Galactosyl Oligosaccharides Research Articles

Galactinol in the leaves of the resurrection plant Boea hygroscopica

The content of low molecular weight substances was analysed in leaf samples of the resurrection plant Boea hygroscopica F. Muell. submitted to dehydration. Drying treatment caused a variation in the carbohydrate pool, with a decrease of all sugars except sucrose which notably increased, becoming the prevalent one in dried leaves. Rehydration almost restored the pre-treatment sugar composition. Along with more common sugars galactinol and some higher oligosaccharides of the raffinose family were detected. Their structures were assigned by NMR and GC–MS analyses after acetylation. To our knowledge, this is the first finding in resurrection plants of significant amounts of the galactosyl donor galactinol and of higher galactosyl oligosaccharides, which may have a role in restoring the pre-drying functions upon rehydration.

Recent Progress in Glycochemistry and Green Chemistry

Abstract: This two-part article will focus on the most recent research efforts from the Wang's group in areas of glycochemistry and green chemistry. In the first part, we wish to address the progress in synthesis, conformational analysis, and transformations of some biologically active a­ galactosyl oligosaccharides. Carbohydrate structures containing Gala1-3Galterminus (a-Gal epitopes) are xenoactive antigens and are considered to be the major cause for hyperacute rejection in xenotransplantation. The latest chemo-enzymatic synthesis of a-galactosyl epitopes using a recombinant a1,3-galactosyltransferase is described. Along with an efficient chemical synthesis of an a-Gal trisaccharide, these synthetic approaches provide an easy access to this important class of oligosaccharides. Conformational analysis of an a-Gal epitope using various NMR techniques and molecular modeling is discussed. These studies provide important information in structure-function relationship and binding conformation of a-Gal epitopes and their interaction with anti-Gal antibody. In another aspect, novel glycoconjugates containing receptor-specific ligands and a-Gal epitopes have been synthesized as potential immunotherapeutic agents.

Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on α-amylases and oligosaccharidases

Determinations of carbohydrases, aminopeptidases and acid phosphatase in the larval midgut cells and in the luminal contents of Callosobruchus maculatus and Zabrotes subfasciatus have been carried out. The results showed that larvae of both species displayed similar distribution of digestive enzymes in the intestinal compartments. Most larval digestive enzyme activities were found in the luminal contents. Of the activities found in the midgut tissue, only aminopeptidase is predominant in the membrane fraction. Comparisons of activities recovered from a seed flour mass equivalent to the midgut mass showed that a high percentage of the luminal aminopeptidase activity and, to a lesser extent, α-galactosidase activity, can be derived from the seeds, whereas the other enzymes are produced by the insects. Activities against starch, maltose and maltodextrins were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on differences in elution profiles on hydrophobic chromatography and banding patterns in mildly denaturing electrophoresis, both species showed a multiplicity of glycosidases. The data suggest that the majority of carbohydrate digestion occurs in the midgut lumen, whereas protein digestion should take place partly in the lumen and partly at the cell surface. Larvae of Z. subfasciatus can modulate the levels of α-amylases and α-glucosidase in response to different diets. The complex of carbohydrases found is qualitatively appropriate to digest the free oligosaccharides and oligomaltodextrins produced by α-amylases from the starch granules of host seeds.

Effect of Germination on Chemical Composition, Biochemical Constituents and Antinutritional Factors of Soya Bean (Glycine max) Seeds

The published scientific data concerning the effects of germination on chemical composition, biochemical constituents and anti-nutritional factors of soya bean are reviewed. The amino acid profile did not change to a great extent; only a noticeable increase in aspartic acid was observed whereas there was a gradual decrease in the available lysine level and lipid content as germination progressed. Both the total protein content and the nonprotein nitrogen increased after 5 days of germination. Dietary fibres are partially degraded in germinated seeds. Germinated soya bean is an excellent source of ascorbic acid and riboflavin. Niacin contents increased distinctly after germination. Germination induced a reduction in lipase inhibitor activity. The galactosyl oligosaccharides drastically decreased in germinated seeds. After 4 days of germination, the activity of certain lectins decreased to 4% of that of ungerminated soya beans. The phytic acid in the seeds was degraded by the phytase activated during germination, thus increasing the availability of the minerals present in the germinated seeds. Germination can degrade both Kunitz soya bean trypsin inhibitor and the major Bowman–Birk soya bean trypsin inhibitor; the degradation is enhanced, if germination process lasts more than 4 days. © 1997 SCI.

Effect of Germination on Chemical Composition, Biochemical Constituents and Antinutritional Factors of Soya Bean (Glycine max) Seeds

The published scientific data concerning the effects of germination on chemical composition, biochemical constituents and anti-nutritional factors of soya bean are reviewed. The amino acid profile did not change to a great extent; only a noticeable increase in aspartic acid was observed whereas there was a gradual decrease in the available lysine level and lipid content as germination progressed. Both the total protein content and the nonprotein nitrogen increased after 5 days of germination. Dietary fibres are partially degraded in germinated seeds. Germinated soya bean is an excellent source of ascorbic acid and riboflavin. Niacin contents increased distinctly after germination. Germination induced a reduction in lipase inhibitor activity. The galactosyl oligosaccharides drastically decreased in germinated seeds. After 4 days of germination, the activity of certain lectins decreased to 4% of that of ungerminated soya beans. The phytic acid in the seeds was degraded by the phytase activated during germination, thus increasing the availability of the minerals present in the germinated seeds. Germination can degrade both Kunitz soya bean trypsin inhibitor and the major Bowman–Birk soya bean trypsin inhibitor; the degradation is enhanced, if germination process lasts more than 4 days. © 1997 SCI.

The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera.

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.

The reducing end of αGal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera

Abstract Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-α-Gal gave weak inhibition, Galal-3Gal substantially inhibited the reaction (400 µM), and Galα1-3Galβ 1-4GlcNAc was ten times more efficient (30 µM). The modification from a to β anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-αGal antibodies recognize the epitope from its non-reducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccha-ride but not as good as Galα1-3Galβ 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.

Effect of galactooligosaccharides on calcium absorption and preventing bone loss in ovariectomized rats.

The effects of galactooligosaccharides (GOS), a mixture of galactosyl oligosaccharides formed from lactose by the transgalactosyl reaction of beta-D-galactosidase derived from Bacillus circulans, on calcium absorption and prevention of bone loss were examined in ovariectomized (OVX) Wistar rats. Rats fed on a diet containing GOS absorbed calcium more efficiently than those on the control diet after 8-10 days and 18-20 days, and the bone (femur and tibia) ash weight and tibia calcium content of OVX rats fed on the GOS diet were significantly higher than those of the control animals. Although the serum total cholesterol of the ovariectomized rats was significantly elevated, GOS produced a significant hypocholesterolemic effect in the OVX rats. GOS, which is fermented by bacteria in the lower part of the intestine, enhanced volatile fatty acid production, and thus prevented bone loss and lower serum total cholesterol concentration in the ovariectomized rats.

Urinary oligosaccharide excretion and severity of galactosialidosis and sialidosis

Urinary oligosaccharides of galactosialidosis and sialidosis patients were investigated by the Bio-Gel P-4 column chromatography in order to delineate each clinical subtype biochemically. The levels of urinary oligosaccharides (nmol/mg creatinine) in late infantile-type galactosialidosis patients with early onset and severe clinical manifestations were 2.5 times the levels in juvenile/adult-type patients. Galactosyl oligosaccharides which had not been reported in the analysis of galactosialidosis urine were identified in the urine of patients of both subtypes. The structures of the urinary oligosaccharides in late infantile-type patients were the same as those in the juvenile/adult-type patients. The levels of urinary oligosaccharides in the infantile-type sialidosis patients were 3.5 times the levels in late-type patients who were less severely affected. The structures of oligosaccharides in the urine of infantile-type patients were the same as those in the urine of late-type patients, but more high-molecular weight sialyl oligosaccharides were excreted in the urine of infantile-type patients than in the urine of late-type patients. The structures of 7 different kinds of sialyl oligosaccharides that have not been previously reported were identified in the urine of both galactosialidosis and sialidosis patients. Sialidosis patients excreted relatively more sialyl oligosaccharides than did galactosialidosis patients.

Diagnosis of subtypes of GM1 gangliosidosis in vitro and in vivo — using urinary oligosaccharides as substrates

In order to delineate clinical subtypes of GM1 gangliosidosis enzymologically, we prepared galactosyl oligosaccharides from the urine of patients, as substrates, and established the method of the galactosyl oligosaccharide β-galactosidase assay. Galactosyl oligosaccharides β-galactosidase activities (nmol/mg protein/20 h) in vitro, using substrates without repeating structures were; type 1, 1.0 ± 0.5 ( n = 6), type 2A, 2.1, type 2B, 3.4 ± 0.7 ( n = 5), type 3, 4.9 ± 0.2 ( n = 2). The activities in vitro using substrates with repeating structures were: type 1, 0.3 ± 0.2 ( n = 5), type 2A, 1.2, type 2B, 2.2 ± 0.5 ( n = 4), type 3, 4.2 ± 0.3 ( n = 2). The activities using substrates with and without repeating structures were affected in the fibroblasts of patients, and the residual activities in each subtype were reduced progressively with the increasing severity of the clinical features. The ratio between activities using substrates without repeating structures and activities using substrates with repeating structures indicated that β-galactosidase activities toward Galβ1- of repeating structures were reduced progressively with the increasing severity of the clinical features. The activities in vivo (pmol/mg protein per 24 h) were: type 1, 11.8 ± 1.8 ( n = 2), type 2A, 24.8, type 2B, 40.0 ± 9.7 ( n = 2), type 3, 63.2. The activities in vivo were affected in the fibroblasts of patients and the residual activities were reduced in proportion to the severity of the clinical features. These differences of residual activities among each subtype make it possible to delineate clinical subtypes enzymologically.

Intestinal lactase (beta-galactosidase) and other disaccharidase activities of suckling and adult common brushtail possums, Trichosurus vulpecula (Marsupialia:Phalangeridae)

Small-intestinal disaccharidase activities of eight suckling T. vulpecula, aged from 34 to 150 days, and of two adult animals were investigated. Intestinal maltase, isomaltase and sucrase activities increased with age, whereas lactase activities decreased. Trehalase activities were relatively high in all animals and showed no obvious age-related changes. Three separate beta-galactosidase activities, one neutral and two acid, acted on lactose. The neutral beta-galactosidase activity appeared to be due to a brush border enzyme similar to that of eutherian mammals, whereas the acid beta-galactosidases were soluble and probably of lysosomal origin. One of these, acid beta-galactosidase-1, had similar properties to the sole intestinal beta-galactosidase of macropodid marsupials, whereas the other, acid beta-galactosidase-2, has not previously been described. Galactosyl oligosaccharides isolated from macropodid milk were readily hydrolysed by both acid beta-galactosidases but not by the neutral beta-galactosidase. The total intestinal lactase activity in animals aged up to 125 days was due mainly to acid beta-galactosidase-1, whereas in older animals it was due mostly to the neutral beta-galactosidase; this suggests that late in lactation the young T. vulpecula change from a macropodid mode of digestion of galactosyl oligosaccharides to a eutherian mechanism for the digestion of lactose. These findings may have implications for the hand-rearing of orphaned T. vulpecula.

Measurement of some antinutritive factors in meat products containing texturated vegetable protein (TVP).

The alpha-(1,6)-galactosyl oligosaccharide and phytic acid contents of sausages containing TVP were examined. The results show how the meat industrial technologies affect the oligosaccharide and phytic acid contents of the end-products. According to these experiences, the small quantities present in the end-products are not considered to be harmful to health but may show unexpected effects when compared to traditional products. With the knowledge of the concentrations of the measured oligosaccharides and phytic acid, after adequate calibration, the real TVP content of the finish-product can be determined according to the method elaborated by the authors.

Probing the combining site of monoclonal IgA J539 using deoxyfluoro- and other galactosides as ligands

IgA J539 is a monoclonal anti β-(1→ 6)- d-galactopyranan. Measurement of its affinity with a number of synthetic galactosyl oligosaccharides of that linkage type, in which one or more hydroxyl groups have been replaced by a fluorine atom, confirm the assignment of the relative binding strength of the subsites in the protein for the individual galactosyl residues of the antigen.

Glycolipids and glycoproteins formed from UDP-galactose by pea membranes

Pea membranes were incubated with UDP-[ 14C]galactose and sequentially extracted with lipid solvents and 2% sodium dodecyl sulfate (SDS). At least three-quarters of the products were SDS-soluble. All fractions contained some [ 14C]glucose, indicating the presence of an active epimerase which, however, could be inhibited by ADP-ribose. The chloroform-methanol extract contained mainly neutral galactosyl lipids and a small amount of dolicyl monophosphoryl glucose. The chloroform-methanol-water extract contained trace amounts of lipid-linked galactosyl oligosaccharide with properties comparable to polyisoprenyl pyrophosphoryl derivatives. Polyacrylamide gel electrophoresis of SDS-soluble products indicated the formation of both immobile and mobile components with similar size distribution (Sepharose CL-6B). The mobile component only was susceptible to hydrolysis by protease. Periodate oxidation analysis of SDS-soluble and -insoluble products indicated that they were composed mainly of 1 → 6 galactosyl residues, i.e. as in many arabinogalactan proteins and arabinogalactans.

Evidence for Spider Mite (Acari: Tetranychidae) Injury-Induced Leaf Water Deficits and Osmotic Adjustment in Peppermint1

Studies were conducted to examine the physiological response of peppermint, Mentha piperita L., to leaf water stress induced by feeding of the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Levels of mite injury were estimated by a leaf injury index developed for these studies. Symptoms of water stress in mite-injured leaves were: a significant reduction in leaf fresh weight; reduced leaf specific weight (mg of fresh weight per cm2 of leaf area); and accumulation of soluble leaf carbohydrates. Soluble leaf carbohydrates of peppermint are sucrose (ca. 50%) and the galactosyl oligosaccharides raffinose and stachyose. Leaf starch content was unaffected by injury at all injury levels. Accumulation of soluble leaf carbohydrate in water-stressed leaves may help mite-injured leaves maintain cell turgor pressure through an osmotic adjustment (osmoregulation) mechanism. A new method for in situ starch hydrolysis for quantitative analysis of leaf starch also is presented.

Distribution of α-Galactosidase in Cucurbita pepo

The distribution of alpha-galactosidase (alpha-d-galactoside galactohydrolase [EC 3.2.1.22]) in Cucurbita pepo has been determined in an attempt to assess its involvement in hydrolysis of transport sugars of the raffinose oligosaccharide series ([alpha-1-6-0-galactopyranosyl](n) sucrose). Extracts prepared from leaves and petioles at different stages of development, roots, flowers, dry and germinating seeds, all contained appreciable levels of alpha-galactosidase activity. Chromatography of these extracts on DEAE-Sephadex resolved the enzyme into three active isozymic forms. These isozymes were present in all regions of the plant analyzed but their relative proportions varied between tissues and changed within leaf and petiole tissues during development and in seeds during their germination. The level of total alpha-galactosidase activity in the leaf blade measured on a fresh weight or total protein basis remained constant at all developmental stages analyzed. The occurrence of these isozymes in mature exporting leaves indicates an effective intracellular compartmentation between their location and the sites of galactosyl oligosaccharide biosynthesis, accumulation and movement in the tissue. We have used these results to comment on the transport pathway of galactosyl oligosaccharides between the phloem and surrounding tissues in this plant.

Existence of multiple immunodeterminants in the type-specific capsular substance of group B type Ia streptococci.

Rabbits immunized with group B, type Ia streptococci produce two distinct populations of protective antibodies. Evidence is presented indicating that these antibodies are directed against two major immunodeterminants which coexist in the specific type Ia antigen. Immunochemical data, using purified antibody preparations, indicate that the type substance, a sialic acid polymer consisting of galactose, glucose, glucosamine, and sialic acid, possesses two distinct immunodominant determinants, terminal sialic acid residues and a galactosyl oligosaccharide. Antibodies directed against either of these determinants were shown to possess in vivo and in vitro opsonic capabilities.

Release of galactosyl oligosaccharides by endo-β-N-acetylglucosaminidase D

Endo-β-N-acetylglucosaminidase D from Diplococcus pneumoniae released galactosyl oligosaccharides from IgG glycopeptides treated with β-N-acetylglucosaminidase. The structure of the major oligosaccharide was proposed to be as follows. ▪Since α-mannosidase digestion of the β-N-acetylglucosaminidase-treated glycopeptides made them again resistant to the endoglycosidase, we concluded that an unsubstituted α-mannosyl residue was required for the enzymic action.

The use of o-nitrophenyl β- d-galactoside for the synthesis of galactosyl oligosaccharides

o-Nitrophenyl β- d-galactoside was found to serve as a donor of galactosyl units in enzymic transgalactosylation to d-galactose and d-arabinose. The oligosaccharides produced in the reactions were isolated by chromatographic procedures, and structural information on the compounds was obtained. Two oligosaccharides were characterized as 3-β- d-galactopyranosyl- d-arabinose and 6-β- d-galactopyranosyl- d-galactose. Tentative structures, 5-β- d-galactopyranosyl- d-arabinofuranose and 3-β- d-galactopyranosyl- d-galactose, were assigned to two other oligosaccharides. A new chromatographic procedure termed column-paper chromatography was found to be effective for separating compounds with small differences in R f values.

Cosubstrate Specificity of the Transgalactosylase of Saccharomyces fragilis

Galactosyl oligosaccharides are produced from lactose by enzyme preparations from yeasts, molds, and bacteria (1, 2). It has been suggested that these compounds are synthesized via a transgalactosylation mechanism in which galactosyl units of lactose are transferred to accepting or cosubstrate molecules (3-5). That the transferring enzyme of Saccharomyces jragilis acts by the transgalactosylation mechanism has now been verified. In addition, a number of saccharides have been tested as cosubstrates for the enzyme with a view to determining its cosubstrate specificity. The latter experiments have resulted in the preparation of several new oligosaccharides.