Programmable reactivity-based one-pot oligosaccharide synthesis

A detailed protocol is described for the application of a programmable one-pot oligosaccharide synthesis methodology to the synthesis of fucosyl GM1. This serves as a general example of the application of this method to the synthesis of any desired oligosaccharide. The method relies on a large database of relative reactivities for differentially protected tolyl thioglycoside donor molecules and a computer program to suggest the best order of addition for assembly of the oligosaccharide in optimal yield and with the fewest operations. The product is a protected form of the desired oligosaccharide isolated in 47% yield, which is then deprotected using standard procedures to provide fucosyl GM1 oligosaccharide (1) in 44% yield. The total time for synthesis of 1 from building blocks 3, 4 and 5 is approximately 4 d, whereas synthesis of the same compound by traditional stepwise procedures would take significantly longer. Protocols for the synthesis of thioglycoside building blocks 3 and 4 are also described.     

Transfer of glucose in the biosynthesis of thyroid glycoproperties I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar.Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1μM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them.Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to acr as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was foun that the presence of glucose did not signifantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.     

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly

The formation of N-glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.     

The synthesis of complex-type oligosaccharides. I. Structure of the lipid-linked oligosaccharide precursor of the complex-type oligosaccharides of the vesicular stomatitis virus G protein.

The synthesis of the complex-type oligosaccharide unit of the vesicular stomatitis virus G protein is initiated by the en bloc transfer of a high molecular weight oligosaccharide from a lipid carrier to the nascent polypeptide. Following transfer the oligosaccharide is "processed" by removal of glucose and mannose residues and the sugars that constitute the outer branches of the complex-type oligosaccharide are added. The structure of the oligosaccharide moiety of the lipid-linked precursor has been elucidated in order to further define the steps involved in processing. Since it was not feasible to obtain adequate amounts of material for standard structural studies, most of the structural studies were performed on radiolabeled material, with radioactivity incorporated differentially into glucose, mannose, and N-acetylglucosamine. Based on endo-beta-N-acetylglucosaminidase CII digestion, alpha-mannosidase digestion, acetolysis, Smith periodate degradation, methylation analysis, and periodate oxidation, we propose the following structure for the oligosaccharide moiety of the lipid-linked oligosaccharide.     

Large scale enzymatic synthesis of oligosaccharides and a novel purification process

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study represents a facile enzymatic synthesis of and novel purification process of oligosaccharide.     

Multifunctionality of Campylobacter jejuni sialyltransferase CstII: characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities

CstII from bacterium Campylobacter jejuni strain OH4384 has been previously characterized as a bifunctional sialyltransferase having both alpha2,3-sialyltransferase (GM3 oligosaccharide synthase) and alpha2,8-sialyltransferase (GD3 oligosaccharide synthase) activities which catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophosphate (CMP)-Neu5Ac to C-3' of the galactose in lactose and to C-8 of the Neu5Ac in 3'-sialyllactose, respectively (Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM, Wakarchuk WW. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 277:327-337). We report here the characterization of a truncated CstII mutant (CstIIDelta32(I53S)) cloned from a synthetic gene whose codons are optimized for an Escherichia coli expression system. In addition to the alpha2,3- and alpha2,8-sialyltransferase activities reported before for the synthesis of GM3- and GD3-type oligosaccharides, respectively, the CstIIDelta32(I53S) has alpha2,8-sialyltransferase (GT3 oligosaccharide synthase) activity for the synthesis of GT3 oligosaccharide. It also has alpha2,8-sialidase (GD3 oligosaccharide sialidase) activity that catalyzes the specific cleavage of the alpha2,8-sialyl linkage of GD3-type oligosaccharides and alpha2,8-trans-sialidase (GD3 oligosaccharide trans-sialidase) activity that catalyzes the transfer of a sialic acid from a GD3 oligosaccharide to a different GM3 oligosaccharide (3'-sialyllactoside). The donor substrate specificity study of the CstIIDelta32(I53S) GD3 oligosaccharide synthase activity indicates that the enzyme is flexible in using different CMP-activated sialic acids and their analogs for the synthesis of GD3 oligosaccharides containing natural and nonnatural modifications at the terminal sialic acid.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Incorporation of glucose into lipid-linked saccharides in aorta and its inhibition by amphomycin

The particulate enzyme from pig aorta catalyzed the transfer of glucose from UDP-glucose into glucosyl-phosphoryl-dolichol, into lipid-linked oligosaccharides, and into glycoprotein. Radioactive lipid-linked oligosaccharides were prepared by incubating the extracts with GDP-[¹⁴C]mannose and UDP-[³H]glucose. When the labeled oligosaccharides were run on Bio-Gel P-4, the two different labels did not exactly coincide; the ³H peak eluted slightly earlier indicating that it was of higher molecular weight than the ¹⁴C material, but there was considerable overlap. The purified oligosaccharide(s) contained glucose, mannose, and N-acetylglucosamine but the ratios of these sugars varied from one enzyme preparation to another, probably depending on the endogenous oligosaccaride-lipids present in the microsomal preparation. Treatment of the [³H]glucose-labeled oligosaccharide with α-mannosidase gave rise to a ³H-labeled oligosaccharide which moved somewhat faster on Bio-Gel P-4 than the original oligosaccharide, suggesting it had lost one or two sugar residues. These data indicate that mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibited the transfer of glucose from UDP-glucose into the lipid-linked saccharides. However the synthesis of glucosyl-phosphoryl-dolichol was much more sensitive then was the synthesis of lipid-linked oligosaccharides. The glucose-labeled oligosaccharide produced in the absence of amphomycin was of high molecular weight based on paper chromatography. But in the presence of partially inhibitory concentrations of antibiotic, the oligosaccharide migrated more rapidly on paper chromatograms. However, amphomycin had no effect on the synthesis of glucosyl-ceramide by the aorta extracts. In fact, the antibiotic may stimulate glucosyl-ceramide by making more of the substrate, UDP-glucose, available for synthesis of this lipid.     

酶法合成功能性低聚糖

功能性低聚糖具有无毒、无残留、稳定性强等特点,作为新型绿色添加剂被广泛应用在食品、饲料、医药行业。国际市场上10余种低聚糖产品中除大豆低聚糖、棉籽糖外,主要采用酶法制备。用于合成功能性低聚糖的酶包括糖苷酶、糖基转移酶和磷酸化酶。本文综述了功能性低聚糖种类、性质和制备方法,分析了酶法合成低聚糖的优缺点,阐述了磷酸化酶种类、催化特性和低聚糖产物。多酶法合成策略和目标酶的分子改造将是酶法合成功能性低聚糖的发展方向。     

Effect of Leuconostoc mesenteroides NRRL B-512F Dextransucrase Carboxy-Terminal Deletions on Dextran and Oligosaccharide Synthesis

Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3′ end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K_m for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.     

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

Effect of dexamethasone on the synthesis of dolichol-linked saccharides and glycoproteins in hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. The incorporation of [14C]mannose into protein was increased in inflamed compared with control hepatocytes. The incorporation of [14C]mannose into protein was also increased when the hepatocytes were cultured in presence of dexamethasone (1 microM), either from control or inflamed rats. At the same time the incorporation of [14C]mannose into dolichol phosphate mannose and dolichol-linked oligosaccharide was increased due to inflammation. The presence of dexamethasone in the hepatocyte culture caused an increased formation of these two products; in particular its effect on oligosaccharide lipid formation was very pronounced. The ratios of activities of formation of [14C]mannose-labelled oligosaccharide lipid in inflamed over control hepatocytes gradually decrease when increasing amounts of exogenous dolichol phosphate was added in cell homogenate assay mixture. These results suggest that the increase of oligosaccharide lipid formation in inflammation could be due to a higher concentration of endogenous dolichol phosphate, as was shown for dolichol phosphate mannose formation in inflammation [Sarkar & Mookerjea (1984) Biochem. J. 219, 429-436]. In contrast, the ratio of activities of [14C]mannose-labelled oligosaccharide lipid between dexamethasone-treated and untreated hepatocytes shows only a slight increase when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the stimulation of dolichol pyrophosphate oligosaccharide synthesis observed in dexamethasone treatment is probably due to the higher level of enzymes involved in oligosaccharide synthesis rather than higher level of endogenous dolichol phosphate in these cells.     

Expeditious oligosaccharide synthesis via selective, semi-orthogonal, and orthogonal activation

Traditional strategies for oligosaccharide synthesis often require extensive protecting and/or leaving group manipulations between each glycosylation step, thereby increasing the total number of synthetic steps while decreasing the efficiency of the synthesis. In contrast, expeditious strategies allow for the rapid chemical synthesis of complex carbohydrates by minimizing extraneous chemical manipulations. Oligosaccharide synthesis by selective activation of one leaving group over another is one such expeditious strategy. Herein, the significant improvements that have recently emerged in the area of the selective activation are discussed. The development of orthogonal strategy further expands the scope of the selective activation methodology. Surveyed in this article, are representative examples wherein these excellent innovations have been applied to the synthesis of various oligosaccharide sequences.     

不同发育时期小鼠胚泡表面Lewis寡糖抗原的表达

在胚泡表面表达的Lewis寡糖抗原 (LewisX ,LewisY)在胚胎发育以及着床过程中起重要作用 .应用免疫印迹和免疫荧光等方法对着床前小鼠胚泡表面的Lewis寡糖抗原进行分析 .结果发现 :小鼠胚泡LewisX寡糖蛋白有 2 7kD、2 9kD、6 8kD和 80kD 4种 ,LewisY寡糖蛋白有 70kD和 90kD 2种 ;2种寡糖抗原均在 8细胞时期开始表达 ,其中 ,LewisY寡糖抗原在胚泡表面的表达持续升高 ,直至胚泡着床 ;而LewisX寡糖抗原的表达则在桑椹期后逐渐降低 ,但仍在胚胎期的囊胚腔侧的顶端可见有部分表达 ;应用RT PCR的分析结果显示 ,LewisX合成的关键糖基转移酶FUT9基因在 4细胞及桑椹期高表达 ,到胚泡期虽然强度明显减弱 ,但仍有表达 ;而LewisY合成关键酶FUT1基因在 4细胞未见表达 ,在桑椹和胚泡阶段均有表达并逐渐升高 ,表达趋势与相应寡糖的表达趋势基本一致 .结果说明 ,在小鼠胚泡表面表达的Lewis寡糖抗原的表达受到相应糖基转移酶基因转录的调控     

Synthesis of Oligosaccharides by β-Fructofuranosidase in Biphasic Medium Containing Organic Solvent as Bulk Phase

The effect of four organic solvents on β-fructofuranosidase mediated synthesis of oligosaccharides from sucrose were investigated. Amongst the solvents examined, butyl acetate proved to be the best for oligosaccharide synthesis. Starting with the equivalent of 44.6 g/L of sucrose, 247 U of enzyme and 91.6% (by vol.) of butyl acetate results in the production of 8.8 g/L of oligosaccharides within 30 min, with trisaccharides constituting more than 60% of the oligosaccharides. The efficiency for conversion of sucrose to oligosaccharides is greater than 19%, and this exceeds the 11.6% (in 24 h) previously achieved with 1271 U of the same enzyme in aqueous medium. Use of butyl acetate as the bulk phase therefore modifies the reaction environment in favour of enhanced and accelerated rate of oligosaccharide synthesis by this β-fructofuranosidase.     

Decreased transfer of oligosaccharide from oligosaccharide-lipid to protein acceptors in regenerating rat liver

The transfer of [14C]glucose from UDP-[14C]glucose to lipid intermediates and glycoproteins was decreased in regenerating rat liver microsomes 24 h after partial hepatectomy. In regenerating liver microsomes, the concentration of free dolichyl phosphate (Dol-P) was significantly decreased. However, it was only about 10% of total Dol-P, which was not significantly changed. On the addition of exogenous Dol-P, the transfer of [14C]glucose to glycoproteins was still decreased, while the decrease of the transfer to lipid intermediates was no longer observed. These results suggest that the glycoprotein synthesis is not regulated by the amount of Dol-P in regenerating liver microsomes. Oligosaccharide obtained from [14C]glucosyl-oligosaccharide-lipid was not distinguishable between regenerating liver and control by paper chromatography. The oligosaccharide transfer to protein in microsomes was compared by using [14C]glucosyl-oligosaccharide-lipid as oligosaccharide donor. The transfer of oligosaccharide to endogenous proteins decreased to 77% of control in regenerating liver and the transfer to exogenously added denatured alpha-lactalbumin decreased to 59% of control. Therefore, it is unlikely that the acceptor capacity of endogenous protein is decreased in regenerating liver. Neither the change in oligosaccharide-lipid under the condition for oligosaccharide transfer assay nor the stability of oligosaccharide transferase was different between regenerating liver and control. These results strongly suggest that the decrease in the activity of the oligosaccharide transferase in microsomes causes the decrease of glycoprotein synthesis in regenerating liver, which was shown in our previous studies.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Enzymatic synthesis of oligosaccharides

As the importance of the oligosaccharide moieties of glycoproteins and glycolipids is being increasingly recognized, efforts to synthesize them are expanding. The number of functional groups of carbohydrate monomers and the variety of configurations that oligomers can adopt is greater than with nucleotides/nucleic acids or amino acids/peptides. By reversing the hydrolytic action of glycosidases and by using highly regiospecific glycosyltransferases, enzymatic oligosaccharide synthesis can be performed.     

Erythroglycan biosynthesis in K-562 cells. Inhibition of synthesis by tunicamycin and lack of attachment to the G-protein of vesicular-stomatitis virus.

K-562 cells, which express foetal erythroglycan, are shown to synthesize the lipid-linked oligosaccharide intermediates commonly found in tissues and cultured fibroblasts. The addition of tunicamycin, which blocks the formation of these intermediates and thus of asparagine-linked oligosaccharides, inhibits the synthesis of erythroglycan (Mr 7000-11 000). Vesicular-stomatitis-virus infection of K-562 cells results in the glycosylation of the G-protein with the transferrin-type oligosaccharide (Mr 3000), but not with the larger erythroglycan. These results suggest that, in K-562 cells, the early stages of erythroglycan biosynthesis are the same as those of the transferrin-type oligosaccharides. However, maturation of the oligosaccharide is influenced by protein structure such that erythroglycan is only expressed on specific glycoproteins.