Glycobiology - current issue

Glycobiology

2009-07-01

Subscriptions

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 - current issue

Glycobiology

Contents

Subscriptions

Meeting Announcements

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