Thr + Ser residues in the glycoprotein were estimated as O-glycosidically linked with the disaccharide units.

An alpha-L-fucosidase from Thermus sp. with unusually broad specificity.

An alpha-L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving alpha1 --> 6-, alpha1 -->3-, alpha1 --> 4-, and alpha1 --> 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The alpha-L-fucosidase hydrolyzes p-nitrophenyl alpha-L-fucopyranoside with V(max) of 12.0 +/- 0.1 microM/min/mg and K(m) = 0.20 +/- 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 +/- 10 kDa consisting of four identical subunits with a molecular mass of 61.0 +/- 0.5 kDa. The N-terminal sequence showed homology to some alpha-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl alpha-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the alpha-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl alpha-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.

[A simple method of isolating monoclonal immunoglobulin M, possessing rheumatoid activity]. / Prostoi metod vydeleniia monoklonal'nogo immunobloulina M, obladaiushchego revmatoidnoi aktivnost'iu.

A simple procedure for obtaining highly purified preparations of native monoclonal (Waldenström's disease) immunoglobulin M possessing a rheumatoid activity (IgM-RF) has been developed. The method is based on the use of affinity chromatography with a new readily available adsorbent (immunoglobulin G-porous glass) and 3 M LiCl in Tris-buffer pH 8.3-8.4 able to induce the dissociation of the IgM-RF-IgG complex. The IgM-RF preparation thus obtained was characterized in terms of amino acid composition (relative to conventional monoclonal IgM), carbohydrate composition and structure of oligosaccharide moieties of a complex type. It was shown that some dissociation conditions for the IgM-RF-IgG complex routinely used to isolate IgM-RF provoke irreversible denaturation of IgM-RF when applied to a preliminarily purified complex.