Effect of molecule branching and glycosidic linkage on the degradation of polydextrose by gut microbiota

Polydextrose is a randomly linked complex glucose oligomer that is widely used as a sugar replacer, bulking agent, dietary fiber and prebiotic. Polydextrose is poorly utilized by the host and, during gastrointestinal transit, it is slowly degraded by intestinal microbes, although it is not known which parts of the complex molecule are preferred by the microbes. The microbial degradation of polydextrose was assessed by using a simulated model of colonic fermentation. The degradation products and their glycosidic linkages were measured by combined gas chromatography and mass spectrometry, and compared to those of intact polydextrose. Fermentation resulted in an increase in the relative abundance of non-branched molecules with a concomitant decrease in single-branched glucose molecules and a reduced total number of branching points. A detailed analysis showed a preponderance of 1,6 pyranose linkages. The results of this study demonstrate how intestinal microbes selectively degrade polydextrose, and provide an insight into the preferences of gut microbiota in the presence of different glycosidic linkages.     

Effect of Molecule Branching and Glycosidic Linkage on the Degradation of Polydextrose by Gut Microbiota

Polydextrose is a randomly linked complex glucose oligomer that is widely used as a sugar replacer, bulking agent, dietary fiber and prebiotic. Polydextrose is poorly utilized by the host and, during gastrointestinal transit, it is slowly degraded by intestinal microbes, although it is not known which parts of the complex molecule are preferred by the microbes. The microbial degradation of polydextrose was assessed by using a simulated model of colonic fermentation. The degradation products and their glycosidic linkages were measured by combined gas chromatography and mass spectrometry, and compared to those of intact polydextrose. Fermentation resulted in an increase in the relative abundance of non-branched molecules with a concomitant decrease in single-branched glucose molecules and a reduced total number of branching points. A detailed analysis showed a preponderance of 1,6 pyranose linkages. The results of this study demonstrate how intestinal microbes selectively degrade polydextrose, and provide an insight into the preferences of gut microbiota in the presence of different glycosidic linkages.     

Analysis of protein glycosylation by mass spectrometry

There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, and only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.     

On the structure of cytolipin R, a ceramide tetrahexoside hapten from rat lymphosarcoma

Cytolipin R, a ceramide tetrahexoside isolated from rat lymphosarcoma, was studied by sequential hydrolysis with specific glycosidases which revealed the anomeric configurations of the glycosidic bonds. Sugar linkages were established by combined gas-liquid chromatography and mass spectrometry of the partially methylated alditol acetates prepared after permethylation and hydrolysis of the intact lipid. Results indicated the structure of cytolipin R to be N-acetylgalactosaminyl(beta1-->3)galactosyl(alpha1-->3) galactosyl(beta1-->4)glucosyl ceramide. Cytolipin K (globoside I) differs in having a -galactosyl(alpha1-->4)galactosyl- internal linkage, and this difference must account for the immunological differences between cytolipin K and cytolipin R.     

Nuclear Overhauser effects and conformations of branched trisaccharide methyl beta-glycosides that contain a 3,4-disubstituted galactose residue

N.O.e. data after pre-irradiation of the anomeric protons and the 3JC,H values associated with the glycosidic linkages for a series of methyl beta-glycosides of trisaccharides that contain 3,4-disubstituted galactose residues have been measured. On the basis of the experimental results and theoretical calculations, it was shown that one preferred conformer (less than 90%) was present for each trisaccharide derivative.     

Immunochemistry of the Lewis blood-group system: isolation and structures of Lewis-c active and related glycosphingolipids from the plasma of blood-group O Le(a-b-) nonsecretors

Five different glycosphingolipid fractions (GL-3, 285 micrograms; GL-5, 1090 micrograms; GL-6, 615 micrograms; GL-7, 555 micrograms; and GL-8, 155 micrograms) have been isolated from 25 liters of plasma of O Le(a-b-) nonsecretors by means of ethanol extraction, several steps of Folch distribution, and reversed-phase, silicic acid, and ion-exchange column chromatography of native or peracetylated substances. Final purification, accomplished by preparative silica gel high-performance thin-layer chromatography, led to chromatographic homogeneity of GL-3 and GL-6. In the hemagglutination inhibition as well as quantitative passive hemagglutination techniques two of these substances (GL-3, GL-5) exhibited distinct, and the other three (GL-6-GL-8) very strong, Lec blood-group activities when tested against two different Lec antisera of human or goat origin. The fragments' structures were elucidated by fast atom bombardment and electron impact mass spectrometry of permethylated derivatives in order to determine molecular weight, sugar sequence, position of branching points, and type of oligosaccharide chains, as well as fatty acid and sphingosine patterns of the ceramide residue. Combined gas-liquid chromatography and mass spectrometry of partially methylated alditol acetates identified sugar composition and glycosidic linkages. Thus, the following structures could be established: (formula; see text) In contrast to the structurally homogeneous GL-3, minor amounts of 4-O-substituted GlcNAc pointed to a small contamination of GL-6 by branched type 2 ceramide nonasaccharide analogs. Glycolipids containing hepta- or nonasaccharides as in GL-3 or GL-6 could also be identified in fractions GL-5 (ceramide heptasaccharide) and GL-7 and GL-8 (ceramide nonasaccharide). These latter fractions revealed, however, distinct heterogeneity due to the presence of a small amount of either a type 2 analog of GL-3 (GL-5) or linear, mainly type 2, ceramide hexa- (GL-5, GL-7) or octasaccharides (GL-8). In addition to previous immunochemical communications the presented Lec active structures of GL-3 and GL-6 provide evidence that 3-fucosyl-N-acetyllactosamine in combination with a type 1 based oligosaccharide sequence and a 3,6-galactosyl branching point are essential parts of the Lec antigenic determinant (as marked in the formula of GL-6).     

The branching and linear portions of poly(adenosine diphosphate ribose) have the same alpha(1 leads to 2) ribose-ribose linkage

The structure of the branching site of poly(ADP-ribose) was determined as O-alpha-D-ribofuranosyl-(1"' leads to 2")-O-alpha-D-ribofuranosyl-(1" leads to 2')-adenosine-5',5",5"'-tris(phosphate) by gas chromatography, mass spectrometry, and 1H-NMR measurements. Thus the structures of all the ribose-ribose linkages known in poly(ADP-ribose) are uniformly alpha(1 leads to 2)glycosidic bond. This indicates that branching ADP-ribosylation and elongating ADP-ribosylation of poly(ADP-ribose) synthesis are catalyzed by similar alpha(1 leads to 2)-specific ADP-ribosyl transferases or the same enzyme. Poly(ADP-ribose) glycohydrolase, which specifically hydrolyzes the ribose-ribose bonds of poly(ADP-ribose), also cleaves the ribose-ribose-ribose bonds at the site of branching.     

Structure of a streptococcal adhesin carbohydrate receptor

Interactions between complementary protein and carbohydrate structures on different genera of human oral bacteria have been implicated in the formation of dental plaque. The carbohydrate receptor on Streptococcus sanguis H1 (one of the primary colonizing species) that is specific for the adhesin on Capnocytophaga ochracea ATCC 33596 (a secondary colonizer) has been isolated from the streptococcal cell wall, purified, and structurally characterized. The hexasaccharide repeating unit of the polysaccharide was purified by reverse-phase, amino-bonded silica, and gel permeation high performance liquid chromatography. Earlier studies established that the repeating unit was a hexasaccharide composed of rhamnose, galactose, and glucose in the ration of 2:3:1, respectively. In the present study, determination of absolute configuration by gas chromatography of the trimethylsilyl (+)-2-butyl glycosides revealed that the rhamnose residues were of the L configuration while the hexoses were all D. 252Californium plasma desorption mass spectrometry of the native, the acetylated and the reduced and acetylated hexasaccharide determined that the molecular mass of the native hexasaccharide was 959, and that the 2 rhamnose residues were linked to each other at the nonreducing terminus of the linear molecule. Methylation analysis revealed the positions of the glycosidic linkages in the hexasaccharide and showed that a galactose residue was present at the reducing end. The structural characterization of the hexasaccharide was completed by one and two dimensional 1H and 13C NMR spectroscopy. Complete 1H and 13C assignments for each glycosyl residue were established by two-dimensional (1H,1H) correlation spectroscopy, homonuclear Hartmann-Hahn, and (13C,1H) correlation experiments. The configurations of the glycosidic linkages were inferred from the chemical shifts and coupling constants of the anomeric 1H and 13C resonances. The sequence of the glycosyl residues was determined by a heteronuclear multiple bond correlation experiment. These data show that the structure of the hexasaccharide repeating unit derived from the cell wall polysaccharide of S. sanguis H1 is: alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-D-Galp- (1----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----3)-alpha/beta-D-Gal.     

The occurrence of batatasins in the dioscoreaceae

The tubers and/or aerial bulbils of 13 species of Dioscorea, mostly of tropical origin, and one species each of Rajania and Tamus, were examined for the presence of batatasins. These phenolics have previously been reported in the temperate species D. opposite. One or more batatasins were found in either tubers or bulbils of most species examined, including taxa from both Old and New Worlds, and with annual or perennial tubers. Batatasin I was the most widespread in occurrence, followed by B-II, while B-III, B-IV and B-V were comparatively rare. Only the buibils of D. opposite contained all five batatasins.     

Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24.

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.     

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.     

Studies on glycosphingolipids of fresh-water bivalves. V. The structure of a novel ceramide octasaccharide containing mannose-6-phosphate found in the bivalve, Corbicula sandai.

A ceramide octasaccharide containing mannose-6-phosphate was isolated from the fresh-water bivalve Corbicula sandai by solvent fractionation, followed by two types of silicic acid column chromatography, and finally QAE-Sephadex column chromatography. The structural analysis involved the following steps. (a) Gas-liquid chromatography of the component sugars, fatty acids, and long-chain bases. (b) Degradation with HCl and HF to elucidate the sugar sequence. (c) Permethylation analysis coupled with GC-MS to identify the positions of the glycosidic linkages between the sugar units. (d) Chromium trioxide oxidation to determine the anomeric configuration. (e) Smith degradation to determine the site of linkage of the ethanolamine residue. The structure of this novel glycolipid was determined to be: 4-O-MeGalp(bets1 yields 3)-GalNAcp(beta1 yields3)Fucp(alpha1 yields 4)GlcNAcp(beta1 yields 2)Manp(alpha1 yields 3)[Xylp(alpha1 yields 2)][2'-aminoethylphosphoryl(yields 6)]Manp(beta1yields 4)Glcp(beta1 yields 1)ceramide. It is very interesting that fucose was found to be internally linked in this sugar chain. To our knowledge, this is the first example of internal fucose in a glycolipid. The ceramide moiety consisted of normal saturated fatty acids, among which stearic acid was pr     

Characterization of gangliosides by direct inlet chemical ionization mass spectrometry

Permethylated derivatives of N-acetylneuraminic acid-containing GM3, N-glycolylneuraminic acid-containing GM3, GD1a, and GD1b, were analyzed by direct inlet ammonia chemical ionization (CI) mass spectrometry. These compounds were found to yield simple fragmentations, prominent fragment ions in the high mass region, and characteristic fragment ions corresponding to the cleavage of glycosidic linkages. This method also provides detailed information on the carbohydrate sequence and lipophilic composition in gangliosides.     

Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis. Relation to trehalose-based oligosaccharides attached to lipid.

A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.     

Differentiation of the anomeric configuration and ring form of glucosyl-glycolaldehyde anions in the gas phase by mass spectrometry: isomeric discrimination between m/z 221 anions derived from disaccharides and chemical synthesis of m/z 221 standards

Mass spectrometry of disaccharides in the negative-ion mode frequently generates product anions of m/z 221. With glucose-containing disaccharides, dissociation of isolated m/z 221 product ions in a Paul trap yielded mass spectra that easily differentiated between both anomeric configurations and ring forms of the ions. These ions were shown to be glucosyl-glycolaldehydes through chemical synthesis of their standards. By labeling the reducing carbonyl oxygen of disaccharides with 18O to mass discriminate between monosaccharides, it was established that the m/z 221 ions are comprised solely of an intact nonreducing sugar with a two-carbon aglycon derived from the reducing sugar, regardless of the disaccharide linkage position. This enabled the anomeric configuration and ring form of the ion to be assigned and the location of the ion to the nonreducing side of a glycosidic linkage to be ascertained. Detailed studies of experimental factors necessary for reproducibility in a Paul trap demonstrated that the unique dissociation patterns that discriminate between the isomeric m/z 221 ions could be obtained from month-to-month in conjunction with an internal energy-input calibrant ion that ensures reproducible energy deposition into isolated m/z 221 ions. In addition, MS/MS fragmentation patterns of disaccharide m/z 341 anions in a Paul trap enabled linkage positions to be assigned, as has been previously reported with other types of mass spectrometers.     

Methylation analysis of the major gangliosides of the human alimentary mucosa.

The five major gangliosides of the human alimentary mucosa were purified with silicic acid column chromatography and with thin-layer chromatography. The linkages in the carbohydrate portion were analysed by permethylation with gas chromatography-mass spectrometry. A succesful analysis of the linkages of two hematosides and three tetraglycosylceramides was performed. Two of the tetraglycosylamides had a galactosamine in their chain and one had a glucosamine.     

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)     

Sequence determination of a non-sulfated glycosaminoglycan-like polysaccharide from melanin-free ink of the squid <Emphasis Type="Italic">Ommastrephes bartrami</Emphasis> by negative-ion electrospray tandem mass spectrometry and NMR spectroscopy

A non-sulfated polysaccharide was isolated from the ink sac of squid Ommastrephes bartrami after removal of the melanin granules. The carbohydrate sequence of this polysaccharide was assigned by negative-ion electrospray tandem mass spectrometry with collision-induced dissociation of the oligosaccharide fractions produced by partial acid hydrolysis of the polysaccharide. The structural determination was completed by NMR for assignment of anomeric configuration and confirmation of linkage information and it was unambiguously identified as a glycosaminoglycan-like polysaccharide containing a glucuronic acid-fucose (GlcA-Fuc) disaccharide repeat in the main chain and a N-acetylgalactosamine (GalNAc) branch at Fuc position 3: -[3GlcAbeta1-4(GalNAcalpha1-3)Fucalpha1](n)-. Partial hydrolysis of the polysaccharide to obtain several oligosaccharide fractions with different numbers of the repeating unit assisted the assignment. In the negative-ion tandem mass spectrometric analysis, the unique (0,2)A type fragmentation was important to establish the presence of a 4-linked fucose in the main polysaccharide chain and a GalNAc branch at the Fuc position-3 of the disaccharide repeat.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.