Glycobiology - current issue

Glycobiology

Tue, 24 Nov 2009 23:57:37 PST

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Tue, 24 Nov 2009 23:57:37 PST

Meeting Announcements

Tue, 24 Nov 2009 23:57:37 PST

Glycan gimmickry by parasitic helminths: A strategy for modulating the host immune response?

van Die, I., Cummings, R. D — Tue, 24 Nov 2009 23:57:37 PST

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le^X (Galβ1-4[Fuc1-3]GlcNAc-), LDNF (GalNAcβ1-4[Fuc1-3]GlcNAc-), LDN (GalNAcβ1-4GlcNAc-), and Tn (GalNAc1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays

Saravanan, C., Cao, Z., Head, S. R, Panjwani, N. — Tue, 24 Nov 2009 23:57:37 PST

It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occuring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of ~2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, β3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counter-receptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.

Characterization of gene-activated human acid-{beta}-glucosidase: Crystal structure, glycan composition, and internalization into macrophages

Tue, 24 Nov 2009 23:57:37 PST

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated^TM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid–base hydrolysis. Kinetic parameters (K_m and V_max) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Hypoxic regulation of secreted proteoglycans in macrophages

Tue, 24 Nov 2009 23:57:37 PST

Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1 and HIF-2). We found that versican co-localized with HIF-1 in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O₂ (hypoxia) compared with 21% O₂ (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1 and HIF-2 in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1 and HIF-2 but expression of perlecan was influenced only by HIF-1 and not by HIF-2 knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I)

Tue, 24 Nov 2009 23:57:37 PST

LT-IIb, a type II heat-labile enterotoxin of Escherichia coli, is a potent immunologic adjuvant with high affinity binding for ganglioside GD1a. Earlier study suggested that LT-IIb bound preferentially to the terminal sugar sequence NeuAc2-3Galβ1-3GalNAc. However, studies in our laboratory suggested a less restrictive binding epitope. LT-IIb(T13I), an LT-IIb variant, engineered by a single isoleucine-threonine substitution, retains biological activity, but with less robust inflammatory effects. We theorized that LT-IIb has a less restrictive binding epitope than previously proposed and that immunologic differences between LT-IIb and LT-IIb (T13I) correlate with subtle ganglioside binding differences. Ganglioside binding epitopes, determined by affinity overlay immunoblotting and enzymatic degradation of ganglioside components of RAW264.7 macrophages, indicated that LT-IIb bound to a broader array of gangliosides than previously recognized. Each possessed NeuAc2-3Galβ1-3GalNAc, although not necessarily as a terminal sequence. Rather, each had a requisite terminal or penultimate single sialic acid and binding was independent of ceramide composition. RAW264.7 enterotoxin-binding and non-binding ganglioside epitopes were definitively identified as GD1a and GM1a, respectively, by enzymatic degradation and mass spectroscopy. Affinity overlay immunoblots, constructed to the diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIb bound NeuAc- and NeuGc-gangliosides with nearly equal affinity. However, LT-IIb(T13I) exhibited enhanced affinity for NeuGc-gangliosides and more restrictive binding. These studies further elucidate the binding epitope for LT-IIb and suggest that the diminished inflammatory activity of LT-IIb(T13I) is mediated by a subtle shift in ganglioside binding. These studies underscore the high degree of specificity required for ganglioside–protein interactions.

Endomannosidase undergoes phosphorylation in the Golgi apparatus

Torossi, T., Guhl, B., Roth, J., Ziak, M. — Tue, 24 Nov 2009 23:57:37 PST

Glucose residues from N-linked oligosaccharides are removed by glucosidases I and II in the endoplasmic reticulum (ER) or by the alternate endomannosidase pathway in the Golgi apparatus. Our morphological analysis demonstrates that recombinant rat endomannosidase exhibited a cis- and medial-Golgi localization alike the endogenous enzyme and its ER to Golgi transport is COP II mediated. Recombinant endomannosidase undergoes a posttranslational modification, which is not related to N-or O-glycosylation. A shift in molecular mass of recombinant endomannosidase was observed upon phosphatase digestion but not for ER-retained CHO cell endomannosidase. Furthermore, immunoprecipitation of ³⁵S- and ³³P-labeled endomannosidase expressed in CHO-K1 cells suggests that recombinant endomannosidase undergoes phosphorylation. Substitution of the single cytoplasmic threonine residue of rat endomannosidase by either an alanine or valine residue resulted in the same posttranslational modification alike the wild-type enzyme. The subcellular localization and the in vivo activity of the mutant endomannosidase were not affected. Thus, endomannosidase phosphorylation is occurring in luminal sequences. Modification was prevented when endomannosidase was synthesized using reticulocyte lysates in the presence of canine microsomes. Treatment of cells with brefeldin A blocked the posttranslational modification of endomannosidase, suggesting that phosphorylation is occurring in the Golgi apparatus, the residence of endomannosidase.

GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells

Tue, 24 Nov 2009 23:57:37 PST

In this paper, we describe the effects of the expression of GM3 synthase at high levels in human ovarian carcinoma cells. Overexpression of GM3 synthase in A2780 cells consistently resulted in elevated ganglioside (GM3, GM2 and GD1a) levels. GM3 synthase overexpressing cells had a growth rate similar to wild-type cells, but showed a strongly reduced in vitro cell motility accompanied by reduced levels of the epithelial-mesenchymal transition marker smooth muscle actin. A similar reduction in cell motility was observed upon treatment with exogenous GM3, GM2, and GM1, but not with GD1a. A photolabeling experiment using radioactive and photoactivable GM3 highlighted several proteins directly interacting with GM3. Among those, caveolin-1 was identified as a GM3-interacting protein in GM3 synthase overexpressing cells. Remarkably, caveolin-1 was markedly upregulated in GM3 synthase overexpressing cells. In addition, the motility of low GM3 synthase expressing cells was also reduced in the presence of a Src kinase inhibitor; on the other hand, higher levels of the inactive form of c-Src were detected in GM3 synthase overexpressing cells, associated with a ganglioside- and caveolin-rich detergent insoluble fraction.

Characterization of GD3 ganglioside as a novel biomarker of mouse neural stem cells

Nakatani, Y., Yanagisawa, M., Suzuki, Y., Yu, R. K — Tue, 24 Nov 2009 23:57:37 PST

Neural stem cells (NSCs) are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency. Over the past two decades, there has been a huge effort to identify NSCs morphologically, genetically, and molecular biologically. It is still controversial, however, what bona fide NSCs are. To define and characterize NSCs more systematically, it is crucial to explore novel cell-surface marker molecules of NSCs. In this study, we focused on GD3, a b-series ganglioside that is enriched in the immature brain and the subventricular zone (SVZ) of the postnatal and adult brain, and evaluated the usefulness of GD3 as a cell-surface biomarker for identifying NSCs. We demonstrated that GD3 was expressed in more than 80% of NSCs prepared from embryonic, postnatal, and adult mouse brain tissue by the neurosphere culture method. The percentage of GD3-expressing NSCs in neurospheres was nearly the same as it was in neurospheres derived from embryonic, postnatal, and adult brains but decreased drastically to about 40% after differentiation. GD3⁺ cells isolated from embryonic mouse striata, postnatal, and adult mouse SVZs by fluorescence-activated cell sorting with an R24 anti-GD3 monoclonal antibody efficiently generated neurospheres compared with GD3^– cells. These cells possessed multipotency to differentiate into neurons, astrocytes, and oligodendrocytes. These data indicate that GD3 is a unique and powerful cell-surface biomarker to identify and isolate NSCs.

Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin

Tue, 24 Nov 2009 23:57:37 PST

The opportunistic pathogen Burkholderia cenocepacia contains three soluble carbohydrate-binding proteins, related to the fucose-binding lectin PA-IIL from Pseudomonas aeruginosa. All contain a PA-IIL-like domain and two of them have an additional N-terminal domain that displays no sequence similarities with known proteins. Printed arrays screening performed on the shortest one, B. cenocepacia lectin A (BC2L-A), demonstrated the strict specificity for oligomannose-type N-glycan structures (Lameignere E, Malinovská L, Sláviková M, Duchaud E, Mitchell EP, Varrot A, Sedo O, Imberty A, Wimmerová M. 2008. Structural basis for mannose recognition by a lectin from opportunistic bacteria Burkholderia cenocepacia. Biochem J. 411:307–318.). The disaccharides Man1-2Man, Man1-3Man, and Man1-6Man and the trisaccharide Man1-3(Man1-6)Man were tested by titration microcalorimetry in order to evaluate their affinity for BC2L-A in solution and to characterize the thermodynamics of the binding. Oligomannose analogs presenting two mannoside residues separated by either flexible or rigid spacer were also tested. Only the rigid one yields to high affinity binding with a fast kinetics of clustering, while the flexible analog and the trimannoside display moderate affinities and no clustering effect on short time scale. The crystal structures of BC2L-A have been obtained in complex with Man1-3Man disaccharide and Man1(Man1-6)-3Man trisaccharide. The lengthy time required for the co-crystallization with the trisaccharide allowed for the formation of cluster since in the BC2L-A-trimannose complex solved at 1.1 Å resolution, the sugar creates a bridge between two adjacent dimers, yielding to molecular strings. AFM experiments were performed in order to visualize the filaments formed in solution by this type of interaction.

Protective effect of N-glycan bisecting GlcNAc residues on {beta}-amyloid production in Alzheimer's disease

Tue, 24 Nov 2009 23:57:37 PST

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, β-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, β-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of β-amyloid. Both β-amyloid 40 and β-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, -secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional β-amyloid production.

Lessons from GNE-deficient embryonic stem cells: sialic acid biosynthesis is involved in proliferation and gene expression

Weidemann, W., Klukas, C., Klein, A., Simm, A., Schreiber, F., Horstkorte, R. — Tue, 24 Nov 2009 23:57:37 PST

Sialic acids are widely expressed as terminal carbohydrates on glycoconjugates of eukaryotic cells. They are involved in a variety of cellular functions, such as cell adhesion or signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyzes the first two steps of sialic acid biosynthesis in the cytosol. Inactivation of GNE causes early embryonic lethality. In this study, we analyzed wild-type and GNE-deficient embryonic stem cells from mice. We found for the first time that proliferation is directly correlated with GNE-expression and the cellular sialic acid concentration. Furthermore, we identified growth-related genes that are differentially expressed in GNE-deficient embryonic stem cells compared to wild-type embryonic stem cells.

Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines

Zarei, M., Muthing, J., Peter-Katalinic, J., Bindila, L. — Tue, 24 Nov 2009 23:57:37 PST

Monosialoganglioside fraction of YAC-1 lymphoma cells was comprehensively analyzed and structurally defined by nano-high-performance liquid chromatography (nanoHPLC) in on-line conjunction with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS). An efficient separation and sensitive detection of Neu5Gc-containing gangliosides from Neu5Ac-containing analogues was for the first time accomplished in a single nanoHPLC/ESI-QTOF MS run, as demonstrated for mouse hybridoma cell GM3 fraction containing GM3(Neu5Ac) and GM3(Neu5Gc) species and further applied for the analysis of YAC-1 lymphoma cell monosialoganglioside fraction. New insights into YAC-1 monosialoganglioside mixture heterogeneity were obtained: 31 distinct species, comprising 18 Neu5Gc-containing gangliosides and 13 Neu5Ac-containing species of GM1b and GalNAc-GM1b type were found to be expressed by YAC-1 cell line. On-line structural elucidation of individually separated Neu5Ac- and Neu5Gc-containing gangliosides provided strong evidence on the "GM1b-pathway" sourcing for monosialoganglioside synthesis. Such an analytical method is documented as superior to the classical approaches by increased speed of analysis, sensitivity and level of information, being thus a viable glycolipidomic tool.

An Echinococcus multilocularis coproantigen is a surface glycoprotein with unique O-gycosylation

Hulsmeier, A. J, Deplazes, P., Naem, S., Nonaka, N., Hennet, T., Kohler, P. — Tue, 24 Nov 2009 23:57:37 PST

A major surface constituent of Echinococcus multilocularis adult worms, referred to as an EmA9 antigen, was immunoaffinity purified and identified as a high-molecular-weight glycoconjugate. Labeling studies using the monoclonal antibody MAbEmA9 indicated that this antigen undergoes a regulated expression during the development from the larval to the adult parasite. Chemical modification of carbohydrate by periodate oxidation resulted in a reduced reactivity with antigen-specific antibodies. Non-reductive β-elimination of the purified molecule indicated the presence of O-linked glycans attached to threonine residues. Carbohydrate compositional analyses indicated the presence of N- and O-glycans with the ratio of carbohydrate to protein being 1.5:1 (w/w). N- and O-linked glycans were released by hydrazinolysis and analyzed as 2-aminobenzamide derivatized glycans by mass spectrometry together with HPLC and enzymatic sequencing. Novel linear O-linked saccharides with multiple β-HexNAc extensions of reducing end Gal were identified. N-Linked glycans were also detected with oligomannose and mono-, bi-, tri- and tetra-antennary-type structures, most of which were found to be core-fucosylated. Taken together, the results indicate that the EmA9 antigen is a glycoprotein located at the outer surface of the adult E. multilocularis. The observation that the EmA9 antigen expression is developmentally regulated suggests an involvement of this glycoprotein in the establishment of the parasite in its canine host.

Glycobiology - current issue

Glycobiology

Contents

Subscriptions

Meeting Announcements

Glycan gimmickry by parasitic helminths: A strategy for modulating the host immune response?

Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays

Characterization of gene-activated human acid-{beta}-glucosidase: Crystal structure, glycan composition, and internalization into macrophages

Hypoxic regulation of secreted proteoglycans in macrophages

Mammalian cell ganglioside-binding specificities of E. coli enterotoxins LT-IIb and variant LT-IIb(T13I)

Endomannosidase undergoes phosphorylation in the Golgi apparatus

GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells

Characterization of GD3 ganglioside as a novel biomarker of mouse neural stem cells

Structural basis of the affinity for oligomannosides and analogs displayed by BC2L-A, a Burkholderia cenocepacia soluble lectin

Protective effect of N-glycan bisecting GlcNAc residues on {beta}-amyloid production in Alzheimer's disease

Lessons from GNE-deficient embryonic stem cells: sialic acid biosynthesis is involved in proliferation and gene expression

Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines

An Echinococcus multilocularis coproantigen is a surface glycoprotein with unique O-gycosylation