Analysis of the oligosaccharides from the roots of Arnica montana L., Artemisia absinthium L., and Artemisia dracuncula L.

The oligosaccharides extracted from the roots of Arnica montana L., Artemisia absinthium L. and Artemisia dracunculus L. have been analysed by thin-layer and gel-permeation chromatography to assess their applicability as ‘guide’ substances for pharmacological activity. Differences observed in the oligosaccharide component composition of such extracts might be more related to the vegetative stage of the plants at time of harvest than to the species themselves. In addition to a series of non-reducing oligofructosides, a series of reducing inulin-type oligosaccharides was found at the initial stages of growth, whereas in later stages of growth only non-reducing oligofructosides were present. These differences have been related to different stages of biosynthetic activity within the plants.     

Health benefit application of functional oligosaccharides

There is no doubt that the functional oligosaccharides have positive effects on human health, both in the prevention and in treatment of chronic diseases. Therefore, there is great interest in health benefits of the functional oligosaccharides. The functional oligosaccharides of various origins (viruses, bacteria, plants and fungi) have been used extensively both as pharmacological supplements, food ingredients, in processed food to aid weight control, to regulation of glucose control for diabetic patients and reducing serum lipid levels in hyperlipidemics and other some acute and chronic diseases. Keeping in view, the pharmacological importance of the functional oligosaccharides and its derivatives, this article discusses the potential of the functional oligosaccharides to modulate the gut flora, to affect different gastrointestinal activities and lipid metabolism, to enhance immunity, and to reduce diabetes, obesity and cardiovascular risk for further exploitation of health benefits of the functional oligosaccharides.     

Metabolic oligosaccharide engineering: perspectives, applications, and future directions

Many adhesion and signaling molecules critical for development, as well as surface markers implicated in diseases ranging from cancer to influenza, contain oligosaccharides that modify their functions. Inside a cell, complex glycosylation pathways assemble these oligosaccharides and attach them to proteins and lipids as they traffic to the cell surface. Until recently, practical technologies to manipulate glycosylation have lagged unlike the molecular biologic and genetic methods available to intervene in nucleic acid and protein biochemistry; now, metabolic oligosaccharide engineering shows promise for manipulating glycosylation. In this methodology, exogenously-supplied non-natural sugars intercept biosynthetic pathways and exploit the remarkable ability of many of the enzymes involved in glycosylation to process metabolites with slightly altered chemical structures. To date, non-natural forms of sialic acid, GalNAc, GlcNAc, and fucose have been incorporated into glycoconjugates that appear on the cell surface; in addition O-GlcNAc protein modification involved in intracellular signaling has been tagged with modified forms of this sugar. Reactive functional groups, including ketones, azides, and thiols, have been incorporated into glycoconjugates and thereby provide chemical 'tags' that can be used for diverse purposes ranging from drug delivery to new modes of carbohydrate-based cell adhesion that can be used to control stem cell destiny. Finally, strategies for further engineering non-natural sugars to improve their pharmacological properties and provide complementary biological activities, such as addition of short chain fatty acids, are discussed in this article.     

Chemical characterization of the oligosaccharides in milk of high Arctic harbour seal (Phoca vitulina vitulina)

Carbohydrates were extracted from high Arctic harbour seal milk, Phoca vitulina vitulina (family Phocidae). Free neutral oligosaccharides were separated by gel filtration and preparative thin layer chromatography, while free sialyl oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and high performance liquid chromatography. Oligosaccharide structures were determined by 1H-NMR spectroscopy. The structures of the neutral oligosaccharides were as follows: lactose, 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-neohexaose, monofucosyl lacto-N-neohexaose and difucosyl lacto-N-neohexaose. Thus, all of the neutral saccharides contained lactose or lacto-N-neotetraose or lacto-N-neohexaose as core units and/or non-reducing alpha(1-2) linked fucose. These oligosaccharides have also been found in hooded seal milk. The structures of the silalyl oligosaccharides were: monosialyl lacto-N-neohexaose, monosialyl monofucosyl lacto-N-neohexaose, monosialyl difucosyl lacto-N-neohexaose and disialyl lacto-N-neohexaose. These oligosaccharides contained lacto-N-neohexaose as core units, and one or two alpha(2-6) linked Neu5Ac, and/or non-reducing alpha(1-2) linked Fuc. The Neu5Ac residues were found to be linked to GlcNAc or penultimate Gal residues. The acidic oligosaccharides are the first to have been characterized in the milk of any species of seal.     

In vivo kinetics and biodistribution analysis of neoglycoproteins: effects of chemically introduced glycans on proteins

Biodistribution and in vivo kinetics analysis of chemically prepared neoglycoproteins are reviewed. Various mono- and oligosaccharides were conjugated onto the protein surface by use of chemical methods. Their kinetic and organ-specific accumulation have extensively been studied after intravenous injection and analyzed by conventional dissection studies, as well as noninvasive methods, such as SPECT, PET, or fluorescence imaging. These studies clearly show the glycan-structure dependency on protein kinetics, which will provide promising possibilities for pharmacological and diagnostic applications.     

The influence of small oligosaccharides on the immune system

In this study, oligosaccharides known to enhance the synthesis of penicillin by Penicillium chrysogenum have been presented to human immune cells and their effect measured. In addition a range of commercially available oligosaccharides have been tested. Results obtained indicate that oligosaccharides with a degree of polymerisation greater than 6 and with a tendency to form helical structures are most effective at influencing the immune system as measured by the production of reactive oxidising species. Laminariheptaose has been shown to increase reactive oxidising species production by up to 25%, whilst mannan-oligosaccharides with a DP of 6 to 7 decrease production by up to 44%. These and other results show that the immune system can recognise subtle differences in oligosaccharides and that these oligosaccharides could potentially be used to modulate the immune response.     

Monosaccharide composition, chain length and linkage type influence the interactions of oligosaccharides with dry phosphatidylcholine membranes

Sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms and disaccharides have been extensively investigated for their ability to stabilize model membranes in the dry state. Much less is known about the ability of oligosaccharides to protect dry membranes. However, it has been shown that different structural families of oligosaccharides have different efficacies to interact with and protect membranes during drying. Here, we have compared three families of linear oligosaccharides (fructans, malto-oligosaccharides, manno-oligosaccharides) for their chain-length dependent lyoprotective effect on egg phosphatidylcholine liposomes. We found increased protection with chain length for the fructans, a moderate decrease in protection with chain length for malto-oligosaccharides, and a strong decrease for manno-oligosaccharides. Using Fourier-transform infrared spectroscopy and differential scanning calorimetry, we show that the degree of lyoprotection of the different sugars is closely related to their influence on the gel to liquid-crystalline phase behavior of the dry membranes and to the extent of H-bonding to different groups (C=O, P=O, choline) in the lipids. Possible structural characteristics of the different oligosaccharides that may determine the extent to which they are able to interact with and protect membranes are discussed.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

糖苷合成酶——— 一类新型的寡糖高效合成工具

寡糖是哺乳动物细胞表面糖蛋白和糖脂以及微生物来源的生理活性物质的要素之一,其应用于医药的巨大潜能至今还没有得到充分体现,主要原因是合成足够于临床使用的寡糖非常困难.传统的化学法和酶法在大规模合成寡糖方面都有一定局限性.近年来,分子生物学技术大大推动了糖苷酶合成寡糖的研究,将糖苷酶催化中心亲核体氨基酸定点突变为非亲核体氨基酸,导致酶的原有水解活性丧失,只催化糖苷键合成反应,寡糖产量最高可达99%,人工产生了一类新酶——糖苷合成酶(glycosynthases),随后又产生了硫代糖苷酶(thioglycoligases)和硫代糖苷合成酶(thioglycosynthases).糖苷合成酶的高通量筛选可用双质粒系统和酵母三杂交系统进行,其活性的进一步改进可通过亲核体氨基酸位点不同氨基酸取代、其他位点氨基酸突变、反应条件优化等方法进行,其区域选择性的改变或增强可通过改变糖基受体分子达到.糖苷合成酶作为一种新型高效的生物催化剂,对寡糖的工业化合成有着重要意义,它的出现对糖生物学的发展必将起到巨大的推动作用.     

Structure/function relationship of homopolysaccharide producing glycansucrases and therapeutic potential of their synthesised glycans

The capability of lactic acid bacteria (LAB) to produce exopoly- and oligosaccharides was and is the subject of expanding research efforts. Due to their physicochemical properties and health-promoting potential, exopoly- and oligosaccharides from food-grade LAB can be used in the food and other industries and may have additional medical applications. In the last years, many LAB have been screened for their ability to produce exopoly- and oligosaccharides, and several glycosyltransferases involved in their biosynthesis have been characterised at biochemical and genetic levels. These research efforts aim to exploit the full potential of these organisms and to understand the structure/function relationship of glycosyltransferases. The latter knowledge is a prerequisite for the production of tailored exopoly- and oligosaccharides for the diverse applications. This review will survey the results of recent works on the structure/function relationship of homopolysaccharide producing glycosyltransferases and the therapeutic potential of their synthesised exopoly- and oligosaccharides.     

Structural analyses of asparagine-linked oligosaccharides of porcine pancreatic kallikrein

The structures of asparagine-linked oligosaccharides of porcine pancreatic beta-kallikrein are reported. Asparagine-linked neutral oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The mixture of pyridylamino oligosaccharides was separated by reverse-phase and amide-adsorption high-performance liquid chromatography. The pyridylamino oligosaccharides were separated into more than 50 kinds of oligosaccharides. The structures of 5 kinds of triantennary and 12 kinds of tetraantennary oligosaccharides were determined by the use of high-resolution proton nuclear magnetic resonance spectroscopy and methylation analysis. Furthermore, the structures of five kinds of oligomannose-type oligosaccharides were elucidated by a combination of exoglycosidase digestion and high-performance liquid chromatography. 1H NMR data for 14 out of the 17 kinds of N-acetyllactosamine-type oligosaccharides reported here have not previously been described in the literature. (1) It has been shown that fucose containing tri- and tetraantennary oligosaccharides is predominant in porcine pancreatic beta-kallikrein B. (2) It has also been shown that the heterogeneity of the structure in these types of oligosaccharides is derived from the variety of the positions of galactose residues linked to outer N-acetylglucosamine residues. (3) The distribution of oligosaccharides into two glycosylation sites, asparagine-95 and asparagine-239, of beta-kallikrein B was determined. It has been found that oligomannose-type oligosaccharides are exclusively present at asparagine-239, although N-acetyllactosamine-type oligosaccharides occur at both glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monolith enzymatic microreactor at the frontier of glycomic toward a new route for the production of bioactive oligosaccharides

Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.     

Localization of water-soluble carbohydrates in wheat stems using imaging matrix-assisted laser desorption ionization mass spectrometry

The pool of endogenous water-soluble oligosaccharides found in the stems of wheat (Triticum aestivum) is being investigated as a potential indicator of grain yield. Techniques such as liquid chromatography with mass spectrometry (LC-MS) can profile these analytes but provide no spatial information regarding their distribution in the wheat stem. The imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique has not been utilized for the analysis of oligosaccharides in plant systems previously. Imaging MALDI mass spectrometry was used to analyse cross and longitudinal sections from the stems of Triticum aestivum. A range of oligosaccharides up to Hex(11) were observed. Water-soluble oligosaccharides were ionized as potassiated molecules, and found to be located in the stem pith that is retained predominantly around the inner stem wall. Imaging MALDI analyses provided spatial information on endogenous oligosaccharides present in wheat stems. The technique was found to offer comparable sensitivities for oligosaccharide detection to those of our established LC-MS method, and has potential for broad application in studying the in situ localization of other compound types in plant material.     

药物分子伴侣: 蛋白质折叠运输缺陷的新疗法

大量遗传性疾病的发生是由于基因突变引起蛋白质错误折叠而不能运输到作用位点,从而导致功能缺陷．近年来兴起的药物分子伴侣是恢复蛋白质折叠运输缺陷的新疗法,这类化合物一般为目的蛋白的底物类似物、受体配基或酶抑制剂等化学小分子,具细胞通透性,能在内质网中特异性识别并结合突变蛋白,校正并稳定其正确构象,协助其运输到正确位点,直接恢复突变蛋白功能,可治疗各种南蛋白质折叠运输缺陷导致的内分泌及代谢疾病．目前已报道的由药物分子伴侣恢复功能的突变蛋白主要为质膜蛋白及细胞器蛋白,如ATP结合盒转运蛋白、G-蛋白耦联受体及溶酶体酶等．大量的细胞及动物实验结果显示了药物分子伴侣的临床应用前景广阔,目前已有一例临床实验获得了成功．     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

The conformational effects of N-glycosylation on the tailpiece from serum IgM

1H-NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22-residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n = 6, 45% having n = 8 and 10% having n = 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen-exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N-glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N-linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.     

Molecular cloning, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.     

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

Oligosaccharides in molecular recognition

The development, organization and growth of complex organisms as well as their interactions with the environment involve an intricate array of molecular recognition events. There is an increased awareness of the involvement of oligosaccharides in many of these processes. In this article, studies of oligosaccharide antigenicity, and the way these have been interpreted with respect to oligosaccharide function will be discussed. In addition, examples of oligosaccharides as receptor, first, as receptors and determinants of susceptibility to an exogenous infective agent and secondly, as recognition structures possibly involved in endogenous interactions, will be described. This will be followed by a discussion of the recent hypothesis in which oligosaccharides are envisaged as recognition structures and integral components of cell growth-regulating networks. Finally, an outline of new strategies for decoding the information content in glycoprotein oligosaccharides will be given.