Anti-tumor activity of an enzymatically synthesized alpha-1,6 branched alpha-1,4-glucan, glycogen

Oral administration of an enzymatically synthesized alpha-1,4:1,6-glycogen (ESG) at a dose of 50 mug/ml significantly prolonged the survival time of Meth A tumor-bearing mice. ESG also significantly stimulated macrophage-like cells (J774.1), leading to augmented production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). The weight-average degree of polymerization (DPw) and the ratio of branch linkage (BL) of ESG were 149,000 and 8.1% respectively. beta-Amylase-treated ESG, however, lost J774.1-activating activity although inhibited subcutaneous growth of Meth A tumor cells admixed with it. Its DPw and BL changed to 126,000 and 20% respectively. Partially degraded amylopectin [(AP), DPw: 110,000, BL; 5.1] was also effective at stimulating J774.1, but its activity was lower than that of ESG. Other alpha-glucans [cycloamylose (CA), enzymatically synthesized amylose (ESA), highly branched cyclic dextrin (HBCD), and beta-amylase-treated HBCD], of which DPw was lower than that of ESG, showed no J774.1-activating activity and weaker anti-tumor activity.     

Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the α-1,6-glucosidic linkage of isomaltooligosaccharides from their non-reducing ends to produce α-glucose. By using the mutant of catalytic acid Glu236→Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 Å resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (β/α)₈-barrel in domain A, which is common to the α-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites −1 to +2. The environment of the glucose residue at subsite −1 is similar to the environment of this residue in the α-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite −1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile α-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile α-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the α-amylase structure from Bacillus subtilis. The comparison with the α-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of α-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes.     

Glycosyl phosphatidylinositol-dependent cross-linking of alpha- agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell- cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross- linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825- 4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6- glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha- agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.     

Interaction of spinach leaf adenosine diphosphate glucose alpha-1,4-glucan alpha-4-glucosyl transferase and alpha-1,4-glucan, alpha-1,4-glucan-6-glycosyl transferase in synthesis of branched alpha-glucan

Salmonella newington lipopolysaccharide extracted from a cell paste grown up from a single smooth clone was fractionated by chromatography on DEAE-cellulose in the presence of 1% Triton X-100 into seven lipopolysaccharide fractions which differed in their degrees of polymerization of the repeating unit of the O-antigen side chain and in their substitution with ester phosphate. Several of the lipopolysaccharide fractions were hydrolyzed in 1% acetic acid at 100 °C to cleave the linkage between the polysaccharide and lipid A parts of the structure. The polysaccharide fractions from each of the purified lipopolysaccharides could be further fractionated on DEAE-cellulose columns to yield a number of peaks of polysaccharide having monosaccharide ratios quite distinct from those of the parent lipopolysaccharide. The results show a high degree of structural heterogeneity in the original lipopolysaccharide.     

Enzyme kinetics and chemical modification of alpha-1,4-glucan lyase from Gracilariopsis sp

The kinetic properties and active site amino acids of alpha-1,4-glucan lyase from the marine red macroalga Gracilariopsis sp. were examined. Using 1H NMR spectroscopy the alpha-1,4-glucan lyase was found to degrade alpha- and beta-maltose at different rates. The effect of pH on the kinetic constants suggested the presence of two catalytically important amino acids in the active site with pKa values of 3.5 and 6.2. The former indicated the presence of an ionised aspartate or glutamate residue in the active site. This was tested using the carboxyl specific reagent EDAC, which inhibited enzyme activity in a time dependent manner when an external nucleophile was added. No protection against the inactivation was obtained by addition of amylopectin, maltitol or 1-deoxinojirimycin. Inactivation decreased Vmax over 2.5-fold with little effect on Km which supports the direct involvement of a carboxyl group in catalysis.     

Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae

Beta1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, beta1,3-glucan, and chitin. It has been postulated that the synthesis of beta1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of beta1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular beta1,6-glucan in cryofixed sec1-1 cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of beta1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of beta1, 6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.     

Action of lysozyme on oligosaccharides from peptidoglycan N-unacetylated at glucosamine residues

On N-acetylmuramyl-L-alanine-amidase treatment followed by lysozyme digestion, peptidoglycan N-unacetylated partially at glucosamine residues yielded three oligosaccharides with N-unacetylated glucosamine residues, GlcN-MurNAc-GlcNAc-MurNAc¹, GlcN-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc, and GlcNAc-MurNAc-GlcN-MurNAc-GlcNAc-MurNAc. Lysozyme can not hydrolyze the former two saccharides, whereas it cleaves the latter one into GlcNAc-MurNAc and GlcN-MurNAc-GlcNAc-MurNAc. This confirms requirement for the acetamido group of the N-acetylglucosamine in the interaction with subsite C of lysozyme.     

Structural studies on kappa-carrageenan derived oligosaccharides

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum carrageenan. Three oligosaccharides were purified by strong-anion exchange high-performance chromatography. Their structure was elucidated using mass spectral and NMR data. Negative-ion electrospray ionization (ESI) mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the kappa-carrageenan oligosaccharides. 2D NMR techniques, including 1H-(1)H COSY, 1H-(1)H TOCSY and 13C-(1)H HMQC, were performed to determine the structure of a trisulfated pentasaccharide. 1D NMR and ESIMS were used to determine the structures of a kappa-carrageenan-derived pentasaccharide, heptasaccharide, and an undecasaccharide. All the oligosaccharides characterized have a 4-O-sulfo-D-galactopyranose residue at both the reducing and nonreducing ends.     

Low-sulphated oligosaccharides derived from heparan sulphate inhibit normal angiogenesis

Heparin, with or without the addition of an adrenocorticosteroid,can inhibit normal angiogenesis in the chick embryo chorioallantoicmembrane. Low- or non-sulphated heparin fragments also haveanti-angiogenic effect. Attempts to define a saccharide structureresponsible for the anti-angiogenic effect implicated a -[GlcAß1,4-GlcNAc     

Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)     

Manufacture and prebiotic potential of oligosaccharides derived from industrial solid wastes

The solid waste obtained in malting industries when dehulling barley grains, which was mainly made up of barley husks, spent grains and grain fragments, was subjected to a double hydrothermal processing under selected conditions. The liquor from the second stage (containing xylooligosaccharides, XOS) was refined by membrane and ion exchange processing (with or without a previous endoxylanase treatment to reduce the XOS molecular weight). Three XOS concentrates with different purity and/or molecular weight distribution were fermented in vitro with faecal inocula to assess their prebiotic potential. Succinate, lactate, formiate, acetate, propionate and butyrate were generated in fermentations, confirming the prebiotic potential of the various products assayed. The purity of XOS concentrates did not play a significant role in fermentation, whereas the sample with shorter average degree of polymerization presented a faster fermentation kinetics and led to the highest concentration of lactic acid.     

Preparation and structural determination of large oligosaccharides derived from acharan sulfate

The structures of a series of large oligosaccharides derived from acharan sulfate were characterized. Acharan sulfate is an unusual glycosaminoglycan isolated from the giant African snail, Achatina fulica. Oligosaccharides from decasaccharide to hexadecasaccharide were enzymatically prepared using heparin lyase II and purified. Capillary electrophoresis and gel electrophoresis confirmed the purity of these oligosaccharides. Their structures, determined by ESI-MS and NMR, were consistent with the major repeating sequence in acharan sulfate, -->4)-alpha-d-GlcN(p)Ac-(1-->4)-alpha-l-IdoA(p)2S-(1-->, terminated by 4-linked alpha-d-GlcN(p)Ac residue at the reducing end and by 4,5-unsaturated pyranosyluronic acid 2-sulfate at the non-reducing end.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.