Stimulation of clover root hair infection by lectin-binding oligosaccharides from the capsular and extracellular polysaccharides of Rhizobium trifolii

A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection. The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy. The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R. trifolii 0403.     

The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size.

Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.     

A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities

A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-α-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.     

Conformational changes induced in a homogeneous anti-type III pneumococcal antibody by oligosaccharides of increasing size.

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.     

Biosynthetic studies on N-acetylmannosaminuronic acid containing teichuronic acid in Bacillus megaterium

The particulate enzymes obtained from four strains of Bacillus megaterium AHU 1240, AHU 1373, AHU 1375, and T catalyzed the synthesis of a polysaccharide and glycolipids from UDP-N-acetylmannosaminuronic acid, UDP-N-acetylglucosamine, and UDP-glucose. Chemical studies involving Smith degradation, acid hydrolysis, and N-acetylation revealed that the polysaccharide product has a backbone made up of trisaccharide repeating units comprising glucose, N-acetylmannosaminuronic acid, and N-acetylglucosamine and that the main oligosaccharide moieties of the glycolipids were identical with N-acetylmannosaminuronosyl-N-acetylglucosamine and glucosyl-N-acetylmannosaminuronosyl-N-acetylglucosamine. Incubation of the disaccharide-linked lipid with each particulate enzyme in the presence of UDP-glucose produced the trisaccharide-linked lipid and a polysaccharide. It is therefore suggested that in this polysaccharide-synthesizing system the repeating unit is formed on a carrier lipid from appropriate nucleotide derivatives first and the polymerization of the units then occurs to synthesize the backbone while the growing chain remains in pyrophosphate linkage to the carrier lipid presumed to be undecaprenol.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from P. aeruginosa O13 (Lányi)

The O-specific polysaccharide, obtained on mild acid degradation of lipopolysaccharide of Pseudomonas aeruginosa O13 (Lányi classification), is built up of trisaccharide repeating units involving 2-acetamidino-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), 2-acetamidino-2,6-dideoxy-L-galactose (L-fucosacetamidine, L-FucAm), and a new sialic-acid-like sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso n ic acid (Sug), and thus contains simultaneously both acidic and basic functions. Cleavage of the polysaccharide with hydrogen fluoride in methanol revealed the high stability of the glycosidic linkage of the ulosonic acid and afforded methyl glycosides of a disaccharide and a trisaccharide. The structures of the new ulosonic acid and acetamidino group were established by analysing the oligosaccharide fragments by 1H, 13C nuclear magnetic resonance spectrometry, as well as on the basis of their chemical conversions: alkaline hydrolysis of the acetamidino group into acetamido group, reductive deamination with lithium borohydride into the ethylamino group and acetylation with acetic anhydride in pyridine accompanied by intramolecular acylation of the acetamidino function by the ulosonic acid to form a six-membered lactam ring. Identification of the oligosaccharide fragments and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and polysaccharide revealed the following structure of the repeating unit: ----3)D-QuiNAcp(alpha 1----3)Sugp(alpha 2----3)L-FucAmp(alpha 1----.     

Spatial Structure of Glycogen Molecules in Cells

Glycogen is a strongly branched polymer of α-D-glucose, with glucose residues in the linear chains linked by 1→4-bonds (~93% of the total number of bonds) and with branching after every 4-8 residues formed by 1→6-glycosidic bonds (~7% of the total number of bonds). It is thought currently that a fully formed glycogen molecule (β-particle) with the self-glycosylating protein glycogenin in the center has a spherical shape with diameter of ~42 nm and contains ~ 55,000 glucose residues. The glycogen molecule also includes numerous proteins involved in its synthesis and degradation, as well as proteins performing a carcass function. However, the type and force of bonds connecting these proteins to the polysaccharide moiety of glycogen are significantly different. This review presents the available data on the spatial structure of the glycogen molecule and its changes under various physiological and pathological conditions.     

Isolation of oligosaccharides from a partial-acid hydrolysate of pneumococcal type 3 polysaccharide for use in conjugate vaccines

A series of well-defined oligosaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 3 has been generated. Partial-acid hydrolysis of the capsular polysaccharide, followed by fractionation of the oligosaccharide mixture by Sepharose Q ion-exchange chromatography yielded fragments containing one to seven [-->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1-->] repeating units. The isolated fragments were analysed for purity by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an IonPac AS11 column, and their structures were verified by 1H NMR spectroscopy and nano-electrospray mass spectrometry. The oligosaccharides can be used to produce neoglycoprotein vaccines with a defined carbohydrate part.     

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.     

Relative susceptibilities of the glucosamine-glucuronic acid and N-acetylglucosamine-glucuronic acid linkages to heparin lyase III

Heparin lyases are valuable tools for generating oligosaccharide fragments and in sequence determination of heparan sulfate (HS). Heparin lyase III is known to cleave the linkages between N-acetylglucosamine (GlcNAc) or N-sulfated glucosamine (GlcNS) and glucuronic acid (GlcA) as the primary sites and the linkages between GlcNAc, GlcNAc(6S), or GlcNS and iduronic acid as secondary sites. N-Unsubstituted glucosamine (GlcN) occurs as a minor component in HS, and it has been associated with various bioactivities. Here we investigate the specificity of heparin lyase III toward the GlcN-GlcA linkage using a recombinant enzyme of high purity and as substrates the partially de-N-acetylated polysaccharide of Escherichia coli K5 strain and derived hexasaccharides. The specificity of lyase III toward the GlcN-GlcA linkage is deduced by sequencing of the oligosaccharide products using electrospray mass spectrometry with collision-induced dissociation and MS/MS scanning. The results demonstrate that under controlled conditions for partial digestion, lyase III does not act at the GlcN-GlcA linkage, whereas GlcNAc-GlcA is cleaved. Even under forced conditions for exhaustive digestion, the GlcN-GlcA linkage is only partly cleaved. It is this property of lyase III that has enabled the isolation of a unique, nonsulfated antigenic determinant DeltaUA-GlcN-UA-GlcNAc from HS and from partially de-N-acetylated K5 polysaccharide. It was unexpected that pentasaccharide fragments were also detected among the digestion products of the K5 polysaccharide used. It is possible that these are products of an additional glycosidase activity of lyase III, although other mechanisms cannot be completely ruled out.     

Binding of N-Acetylglucosamine (GlcNAc) β1-6-branched Oligosaccharide Acceptors to β4-Galactosyltransferase I Reveals a New Ligand Binding Mode

N-Acetyllactosamine is the most prevalent disaccharide moiety in the glycans on the surface of mammalian cells and often found as repeat units in the linear and branched polylactosamines, known as i- and I-antigen, respectively. The β1-4-galactosyltransferase-I (β4Gal-T1) enzyme is responsible for the synthesis of the N-acetyllactosamine moiety. To understand its oligosaccharide acceptor specificity, we have previously investigated the binding of tri- and pentasaccharides of N-glycan with a GlcNAc at their nonreducing end and found that the extended sugar moiety in these acceptor substrates binds to the crevice present at the acceptor substrate binding site of the β4Gal-T1 molecule. Here we report seven crystal structures of β4Gal-T1 in complex with an oligosaccharide acceptor with a nonreducing end GlcNAc that has a β1-6-glycosidic link and that are analogous to either N-glycan or i/I-antigen. In the crystal structure of the complex of β4Gal-T1 with I-antigen analog pentasaccharide, the β1-6-branched GlcNAc moiety is bound to the sugar acceptor binding site of the β4Gal-T1 molecule in a way similar to the crystal structures described previously; however, the extended linear tetrasaccharide moiety does not interact with the previously found extended sugar binding site on the β4Gal-T1 molecule. Instead, it interacts with the different hydrophobic surface of the protein molecule formed by the residues Tyr-276, Trp-310, and Phe-356. Results from the present and previous studies suggest that β4Gal-T1 molecule has two different oligosaccharide binding regions for the binding of the extended oligosaccharide moiety of the acceptor substrate.     

Confirmation of the D configuration of the 2-substituted arabinitol 1-phosphate residue in the capsular polysaccharide from Streptococcus pneumoniae Type 17F

The absolute configuration of the 2-substituted arabinitol 1-phosphate residue present in the repeating unit of the capsular polysaccharide (CPS) from Streptococcus pneumoniae Type 17F is confirmed as D, based on a comparison of proton and carbon chemical shifts in a synthetic oligosaccharide and in an oligosaccharide derived from the CPS by degradation.     

Structural investigation of the capsular polysaccharide from Escherichia coli O9:K37 (A 84a)

The structure of the capsular polysaccharide from E. coli O9:K37 (A 84a) has been studied, using methylation analysis, Smith degradation, and graded acid hydrolysis. The configurations at the anomeric centres were assigned by 1H-n.m.r. spectroscopy of the polysaccharide and its derivatives and oligosaccharide fragments. The polysaccharide has the following trisaccharide repeating-unit which is unique in the E. coli series of capsular polysaccharides in possessing a 1-carboxyethylidene group as the sole acidic function. (Formula: see text) E. coli capsular polysaccharides have been classified into seventy-four serotypes. The structures of about twenty of these polysaccharides have been elucidated, one of which, K29, has been reported to contain a 1-carboxyethylidene group. In continuation of a programme aimed at establishing the structural basis for the serology and immunochemistry of the E. coli capsular antigens, we now report on the structure of the capsular polysaccharide from E. coli O9:K37.     

Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen

We have derived oligosaccharides from the capsular polysaccharide of type III group B Streptococcus by enzymatic hydrolysis of a specific backbone glycosidic bond utilizing an endo-beta-galactosidase from Flavobacterium keratolyticus. Enzymatic digestion of the polysaccharide produced oligosaccharide fragments of one or more pentasaccharide repeating units. On the basis of 13C NMR, 1H NMR, and methylation analyses, it was established that the smallest digestion fragment was alpha-D-NeupNAc-(2----3)-beta-D-Galp-(1----4)-[beta-D-Glcp-(1----6 )]- beta-D-GlcpNAc-(1----3)-beta-D-Gal. The isolation of this oligosaccharide is consistent with the susceptibility of the beta-D-Galp-(1----4)-beta-D-Glcp linkage in the backbone of the type III group B streptococcal polysaccharide and confirms that the polysaccharide is composed of a pentasaccharide repeating unit. High resolution 13C NMR spectroscopic studies indicated that, as in the case of the pentasaccharide, the terminal sialic acid residues of the type III group B streptococcal polysaccharide were linked to O-3 and not to O-6 of its branch beta-D-galactopyranosyl residues as had been previously reported (Jennings, H. J., Rosell, K.-G., and Kasper, D. L. (1980) Can. J. Chem. 58, 112-120). This linkage was confirmed in an independent methylation analysis of the type III group B streptococcal polysaccharide. Thin layer chromatogram binding assay and radioactive antigen binding assays with radiolabeled oligosaccharides demonstrated the single repeating unit pentasaccharide oligosaccharide to be poorly antigenic. Increasing oligosaccharide size to a decasaccharide consisting of two repeating units resulted in an 8-fold increase in antigen binding in the direct radioactive antigen binding assay. The results suggest that a region of the immunodeterminant site critical for antibody binding is located in the backbone of the polysaccharide and involves the beta-D-galactopyranose-(1----4) beta-D-glucopyranose bond.     

Reexamination of the presence and linkage of 3-hydroxybutyryl substituents in the acidic capsular polysaccharide of Rhizobium trifolii 0403.

We resolved previous conflicting results concerning the presence of 3-hydroxybutyryl substituents on the extracellular acidic polysaccharide from Rhizobium trifolii 0403. These substituents were indeed present in the polysaccharide and in the oligosaccharide fragments obtained by hydrogen fluoride solvolysis of the extracellular and capsular polysaccharides of the bacteria grown on plates. The 3-hydroxybutyrate substituent could be removed from the polysaccharide by 10 mM sodium deuteroxide without evidence of elimination, indicating that this substituent was ester linked.     

Structural Characterization and Immunomodulating Activity of a Complex Glucan from Spores of Ganoderma lucidum

A polysaccharide with a molecular weight of 1.26×10⁵, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1D, 2D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Deciphering Mode of Action of Heparanase Using Structurally Defined Oligosaccharides

Heparan sulfate (HS) is a highly sulfated polysaccharide that serves many biological functions, including regulating cell growth and inflammatory responses as well as the blood coagulation process. Heparanase is an enzyme that cleaves HS and is known to display a variety of pathophysiological effects in cancer, diabetes, and Alzheimer disease. The link between heparanase and diseases is a result of its selective cleavage of HS, which releases smaller HS fragments to enhance cell proliferation, migration, and invasion. Despite its importance in pathological diseases, the structural cues in HS that direct heparanase cleavage and the steps of HS depolymerization remain unknown. Here, we sought to probe the substrate specificity of heparanase using a series of structurally defined oligosaccharide substrates. The sites of heparanase cleavage on the oligosaccharide substrates were determined by mass spectrometry and gel permeation chromatography. We discovered that heparanase cleaves the linkage of glucuronic acid linked to glucosamine carrying 6-O-sulfo groups. Furthermore, our findings suggest that heparanase displays different cleavage modes by recognizing the structures of the nonreducing ends of the substrates. Our results deepen the understanding of the action mode of heparanase.     

Domainal organization of the molecules of Lys-plasminogen

The intramolecular melting of the human Lys-plasminogen and its different fragments were studied by the differential scanning microcalorimetry method. Thermodynamical analysis of melting curves showed that the Lys-plasminogen molecule consists of 7 domains. Five of them are formed by five homologeus regions of the polypeptide chain (kringle), while two domains are formed by the part of the polypeptide chain corresponding to the plasmin light chain. The domains included in the fragments seem to be rather independent, since fragmentation does not lead to noticeable changes of their stability in comparison to that of the intact molecule. It has been shown also that plasminogen-plasmin conversion is accompanied by structural transformation of the molecule which results in the destabilization of one of the light chain domains.