Glycobiology - Advance Access

{alpha}1,4GlcNAc-capped mucin-type O-glycan inhibits cholesterol {alpha}-glucosyltransferase from Helicobacter pylori and suppresses H. pylori growth

2008-05-05

Helicobacter pylori infects over half the world's population and is thought to be a leading cause of gastric ulcer, gastric carcinoma, and gastric malignant lymphoma of mucosa-associated lymphoid tissue type. Previously, we reported that a gland mucin (MUC6) present in the lower portion of the gastric mucosa containing 1,4GlcNAc-capped core 2-branched O-glycans suppresses H. pylori growth by inhibiting synthesis of -glucosyl cholesterol, a major constituent of the H. pylori cell wall (Kawakubo et al. 2004 Science 305:1003–1006). Therefore, we cloned the genomic DNA encoding cholesterol -glucosyltransferase (HP0421), and expressed its soluble form in Escherichia coli. Using this soluble HP0421, we show herein that HP0421 sequentially acts on UDP-Glc and cholesterol in an ordered Bi-Bi manner. We found that competitive inhibition of HP0421 by 1,4GlcNAc-capped core 2-branched O-glycan is much more efficient than non-competitive inhibition by newly synthesized -glucosyl cholesterol. Utilizing synthetic oligosaccharides, -glucosyl cholesterol and monosaccharides, we found that 1,4GlcNAc-capped core 2-branched O-glycan most efficiently inhibits H. pylori growth. These findings together indicate that 1,4GlcNAc-capped O-glycans suppress H. pylori growth by inhibiting HP0421, and that 1,4GlcNAc-capped core 2 O-glycans may be useful to treat patients infected with H. pylori.

Glycation does not modify bovine serum albumin (BSA)-induced reduction of rat aortic relaxation. The response to glycated and non-glycated BSA is lost in metabolic syndrome

2008-05-05

The effects of non-glycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37°C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six months old animals were used. Blood pressure, insulin, triglycerides and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of non-glycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Non-glycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using non-glycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.

Analysis of the N-glycans of Recombinant Human Factor IX Purified from Transgenic Pig Milk

Gil, G.-C., Velander, W. H., Cott, K. E. V. — 2008-05-02

Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of tg-FIX are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. Tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Gal(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

Adjuvant potential of archaeal synthetic glycolipid mimetics critically depend on glyco head group structure

Sprott, G.D., Dicaire, C.J., Cote, J.-P., Whitfield, D.M. — 2008-05-01

Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide-archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8⁺ T cell activity, with highest responses to antigen entrapped in archaeosomes containing disaccharides of glucose in β or 1–6 linkage (β-gentiobiose, β-isomaltose), or of β-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive and potent glyco-adjuvant.

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus

2008-05-01

Edible fungi, mushrooms, are a popular food in Japan and over fifteen cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (Basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Man1–2Ins1-[PO₄]-Cer and extension of Man, Gal and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO₄]-Cer (AGL0), Man1–2Ins1-[PO₄]-Cer (AGL1), Galβ1–6Man1–2Ins1-[PO₄]-Cer (AGL2), Fuc1–2Galβ1–6Man1–2Ins1-[PO₄]-Cer (AGL3), Gal1–3(Fuc1–2)Galβ1–6Man1–2Ins1-[PO₄]-Cer (AGL4), Gal1–2Gal1–3(Fuc1–2)Galβ1–6Man1–2Ins1-[PO₄]-Cer (AGL5) and Gal1–2Gal1–2Gal1–3(Fuc1–2)Galβ1–6Man1–2Ins1-[PO₄]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatograph-mass spectrometry (GC-MS), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ¹H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids.

By immunochemical detection, the terminal structure of AGL4, Gal1–3(Fuc1–2)Galβ-, was shown to have blood group type B activity. Gal1–2 and its repeating sequence in AGL5 and AGL6 are novel structures on the non-reducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

Comparative Structural Analyses of the {alpha}-Glucan and Glycogen from Mycobacterium bovis

Dinadayala, P., Sambou, T., Daffe, M., Lemassu, A. — 2008-04-24

Pathogenic mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis, are surrounded by a non-covalently bound capsule, whose major carbohydrate constituent is a glycogen-like -glucan. In the present study we compared the structures of the extracellular polysaccharide to that of the ubiquitous intracellular glycogen. The -glucan was isolated from the culture medium of Mycobacterium bovis BCG, the vaccine strain, in which it is released whereas the intracellular glycogen was obtained after disruption of cells. The two purified polysaccharides were eluted from permeation gel at a similar position but glycogen was less soluble and gave a more opalescent solution in water than -glucan. Combination of gas chromatography-mass spectrometry analysis of partially O-methylated partially O-acetylated alditols and NMR analysis confirmed that both polysaccharides were composed of ->4--D-Glcp-1-> core, substituted at some 6 positions with short chains. Degradation of polysaccharides with pullulanase, followed by mass spectrometry analysis of the resulting products also showed that the lengths of the branched chains do not differ from one another polysaccharide. Interestingly, application of analytical ultracentrifugation and dynamic light scattering to the mycobacterial -glucan and glycogen and their enzymatic degradative products indicated that the -glucan possessed a higher molecular mass and was more compact than the glycogen from the same species, allowing the formulation of working structural models for the two polysaccharides. Consistent with the models, the -glucan was found to be less accessible to pullulanase, a debranching enzyme, than glycogens.

Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants

2008-04-09

Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le^b) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le^b positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le^a, Le^b, Le^x and Le^y) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of Helicobacter pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le^a and Le^b positive. 1C5 expressed Le^b on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le^b on N-, O-glycans and GLs. Furthermore, both clones expressed Le^a on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le^y on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le^x on GLs. A Le^b-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le^b-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.

Structural Glycobiology: A Game of Snakes and Ladders

DeMarco, M. L., Woods, R. J. — 2008-04-04

Oligo- and polysaccharides are infamous for being extremely flexible molecules, populating a series of well-defined rotational isomeric states under physiological conditions. Characterization of this heterogeneous conformational ensemble has been a major obstacle impeding high-resolution structure determination of carbohydrates and acting as a bottleneck in the effort to understand the relationship between carbohydrate structure and function. This challenge has compelled the field to develop and apply theoretical and experimental methods that can explore conformational ensembles by both capturing and deconvoluting the structural and dynamic properties of carbohydrates. This review focuses on computational approaches that have been successfully used in combination with experiment to detail the three-dimensional structure of carbohydrates in solution and in complex with proteins. In addition, emerging experimental techniques for three-dimensional structural characterization of carbohydrate—protein complexes and future challenges in the field of structural glycobiology are discussed. The review is divided into to five sections: (1) The Complexity and Plasticity of Carbohydrates, (2) Predicting Carbohydrate—Protein Interactions, (3) Calculating Relative and Absolute Binding Free Energies for Carbohydrate—Protein Complexes, (4) Emerging and Evolving Techniques for Experimental Characterization of Carbohydrate—Protein Structures, and (5) Current Challenges in Structural Glycoscience.

Egg box conformation of oligogalacturonides. The time-dependent stabilization of the elicitor-active conformation increases its biological activity

Cabrera, J. C., Boland, A., Messiaen, J., Cambier, P., Van Cutsem, P. — 2008-04-01

Circular dichroism spectrometry was used on oligogalacturonides (OGA) and showed the existence of a calcium/sodium induced conformational state that is intermediate between single isolated chains and calcium-associated multimer chains. This conformation is interpreted as being egg box dimers. Using the 2F4 monoclonal antibody that specifically binds such an egg box dimer conformation of pectin, the stability of OGA dimers was investigated over a period of several days. The extent to which egg box dimers were recognized by the antibody was dependent on temperature and duration of pre-incubation of the OGA. This suggests a "maturation" process of the egg-box structure that consists in a progressive increase in length of the junction sequences between two chains that slide along each other in order to form a maximum number of calcium bridges and dimer ends. The maturation of egg boxes induced both a significant increase in their binding to Wall Associated Kinase 1 (WAK1) and an increased extracellular alkalinization when applied to Arabidopsis thaliana cell suspensions. Chemical modification of the reducing end of the oligogalacturonides largely diminished their elicitating activity but did not hinder neither dimerization nor binding of these end-reduced egg boxes to WAK1. We conclude that there are at least two different perception systems for egg box dimers. One binds egg box junctions and the other one binds egg box ends. The relevance of these results is discussed in terms of pectic signal perception and plant-pathogen interaction.

A Simplified and Sensitive Fluorescent Method for Disaccharide Analysis of both Heparan Sulphate and Chondroitin / Dermatan Sulphates from Biological Samples

Deakin, J. A., Lyon, M. — 2008-03-31

Sulphated glycosaminoglycans regulate the biological functions of a wide variety of proteins, primarily through high affinity interactions mediated by specific sugar sequences or patterns / densities of sulphation. Disaccharide analysis of such glycosaminoglycans yields important diagnostic and comparative structural information on sulphate patterning. When applied to specific oligosaccharides it can also make a vital contribution to sequence elucidation. Standard UV detection of lyase-generated disaccharides resolved by HPLC can lack sufficient sensitivity, and be compromised by contaminating UV signals, when dealing with scarce tissue- or cell culture-derived material. Various methods exist for improved detection, but usually involve additional HPLC hardware and often necessitate different procedures for analysing different glycosaminoglycans. We describe a simple procedure, requiring only standard HPLC instrumentation, involving pre-derivatisation of disaccharides with 2-amino-acridone with no clean-up of samples, followed by separation by reverse-phase HPLC, that is sensitive to as little as ~100pg (~10^–13 mol) of individual disaccharide, thereby allowing analyses of >10ng of total glycosaminoglycan. Importantly, separate analysis of both HS/heparin and CS/DS species within a mixed GAG pool can be performed using the same procedure on a single column. We demonstrate its applicability in dealing with small quantities of material derived from rat liver (where we demonstrate a high abundance of the unusual CS-E species within the CS/DS pool), and from MDCK cells (which revealed a HS species of relatively low N-sulphation, but high O- sulphation). This simplified method should find widespread utility for analysing glycosaminoglycans from limited animal and cell culture samples.

Analyzing the functions of large glycoconjugates through the dissipative properties of their absorbed layers using the gel-forming mucin MUC5B as an example

Kesimer, M., Sheehan, J. K. — 2008-03-13

Glyconjugates such as mucins, proteoglycans and polysaccharides form the structural basis of protective cell-surface layers. In particular gel-forming mucins define a zone between the epithelial cell layer and the environment. Such molecules are of extreme molecular weight 5–100x10⁶ and size (Rg 20–300nm). On this account their biochemistry is inseparable from their physical biochemistry. Combining laser light scattering and quartz crystal mass balance with dissipation methods (QCM-D) we have investigated the properties of the MUC5B mucin and its cognate fragments when bound to a hydrophobic surface. MUC5B forms the basis of gels responsible for the protection of the oral cavity, lung and cervical canal surfaces. Here we show, by analyzing dissipative interactions of hydrophobic, gold and polystyrene surfaces, with the intact MUC5B molecule, its reduced subunits and glycosylated tryptic fragments (obtained after reduction) the formation of 40–100nm thick highly structured, hydrated interfaces. These interfaces are dominated in their geometry and dissipative properties by the negatively charged carbohydrate-rich domains of the molecule, the naked protein domains being responsible for attachment. These carbohydrate-rich surfaces have well defined absorptive properties and permit the entry and entrapment of albumin coated micro-beads into the absorbed layer at and below a size of 60nm. However beads larger than 100nm are completely excluded from the surfaces. These absorptive phenomena correlate with large changes in film dissipation and thus may not only be important in biological functions e.g. binding viruses but could well be informative to the surfaces (often ciliated) onto which such mucus films are attached.

A Unique Glycosyltransferase Involved in the Initial Assembly of Moraxella catarrhalis Lipooligosaccharides

2008-03-12

Moraxella catarrhalis express three predominant forms of lipooligosaccharide (LOS) molecules on the bacterial surface. These major glycolipids contain specific carbohydrate epitopes that distinguish each glycoform into serotype A, B, or C LOS. All three serotypes, however, share a common glucose containing inner-core structure, consisting of an -glucose attached to 2-keto-3-deoxyoctulosonic acid (KDO), which is unique among Gram-negative bacteria. Many of the LOS glycosyltransferase genes (lgt) responsible for assembly of the extended M. catarrhalis LOS structure have been identified. In this report, we now describe the identification and characterization of Lgt6, a unique glycosyltransferase that is responsible for the addition of the first glucose to the inner core thus initiating the assembly of full length LOS. Isogenic mutants defective in the expression of lgt6 were constructed in all three M. catarrhalis LOS serotypes and the resulting LOS glycoforms consisted of KDO₂-lipidA-OH as analyzed by urea SDS-PAGE and mass spectrometry. In addition, the expression of lgt6 in trans in a heptose deficient Neisseria meningitidis NMB gmhX mutant resulted in the addition of a hexose to the LOS of this strain. These studies demonstrate that Lgt6 functions as an –(1–5)-glucosyltransferase in M. catarrhalis adding the primary glucose to the KDO₂-lipidA-OH in LOS biosynthesis. The function of Lgt6 is required for the completion of both the major and minor oligosaccharide chains in M. catarrhalis.

The O-linked glycosylation of secretory/shed MUC1 from advanced breast cancer patient serum

2008-03-10

MUC1 is a high molecular weight glycoprotein that is over-expressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and by high performance liquid chromatography (HPLC) coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or tri-sialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Gal1–3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid 2--8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient derived material.

PSGL-1 from the Murine Leukocytic Cell Line WEHI-3 is Enriched for Core 2-Based O-glycans with Sialyl Lewis x Antigen

Kawar, Z. S., Johnson, T. K., Natunen, S., Lowe, J. B., Cummings, R. D. — 2008-02-29

Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x antigen (SLe^x). However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contribute to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe^x, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with ³H-monosaccharide precursors to detect low abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe^x antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing SLe^x antigen.

The "Glyconutrient Sham"

2007-10-11

Glycobiology Advance Access originally published on September 13, 2007. Subsequent version published September 14, 2007.

doi:10.1093/glycob/cwm098

The above article has been temporarily taken down from the Glycobiology Advance Access site at the Editor-in-Chief's request and with the authors' consent. It is currently undergoing further review and will be republished along with other perspectives on this topic in due course.

The Editor-in-Chief of Glycobiology, and the publisher, Oxford Journals, a division of Oxford University Press, sincerely apologize for any inconvenience caused.