Glycobiology - recent issues

Glycobiology

2009-07-01

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2009-07-01

Meeting Announcements

2009-07-01

Natural ligands for CD33-related Siglecs?

Varki, A. — 2009-07-01

For intra-articular delivery of chondroitin sulfate

David-Raoudi, M, Mendichi, R, Pujol, J P — 2009-07-01

Protein O-mannosylation: Conserved from bacteria to humans

Lommel, M., Strahl, S. — 2009-07-01

Protein O-mannosylation is an essential modification in fungi and animals. Different from most other types of O-glycosylation, protein O-mannosylation is initiated in the endoplasmic reticulum by the transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine residues of secretory proteins. In recent years, it has emerged that even bacteria are capable of O-mannosylation and that the biosynthetic pathway of O-mannosyl glycans is conserved between pro- and eukaryotes. In this review, we summarize the observations that have opened up the field and highlight characteristics of O-mannosylation in the different domains/kingdoms of life.

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum

Condac, E., Dale, G. L, Bender-Neal, D., Ferencz, B., Towner, R., Hinsdale, M. E — 2009-07-01

Circulating glycosyltransferases including xylosyltransferases I (XylT1) and II (XylT2) are potential serum biomarkers for various diseases. Understanding what influences the serum activity of these enzymes as well as the sources of these enzymes is important to interpreting the significance of alterations in enzyme activity during disease. This article demonstrates that in the mouse and human the predominant XylT in serum is XylT2. Furthermore, that total XylT levels in human serum are approximately 200% higher than those in plasma due in part to XylT released by platelets during blood clotting in vitro. In addition, the data from Xylt2 knock-out mice and mice with liver neoplasia show that liver is a significant source of serum XylT2 activity. The data presented suggest that serum XylT levels may be an informative biomarker in patients who suffer from diseases affecting platelet and/or liver homeostasis.

Genetic analysis of glucosidase II {beta}-subunit in trimming of high-mannose-type glycans

Watanabe, T., Totani, K., Matsuo, I., Maruyama, J.-i., Kitamoto, K., Ito, Y. — 2009-07-01

Glucosidase II (G-II) is a glycoprotein-processing enzyme that successively cleaves two 1,3-linked glucose residues from N-linked oligosaccharides in the endoplasmic reticulum. G-II is a heterodimer whose -subunit contains a glycosidase active site, but the function(s) of the β-subunit remain poorly defined. We report here an in vivo enzymatic analysis using gene disruptants lacking either the G-II - or β-subunit in the filamentous fungus Aspergillus oryzae. Using synthetic oligosaccharides as probes, G-II activity of the membranous fraction of the gene disruptants was investigated. The fraction lacking the β-subunit retained hydrolytic activity toward p-nitrophenyl -d-glucopyranoside but was inactive toward both Glc₂Man₉GlcNAc₂ and Glc₁Man₉GlcNAc₂. When the fraction containing the β-subunit was added to the one including the -subunit, the glucosidase activity was restored. These results suggested that the β-subunit confers the substrate specificity toward di- and monoglucosylated glycans on the glucose-trimming activity of the -subunit.

Deletion polymorphism of SIGLEC14 and its functional implications

Yamanaka, M., Kato, Y., Angata, T., Narimatsu, H. — 2009-07-01

Human Siglec-14, a member of the Siglec family of sialic acid-binding lectins, shows extensive sequence similarity to human Siglec-5. To analyze respective expression patterns of Siglec-14 and Siglec-5, we developed specific antibodies against each of them. We found that the former was expressed on granulocytes and monocytes, while the latter was on granulocytes and B-cells. Surprisingly, some individuals lacked the expression of Siglec-14, while they all expressed Siglec-5. We found that a fusion between SIGLEC14 and SIGLEC5 genes, resulting in the functional deletion of SIGLEC14, underlies this phenotype. The presence of the "SIGLEC14 null" allele in all human populations we tested implies an ancient origin, while its allelic frequency is higher in Asians compared with Africans and Europeans. The forced expression of Siglec-14 in a monocytic cell line-enhanced TNF- secretion elicited by lipopolysaccharide. These results imply that Siglec-14 may play some role in bacterial infection.

Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes

Nystrom, K., Norden, R., Muylaert, I., Elias, P., Larson, G., Olofsson, S. — 2009-07-01

We have previously shown that varicella-zoster virus (VZV) and cytomegalovirus (CMV) infection of diploid human fibroblasts (HEL) results in neo-expression of Lewis antigens sialyl Lewis x (sLe^x) and Lewis y (Le^y), respectively, after transcriptional activation of different combinations of dormant human fucosyltransferase genes (FUT1, FUT3, FUT5, and FUT6), whose gene products are responsible for the synthesis of Le antigens. Here, we show that herpes simplex virus type 1 (HSV-1) also induces sLe^x expression dependent on induction of FUT3, FUT5, and FUT6 transcription in infected cells. HSV-1 induction of FUT5 was subsequently used as a model system for analyzing the mechanism of viral activation of dormant fucosyltransferase genes. We show that this is a rapid process, which gives rise to elevated FUT5 RNA levels already at 90 min postinfection. Augmented FUT5 transcription was found to be dependent on transcription of viral genes, but not dependent on the immediate early proteins ICP0 and ICP4, as demonstrated by experiments with HSV-1 mutants defective in expression of these genes. Augmented FUT5 transcription takes place in cycloheximide-treated HSV-1-infected cells, suggesting a more direct role for IE viral RNA during activation of cellular FUT5.

Sulfated polysaccharides from marine sponges (Porifera): an ancestor cell-cell adhesion event based on the carbohydrate-carbohydrate interaction

Vilanova, E., Coutinho, C. C, Mourao, P. A S — 2009-07-01

Marine sponges (Porifera) are ancient and simple eumetazoans. They constitute key organisms in the evolution from unicellular to multicellular animals. We now demonstrated that pure sulfated polysaccharides from marine sponges are responsible for the species-specific cell–cell interaction in these invertebrates. This conclusion was based on the following observations: (1) each species of marine sponge has a single population of sulfated polysaccharide, which differ among the species in their sugar composition and sulfate content; (2) sulfated polysaccharides from sponge interact with each other in a species-specific way, as indicated by an affinity chromatography assay, and this interaction requires calcium; (3) homologous, but not heterologous, sulfated polysaccharide inhibits aggregation of dissociated sponge cells; (4) we also observed a parallel between synthesis of the sulfated polysaccharide and formation of large aggregates of sponge cells, known as primmorphs. Once aggregation reached a plateau, the demand for the de novo synthesis of sulfated polysaccharides ceased. Heparin can mimic the homologous sulfated polysaccharide on the in vitro interaction and also as an inhibitor of aggregation of the dissociated sponge cells. However, this observation is not relevant for the biology of the sponge since heparin is not found in the invertebrate. In conclusion, marine sponges display an ancestor event of cell–cell adhesion, based on the calcium-dependent carbohydrate–carbohydrate interaction.

Regulated expression of the HNK-1 carbohydrate is essential for medaka (Oryzias latipes) embryogenesis

Anzai, D., Tonoyama, Y., Ikeda, A., Kawasaki, T., Oka, S. — 2009-07-01

Carbohydrates are known to play essential roles in various biological processes including development. However, it remains largely unknown which carbohydrate structure takes part in each biological event. Here, we examined the roles of the human natural killer-1 (HNK-1) carbohydrate in medaka embryogenesis. We first cloned two medaka glucuronyltransferases, GlcAT-P and GlcAT-S, key enzymes for HNK-1 biosynthesis. Overexpression of these glucuronyltransferases affected morphogenetic processes. In addition, loss-of-function experiments revealed that GlcAT-P is physiologically indispensable for head morphogenesis and GlcAT-P depletion also led to markedly increased apoptosis. However, even when the apoptosis was blocked, abnormal head morphogenesis caused by GlcAT-P depletion was still observed, indicating that apoptosis was not the main cause of the abnormality. Moreover, in situ hybridization analyses indicated that GlcAT-P depletion resulted in the abnormal formation of the nervous system but not in cell specification. These results suggest that tight regulation of HNK-1 expression is essential for proper morphogenesis of medaka embryos.

Glycosylation-related gene expression profiling in the brain and spleen of scrapie-affected mouse

2009-07-01

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrP^C, into a pathogenic isoform, PrP^Sc. The molecular requirements for efficient PrP conversion remain unknown. Altered glycosylation has been linked to various pathologies and the N-glycans harbored by two prion protein isoforms are different. In order to search for glycosylation-related genes that could mark prion infection, we used a glycosylation-dedicated microarray that allowed the simultaneous analysis of the expression of 165 glycosylation-related genes encoding proteins of the glycosyltransferase, glycosidase, lectin, and sulfotransferase families to compare the gene expression profiles of normal and scrapie-infected mouse brain and spleen. Eight genes were found upregulated in "scrapie brain" at the final state of the disease. In the spleen, five genes presented a modified expression. Three genes were also upregulated in the spleen of infected mice, and two (Pigq and St3gal5) downregulated. All changes were confirmed by qPCR and biochemical analyses applied to Pigq and St3gal5 proteins.

Size-dependent regulation of Snail2 by hyaluronan: Its role in cellular invasion

Craig, E. A, Parker, P., Camenisch, T. D — 2009-07-01

Hyaluronan (HA) induces changes in cellular behavior that are crucial during both embryonic development and cancer progression. However, the biological effects of varying sizes of HA and the signal transduction mechanisms that these polymers may activate remain unclear. In this study, we demonstrate that pulse stimulation of mouse embryonic fibroblasts with high-molecular-weight (HMW) HA, but not HA of lower molecular sizes, leads to increases in Snail2 protein which are dependent on NFB activity. Involvement of CD44, the main HA receptor, in these responses was determined by use of a CD44 blocking antibody and CD44 siRNA. Both the blockade and silencing of CD44 significantly abrogate the increases in nuclear factor kappaB (NFB) activity and Snail2 protein following HMW-HA stimulation. Furthermore, we show that HMW-HA induces cellular invasion and that inhibition of CD44, Snail2, or NFB significantly decreases this response. These studies elucidate a novel HA/Snail2 functional connection through CD44 and NFB that is important for the induction of cellular invasion and is dependent on HA size.

Targeted glycoproteomic identification of cancer cell glycosylation

2009-07-01

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewis^x and Lewis^y structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewis^x antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Quality control of glycoproteins bearing truncated glycans in an ALG9-defective (CDG-IL) patient

2009-07-01

We describe an ALG9-defective (congenital disorders of glycosylation type IL) patient who is homozygous for the p.Y286C (c.860A>G) mutation. This patient presented with psychomotor retardation, axial hypotonia, epilepsy, failure to thrive, inverted nipples, hepatomegaly, and pericardial effusion. Due to the ALG9 deficiency, the cells of this patient accumulated the lipid-linked oligosaccharides Man₆GlcNAc₂-PP-dolichol and Man₈GlcNAc₂-PP-dolichol. It is known that the oligosaccharide structure has a profound effect on protein glycosylation. Therefore, we investigated the influence of these truncated oligosaccharide structures on the protein transfer efficiency, the quality control of newly synthesized glycoproteins, and the eventual degradation of the truncated glycoproteins formed in this patient. We demonstrated that lipid-linked Man₆GlcNAc₂ and Man₈GlcNAc₂ are transferred onto proteins with the same efficiency. In addition, glycoproteins bearing these Man₆GlcNAc₂ and Man₈GlcNAc₂ structures efficiently entered in the glucosylation/deglucosylation cycle of the quality control system to assist in protein folding. We also showed that in comparison with control cells, patient's cells degraded misfolded glycoproteins at an increasing rate. The Man₈GlcNAc₂ isomer C on the patient's glycoproteins was found to promote the degradation of misfolded glycoproteins.

Molecular analysis of a UDP-GlcNAc:polypeptide {alpha}-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi

2009-07-01

Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, -N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide -GlcNAc-transferase (pp-GlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[³H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[³H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that ³H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-GalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-GalNAcTs that initiate mucin-type O-glycosylation in animals.

Glycobiology

2009-05-29

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2009-05-29

Meeting Announcements

2009-05-29

Antiganglioside antibodies and their pathophysiological effects on Guillain-Barre syndrome and related disorders--A review

Kaida, K., Ariga, T., Yu, R. K — 2009-05-29

Guillain–Barré syndrome (GBS) is an acute immune-mediated polyradiculoneuropathy which can cause acute quadriplegia. Infection with micro-organisms, including Campylobacter jejuni (C. jejuni), Haemophilus influenzae, and Cytomegalovirus (CMV), is recognized as a main triggering event for the disease. Lipooligosaccharide (LOS) genes are responsible for the formation of human ganglioside-like LOS structures in infectious micro-organisms that can induce GBS. Molecular mimicry of LOSs on the surface of infectious agents and of ganglioside antigens on neural cells is thought to induce cross-reactive humoral and cellular immune responses. Patients with GBS develop antibodies against those gangliosides, resulting in autoimmune targeting of peripheral nerve sites, leading to neural damage. Heterogeneity of ganglioside expression in the peripheral nervous system (PNS) may underlie the differential clinical manifestation of the GBS variants. Recent studies demonstrate that some GBS sera react with ganglioside complexes consisting of two different gangliosides, such as GD1a and GD1b, or GM1 and GD1a, but not with each constituent ganglioside alone. The discovery of antiganglioside complex antibodies not only improves the detection rate of autoantibodies in GBS, but also provides a new concept in the antibody–antigen interaction through clustered carbohydrate epitopes. Although ganglioside mimicry is one of the possible etiological causes of GBS, unidentified factors may also contribute to the pathogenesis of GBS. While GBS is not considered a genetic disease, host factors, particularly human lymphocyte antigen type, appear to have a role in the pathogenesis of GBS following C. jejuni infection.

Structural analysist of N-glycans from gull egg white glycoproteins and egg yolk IgG

Suzuki, N., Su, T.-H., Wu, S.-W., Yamamoto, K., Khoo, K.-H., Lee, Y. C — 2009-05-29

We previously showed that the expression of (Gal1-4Gal)-bearing glycoproteins among birds is related to their phylogeny. However, precise structures of (Gal1-4Gal)-containing N-glycans were only known for pigeon egg white glycoproteins and IgG. To compare structural features of (Gal1-4Gal)-containing N-glycans from other species, we analyzed N-glycans of gull egg white (GEW)-glycoproteins, ovomucoid, and ovotransferrin, and gull egg yolk IgG by HPLC, mass spectrometry (MS), and MS/MS analyses. GEW-glycoproteins included neutral, monosialyl, and disialyl N-glycans, and some of them contained Gal1-4Gal sequences. Bi-, tri-, and tetra-antennary oligosaccharides that lacked bisecting GlcNAc were the major core structures, and incomplete -galactosylation and sialylation as well as the presence of diLacNAc on the branches generated microheterogeneity of the N-glycan structures. Moreover, unlike pigeon egg white glycoproteins, the major sialylation in GEW-glycoproteins is 2,3-, but not 2,6-linked sialic acids (NeuAc). In addition to the complex-type oligosaccharide, hybrid-type oligosaccharides that lack bisecting GlcNAc were also abundant in GEW-glycoproteins. Gull egg yolk IgG also contained Gal1-4Galβ1-4GlcNAcβ1- sequences, but unlike pigeon IgG, no Gal1-4Galβ1-4Galβ1-4GlcNAcβ1- sequence was detected. Bi- and tri-antennary complex-type oligosaccharides with bisecting GlcNAc and with core fucosylation as well as high-mannose-type oligosaccharides were the major structures in gull IgG. Our data indicated that some N-glycans from both GEW-glycoproteins and gull IgG contain the Gal1-4Galβ1-4GlcNAcβ1- sequence, but the ratio of -Gal-capped residues to non--Gal-capped residues in the nonreducing termini of N-glycans is much lower than that in those of pigeon glycoproteins.

De novo glycan structure search with the CID MS/MS spectra of native N-glycopeptides

Peltoniemi, H., Joenvaara, S., Renkonen, R. — 2009-05-29

The aim of our study is to automatically analyze the glycan and peptide structures of N-glycopeptides without a need to release glycans from the glycopeptides. Our wet laboratory raw data represent a series of MS/MS mass spectra obtained from a reverse-phase liquid chromatography run of size-exclusion-enriched tryptic-digested glycopeptides from glycoproteins. The MS/MS spectra are first analyzed in order to identify glycosylated peptides and N-glycan monosaccharide compositions present on each glycopeptide. We further developed a Branch-and-Bound algorithm to search de novo N-glycan structures, i.e., monosaccharide compositions and their ordered sequences from native glycopeptides. Our de novo algorithm is based on iterative growth and selection of a population of glycan structures and it does not use databases of known glycan structures. We validate the algorithm with (i) in silico-generated spectra, with or without deteriorating deletions, (ii) with a purified glycoprotein transferrin, and (iii) with a complex mixture of N-glycopeptides enriched from human plasma. Our Branch-and-Bound algorithm depicted glycan structures from all the above-mentioned three input data types. Due to the large diversity of glycan structures, the results typically contained several proposed structures matching almost equally well to the spectra. In conclusion, this algorithm automatically identifies glycopeptides and their structures from the MS/MS spectra and thus greatly reduces the number of possible glycan structures from the vast amount of potential ones.

The CMP-legionaminic acid pathway in Campylobacter: Biosynthesis involving novel GDP-linked precursors

Schoenhofen, I. C, Vinogradov, E., Whitfield, D. M, Brisson, J.-R., Logan, S. M — 2009-05-29

The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto-nonulosonic acid, or legion- aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaire's disease, contain an 2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same d-glycero-d-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

Compensation of loss of protein function in microsatellite-unstable colon cancer cells (HCT116): A gene-dependent effect on the cell surface glycan profile

2009-05-29

Tumors that display a high level of microsatellite instability (MSI-H) accumulate somatic frameshift mutations in several genes. The compensation of this loss of function by transfection represents a suitable approach to tie respective gene deficiency to alterations in cellular characteristics. In view of the emerging significance of cell surface glycans as biochemical signals for presentation/activity of various receptors/integrins and for susceptibility to adhesion/growth-regulatory tissue lectins, we examined the glycophenotype in the MSI-H colon cancer cell line HCT116 for activin type 2 receptor (ACVR2), absent in melanoma 2 (AIM2), and transforming growth factor β-type 2 receptor (TGFBR2) known to be associated with MSI colorectal carcinogenesis. A panel of probes specific for functional carbohydrate epitopes including human lectins was used to trace changes in cell surface levels, thereby initiating glycan analysis related to MSI. In particular, the presence of core substitutions and branching in N-glycans, the sialylation status of N- and O-glycans, and the presence of Le^a/x-epitopes were profiled. Transient transfection affected the glycophenotype, depending on the nature of the gene and the probe. The TGFBR2 presence reduced binding of probes specific for a core substitution and increased branch length in N-glycosylation, even reaching a P-value of 0.0016. ACVR2/AIM2 influenced core 1 mucin-type O-glycosylation differentially, upregulation by ACVR2, and downregulation by AIM2. These alterations of cell surface glycosylation by gene products that are not directly associated with the machinery for glycan generation direct attention to pursue analysis of glycosylation in MSI tumor cells on the level of target glycoproteins and open the way for functional studies.

Involvement of chondroitin sulfate E in the liver tumor focal formation of murine osteosarcoma cells

2009-05-29

Cell surface heparan sulfate plays a critical role in regulating the metastatic behavior of tumor cells, whereas the role of chondroitin sulfate/dermatan sulfate (CS/DS) has been little understood in this context. Here, we characterized CS/DS chains from the murine osteosarcoma cell line LM8G7, which forms tumor nodules in liver. Structural analysis of the CS/DS chains showed a higher proportion of GlcUAβ1-3GalNAc(4,6-O-disulfate) (E-units) in LM8G7 (12%) than in its parental cell line LM8 (6%), which rarely forms tumors in the liver. Immunostaining with GD3G7, an antibody specific to E-units, confirmed the higher expression of the epitope in LM8G7 than LM8 cells. The tumor focal formation of LM8G7 cells in the liver in mice was effectively inhibited by the preadministration of CS-E (rich in E-unit) or the preincubation of the antibody GD3G7 with the tumor cells. CS-E or GD3G7 inhibited the adhesion of LM8G7 cells to a laminin-coated plate in vitro. In addition, the invasive ability of LM8G7 cells in vitro was also reduced by the addition of CS-E or the antibody. Further, CS-E or the antibody inhibited the proliferation of LM8G7 cells dose dependently. The binding of LM8G7 cells to VEGF in vitro was also significantly reduced by CS-E and GD3G7. Thus, the present study reveals the significance of highly sulfated CS/DS structures in the liver colonization of osteosarcoma cells and also provides a framework for the development of GAG-based anticancer molecules.

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death

2009-05-29

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal -[1->2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest. An important gene described in the biosynthesis of these molecules is the mannosyltransferase pimB (Rv0557). Here, we disrupted pimB in a virulent strain of M.tb. We demonstrate that the inactivation of pimB in M.tb does not abolish the production of any of its cell wall mannosylated lipoglycans; however, it results in a quantitative decrease in the ManLAM and LM content without affecting higher order PIMs. This finding indicates gene redundancy or the possibility of an alternative biosynthetic pathway that may compensate for the PimB deficiency. Furthermore, infection of human macrophages by the pimB mutant leads to an alteration in macrophage phenotype concomitant with a significant increase in the rate of macrophage death.

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Holmen Larsson, J. M, Karlsson, H., Sjovall, H., Hansson, G. C — 2009-05-29

High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (~5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS–agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive β-elimination and analyzed by nanoLC/mass spectrometry and MSⁿ. The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Galβ1-3/4-GlcNAcβ1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase

2009-05-29

We report here a method of simultaneously quantifying glucosylceramide (GlcCer) and galactosylceramide (GalCer) by normal-phase HPLC using O-phtalaldehyde derivatives. Treatment with sphingolipid ceramide N-deacylase which converts the cerebrosides in the sample to their lyso-forms was followed by the quantitative labeling of free NH₂ groups of the lyso-cerebrosides with O-phtalaldehyde. Using this method, 14.1 pmol of GlcCer and 10.4 pmol of GalCer, and 108.1 pmol of GlcCer and 191.1 pmol of GalCer were detected in zebrafish embryos and RPMI 1864 cells, respectively, while 22.2 pmol of GlcCer but no GalCer was detected in CHOP cells using cell lysate containing 100 µg of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 µg of protein under the conditions used, which corresponds to approximately 10³ to 10⁵ RPMI cells and 5 to 80 zebrafish embryos. The present method clearly revealed that the treatment of RPMI cells with a GlcCer synthase inhibitor P4 resulted in a marked decrease in GlcCer but not GalCer, concomitantly with a significant decrease in the GlcCer synthase activity. On the other hand, GlcCer but not GalCer increased 2-fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, the treatment of CHOP cells with ciclosporin A increased GlcCer possibly due to the inhibition of LacCer synthase. A significant increase in levels of GlcCer in fibroblasts from patients with Gaucher disease was clearly shown by the method. The proposed method is useful for the determination of GlcCer and GalCer levels in various biological samples.

Activation of host antiviral RNA-sensing factors necessary for herpes simplex virus type 1-activated transcription of host cell fucosyltransferase genes FUT3, FUT5, and FUT6 and subsequent expression of sLex in virus-infected cells

Norden, R., Nystrom, K., Olofsson, S. — 2009-05-29

Herpes simplex virus type 1 (HSV-1) induces expression of a selectin receptor, the carbohydrate epitope sialyl Lewis X (sLe^x), at the surface of infected cells. The molecular background to this phenomenon is that a viral immediate early RNA interacts with as yet unidentified host factors, eventually resulting in transcription of three dormant host fucosyltransferase genes (FUT3, FUT5, and FUT6), whose gene products are rate-limiting for synthesis of sLe^x. The aim of the present study was to define the immediate targets for the viral RNA in this process. We found that the Protein Kinase R (PKR) inhibitors 2-aminopurine (2-AP) and C16 inhibited FUT3, FUT5, and FUT6 expression as well as HSV-1-induced expression of sLe^x, indicating a primary role of PKR as a viral RNA target. The PKR-dependent activation of the FUT genes seemed neither to involve PKR effects on translation nor to involve NF-B- or JNK-dependent activation. IMD-0354, known as an inhibitor of the NF-B-activating factor IKK-2, induced FUT transcription via a novel IKK-2-independent mechanism, irrespective of whether the cells were virus-infected or not. Altogether, the results suggested that PKR is the primary target for HSV-1 early RNA during induction of FUT3, FUT5, and FUT6, and that the subsequent steps in the transcriptional activation of these host genes involve a hitherto unknown IMD-0354, yet IKK-2-independent, pathway.

Analysis of lectin binding to glycolipid complexes using combinatorial glycoarrays

2009-05-29

Glycolipids are major components of the plasma membrane, interacting with themselves, other lipids, and proteins to form an array of heterogeneous domains with diverse biological properties. Considerable effort has been focused on identifying protein binding partners for glycolipids and the glycan specificity for these interactions, largely achieved through assessing interactions between proteins and homogenous, single species glycolipid preparations. This approach risks overlooking both the enhancing and attenuating roles of heterogeneous glycolipid complexes in modulating lectin binding. Here we report a simple method for assessing lectin–glycolipid interactions. An automatic thin-layer chromatography sampler is employed to create easily reproducible arrays of glycolipids and their heterodimeric complexes immobilized on a synthetic polyvinyl-difluoride membrane. This array can then be probed with much smaller quantities of reagents than would be required using existing techniques such as ELISA and thin-layer chromatography with immuno-overlay. Using this protocol, we have established that the binding of bacterial toxins, lectins, and antibodies can each be attenuated, enhanced, or unaffected in the presence of glycolipid complexes, as compared with individual, isolated glycolipids. These findings underpin the wide-ranging influence and importance of glycolipid–glycolipid cis interactions when the nature of protein–carbohydrate recognition events is being assessed.

Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase

2009-05-29

Although 6-gala series glycosphingolipids possessing R-Gal (/β) 1-6Galβ1-1'Cer have been found in some mollusks, pathogenic parasites, and fungi, their physiological functions and metabolic pathway are not fully understood. We described a novel method of detecting 6-gala series glyco- sphingolipids utilizing the specificity of endogalactosylceramidase (EGALC), which is capable of hydrolyzing 6-gala series glycosphingolipids to produce intact oligosaccharides and ceramides. EGALC catalyzes not only hydrolysis but also a transglycosylation reaction. In the latter reaction, EGALC transfers oligosaccharides from the glycosphingolipids to acceptors such as fluorescent 1-alkanols. Based on the transglycosylation reaction of EGALC, a specific, easy, fast, sensitive, and reproducible method of detecting 6-gala series glycosphingolipids was developed using NBD-pentanol as an acceptor. The fluorescent products, NBD-pentanol-conjugated 6-gala oligosaccharides, were separated and detected by TLC or HPLC with a fluorescent detector. Moreover, it was revealed that as well as glycosphingolipids, a glycoglycerolipid, digalactosyldiacylglycerol, was utilized by EGALC as a donor substrate. This method was successfully applied to detect 6-gala series glycosphingolipids in a fungus, Rhizopus oryzae, and a parasite, Taenia crassiceps. The method would be useful for studying glycosphingolipids and galactosyl glycerolipids which share the Gal (/β) 1-6Gal structure.

2009-05-14

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2009-05-14

Glycobiology

2009-05-14

Meeting Announcements

2009-05-14

Obituary: Annette Herscovics (1938-2008)

Schachter, H. — 2009-05-14

Dietary glucosamine under question

Silbert, J. E — 2009-05-14

Annual sales of glucosamine as a neutraceutical for affecting cartilage in treatment of osteoarthritis are close to a billion dollars, but recent clinical studies have currently raised severe criticism regarding its functional value. Additional doubts can be raised by the knowledge of the well-defined cellular steps in glucosamine formation and production of glycosaminoglycans such as chondroitin. Glucosamine is produced in an activated state from glucose by essentially all cells for incorporation into glycosaminoglycans and glycoproteins, and there have been no reports of any deficiencies in its production under any conditions. Nevertheless, many investigations of glucosamine, using cells or tissues, have claimed effects on cartilage and chondroitin sulfate. The significance of these studies is questionable since they have invariably been with concentrations that were 10- to 1000-fold higher than has been found in human serum or plasma after glucosamine ingestion. Experiments with cells or tissues using glucosamine in the low concentrations found after ingestion need to be examined before any conclusions are drawn concerning its direct action on cartilage and its potential for modifying osteoarthritis.

Recognition of non-self-polysaccharides by C-type lectin receptors dectin-1 and dectin-2

Hollmig, S T., Ariizumi, K., Cruz, P. D — 2009-05-14

The discovery of several transmembrane receptors expressed by antigen presenting cells, including those that detect and interact with specific sugar moieties on the surface of microbes, has improved our understanding of how immunity against infection is generated. This knowledge, in turn, prompted us to review such interactions with emphasis on C-type lectin receptors and a focus on the roles of dectin-1 and dectin-2 in anti-fungal immunity.

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells

2009-05-14

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

Human pseudogenes of the ABO family show a complex evolutionary dynamics and loss of function

2009-05-14

The GT6 glycosyltransferases gene family, that includes the ABO blood group, shows a complex evolution pattern, with multiple events of gain and loss in different mammal species. In humans the ABO gene is considered the sole functional member although the O allele is null and is fixed in certain populations. Here, we analyze the human GT6 pseudogene sequences (Forssman, IGB3, GGTA1, GT6m5, GT6m6, and GT6m7) from an evolutionary perspective, by the study of (i) their diversity levels in populations through the resequencing analysis of European and African individuals; (ii) the interpopulation differentiation, with genotyping data from a survey of populations covering most of human genetic diversity; and (iii) the interespecific divergence, by the comparison of the human and some other primate species sequences. Since pseudogenes are expected to evolve under neutrality, they should show an evolutionary pattern different to that of functional sequences, with higher levels of diversity as well as a ratio of nonsynonymous to synonymous changes close to 1. We describe some departures from these expectations, including selection for inactivation in IGB3, GGTA1, and the interesting case of FS (Forssman) with a probable shift of its initial function in the primate lineage, which put it apart from a pure neutral pseudogene. These results suggest that some of these GT6 human pseudogenes may still be functional and retain some valuable unknown function in humans, in some case even at the protein level. The evolutionary analysis of all members of the GT6 family in humans allows an insight into their functional history, a process likely due to the interaction of the host glycans that they synthesize with pathogens; the past process that can be unraveled through the footprints left by natural selection in the extant genome variation.

Negative-ion MALDI-QIT-TOFMSn for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative

Amano, J., Sugahara, D., Osumi, K., Tanaka, K. — 2009-05-14

Oligosaccharides have many isomers and MALDI-QIT-TOFMSⁿ analysis is effective for determining their structures. However, it is difficult to elucidate in detail the structures of fucosylated and/or sialylated oligosaccharides that are known to be disease markers because fucose and sialic acid residues are easily released. We have introduced a technique of labeling oligosaccharides with a pyrene derivative prior to negative-ion MALDI-QIT-TOFMSⁿ, and we have established a reliable method using this technique for the analysis of neutral oligosaccharides, such as fucosylated oligosaccharides containing blood group antigens H, Le^a, and Le^x. Intense and stable ionization in both positive and negative modes was achieved by derivatization with pyrene. As little as 10 fmol of pyrene-labeled oligosaccharides gave sufficient signals for analysis. Specific A-, D- or Y-type ions that depend on the structures of branching antennae could be detected by MSⁿ and were useful for rapid and easy structural determination. These specific fragmentations resulting from collision-induced dissociation can be used to elucidate the structures of unknown oligosaccharides even if authentic oligosaccharides are not available as standards. By using this method, we identified and quantitated isomeric oligosaccharides with different fucosyl linkages from their mixtures. Moreover, sialylated oligosaccharide was converted to the corresponding neutral oligosaccharide by amidation, and the negative-ion spectrum was shown to be more informative than that of the original acidic oligosaccharide. Structural determination of both fucosylated and sialylated isomers, such as sialylfucosyllacto-N-hexaose I and monosialyl monofucosyllacto-N-neohexaose, was successful because fragment ions bearing fucose or amidated sialic acid were obtained on negative-MSⁿ.

Structural determination by negative-ion MALDI-QIT-TOFMSn after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk

Amano, J., Osanai, M., Orita, T., Sugahara, D., Osumi, K. — 2009-05-14

We prepared neutral oligosaccharide fraction from milk of a woman (blood type A, Le^b+) by anion-exchange column chromatography after the removal of lipids and proteins. Further fractionation was performed by means of Aleuria aurantia lectin-Sepharose column chromatography and reverse-phase HPLC after labeling with a pyrene derivative. This pyrene labeling allowed identification by negative-MALDI-TOFMSⁿ analysis of 22 oligosaccharides with decaose cores, among which 21 had novel structures. Negative ions could not be produced from neutral oligosaccharides without labeling on MALDI. Mono-, di-, tri-, and tetrafucosylated decaose fractions contained three, nine, six, and four isomers, respectively. Our method enables easy determination of fucosylated structures on the N-acetyllactosamine branches of these isomers. On negative-MSⁿ the fragment ions included several A and D ions, from which fucosylation on the branches could be elucidated. Other characteristic ions were also detected. Y-type cleavage at the reducing side of -3GlcNAc indicated the occurrence of type 1 chain. Specific fragment ions were produced from H, Le^a, and Le^x antigens. Linkage-specific exoglycosidase digestion confirmed the structures. The results indicate that the diversity of the oligosaccharides is due to combinations of type 1 H, Le^a, Le^x, and Le^b/Le^y on branched decaose cores. In typical oligosaccharides, 6-branches always consist of type 2 chain, while 3-branches, such as β and chains, are fucosylated type 1 chains. From the viewpoint of biosynthesis, the presence of fucosylation and type 1 chain may halt elongation of the N-acetyllactosamine and promote formation of branched structures.

The family 6 carbohydrate-binding modules have coevolved with their appended catalytic modules toward similar substrate specificity

Michel, G., Barbeyron, T., Kloareg, B., Czjzek, M. — 2009-05-14

The survey of carbohydrate active enzymes in genomic data uncovered the modular architecture of most of these proteins. Many of the additional modules associated with catalytic modules tightly bind carbohydrates. The primary role of these carbohydrate-binding modules (CBMs) is to enhance the enzymatic activity of the ensemble by bringing their appended catalytic module(s) in intimate contact with their substrates. Biochemical and biophysical approaches have unraveled the subtle interplay of the modules and the structural basis for their ligand specificities, but little attention has been paid to the evolutionary mechanisms leading to the appearance of modular architecture in carbohydrate active enzymes. Focusing on the promiscuous family CBM6 modules, we investigated the evolution of substrate specificities in parallel to that of their respectively appended catalytic modules. An extensive phylogenetic analysis of family CBM6 modules indicates that these noncatalytic modules have diverged into clades which coincide with their substrate selectivity. These data as well as the remarkable congruence of the phylogenetic trees inferred from CBM6s on the one hand and their associated catalytic modules on the other hand show that CBM6s and their associated glycoside hydrolases have coevolved to acquire the same substrate specificity. We also propose an evolutionary scenario explaining the emergence of the modular agarases, by which existent alpha-agarases acquired their agar-binding CBM6 module through a lateral transfer from pre-existing beta-agarases. Altogether, this observed coevolution between CBM6s and their catalytic modules will facilitate the prediction of the substrate specificity of uncharacterized CBM6 modules present in genomic data.

Class IIC {alpha}-mannosidase AfAms1 is required for morphogenesis and cellular function in Aspergillus fumigatus

Li, Y., Fang, W., Zhang, L., Ouyang, H., Zhou, H., Luo, Y., Jin, C. — 2009-05-14

The mammalian ER/cytosolic -mannosidase (Man2C1p), yeast vacuolar -mannosidase (Ams1p) and the Aspergillus nidulans -mannosidase are members of Class IIC subgroup, which is involved in oligosaccharide catabolism and N-glycan processing. Unlike their mammalian counterparts, the yeast Ams1p and A. nidulans Class IIC -mannosidase are not essential for morphogenesis and cellular function. In this study, the Afams1, a gene encoding a member of Class IIC -mannosidases, was identified in the opportunistic pathogen Aspergillus fumigatus. Deletion of the Afams1 led to a severe defect in conidial formation, especially at a higher temperature. In addition, abnormalities of polarity and septation were associated with the Afams1 mutant. Our results showed that the Afams1 gene, in contrast to its homolog in yeast or A. nidulans, was required for morphogenesis and cellular function in A. fumigatus.

Characterization of the wheat germ agglutinin binding to self-assembled monolayers of neoglycoconjugates by AFM and SPR

2009-05-14

Carbohydrate–protein interactions govern many crucial life processes involved in cell recognition events, but are often difficult to study because the interactions are weak, and multivalent exposure appears to be crucial for their biological function. We have used self-assembled monolayers (SAMs) of neoglycoconjugates as a model system to probe the specific interactions between the lectin wheat germ agglutinin (WGA) and monosaccharides by surface plasmon resonance (SPR) and atomic force microscopy (AFM) force measurements. SAMs presenting N-acetyl-d-glucosamine (GlcNAc) as a neoglycoconjugate were produced on gold surfaces, where the SAM formation was monitored using a quartz crystal microbalance (QCM) and shown to be a very rapid process. In the AFM force measurements WGA was covalently coupled to flexible polyethylene glycol (PEG) molecules at a probe surface using amine coupling. GlcNAc-specific binding events were detected with a WGA-modified probe on the GlcNAc-neoglycoconjugate SAM at bond rupture forces of 47 ± 15 pN. Additionally, less frequent GlcNAc-specific unbinding events were detected at higher forces (120 ± 20 pN) which are believed to originate from simultaneous detachment of multiple binding sites from the SAM surface. SPR measurements confirmed that WGA has higher affinity toward the immobilized GlcNAc-SAM than toward the soluble free monosaccharide. The binding constants obtained for soluble chitinoligosaccharides suggested up to three subsites within one carbohydrate-binding site of the WGA molecule and also provided further evidence of the multivalent binding character of the WGA dimer.

Specific inhibition of FGF-2 signaling with 2-O-sulfated octasaccharides of heparan sulfate

Ashikari-Hada, S., Habuchi, H., Sugaya, N., Kobayashi, T., Kimata, K. — 2009-05-14

In fibroblast growth factor (FGF)-2 signaling, the formation of a ternary complex of FGF-2, tyrosine-kinase fibroblast growth factor receptor (FGFR)-1, and cell surface heparan sulfate (HS) proteoglycan is known to be critical for the activation of FGFR-1 and downstream signal transduction. Exogenous heparin polymer and some octasaccharides inhibited FGF-2-induced phosphorylation both of FGFR-1 and of extracellular signal-regulated kinase (ERK1/2) in Chinese hamster ovary (CHO)-K1 cells transfected with FGFR-1, which present HS on their cell surface. The inhibitory effect of octasaccharide was dependent on the number of 2-O-sulfate groups within a molecule but independent of the number of 6-O-sulfate groups. Sulfation at the 2-O-position was a prerequisite not only for the binding of HS to FGF-2 but also for regulation of FGF-2 signaling and competitive inhibition with endogenous HS. Interestingly, FGF-4-induced phosphorylation was impeded only by specific octasaccharides containing both 2-O- and 6-O-sulfated groups, which were necessary for binding FGF-4. In CHO-677 cells deficient in HS biosynthesis, heparin enhanced FGF-2-induced phosphorylation of ERK1/2. On the other hand, an FGF-2-binding octasaccharide inhibited the phosphorylation. Our data suggest that the activity of particular heparin-binding factors can be inhibited by distinctive oligosaccharides that can bind the factors but cannot form functional signaling complexes irrespective of whether cells have a normal complement of HS or lack HS.

Association of {beta}-1,3-N-acetylglucosaminyltransferase 1 and {beta}-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis

Lee, P. L, Kohler, J. J, Pfeffer, S. R — 2009-05-14

Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues involved in cellular functions ranging from differentiation to metastasis. PolyLacNAc also serves as a scaffold on which other oligosaccharides such as sialyl Lewis X are displayed. The polymerization of the alternating N-acetylglucosamine and galactose residues is catalyzed by the successive action of UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1) and UDP-Gal:βGlcNAc β-1,4-galactosyltransferase, polypeptide 1 (B4GALT1), respectively. The functional association between these two glycosyltransferases led us to investigate whether the enzymes also associate physically. We show that B3GNT1 and B4GALT1 colocalize by immunofluorescence microscopy, interact by coimmunoprecipitation, and affect each other's subcellular localization when one of the two proteins is artificially retained in the endoplasmic reticulum. These results demonstrate that B3GNT1 and B4GALT1 physically associate in vitro and in cultured cells, providing insight into possible mechanisms for regulation of polyLacNAc production.

Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested

2009-05-14

The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.

Glycobiology - recent issues

Glycobiology

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Natural ligands for CD33-related Siglecs?

For intra-articular delivery of chondroitin sulfate

Protein O-mannosylation: Conserved from bacteria to humans

Xylosyltransferase II is a significant contributor of circulating xylosyltransferase levels and platelets constitute an important source of xylosyltransferase in serum

Genetic analysis of glucosidase II {beta}-subunit in trimming of high-mannose-type glycans

Deletion polymorphism of SIGLEC14 and its functional implications

Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes

Sulfated polysaccharides from marine sponges (Porifera): an ancestor cell-cell adhesion event based on the carbohydrate-carbohydrate interaction

Regulated expression of the HNK-1 carbohydrate is essential for medaka (Oryzias latipes) embryogenesis

Glycosylation-related gene expression profiling in the brain and spleen of scrapie-affected mouse

Size-dependent regulation of Snail2 by hyaluronan: Its role in cellular invasion

Targeted glycoproteomic identification of cancer cell glycosylation

Quality control of glycoproteins bearing truncated glycans in an ALG9-defective (CDG-IL) patient

Molecular analysis of a UDP-GlcNAc:polypeptide {alpha}-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi

Glycobiology

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Antiganglioside antibodies and their pathophysiological effects on Guillain-Barre syndrome and related disorders--A review

Structural analysist of N-glycans from gull egg white glycoproteins and egg yolk IgG

De novo glycan structure search with the CID MS/MS spectra of native N-glycopeptides

The CMP-legionaminic acid pathway in Campylobacter: Biosynthesis involving novel GDP-linked precursors

Compensation of loss of protein function in microsatellite-unstable colon cancer cells (HCT116): A gene-dependent effect on the cell surface glycan profile

Involvement of chondroitin sulfate E in the liver tumor focal formation of murine osteosarcoma cells

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death

A complex, but uniform O-glycosylation of the human MUC2 mucin from colonic biopsies analyzed by nanoLC/MSn

Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase

Activation of host antiviral RNA-sensing factors necessary for herpes simplex virus type 1-activated transcription of host cell fucosyltransferase genes FUT3, FUT5, and FUT6 and subsequent expression of sLex in virus-infected cells

Analysis of lectin binding to glycolipid complexes using combinatorial glycoarrays

Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase

Contents

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Glycobiology

Meeting Announcements

Obituary: Annette Herscovics (1938-2008)

Dietary glucosamine under question

Recognition of non-self-polysaccharides by C-type lectin receptors dectin-1 and dectin-2

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells

Human pseudogenes of the ABO family show a complex evolutionary dynamics and loss of function

Negative-ion MALDI-QIT-TOFMSn for structural determination of fucosylated and sialylated oligosaccharides labeled with a pyrene derivative

Structural determination by negative-ion MALDI-QIT-TOFMSn after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk

The family 6 carbohydrate-binding modules have coevolved with their appended catalytic modules toward similar substrate specificity

Class IIC {alpha}-mannosidase AfAms1 is required for morphogenesis and cellular function in Aspergillus fumigatus

Characterization of the wheat germ agglutinin binding to self-assembled monolayers of neoglycoconjugates by AFM and SPR

Specific inhibition of FGF-2 signaling with 2-O-sulfated octasaccharides of heparan sulfate

Association of {beta}-1,3-N-acetylglucosaminyltransferase 1 and {beta}-1,4-galactosyltransferase 1, trans-Golgi enzymes involved in coupled poly-N-acetyllactosamine synthesis

Secondary cell wall polysaccharides of Bacillus anthracis are antigens that contain specific epitopes which cross-react with three pathogenic Bacillus cereus strains that caused severe disease, and other epitopes common to all the Bacillus cereus strains tested