This review shows introduction of glycoclusters using carbosilanes as core scaffolds, preparations of glycoclusters and their excellent properties as well as functions.Since a dendrimer has unique advantages such as single molecular weight, regularity of structure and easy control of shape and size, dendrimers are utilized in various research areas.Results of syntheses and biological evaluations of the carbosilane dendrimers having carbohydrate moieties for influenza viruses are presented.
The protein structures of most mammalian sialyltransferases have yet to be elucidated. Practical and convenient protein expression systems for soluble and active sialyltransferases will facilitate elucidation of the protein structures and catalytic mechanisms of these enzymes. The present study was performed to establish an efficient expression system for human ST6Gal I (hST6Gal I). cDNA encoding a soluble form of hST6Gal I was introduced into the bacterial expression vector pCold I carrying the cold shock promoter that is inducible by low-temperature conditions. The resultant DNA en- codes the enzyme fused in frame with a maltose-binding protein (MBP) as a purification tag. This expression plasmid was introduced into the E. coli strain pGro7/BL21 harboring the molecular chaperones GroES and GroEL. Combined use of chaperone proteins and low-temperature cultivation during IPTG induction significantly improved the functional enzyme solubility in bacteria. The MBP-tagged hST6Gal I was efficiently purified by affinity chromatography using amylose- conjugated agarose.
Glycans exhibit enormous structural diversity in nature and are of particular importance for self-cell survival and are often targeted by microbes. In this study, N-glycans (374.9 pmol/mg in dry delipidated weight) were enzymatical- ly released from bovine milk whey protein concentrate, and they were isolated and analyzed by a two-dimensional HPLC mapping technique and/or by MALDI-TOF mass spectrometry. A total of 39 identified N-glycans are bi- and tri-antennary sugar chains terminated with multiple mannose residues (Man-Man; molar ratio of 39.5%), N-acetyl-lactosamine (Lac- NAc; Gal1-4GlcNAc; molar ratio of 17.9), di-N-acetylated lactosamine (LacdiNAc; GalNAc1-4GlcNAc; molar ratio of 22.8), GlcNAc (molar ratio of 7.05), Neu5Ac2-6Gal1-4GlcNAc (molar ratio of 5.3), Neu5Ac2-6GalNAc1-4GlcNAc (molar ratio of 1.25), Neu5Gc2-6Gal1-4GlcNAc (molar ratio of 2.5), and Neu5Gc2-3Gal1-4GlcNAc (molar ratio of 0.25), in which some are fucosylated on the proximal core GlcNAc1-N-Asn. Terminal Neu5Ac2-3Gal/GalNAc and Neu5Gc2-3/2-6GalNAc were not detected in the bovine whey protein concentrate. Among the 39 glycans, GalNAcβ1- 4GlcNAcβ1-2Manα1-3(GalNAcβ1-4GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ- and Manα1-2Manα1-2Manα 1-3(Manα1-2Manα1-3(Manα1-2Manα1-6)Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ- were the most abundant types found with molar ratios of 11.3 and 10, respectively. Elucidation of glycan molecular structures will lead to an understanding of their biological roles and functions. Whey contains a variety of glycans and is inexpensive, and it is thus considered to be source of glycans for array glycan libraries to be used for investigations of specific glycan-protein interactions, enabling not only analysis of biological roles of the glycan-binding proteins but also development of molecules affecting these in- teractions. Furthermore, these natural glycans may have therapeutic value in prevention and inhibition of infection of mi- crobes that recognize them.
Sugar-lectin interactions play an important role in viral infections. Many viruses, such as human immunodefi- ciency virus (HIV), Ebola, dengue, cytomegalovirus, and hepatitis C, possess glycans that recognize C-type lectins, espe- cially CD209 (DC-SIGN), for infection. Other viruses possess lectins on their surfaces that recognize glycan epitopes on human epithelial cells for infection. Human and avian influenza viruses recognize different glycan epitopes, sialic acid- 2,6 galactose (SA-2,6Gal) and SA-2,3Gal, respectively, as their receptors, resulting in different host ranges for these two viruses. We and others have shown that sialogalactosides and fucosyllactoses are receptors for enterovirus 71 and no- rovirus infections, respectively; human milk oligosaccharides (HMOs) could block enterovirus 71 and norovirus infec- tions. Several lines of evidence also suggest that HMOs cannot only mimic viral receptors and block viral infections, but also raise immune responses through sugar/lectin (galactosides/galactins and sialylglycans/Siglecs) interactions and im- prove gut ecology by nurturing intestinal cells and/or intestinal microbiota. This review article summarizes how and why HMOs directly or indirectly protect humans from viral infections.
Glycosylation of the surface glycoproteins of influenza A virus is associated with several viral properties such as receptor binding and susceptibility to neuraminidase inhibitors.In this study, we evaluated the detailed structures of N-glycans derived from the same influenza virus strain A/Memphis/1/71 (H3N2) grown in different host cells, i.e., Madin-Darby canine kidney (MDCK) cells and embryonated eggs.Although both influenza virus isolates expressed neutral and sulfated oligosaccharides, their detailed profiles were significantly different.In contrast, N-glycosylation profiles of the influenza virus isolate from MDCK cells were highly homologous with those of desialylated N-glycans derived from its host cells.These data demonstrate that the glycosylation of influenza viruses is governed by their host cells.
The present mini-review aims first at an introduction to two thermodynamic essentials of the binding between the influenza A virus hemagglutinin (HA) and the cell surface receptor sialoside, (1) the equilibrium 1:1 binding of the HA with the sialoside, (2) the polyvalent effect of the HA binding to the polyvalent sialoside.Second, the review introduces the fragment molecular orbital (FMO) studies of the HA-sialoside (1:1) complexes.The recent FMO method with the polarizable continuum model as one of the residue-based energy analysis method has revealed the role of key amino acid residue on the selective HA subtype H3 binding to the sialosides.
Glycosylated derivatives of physiologically active natural compound juglone and related 1,4- naphthoquinones are known as antifungal, immunomodulatory, and antitumor substances. However, their antileukemic properties and struc- ture-activity relationships have been studied insufficiently. Antileukemic effects and structure-activity relationships (SAR) of the 50 1,4- naphthoquinone derivatives were examined using HL-60 human promyelocytic leukemia cells and MTS method of the study of cell viability. As was shown, the substances inhibited viability of HL-60 cells at the wide range of concentrations. SAR study revealed the structure peculiarities which lead to increase or decrease of the antileu- kemic activity of the compounds studied. In conclusion, O- or S- glycosylated derivatives of juglone and related 1,4- naphthoquinones have potential for development of the new antileukemic agents and should be further investigated.
Glucansto a group of biologically active natural compounds called biological response modifiers. These substances represent highly conserved structural components of cell walls in yeast, fungi, grain and seaweed. The effects of � -glucan on immune reactions are well established however direct comparisons of the biological activities of several individual glucans are extremely rare. As this paper will show, we tested sixteen different glucans and evaluated the possibility whether individual glucans will be similarly active against each of the tested biological properties or if each glucan will affect different reactions. No direct connection between source and immunological activities was found. Based on our results, we can conclude that highly purified and highly active glucans have pleotropic effects, whereas poorly iso- lated glucans have only average (if any) biological effect.
A method was developed to isolate mono-and disaccharide-free oligosaccharides from human and bovine milk using a combination of enzymatic digestion of lactose and solid-phase extraction.In the initial trial, 2.5 g of oligosaccharides were isolated from one liter of human milk.In subsequent trials this was increased to over 5 g of oligosaccharides per liter.Compared to filtration-based extraction methods, this procedure allows for further isolation of oligosaccharide fractions via modulation of the column rinsing solvent.Neutral monosaccharide composition of the oligosaccharide polymers was investigated using gas chromatographic analysis of the monosaccharides as alditol acetate derivatives.Results indicate oligosaccharides are approximately made up of 24% fucose, 41% galactose, 22% glucose and 13% glucosamine.Isolated bovine and human milk oligosaccharides were compared to lactose as fermentation substrates for Bifidobacterium longum biovar infantis.Lactose fermentation yielded the greatest production of biomass followed by bovine and human milk oligosaccharides.
The role of advanced glycation end products (AGEs)-damaged immunoglobulin G (AGE-IgG) in type 1 diabetes has been investigated in the present study.IgG was isolated from the normal humans and was subjected to in vitro glycation with glucose.The AGEs caused extensive damaged to IgG.The AGE-IgG was found to be highly immunogenic in rabbits as compared to native IgG.The binding characteristics of circulating autoantibodies in type 1 diabetes mellitus (DM) patients against native and AGE-IgG were assessed.Type 1 DM patients (n=31) were examined by ELISA and their results were compared with healthy age-matched human controls (n=22).High degree of specific binding by 61.3 % of DM sera autoantibodies towards AGE-IgG was observed, in comparison to its native analog (p< 0.05).Sera from those type 1 DM patients having smoking history, high aging with high degree of disease showed substantially stronger binding to AGE-IgG over native IgG in particular.IgG from type 1 DM patients (DM-IgG) contained higher levels of carbonyls as compared to normal human subjects (normal-IgG) (p<0.001).Collectively, the AGEs modification of IgG causes perturbations, resulting in the generation of neo-epitopes, and making it a potential immunogen.The IgG modified with AGEs may be one of the factors for the induction of circulating type 1 diabetes autoantibodies.
The use of natural gums, taken from the exudates and extracts of plants, has been given a strong impulse due to both the many and lucrative possibilities for industrialization and to the excellent international market. A gum or resin of a yellowish color, soluble in water, and which presents a great potential for industrialization, appears on the trunk and branches of the cashew tree; that is known mainly for its nuts that are used as a food ingredient. Cashew gum interacts with water and it has emulsifier, adhesive and stabilizer properties, occurring in the form of pale yellow to reddish am- bers. In cold water, it swells into a jelly like mass but dissolves rapidly when heated. The resin/gum secretory ducts that appear along the cashew tree bark were studied using histological procedures and microscopic observations. The gum was studied at its crude state and then chemically treated in order to obtain a kind of gel (microbiofilm).
Four enzyme preparations produced by fungal species belonging to different genera (Aspergillus niger, Corynascus sp., Penicillium verruculosum, Trichoderma reesei) were used for synthesis of disaccharides by D-glucose (60% w/v) condensation catalyzed by -glucosidase.Effects of pH and temperature on the disaccharide synthesis were studied, and glycoside linkage patterns for enzymes from different sources were determined.The highest concentration of disaccharides (114 and 118 g/l) was achieved in the case of A. niger and Corynascus sp. enzymes after 48 h of the condensation reaction carried out at 70 o C and optimal pH; the P. verruculosum sample slightly conceded them in the yield of products (96 g/l), while the T. reesei preparation displayed the lowest synthetic activity (35 g/l).Gentiobiose was predominantly formed in the reaction catalyzed by the first three enzyme samples, while in the case of T. reesei laminaribiose was the main condensation product.
A fructan named ALF was isolated from the roots of Arctium lappa L.. The structure of ALF was identified by analyses of methylation, GC-MS, and both 1 H and 13 C NMR spectroscopy.The results obtained indicated that ALF, comprised of D-fructose and D-glucose in the molar ratio of 14:1, was an inulin-type fructan, which was confirmed by the composition of 14 fructose residues linked by (2 1) glycosidic bond and 1 glucose residue linked by (1 2) glycosidic bond at the end of linear straight sugar chain.
Highly pathogenic and potentially pandemic H5N1 avian influenza A viruses have become endemic and are now residing in Asia, Europe, Africa, and the Middle East.H5N1 viruses have been shown to cross the species barrier and infect both dogs and cats.Domestic cats and dogs in Thailand, which were naturally infected with H5N1, exhibited severe pulmonary edema and peumonia in lung tissue as well as in other tissue dysfunctions.In order to understand the structure and quantity of influenza A receptor sialyl sugar chains in cats and dogs, especially in lung tissue, glycosylation profiles of N-glycans were determined from lung tissues of dogs and cats susceptible to H5N1 in Thailand by using multidimensional HPLC mapping combined with mass spectrometry.The results demonstrated different N-linked glycans composition ratios between dogs and cats.There were a total of 30 kinds of N-linked glycans from cat lungs, which were comprised of 11 neutral, 13 mono-, 3 di-, and 3 tri-sialyl sugar chains, and 29 kinds from dog lungs, which were comprised of 16 neutral, 11 mono-and 2 di-sialyl sugar chains.Cat lungs exhibited both 5-N-acetylneuraminic acid and 5-Nglycolylneuraminic acid sialic acid (Sia 2-3Gal and Sia 2-6Gal), but dog lungs contained only 5-N-acetylneuraminic (Sia 2-3Gal and Sia 2-6Gal) molecular species.The composition ratios of molar percentage of Sia 2-3Gal for domestic cat and dog lungs were 21.5 and 9.9, respectively, while the composition ratios of Sia 2-6Gal were 47.1 and 59.2, respectively.These results may indicate that domestic cats are more susceptible than dogs to H5N1 influenza virus infection and also cats and dogs play an important role as "mixing vessels" for the virus re-assortment.
Soluble intercellular adhesion molecule-1 (sICAM-1) is elevated in the cerebrospinal fluid of patients with severe brain trauma and mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes.The production of MIP-2 is greatly enhanced when sICAM-1 contains sialylated complex-type N-glycans (sICAM-1-CT) as produced by Chinese hamster ovary (CHO) cells.By contrast, sICAM-1 from the Lec1 mutant of CHO cells (sICAM-1-HM), containing only high mannose-type N-glycans, is relatively inactive.Here we show that the Nglycans of sICAM-1-CT are mostly 2,3-sialylated bi-, tri-, and tetraantennary complex-type structures with varying amounts of core fucosylation.Unexpectedly, sICAM-1-CT and sICAM-1-HM bound equivalently to mouse astrocytes.Enhanced MIP-2 induction by sICAM-1-CT was associated with a more rapid, higher level, and prolonged MIP-2 response as well as sICAM-1-CT accumulation at the plasma membranes of mouse astrocytes.These results show that glycosylation of sICAM-1 contributes to its signaling properties at the astrocyte cell surface, and suggest that altered glycosylation which might arise as a result of inflammation could regulate the bioactivity of sICAM-1.
The fully solid-phase synthesis of chemically well-defined glycoclusters grafted to a topological cyclodecapep- tide template is described. The orthogonally protected peptide backbone was first synthesized and cyclized on solid sup- port using D-glutamic acid as first amino acid linked to the resin. After successive regioselective deprotection steps, bi- otins were coupled to the lower addressable domains of the scaffold, then carbohydrates-binding ligands were assembled as cluster on the upper domain using a chemoselective oxime-based strategy. This provides multitopic labeled glycopep- tides which can be easily immobilized to streptavidin-coated surfaces for studying carbohydrate-protein interactions in glycomic researches.
Although the multi-dimensional HPLC maps of neutral, sialyl, and sulfated N-glycans have been reported and widely used for glycosylation profiling, those of glucuronyl oligosaccharides have not yet been available.In the present study, by in vitro enzymatic reactions, we prepared 55 different glucuronyl PA-oligosaccharides that include 6 kinds of HNK-1-containing N-glycans, and established their HPLC map.Furthermore, we applied this map to the characterization of branch specificity in glucuronylation reaction catalyzed by human GlcAT-S, revealing that this enzyme transfers the glucuronyl residues preferentially onto the Gal 1 4GlcNAc 1 4Man 1 3 and Gal 1 4GlcNAc 1 2Man 1 3 branches of a galactose-terminated tri-antennary oligosaccharide.The HPLC map developed in the present study will be a useful glycomics tool for identification and profiling of glucuronyl N-glycans expressed in the neural and other biological systems.
Epithelial mucin glycoproteins of bladder act as an effective barrier against invasion by pathogenic microorganisms and injury by toxic substances in urine.Although these glycoconjugates play important roles in the pathophysiology of bladder disorders such as intestinal cystisis, cancer, and urinary tract infections, they have not been characterized in detail either in humans or in animals.Rabbits could be useful for developing models for studying bladder disorders.In this study, we purified and partially characterized two major high molecular weight rabbit bladder mucin glycoproteins, designated RBM 1 and RBM 2 , found in urine.Consistent with their mucin characteristics, amino acid compositions showed have high levels of serine, glutamic acid, proline, glycine and alanine, which together comprise 34% and 42% of the total amino acids in RBM 1 and RBM 2 , respectively.Carbohydrate compositional analysis indicated that RBM 1 and RBM 2 consist of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), N-acetylneuraminic acid (NeuAc) and fucose (Fuc) in the molar ratio of 1.0: 0.82: 0.12: 0.30: 0.02 and 1.0: 1.03: 0.46: 0.16: 0.05, respectively; mannose (Man) was not detected in either mucin.Both mucin fractions were strongly reactive to wheat germ agglutinin, but not to Ca2 antibody specific to a human tumor mucin antigen (asialylated carbohydrate linked to protein core), suggesting that most of the galactosyl residues of oligosaccharides are sialylated.Together, the data suggest that rabbit mucin glycoproteins characterized here are distinctively different from MUC1 mucin glycoprotein found in human urine.
Laminaribiose, which is the simplest -(1,3)-glucan, is one of the most powerful agents able to increase germi- nation. Its chemical synthesis was revised in detail starting from peracylated donors and easily available glucofuranose protected by acetal groups in the presence of appropriated catalyst and/or promoter. We particularly focused our attention on the nature of the Lewis acid generally required in glycosidic couplings. Finally, an interesting scale-up was performed which allowed us to prepare laminaribiose on a kilogram scale.
