Anomeric Linkage Research Articles

Structural investigation of Klebsiella K-type 4 capsular polysaccharide

The capsular polysaccharide from Klebsiella K4 contains the tetrasaccharide repeating-sequence →3)-α- d-Glc p-(1→2)-α- d-Glc pA-(1→3)-α-- d-Man p-(1→3)-β- d-Glc p-(1→. P.m.r. spectroscopy and the measurement of optical rotation were used to establish the anomeric linkages in the polysaccharide and in the oligosaccharides derived by partial hydrolysis. The repeating unit also contains one O-acetyl group.

Structure elucidation of marker glycolipids of alloantigen-activated murine T lymphocytes.

DBA/2 mouse spleen cells were stimulated in vitro by (a) alloantigen (mitomycin-treated CBA/J splenocytes), (b) the T cell mitogen concanavalin A (Con A), and (c) the B cell mitogen lipopolysaccharide (LPS). The cultures were pulsed for 10 h with (14)C-labeled galactose and glucosamine. Radiolabeled glycosphingolipids (GSL's) were extracted from the cells and the neutral GSL's isolated and analyzed by high-performance thin-layer chromatography. Two of the radioactive neutral GSL's, 9 and 12a, were found to be prominent in the alloantigen-stimulated cells but not in T cells stimulated by Con A. GSL 9 was also present as a minor component in LPS-stimulated B lymphocytes. GSL's 9 and 12a were purified by preparative column chromatography on Iatrobeads. The sequence and anomeric linkages of the carbohydrate moiety of these glycolipids were determined by successive degradation with exoglycosidases. The structures were shown to be Gal(alpha1-x)Gal(beta1-x)GlcCer (glycolipid 9) and GalNAc(beta1-x)Gal(alpha1-x)Gal(beta1-x)GlcCer (glycolipid 12a), respectively. The latter glycolipid may serve as a marker for alloantigen-activated T cell subpopulations.

Structural investigation of the capsular polysaccharide of klebsiella serotype K26

The structure of the capsular polysaccharide from Klebsiella K26 has been determined by using the techniques of methylation, periodate oxidation, partial hydrolysis, and β-elimination. N.m.r. spectroscopy ( 1H and 13C) was used to establish the nature of the anomeric linkages and to identify oligosaccharides obtained by the different degradative techniques employed. The polysaccharide is comprised of repeating units of the heptasaccharide shown. ▪

Sugar residues on proteins.

Glycoproteins have become increasingly important in the structure and function of many different mammalian systems; for example, membrane glycoproteins and glycoprotein hormones. It is, therefore, important to understand their chemistry, which would include an understanding of both the carbohydrate and protein parts of the molecule. Since the chemical characterization of the protein moiety has been extensively examined and the techniques for its characterization are well worked out, only the carbohydrate portion of glycoproteins will be reviewed in this article. The chemical nature of the carbohydrate moiety of glycoproteins will be examined. First, the types of monosaccharides present in animal systems, especially those in the mammalian systems, will be described. Next, various types of simple and complex carbohydrate chains will be discussed to establish the diversity, size, and number of chains present in the carbohydrate units in different glycoproteins. Then, the type of linkages of the carbohydrate to the protein will be examined to determine if the primary sequence of protein is important in determining the size and type of carbohydrate chains present in glycoproteins. Finally, the current methods of structural elucidation such as monosaccharide sequence, intersugar bonds, and anomeric linkages in the carbohydrate moiety of glycoproteins will be reviewed. These methods include the techniques of periodate oxidation, methylation, partial acid hydrolysis, and specific glycosidase digestion of glycoproteins, as well as the latest techniques using micromethods of carbohydrate quantitation and characterization involving gas chromatography and mass spectrometry. The function of the carbohydrate in glycoproteins will also be considered. First, hormone glycoproteins will be discussed in their relationship to the immunological and biological function of the glycoprotein when the carbohydrate is sequentially removed. Next, the function of the carbohydrate in the turnover of glycoproteins will be discussed. These topics will be considered in order to develop an understanding of a specific function(s) of the carbohydrate in glycoproteins.

Structural investigation of Klebsiella serotype K46 poly-saccharide

The structure of the capsular polysaccharide from Klebsiella type K46 has been investigated by using the techniques of methylation analysis, periodate oxidation, and partial hydrolysis. The anomeric linkages were determined by 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and of derived poly- and oligo-saccharides obtained through degradative procedures. 1H-N.m.r. spectroscopy of the poly-saccharide in D 2O showed clearly a ratio of one (1-carboxyethylidene) group (CH 3δ 1.47) to six anomeric protons (δ 4.62–5.29). The polysaccharide was shown to consist of the following hexasaccharide repeating unit, which is unique in this series in having a (1-carboxyethylidene) acetal group on a lateral, but nonterminal, sugar residue. ▪

Interaction of Mycoplasma gallisepticum with sialyl glycoproteins.

The binding of several glycoproteins to freshly grown and harvested cells of Mycoplasma gallisepticum was examined. Only human glycophorin, the major sialoglycoprotein of the erythrocyte membrane, bound tightly as judged by direct binding assays with 125I-labeled glycoproteins. Neuraminidase-treated glycophorin did not bind, suggesting that binding is mediated through sialic acid groups. Although other sialoglycoproteins did not appear to bind M. gallisepticum by direct binding assays, some inhibited the binding of glycophorin. The best inhibitors had a mucin-like structure, with high molecular weights and high sialic acid contents. N-acetylneuraminic acid appeared to be the favored sialic acid structure for binding, but there was no strict specificity for its anomeric linkage. Neuraminidase activity could not be detected on the surface of M. gallisepticum, suggesting that this enzyme is not involved in the mechanism of adherence of sialoglycoproteins. Binding of sialoglycoproteins was time dependent, however, and markedly diminished with increasing ionic strength, but was largely unaffected between pH 4 and 9.

A novel carbohydrate structure in bovine and ovine luteinizing hormones

Bovine and ovine lutropins (bLH and oLH) have three similar asparagine-linked carbohydrate units made up of Fuc, Gal (present only in oLH), Man, GlcNAc and GalNAc. The structural analyses of these carbohydrate units were performed on the oligosaccharides obtained by the alkaline borohydride treatment of the hormones and on the native hormones. The determination of intersugar and anomeric linkages, monosaccharide sequences and the polypeptide-carbohydrate linkage was carried out by methylation, periodate oxidation and deamination techniques and treatment with exoglycosidases. Based on these studies the structure for the oligosaccharide of bLH and oLH is proposed.

Reactivity of Isolated Lipopolysaccharides of Enterobacterial R Mutants with Complete or Incomplete Core Structures with Lectins

Alkali-treated lipopolysaccharides of a range of enterobacterial R mutants with complete and incomplete core structures were investigated by agar gel and microcapillary precipitation onto their reactivity with three selected commercial lectins, namely Concanavalin A, Ricinus communis agglutinin and Wheat germ agglutinin. R lipopolysaccharides with complete R cores of the six so far established core types were differentiable from each other by their characteristic lectin reactivity pattern. One core type showed no reaction with all the three lectins. R lipopolysaccharides with incomplete core structure showed in general the reactivity pattern predictable from their individual chemical configuration. It was further shown that substitution of complete R cores by haptens (T1, T2 or ECA) or by a single repeating unit (SR mutant) did not change the reactivity pattern, although some interactions were less pronounced. It will be demonstrated that lectin precipitation can be of help for structural studies in recognizing, for example, terminal sugar units and their anomeric linkages.

Carbon 13 NMR spectroscopy of a cerebroside. Proof of the beta-pyranosyl structure of D-glucosylceramide

The first 13C nuclear magnetic resonance spectrum of a cerebroside is reported. All signals of D-glucosylceramide (I), the cerebroside from human Gaucher's diseased liver, have been assigned by comparison of their chemical shifts with those of model compounds. This data has been used to demonstrate for the first time directly: (a) that the glucosyl moiety must exist in the pyranoside ring form and have a beta anomeric linkage, and (b) that the fatty acyl composition of the aglycon is: alpha-hydroxy, 0%; saturated, 86 +/- 2%; unsaturated long chain, 14 +/- 2%; and unsaturated short chain, less than or equal to 2%. The use of 13C signal shifts in the delta 80 to 120 region (anomeric window) for the assignment of glycon anomeric and ring forms demonstrated herein should be applicable to other glycoconjugates.

Isolation and characterization of [formula omitted]- and [formula omitted]- glycosidic carbohydrate units of human chorionic gonadotropin

The detailed structures of the carbohydrate moiety of hCG have been determined. The structural analyses were carried out on all four asparagine-linked glycopeptides as well as serine-linked carbohydrate chains. The glycopeptides were prepared from the tryptic hydrolysates of the reduced-S-carboxamidomethyl hCG-α and hCG-β and were purified by chromatography on Sephadex G-50 and preparative high voltage paper electrophoresis at pH 1.8. The serine-linked carbohydrate chains were cleaved by β-elimination with alkali in the presence of sodium borohydride and were purified by chromatography on Sephadex G-25. All glycopeptides and the oligosaccharide were examined for homogeneity by high voltage paper electrophoresis, paper chromatography, and chemical composition. The structural studies involved the determination of intersugar and anomeric linkages and monosaccharide sequences and were carried out by a combination of several techniques such as periodate oxidation, methylation and sequential enzymatic degradation.

Structural determination and stereospecificity of the choleragen-catalyzed reaction of NAD+ with guanidines.

The products of the choleragen-catalyzed reaction of NAD+ with guanidine HCl or L-arginine have been isolated by high performance liquid chromatography. Analysis of 1H NMR spectra obtained at 360 MHz establishes the structure of the isolated products to be adenosine diphosphoribosyl guanidiniums present as 1:1 mixtures of alpha and beta anomeric forms. Direct observation of the choleragen-catalyzed reaction of NAD+ with L-arginine by 1H NMR spectroscopy establishes that choleragen synthesizes an alpha anomeric linkage that is subject to subsequent anomerization. The stereospecificity of the reaction provides further evidence that choleragen behaves as an enzyme and may be related mechanistically to other ADP-ribosyltransferases.

Structural investigation of Klebsiella serotype K32 polysaccharide

The capsular polysaccharide from Klebsiella K32 has been studied by using methylation, periodate oxidation, and partial-hydrolysis techniques. The polysaccharide is shown to comprise the four-sugar repeating unit below. Features of interest in this structure include the presence of a β-linked l-rhamnosyl residue, and the extreme lability of the 1-carboxyethylidene acetal towards acid. N.m.r. spectroscopy was used extensively to establish the nature of the anomeric linkages and to identify oligosaccharides obtained by the various degradative techniques used.

Structural investigation of Klebsiella serotype K62 polysaccharide

By methylation analysis and, in particular, by characterization of the aldobioto aldotetrao-uronic acids obtained by partial hydrolysis, the polysaccharide has been shown to contain the pentasaccharide repeating unit given. The configuration of the anomeric linkages from the 1H-n.m.r. spectroscopy of the intact polysaccharide and of the isolated oligomers. Serotype K62 is one of ∼ 20 strains of the same chemotype, which includes K7 and K28 (whose structures are already known).

Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells.

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Cytolipin R and Its Possible Precursor Ceramide Trisaccharide from Metastatic Rat Mammary Tumor

Ceramide mono, di-, and trihexoside, two globosides, and hematoside were isolated from metastatic rat mammary tumor (MRMT-1) by column chromatography on silicic acid and preparative thin-layer chrpmatography. Each glycolipid was analyzed for fatty acids, long chain bases, and sugars. A combination of periodate oxidation, specific enzymatic hydrolysis, permethylation, and gas chromatographic studies of the sugar moieties established the arrangement of sugars, their anomeric configurations, and positions of their anomeric linkages in the lipids. The main glycolipids were two globosides having structure identical with cytolipin R; N-acetylgalactosaminyl(β1→3)galactosyl(α1→3)galactosyl(β1→4)glucosyl-ceramide. The only structural differences were in their component fatty acid composition. The structure of ceramide trisaccharide was galactosyl(α1→3)galactosyl(β1→4)glucosyl-ceramide, which is assumed to be a possible precursor of cytolipin R. The hematoside from this tumor was N-acetylneuraminyl-lactosyl-ceramide.

The Structure of the Capsular Polysaccharide of Klebsiella K-type 72; Occurrence of 3,4-O-(1-Carboxyethylidene)-L-rhamnose

Methylation and partial hydrolysis studies on the capsular polysaccharide of Klebsiella K72 show the structure to consist of a repeating unit[Formula: see text]The anomeric linkages were determined in isolated oligosaccharides by p.m.r. spectroscopy which also showed the presence in the polysaccharide of one pyruvate ketal per four sugar residues.

Structure of the Capsular Polysaccharide of Klebsiella K-type 20

Methylation, periodate oxidation, and partial hydrolysis studies on the capsular polysaccharide, and on the carboxyl reduced polymer, of Klebsiella K20 show the structure to consist of a repeating unit[Formula: see text]The anomeric linkages were determined by p.m.r. spectroscopy of the carboxyl reduced polysaccharide, and periodate degraded polysaccharides. The p.m.r. spectroscopy of the original polysaccharide also showed the presence in the polysaccharide of one O-acetyl group per eight sugar residues.

Structure of the Capsular Polysaccharide of Klebsiella K-type 56

Methylation, periodate oxidation, and partial hydrolysis studies on the capsular polysaccharide of Klebsiella K-type 56 show the structure to be a repeating unit consisting of[Formula: see text]The nature of the anomeric linkages was determined by p.m.r. spectroscopy of isolated oligosaccharides. The position of the L-rhamnose side chain was defined by characterization of the di- and tetrasaccharides obtained by partial hydrolysis of the fully methylated polysaccharide.This structure represents the first capsular polysaccharide lacking uronic acid to be studied in the genus Klebsiella.

Structures of fucose-containing glycolipids with H and B blood-group activity and of sialic acid and glucosamine-containing glycolipid of human-erythrocyte membrane.

Fucose‐containing glycolipids with H and B blood group activity as well as sialic acid and glucosamine‐containing glycolipid were isolated from human erythrocytes. The sequences and linkages of carbohydrate residues in these glycolipids were determined by partial acid hydrolysis, periodate oxidation and methylation studies. Anomeric linkage were ascribed to those materials on the basis of their optical rotations, infrared spectra, susceptibility to enzymes and immunological activities.The structures of glycolipid H‐I and H‐II were respectively fucopyranosyl‐α‐(1 → 2)‐galacto‐pyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide; fucopyranosyl‐α‐(1 → 2)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The structure of glycolipid B‐I was galactopyranosyl‐α‐(1 → 3)‐[fucopyranosyl‐α‐(1 → 2)]‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The structure of sialic acid and glucosamine‐containing glycolipid was N‐acetyl‐neuraminosyl‐α‐(2 → 3)‐galactopyranosyl‐β‐(1 → 4)‐2‐acetamido‐2‐deoxy‐glucopyranosyl‐β‐(1 → 3)‐galactopyranosyl‐β‐(1 → 4)‐glucopyranosyl‐β‐(1 → 1)‐ceramide. The exclusive presence of 4′‐galactosyl‐N‐acetylglucosamine in four erythrocyte glycolipids makes it likely that the (1 → 3) linkage between galactose and N‐acetyl‐glucosamine residues is missing from all membrane glycolipids which comprise these two sugars. Therefore the formation of Lea and Leb active structures is not possible in membrane glycolipids and all Lewis activity of erythrocytes must be derived from glycolipids absorbed from blood plasma.

The Structure of the Capsular Polysaccharide of Klebsiella K-type 24

Methylation, periodate oxidation, and partial hydrolysis studies on the capsular polysaccharide, and on the carboxyl reduced polymer, of Klebsiella K24 show the structure to consist of a repeating unit[Formula: see text]The anomeric linkages were determined in isolated oligosaccharides by p.m.r. spectroscopy which also showed the presence in the polysaccharide of one O-acetyl group per seven or eight sugar residues. The O-acetyl is tentatively assigned to one of the D-mannose units.