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Characterization and Engineering of S100A12–Heparan sulfate interactions
Glycobiology (IF 4.194) Pub Date : 2020-01-14
Zhang X, Ong C, Su G, et al.
S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12-HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12–HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single point mutation. Using a HS oligosaccharide microarray we demonstrated that the N43K mutant displayed markedly enhanced selectivity towards longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.
Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?
Obukhova P, Tsygankova S, Chinarev A, et al.
Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialo-glycans (Hanganutziu-Deicher Neu5Gcɑ2–3Galβ1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array (PGA) and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics (CFG). As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galβ1-4Glc, but very few true anti-Neu5Gcɑ2–3Galβ1-4Glc antibodies, i.e. antibodies capable of specifically recognizing the entire trisaccharide.
The Sialyltransferase ST6GAL1 Protects Against Radiation-Induced Gastrointestinal Damage
Punch P, Irons E, Manhardt C, et al.
High dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can re-establish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome, for which there are no FDA approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation induced apoptosis in vitro. Here, we used a C57BL/6 St 6 gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. 12 grays TBI followed by BMT is not lethal to wild-type mice, but St 6 gal1-KO counterparts succumbed within 7 days. Both St 6 gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St 6 gal1-KO animals while wild-type counterparts showed signs of recovery by 120 hours after TBI. Increased apoptosis in the GI tracts of St 6 gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.
Differential Distribution of N- and O-Glycans and Variable Expression of Sialyl-T Antigen on HeLa Cells—Revealed by Direct Fluorescent Glycan Imaging
Wu Z, Person A, Zou Y, et al.
Cells are covered with glycans. The expression and distribution of specific glycans on the surface of a cell are important for various cellular functions. Imaging these glycans is essential to aid elucidation of their biological roles. Here, utilizing methods of direct fluorescent glycan imaging, in which fluorescent sialic acids are directly incorporated into substrate glycans via recombinant sialyltranferases, we report the differential distribution of N- and O-glycans and variable expression of sialyl-T antigen on HeLa cells. While the expression of N-glycans tends to be more peripheral at positions where cell-cell interaction occurs, O-glycan expression is more granular but relatively evenly distributed on positive cells. While N-glycans are expressed on all cells, sialyl-T antigen expression exhibits a wide spectrum of variation with some cells being strongly positive and some cells being almost completely negative. The differential distribution of N- and O-glycans on cell surface reflect their distinctive roles in cell biology.
Characterization of disease-specific chondroitin sulfate non-reducing end accumulation in mucopolysaccharidosis IVA
Lawrence R, Prill H, Vachali P, et al.
Morquio syndrome type A, also known as MPS IVA, is a rare autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfatase, a lysosomal hydrolase critical in the degradation of keratan sulfate and chondroitin sulfate. The chondroitin sulfate that accumulates in MPS IVA patients has a disease-specific non-reducing end terminating with N-Acetyl-D-galactosamine 6-sulfate which can be specifically quantified after enzymatic depolymerization of chondroitin sulfate polysaccharide chains. The abundance of N-Acetyl-D-galactosamine 6-sulfate over other possible non-reducing end structures is diagnostic for MPS IVA. Here, we describe an assay for the liberation and measurement of N-Acetyl-D-galactosamine 6-sulfate and explore its application to MPS IVA patient samples in pilot studies examining disease detection, effects of age, and treatment with enzyme replacement therapy. This assay complements the existing urinary keratan sulfate assay by quantifying chondroitin sulfate-derived substrates, which represent a distinct biochemical aspect of MPS IVA. A more complete understanding of the disease could help to more definitively detect disease across age ranges and more completely measure the pharmacodynamic efficacy of therapies. Larger studies will be needed to clarify the potential value of this chondroitin sulfate derived substrate to manage disease in MPS IVA patients.
Structural and functional analysis of four family 84 glycoside hydrolases from the opportunistic pathogen Clostridium perfringens
Pluvinage B, Massel P, Burak K, et al.
The opportunistic pathogen Clostridium perfringens possesses the ability to colonize the protective mucin layer in the gastrointestinal tract. To assist this, the C. perfringens genome contains a battery of genes encoding glycoside hydrolases (GHs) that are likely active on mucin glycans, including four genes encoding family 84 GHs: CpGH84A (NagH), CpGH84B (NagI), CpGH84C (NagJ) and CpGH84D (NagK). To probe the potential advantage gained by the expansion of GH84 enzymes in C. perfringens, we undertook the structural and functional characterization of the CpGH84 catalytic modules. Here, we show that these four CpGH84 catalytic modules act as β-N-acetyl-D-glucosaminidases able to hydrolyze N- and O-glycan motifs. CpGH84A and CpGH84D displayed a substrate specificity restricted to terminal β-1,2- and β-1,6-linked N-acetyl-D-glucosamine (GlcNAc). CpGH84B and CpGH84C appear more promiscuous with activity on terminal β-1,2-, β-1,3- and β-1,6-linked GlcNAc; both possess some activity toward β-1,4-linked GlcNAc, but this is dependent upon which monosaccharide it is linked to. Furthermore, all the CpGH84s have different optimum pHs ranging from 5.2 to 7.0. Consistent with their β-N-acetyl-D-glucosaminidase activities, the structures of the four catalytic modules revealed similar folds with a catalytic site including a conserved −1 subsite that binds GlcNAc. However, nonconserved residues in the vicinity of the +1 subsite suggest different accommodation of the sugar preceding the terminal GlcNAc, resulting in subtly different substrate specificities. This structure–function comparison of the four GH84 catalytic modules from C. perfringens reveals their different biochemical properties, which may relate to how they are deployed in the bacterium's niche in the host.
Intracellular galectins control cellular responses commensurate with cell surface carbohydrate composition
Hong M, Lin W, Weng I, et al.
Galectins are β-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans.
Influence of labeling on the glycan affinities and specificities of glycan-binding proteins. A case study involving a C-terminal fragment of human galectin-3
Kitova E, Han L, Vinals D, et al.
Glycan interactions with glycan-binding proteins (GBPs) play essential roles in a wide variety of cellular processes. Currently, the glycan specificities of GBPs are most often inferred from binding data generated using glycan arrays, wherein the GBP is incubated with oligosaccharides immobilized on a glass surface. Detection of glycan–GBP binding is typically fluorescence-based, involving the labeling of the GBP with a fluorophore or with biotin, which binds to fluorophore-labeled streptavidin, or using a fluorophore-labeled antibody that recognizes the GBP. While it is known that covalent labeling of a GBP may influence its binding properties, these effects have not been well studied and are usually overlooked when analyzing glycan array data. In the present study, electrospray ionization mass spectrometry (ESI-MS) was used to quantitatively evaluate the impact of GBP labeling on oligosaccharide affinities and specificities. The influence of three common labeling approaches, biotinylation, labeling with a fluorescent dye and introducing an iodination reagent, on the affinities of a series of human milk and blood group oligosaccharides for a C-terminal fragment of human galectin-3 was evaluated. In all cases labeling resulted in a measurable decrease in oligosaccharide affinity, by as much as 90%, and the magnitude of the change was sensitive to the nature of the ligand. These findings demonstrate that GBP labeling may affect both the absolute and relative affinities and, thereby, obscure the true glycan binding properties. These results also serve to illustrate the utility of the direct ESI-MS assay for quantitatively evaluating the effects of protein labeling on ligand binding.
ragp: Pipeline for mining of plant hydroxyproline-rich glycoproteins with implementation in R
Dragićević M, Paunović D, Bogdanović M, et al.
Hydroxyproline-rich glycoproteins (HRGPs) are one of the most complex families of macromolecules found in plants, due to the diversity of glycans decorating the protein backbone, as well as the heterogeneity of the protein backbones. While this diversity is responsible for a wide array of physiological functions associated with HRGPs, it hinders attempts for homology-based identification. Current approaches, based on identifying sequences with characteristic motifs and biased amino acid composition, are limited to prototypical sequences. Ragp is an R package for mining and analysis of HRGPs, with emphasis on arabinogalactan proteins. The ragp filtering pipeline exploits one of the HRGPs key features, the presence of hydroxyprolines which represent glycosylation sites. Main package features include prediction of proline hydroxylation sites, amino acid motif and bias analyses, efficient communication with web servers for prediction of N-terminal signal peptides, glycosylphosphatidylinositol modification sites and disordered regions and the ability to annotate sequences through hmmscan and subsequent GO enrichment, based on predicted Pfam domains. As such, ragp extends R's rich ecosystem for high-throughput sequence data analyses. The ragp R package is available under the MIT Open Source license and is freely available to download from GitHub at: https://github.com/missuse/ragp.
Advanced glycation end products (AGEs), protein aggregation and their cross talk: new insight in tumorigenesis
Haque E, Kamil M, Hasan A, et al.
Protein glycation and protein aggregation are two distinct phenomena being observed in cancer cells as factors promoting cancer cell viability. Protein aggregation is an abnormal interaction between proteins caused as a result of structural changes in them after any mutation or environmental assault. Protein aggregation is usually associated with neurodegenerative diseases like Alzheimer's and Parkinson's, but of late, research findings have shown its association with the development of different cancers like lung, breast and ovarian cancer. On the contrary, protein glycation is a cascade of irreversible nonenzymatic reaction of reducing sugar with the amino group of the protein resulting in the modification of protein structure and formation of advanced glycation end products (AGEs). These AGEs are reported to obstruct the normal function of proteins. Lately, it has been reported that protein aggregation occurs as a result of AGEs. This aggregation of protein promotes the transformation of healthy cells to neoplasia leading to tumorigenesis. In this review, we underline the current knowledge of protein aggregation and glycation along with the cross talk between the two, which may eventually lead to the development of cancer.
Expanding the capillary electrophoresis based glucose unit database of the GUcal app
Jarvas G, Szigeti M, Campbell M, et al.
GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis data in the GlycoStore collection (https://www.glycostore.org) and with in-house measured glucose unit values. The GUcal app is freely available online (https://www.gucal.hu) and readily facilitates capillary electrophoresis based high throughput GU value determination for first line structural elucidation.
An atomistic perspective on ADCC quenching by core-fucosylation of IgG1 Fc N-glycans from enhanced sampling molecular dynamics
Harbison A, Fadda E.
The immunoglobulin type G (IgG) Fc N-glycans are known to modulate the interaction with membrane-bound Fc γ receptors (FcγRs), fine-tuning the antibody's effector function in a sequence-dependent manner. Particularly interesting in this respect are the roles of galactosylation, which levels are linked to autoimmune conditions and aging, of core fucosylation, which is known to reduce significantly the antibody-dependent cellular cytotoxicity (ADCC), and of sialylation, which also reduces ADCC but only in the context of core-fucosylation. In this work we provide an atomistic level perspective through enhanced sampling computer simulations, based on replica exchange molecular dynamics (REMD), to understand the molecular determinants linking the Fc N-glycans sequence to the observed IgG1 function. Our results indicate that the two symmetrically opposed N-glycans interact extensively through their core trimannose residues. At room temperature the terminal galactose on the α(1-6) arm is restrained to the protein through a network of interactions that keep the arm outstretched, meanwhile the α(1-3) arm extends towards the solvent where a terminal sialic acid remains fully accessible. We also find that the presence of core fucose interferes with the extended sialylated α(1-3) arm, altering its conformational propensity and as a consequence of steric hindrance, significantly enhancing the Fc dynamics. Furthermore, structural analysis shows that the core fucose position within the Fc core obstructs the access of N162 glycosylated FcγRs very much like a "door-stop", potentially decreasing the IgG/ FcγR binding free energy. These results provide an atomistic level-of-detail framework for the design of high potency IgG1 Fc N-glycoforms.
Heparan sulfate inhibits transforming growth factor β signaling and functions in cis and in trans to regulate prostate stem/progenitor cell activities
Rai S, Alsaidan O, Yang H, et al.
Prostate stem/progenitor cells (PrSCs) are responsible for adult prostate tissue homeostasis and regeneration. However, the related regulatory mechanisms are not completely understood. In this study, we examined the role of heparan sulfate (HS) in PrSC self-renewal and prostate regeneration. Using an in vitro prostate sphere formation assay, we found that deletion of the glycosyltransferase exostosin 1 (Ext1) abolished HS expression in PrSCs and disrupted their ability to self-renew. In associated studies, we observed that HS loss inhibited p63 and CK5 expression, reduced the number of p63+- or CK5+-expressing stem/progenitor cells, elevated CK8+ expression and the number of differentiated CK8+ luminal cells, and arrested the spheroid cells in the G1/G0 phase of cell cycle. Mechanistically, HS expressed by PrSCs (in cis) or by neighboring cells (in trans) each could maintain sphere formation. Furthermore, HS deficiency up-regulated transforming growth factor β (TGFβ) signaling and inhibiting TGFβ signaling partially restored the sphere-formation activity of the HS-deficient PrSCs. In an in vivo prostate regeneration assay, simultaneous loss of HS in both epithelial cell and stromal cell compartments atteneuated prostate tissue regeneration, whereas, the retention of HS expression in either of the two cellular compartments was sufficient to sustain prostate tissue regeneration. We conclude that HS preserves self-renewal of adult PrSCs by inhibiting TGFβ signaling and functions both in cis and in trans to maintain prostate homeostasis and to support prostate regeneration.
Microarray Analyses of Closely Related Glycoforms Reveal Different Accessibilities of Glycan Determinants on N-Glycan Branches
Li L, Guan W, Zhang G, et al.
Glycans mediate a wide variety of biological roles via recognition by glycan-binding proteins (GBPs). Comprehensive knowledge of such interaction is thus fundamental to glycobiology. While the primary binding feature of GBPs can be easily uncovered by using a simple glycan microarray harboring limited numbers of glycans motifs, their fine specificities are harder to interpret. In this study, we prepared 98 closely related N-glycoforms that contain 5 common glycan epitopes which allowed the determination of the fine binding specificities of several plant lectins and anti-glycan antibodies. These N-glycoforms differ from each other at the monosaccharide level and were presented in an identical format to ensure comparability. With the analysis platform we used, it was found that most tested GBPs have preferences toward only one branch of the complex N-glycans, and their binding toward the epitope-presenting branch can be significantly affected by structures on the other branch. Fine specificities described here are valuable for a comprehensive understanding and applications of GBPs.
The O-GalNAcylating enzyme GALNT5 mediates carcinogenesis and progression of cholangiocarcinoma via activation of AKT/ERK signaling
Detarya M, Sawanyawisuth K, Aphivatanasiri C, et al.
Mucin type O-glycosylation is a post-translational modification of membrane and secretory proteins. Transferring of N-acetylgalactosamine, the first sugar of O-glycosylation, is catalyzed by one of the 20 isoforms of polypeptide N-acetylgalactosaminyltransferases (GALNTs). In this study, Vicia villosa lectin (VVL), a lectin that recognizes O-GalNAcylated glycans, was used to detect VVL-binding glycans (VBGs) in cholangiocarcinoma (CCA). The elevation of VBGs in tumor tissues of the liver fluke associated with CCA from hamsters and patients was noted. VBGs were detected in hyperplastic/dysplastic bile ducts and CCA but not in normal biliary epithelia and hepatocytes, indicating the association of VBGs with CCA development and progression. GALNT5 was shown to be the major isoform found in human CCA cell lines with high VBG expression. Suppression of GALNT5 expression using siRNA significantly reduced VBG expression, signifying the connection of GALNT5 and VBGs observed. Knocked-down GALNT5 expression considerably inhibited proliferation, migration and invasion of CCA cells. Increased expression of GALNT5 using pcDNA3.1-GALNT5 expression vector induced invasive phenotypes in CCA cells with low GALNT5 expression. Increasing of claudin-1 and decreasing of slug and vimentin expression, together with inactivation of Akt/Erk signaling were noted in GALNT5 knocked-down cells. These observations were reversed in GALNT5 over-expressing cells. GALNT5 modulated progression of CCA cells was shown to be, in part, via GALNT5 mediated autocrine/paracrine factors that stimulated activations of Akt/Erk signaling and the EMT process. GALNT5 and its O-GalNAcylated products may have important roles in promoting progression of CCA and could possibly be novel targets for treatment of metastatic CCA.
Directed Evolution of a Remarkably Efficient Kdnase from a Bacterial Neuraminidase
Abadi S, Deen M, Watson J, et al.
N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution, and are relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens, and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M–1 s–1; the best variant displayed an efficiency of >106 M–1 s–1 at its optimal pH.
Fucoidan suppresses the gastric cancer cell malignant phenotype and production of TGF-β1 via CLEC-2
Xu L, Liu F, Li C, et al.
The sulfated polysaccharide fucoidan displays excellent anticancer properties with low toxicity in many kinds of cancers. However, its detailed pharmacological effect and mechanism of action in gastric carcinoma remains unclear. In this study, we found that fucoidan could suppress gastric cancer (GC) cell growth, as well as cell migration and invasion. A cytokine expression screen demonstrated that TGF-β1 secretion was decreased in fucoidan-treated cells. Fucoidan has been reported to be a platelet agonist for the C-type lectin-like receptor 2 (CLEC-2), and our previous research found that upregulation of CLEC-2 inhibited GC progression. Here, we confirmed that fucoidan, combined with CLEC-2, significantly increased CLEC-2 expression in GC cells via the transcription factor CDX2, an important regulator of gut homeostasis. In addition, the inhibitory effect of fucoidan on the gastric cancer cell malignant phenotype and TGF-β1 secretion could be restored by knocking down CLEC-2. Thus, our data suggest that fucoidan targets CLEC-2 to exert anti-tumorigenesis and anti-metastatic activity, suggesting that fucoidan is a promising treatment for gastric carcinoma.
Modulation of hepatocyte sialylation drives spontaneous fatty liver disease and inflammation
Oswald D, Jones M, Cobb B.
Circulatory protein glycosylation is a biomarker of multiple disease and inflammatory states and has been applied in the clinic for liver dysfunction, heart disease, and diabetes. With the notable exception of antibodies, the liver produces most of the circulatory glycoproteins, including the acute phase proteins released as a function of the inflammatory response. Among these proteins is β-galactoside α2,6-sialyltransferase (ST6Gal1), an enzyme required for α2,6-linked sialylation of glycoproteins. Here, we describe a hepatocyte-specific conditional knockout of ST6Gal1 (H-cKO) using albumin promoter-driven Cre-lox recombination. We confirm the loss of circulatory glycoprotein α2,6 sialylation and note no obvious dysfunction or pathology in young H-cKO mice, yet these mice show robust changes in plasma glycoprotein fucosylation, branching, and the abundance of bisecting GlcNAc and marked changes in a number of metabolic pathways. As H-cKO mice aged, they spontaneously developed fatty liver disease characterized by the buildup of fat droplets in the liver, inflammatory cytokine production, and a shift in liver leukocyte phenotype away from anti-inflammatory Kupffer cells and towards pro-inflammatory M1 macrophages. These findings connect hepatocyte and circulatory glycoprotein sialylation to the regulation of metabolism and inflammation, potentially identifying the glycome as a new target for liver-driven disease.
Identification of Tn Antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies
Matsumoto Y, Kudelka M, Hanes M, et al.
The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody, and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors, and represent promising tools for Tn biomarker discovery independently of recognition of IgA1. [191 words].
Glycoxidative profile of cancer patient serum: A clinical result to associate glycation to cancer
Khan H, Alouffi S, Alatar A, et al.
The influence of advanced glycation end products (AGEs) in the biological processes contribute to the life changing complications such as progression of cancer, diabetes and other chronic disorder. The receptor of AGEs while interacting with its ligands causes a never ending irregularity in the cell signaling communication. Hence, AGEs is considered as an important link between progression and contribution to cancer. This study focuses on the presence and/or absence of oxidative and glycative stress in the serum samples of various cancer patients. During analysis of the early and intermediate glycation product in cancer patient's sera, our result indicates an increasing trend of both the adducts as compared to normal healthy subjects (NHS). Similarly, one of the AGEs i.e., carboxymethyllysine (CML) was found to be enhanced in cancer sera as compared to NHS. The binding characteristics of circulating auto-antibodies in cancer patient's sera against HSA-AGEs were assessed through ELISA and further, the maximum percent inhibition against HSA-AGEs was observed as 57–63%, 46–62% and 42–64% in prostate cancer, lung cancer and head & neck cancer. Hence our result successfully assisted the presence of AGEs in all the cancer patient's sera though it is not clear which specific cancer is more potent to AGEs.
Cosmc is required for T cell persistence in the periphery
Cutler C, Jones M, Cutler A, et al.
T lymphocytes, a key arm of adaptive immunity, are known to dynamically regulate O-glycosylation during T cell maturation and when responding to stimuli; however, the direct role of O-glycans in T cell maturation remains largely unknown. Using a conditional knockout of the gene (C1GalT1C1 or Cosmc) encoding the specific chaperone Cosmc, we generated mice whose T cells lack extended O-glycans (T cell conditional Cosmc knock out or TCKO mice) and homogeneously express the truncated Tn antigen. Loss of Cosmc is highly deleterious to T cell persistence, with near-complete elimination of Cosmc-null T cells from spleen and lymph nodes. Total T cell counts are 20% of wild type (WT), among which only 5% express the truncated glycans, with the remaining 95% consisting of escapers from Cre-mediated recombination. TCKO thymocytes were able to complete thymic maturation but failed to populate the secondary lymphoid organs both natively and upon adoptive transfer to WT recipients. Our results demonstrate that extended O-glycosylation is required for the establishment and maintenance of the peripheral T cell population.
Direct fluorescent glycan labeling with recombinant sialyltransferases
Wu Z, Person A, Burton A, et al.
Glycosylation is a common modification found on numerous proteins and lipids. However, direct detection of glycans on these intact biomolecules has been challenge. Here, utilizing enzymatic incorporation of fluorophore-conjugated sialic acids, dubbed as direct fluorescent glycan labeling, we report the labeling and detection of N- and O-glycans on glycoproteins. The method allows detection of specific glycans without the laborious gel blotting and chemiluminescence reactions used in Western blotting. The method can also be used with a variety of fluorescent dyes.
Bottom-up analysis using liquid chromatography–Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers
Yan L, Li L, Li J, et al.
Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure–activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography–Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.
PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells
Legros N, Ptascheck S, Pohlentz G, et al.
Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1–4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1–4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1–4Galβ1–4Glcβ1–1Cer), globotetraosylceramide (Gb4Cer, GalNAcβ1–3Galα1–4Galβ1–4Glcβ1–1Cer) and Forssman GSL (GalNAcα1–3GalNAcβ1–3Galα1–4Galβ1–4Glcβ1–1Cer) revealed balanced adhesion toward the three GSLs for PapG I–mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III–harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface–exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.
Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity
Joshi R, Trinkl J, Haugeneder A, et al.
Uridine diphosphate-dependent glycosyltransferases (UGTs) catalyze the transfer of a diversity of sugars to several acceptor molecules and often exhibit distinct substrate specificity. Modulation of glycosyltransferases for increased catalytic activity and altered substrate or product specificity are the key manipulations for the biotechnological use of glycosyltransferases in various biosynthetic processes. Here, we have engineered the binding pocket of three previously characterized Vitis vinifera glycosyltransferases, UGT88F12, UGT72B27 and UGT92G6, by structure-guided in silico mutagenesis to facilitate the interactions of active site residues with flavonol glucosides and thus modify substrate specificity and activity. Site-directed mutagenesis at selected sites, followed with liquid chromatography–mass spectrometry based activity assays, exhibited that mutant UGTs were altered in product selectivity and activity as compared to the wild-type enzymes. Mutant UGTs produced larger amounts of flavonol di-monosaccharide glucosides, which imply that the mutations led to structural changes that increased the volume of the binding pocket to accommodate a larger substrate and to release larger products at ease. Mutants showed increased activity and modified product specificity. Thus, structure-based systematic mutations of the amino acid residues in the binding pocket can be explored for the generation of engineered UGTs for diverse biotechnological applications.
GAG positioning on IL-1RI; A mechanism regulated by dual effect of glycosylation
Azimzadeh Irani M, Ejtehadi M.
IL-1RI is the signaling receptor for the IL-1 family of cytokines that are involved in establishment of the innate and acquired immune systems. Glycosylated extracellular (EC) domain of the IL-1RI binds to agonist such as IL-1β or antagonist ligands and the accessory protein to form the functional signaling complex. Dynamics and ligand binding of the IL-1RI is influenced by presence of the glycosaminoglycans (GAGs) of the EC matrix. Here a combination of molecular dockings and molecular dynamics simulations of the unglycosylated, partially N-glycosylated and fully N-glycosylated IL-1RI EC domain in the apo, GAG-bound and IL-1β-bound states were carried out to explain the co-occurring dynamical effect of receptor's glycosylation and GAGs. It was shown that the IL-1RI adopts two types of "extended" and "locked" conformations in its dynamical pattern, and glycosylation maintains the receptor in the latter form. Maintaining the receptor in the locked conformation disfavors IL-1β binding by burying its two binding site on the IL-1RI EC domain. Glycosylation disfavors GAG binding to the extended IL-1RI EC domain by sterically limiting the GAGs degrees of freedom in targeting its binding site, while it favors GAG binding to the locked IL-1RI by favorable packing interactions.
Carbo-loading in Coccidioides spp.: a quantitative analysis of CAZyme abundance and resulting glycan populations
Mitchell N, Grys T, Lake D.
Coccidioides spp. are important pneumonia-causing pathogens of the American southwest, but little is known about their glycobiology and how their glycosylations differ from other pneumonia-causing fungi. There is mounting preliminary evidence to suggest genus or even species-specific glycosylations in the fungal kingdom due to the presence of unique Carbohydrate-Active Enzymes (CAZymes) in fungal genomes (Deshpande and others 2008; Karkowska-Kuleta and Kozik 2015). If Coccidioides spp.-specific glycans can be identified, it may be possible to exploit these differences to develop more specific diagnostic approaches and more effective therapeutics. Herein we i) mined Coccidioides spp. and other pathogenic fungal genomes to identify CAZymes specific for Coccidiodes spp., ii) proteomically determined the Coccidioides spp. "CAZome" produced in vivo and in vitro, and iii) utilized glycomics to differentiate Coccidioides genus-specific N-glycans from other pathogenic fungi. As far as we are aware, this is the first proteomic and glycomic comparison of the N-glycomes and CAZomes of different fungal genera during infection in human hosts.
Real- time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells
Detzner J, Steil D, Pohlentz G, et al.
Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the nonDRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the nonDRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins towards DRMs and failure in interaction with nonDRMs. These results support the hypothesis of preferential binding of Stxs towards microdomains harbouring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unravelling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.
Society for Glycobiology Awards-2019.
GlyGen: Computational and Informatics Resources for Glycoscience.
Glycobiology (IF 4.194) Pub Date : null
William S York,Raja Mazumder,Rene Ranzinger,Nathan Edwards,Robel Kahsay,Kiyoko F Aoki-Kinoshita,Matthew P Campbell,Richard D Cummings,Ten Feizi,Maria Martin,Darren A Natale,Nicolle H Packer,Robert J Woods,Gaurav Agarwal,Sena Arpinar,Sanath Bhat,Judith Blake,Leyla Jael Garcia Castro,Brian Fochtman,Jeffrey Gildersleeve,Radoslav Goldman,Xavier Holmes,Vinamra Jain,Sujeet Kulkarni,Rupali Mahadik,Akul Mehta,Reza Mousavi,Sandeep Nakarakommula,Rahi Navelkar,Nagarajan Pattabiraman,Michael J Pierce,Karen Ross,Preethi Vasudev,Jeet Vora,Tatiana Williamson,Wenjin Zhang
The GlySpace Alliance: towards a collaborative global glycoinformatics community.
Kiyoko F Aoki-Kinoshita,Frederique Lisacek,Raja Mazumder,William S York,Nicolle H Packer
Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays.
Chandrassegar Saravanan,Zhiyi Cao,Steven R Head,Noorjahan Panjwani
Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.
Jesús E Serrano-Negrón,Zhenbo Zhang,Andrea P Rivera-Ruiz,Aditi Banerjee,Eva C Romero-Nutz,Neysharie Sánchez-Torres,Krishna Baksi,Dipak K Banerjee
Identification and characterization of a Drosophila melanogaster ortholog of human beta1,4-galactosyltransferase VII.
Nadia Vadaie,Rebecca S Hulinsky,Donald L Jarvis
Drosophila melanogaster is widely considered to be an attractive model organism for studying the functions of the carbohydrate moieties of glycoconjugates produced by higher eukaryotes. However, the pathways of glycoconjugate biosynthesis are not as well defined in insects as they are in higher eukaryotes. One way to address this problem is to identify genes in the Drosophila genome that might encode relevant functions, express them, and determine the functions of the gene products by direct biochemical assays. In this study, we used this approach to identify a putative Drosophila beta4-galactosyltransferase gene and determine the enzymatic activity of its product. Biochemical assays demonstrated that this gene product could transfer galactose from UDP-galactose to a beta-xylosyl acceptor, but not to other acceptors in vitro. The apparent K(m) values for the donor and acceptor substrates indicated that this gene product is a functional galactosyltransferase. Additional assays showed that the enzyme is activated by manganese, has a slightly acidic pH optimum, and is localized in the insect cell Golgi apparatus. These results showed that Drosophila encodes an ortholog of human beta4-galactosyltransferase-VII, also known as galactosyltransferase I, which participates in proteoglycan biosynthesis by transferring the first galactose to xylose in the linkage tetrasaccharide of glycosaminoglycan side chains.
Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase.
C A Stern,T R Braverman,M Tiemeyer
A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.
Since there are PAMPs and DAMPs, there must be SAMPs? Glycan "self-associated molecular patterns" dampen innate immunity, but pathogens can mimic them.
Ajit Varki
Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities.
Christoph Geisler,Donald L Jarvis
Molecular models for mouse sperm-oocyte binding.
Gary F Clark
Binding of sperm to egg in the mouse has been proposed to depend primarily on interactions between lectin-like egg-binding proteins on the sperm plasma membrane and complementary carbohydrates on the specialized extracellular matrix of the egg known as the zona pellucida. An alternative model posits that initial sperm-egg binding depends on the interaction of a sperm surface protein with a supramolecular complex of the three mouse zona pellucida glycoproteins (mZP1, mZP2, mZP3); the role of carbohydrate recognition in this paradigm is thought to be minimal (Gahlay G, Gauthier L, Baibakov B, Epifano O,Dean J. 2010. Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein. Science.329:216-219). This perspective reviews these recent findings,and considers them in light of evidence favoring a major role for lectin-like interactions. An alternative model, the domain specific model for mammalian gamete binding, could reconcile some of the conflicting observations.
Cholera toxin B subunit binding does not correlate with GM1 expression: a study using mouse embryonic neural precursor cells.
Makoto Yanagisawa,Toshio Ariga,Robert K Yu
Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitously expressed in all eukaryotic cells and are primarily localized in the plasma membrane. Cholera toxin B subunit (Ctxb), a component of a heat-labile enterotoxin produced by Vibrio cholerae, has been frequently used as a probe to detect GM1 ganglioside because of its high affinity for this glycolipid. In this study, we evaluated the reactivity of Ctxb and the expression of GM1 in mouse embryonic neuroepithelial cells (NECs). Analysis of Ctxb reactivity of NECs based on flow cytometry revealed that about 80% of the cells are Ctxb positive. A detailed biochemical analysis, however, indicated that GM1 was expressed in NECs in barely detectable quantities. Thus, it was thought that reactivity of Ctxb in the NECs could arise from high-affinity interaction with GM1. Because Ctxb is commonly used as a reagent for flow cytometry and GM1 cell staining, we recommend that using this reagent alone would be inconclusive and that biochemical analysis of GM1 should also be performed to avoid overestimation of GM1 expression and/or mischaracterization of the ganglioside species.
Preparation of CD25(+) and CD25(-) CD4(+) T cells for glycomic analysis--a cautionary tale of serum glycoprotein sequestration.
C R Monk,M Sutton-Smith,A Dell,O A Garden
Asparagine linked glycosylation in Giardia.
Phillips W Robbins,John Samuelson
SphinGOMAP--a web-based biosynthetic pathway map of sphingolipids and glycosphingolipids.
Alfred H Merril
A green thumb and a broad back: a tribute to the late Dr. Yasuo Inoue (1934-2005).
Y C Lee
Letter to the Glyco-Forum: Karl Meyer, lysozyme, and penicilin.
Nathan Sharon
Shooting HARE.
Bård Smedsrød,Staffan Johansson,Sergij Goerdt
Alternative and sources of reagents and supplies of fluorophore-assisted carbohydrate electrophoresis(FACE).
Mark A Lehrman,Ningguo Gao
Expression of histo-blood group A antigen increases resistance to apoptosis and facilitates escape from immune control of rat colon carcinoma cells.
Séverine Marionneau,Béatrice Le Moullac-Vaidye,Jacques Le Pendu
A and B histo-blood group antigens are present on carcinoma cells at the early stages of cancerogenesis and tend to disappear at later stages, but it is not yet clear whether they take part to the process of tumor progression. To gain some insight into this issue, we used a rat colon carcinoma experimental model. To obtain expression of the A antigen, REG cells were cotransfected with the rat A enzyme cDNA and a rat alpha1,2fucosyltransferase cDNA, either FTA or FTB, whereas PRO cells that spontaneously have alpha1,2fucosyltransferase activity were only transfected with the A enzyme cDNA. All A antigen-expressing transfected cells derived from either REG FTA, REG FTB, or PRO parental cells were more resistant to apoptosis induced by either serum deprivation or heat shock than were their respective controls. When injected to syngeneic immunocompetent rats, A enzyme-transfected PRO cells formed tumors that grew faster than those formed by mock-transfected PRO cells. However, in immunodeficient SCID mice, no difference in growth could be observed between the two types of tumors, indicating that the faster tumor growth of the A antigen-positive cells in immunocompetent animals was due to their higher ability to escape immune control and that this was associated with their higher degree of resistance to apoptosis. These results might explain the slightly augmented incidence of carcinomas observed in A and B blood group individuals compared to O individuals.
Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei.
Joseph P M Hui,Theresa C White,Pierre Thibault
Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.
An investigation of the interactions of E-selectin with fuco-oligosaccharides of the blood group family.
María J Martín,Ten Feizi,Christine Leteux,Davor Pavlovic,Vladimir E Piskarev,Wengang Chai
This investigation is concerned with assignments of Lewis(a) (Le(a)) and Le(x) analogs on linear and branched di- to hexasaccharide backbones as components of the recognition motifs for E-selectin. The influence of the location of fucose residue(s) was investigated using 14 structurally defined and variously fucosylated oligosaccharides in biotinylated form or as neoglycolipids in static binding assays, in microwells, and on thin-layer chromatograms. Results of the two assay systems were in agreement overall and showed that the recognition motifs for E-selectin include 4-fucosyl-lacto (Le(a)) and 3-fucosyl-neo-lacto (Le(x)) sequences strictly at capping positions and not Le(x) at an internal position as a part of VIM-2 antigen sequence. There is greater potency of the Le(a) over the Le(x) series. Additional fucose residues alpha1-2-linked to neighboring galactoses or alpha1-3-linked to inner N-acetyglucosamines or to reducing-terminal glucose residues of the tetrasaccharide backbone had little or no effect on the selectin binding. E-selectin binding to the Le(a) or Le(x )capping motif on a 3-linked branch was equivalent to the binding on the corresponding linear backbone. A lack of E-selectin binding to the Le(x) motif capping a 6-linked branch and to the Le(x) trisaccharide linked to biotin via a nine-carbon spacer indicates that the -GlcNAcbeta1-3Gal- sequence on the oligosaccharide backbone adjoining the Le(x) is a part of recognition motif for E-selectin. These findings contribute to understanding the molecular basis of E-selectin recognition and could influence future designs of selectin antagonists as possible therapeutic substances.
Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease.
Yasunori Chiba,Hitoshi Sakuraba,Masaharu Kotani,Ryoichi Kase,Kazuo Kobayashi,Makoto Takeuchi,Satoshi Ogasawara,Yutaka Maruyama,Tasuku Nakajima,Yuki Takaoka,Yoshifumi Jigami
A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.
Identification, cloning, purification, and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.
Ann Marie Bailey,Sebabrata Mahapatra,Patrick J Brennan,Dean C Crick
The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).
Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein.
Tadashi Suzuki,William J Lennarz
When glycoproteins formed in the endoplasmic reticulum (ER) are misfolded, they are generally translocated into the cytosol for ubiquitination and are subsequently degraded by the proteasome. This system, the so-called ER-associated glycoprotein degradation, is important for eukaryotes to maintain the quality of glycoproteins generated in the ER. It has been established in yeast that several distinct proteins are involved in this translocation and degradation processes. Small glycopeptides formed in the ER are exported to the cytosol in a similar manner. This glycopeptide export system is conserved from yeast to mammalian cells, suggesting its basic biological significance for eukaryotic cells. These two export systems (for misfolded glycoproteins and glycopeptides) share some properties, such as a requirement for ATP and involvement of Sec61p, a central membrane protein presumably forming a dislocon channel for export of proteins. However, the machinery of glycopeptide export is poorly understood. In this study, various mutants known to have an effect on export/degradation of misfolded glycoproteins were examined for glycopeptide export activity with a newly established assay method. Surprisingly, most of the mutants were found not to exhibit a defect in glycopeptide export. The only gene that was found to be required on efficient export of both types of substrates was PMR1, the gene encoding the medial-Golgi Ca(2+)/Mn(2+)-ion pump. These results provide evidence that although the systems involved in export of misfolded glycoproteins and glycopeptides share some properties, they have exhibited distinct differences.
Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity.
Brooke D Lazarus,Julie Milland,Paul A Ramsland,Effie Mouhtouris,Mauro S Sandrin
Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.
A complete alpha1,3-galactosyltransferase gene is present in the human genome and partially transcribed.
Marion Lantéri,Valérie Giordanengo,Frédérique Vidal,Patrick Gaudray,Jean-Claude Lefebvre
The synthesis of Galalpha1-3Gal-terminated oligosaccharides (alpha-Gal) epitopes has been interrupted during the course of evolution, starting with Old World primates. Partial sequences similar to the alpha1,3-galactosyltransferase (alpha1,3GalT) gene, which governs the synthesis of alpha-Gal epitopes, have been detected in the human genome and were found to correspond to pseudogenes. We completed the sequence of the human alpha1,3GalT pseudogene present on chromosome 9 and found it to be organized like the murine alpha1,3GalT gene. In human cell lines and several normal and tumor tissues we detected truncated transcripts corresponding to this pseudogene. Considering these mRNAs, translation of an open reading frame containing the first four translated exons but missing the two catalytic exons could predict a truncated alpha1,3GalT polypeptide that should be enzymatically inactive. We show that transcription of human alpha1,3GalT is prematurely terminated at the level of a strong transcriptional stop signal in the middle of intron VII. We were able to reproduce this effect in vitro by subcloning the implicated DNA region upstream from a reporter cDNA. The premature transcriptional arrest of human alpha1,3-GalT gene leads to an ectopic splicing event and to the connection of a short intronic sequence downstream from translated exons. Finally, we show that these truncated transcripts are overexpressed in cell lines with modifications of O-glycans.
Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids.
Tobias Willer,Werner Amselgruber,Rainer Deutzmann,Sabine Strahl
Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans. They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease. Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes. In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed. The shorter transcript is present in all somatic tissues examined. Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa. The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform. Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization. Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.
O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase.
Li Shao,Yi Luo,Daniel J Moloney,Robert Haltiwanger
O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.
The Saccharomyces cerevisiae alg12delta mutant reveals a role for the middle-arm alpha1,2Man- and upper-arm alpha1,2Manalpha1,6Man- residues of Glc3Man9GlcNAc2-PP-Dol in regulating glycoprotein glycan processing in the endoplasmic reticulum and Golgi apparatus.
John F Cipollo,Robert B Trimble
N-glycosylation in nearly all eukaryotes proceeds in the endoplasmic reticulum (ER) by transfer of the precursor Glc(3)Man(9)GlcNAc(2) from dolichyl pyrophosphate (PP-Dol) to consensus Asn residues in nascent proteins. The Saccharomyces cerevisiae alg (asparagine-linked glycosylation) mutants fail to synthesize oligosaccharide lipid properly, and the alg12 mutant accumulates a Man(7)GlcNAc(2)-PP-Dol intermediate. We show that the Man(7)GlcNAc(2) released from alg12Delta-secreted invertase is Manalpha1,2Manalpha1,2Manalpha1,3(Manalpha1,2Manalpha1,3Manalpha1,6)-Manbeta1,4-GlcNAcbeta1-4GlcNAcalpha/beta, confirming that the Man(7)GlcNAc(2) is the product of the middle-arm terminal alpha1,2-mannoslytransferase encoded by the ALG9 gene. Although the ER glucose addition and trimming events are similar in alg12Delta and wild-type cells, the central-arm alpha1,2-linked Man residue normally removed in the ER by Mns1p persists in the alg12Delta background. This confirms in vivo earlier in vitro experiments showing that the upper-arm Manalpha1,2Manalpha1,6-disaccharide moiety, missing in alg12Delta Man(7)GlcNAc(2), is recognized and required by Mns1p for optimum mannosidase activity. The presence of this Man influences downstream glycan processing by reducing the efficiency of Ochlp, the cis-Golgi alpha1,6-mannosyltransferase responsible for initiating outer-chain mannan synthesis, leading to hypoglycosylation of external invertase and vacuolar protease A.
Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides.
N Fraysse,S Jabbouri,M Treilhou,F Couderc,V Poinsot
When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.
Reduction of alpha-Gal expression by relocalizing alpha-galactosidase to the trans-Golgi network and cell surface.
Simon G Taylor,Narin Osman,Ian F C McKenzie,Mauro S Sandrin
Historically, the most effective means of modifying cell surface carbohydrates has required the intracellular overexpression of glycosyltransferases or glycosidases and is dependent on the enzymes occupying a cellular localization close to the carbohydrate structures they modify. We report on relocalizing the lysosomal resident glycosidase human alpha-galactosidase to other regions of the cell, Golgi and cell surface, where it is in closer proximity for cleaving the carbohydrate structure Galalpha(1,3)Gal. Relocalization of alpha-galactosidase was achieved by using the transmembrane and cytoplasmic domains from the human protein furin, which is known to localize in the trans-Golgi network (TGN) and cell surface. Two chimeric forms of alpha-galactosidase were generated, one directing it to the TGN of the cell and the other to the cell surface, as shown by confocal microscopy. The relocalized enzymes have the ability to cleave terminal alpha-galactose as detected by expression on the cell surface. Furthermore, when expressed as a transgene in mice, the TGN form of alpha-galactosidase was more effective at decreasing cell surface terminal alpha-galactose than was the native lysosomal form. When expressed in conjunction with the alpha1,2fucosyltransferase that also decreases Galalpha(1,3)Gal, the reduction was additive. The ability to relocalize enzymes that modify cell surface carbohydrate structures has far-reaching implications in biology and may be useful in such fields as xenotransplantation and treatment of glycosidase disorders.
Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling.
Miriam Ford-Perriss,Scott E Guimond,Una Greferath,Magdalena Kita,Kay Grobe,Hiroko Habuchi,Koji Kimata,Jeffrey D Esko,Mark Murphy,Jeremy E Turnbull
Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.


Advanced Simulation Technology inc. (ASTi), a leading provider of modular, product-based solutions to the simulation market, last week demonstrated a revolutionary low-cost commercial flight training system at the European Airline Training Symposium (EATS) in Berlin. The system, which consists of ASTi's new Simulated Environment for Realistic ATC (SERA) and the Airliner1 Flight Management Computer (FMC) trainer, is poised to address important commercial aviation industry challenges such as the lack of realistic air traffic control (ATC) communications training and overcrowded training centers by introducing a low-cost option that offloads demand from Level D simulators.
At EATS, representatives from the airline industry, flight training centers and flight simulator manufacturers had a chance to test the system. Demonstrations included ASTi's SERA in Airliner1's FMC trainer as well as SERA in Airliner1's fully simulated Boeing 737NG cockpit with a Cobra Simulation mini-dome display. The new FMC trainer addresses two critical training needs. The first is a realistic ATC environment, which provided by SERA, exposes a trainee to the pressures and workload of an aircraft flight deck environment. This includes experiencing and interacting with ATC controllers and other aircraft radio traffic while following a realistic flight plan entered into the Control Display Unit (CDU). To date, ATC training has been limited and unrealistic, largely relying on role-playing by an instructor. Second, type-conversion training on a specific FMC/CDU typically takes place in an FFS Level D cockpit, which is the only place a fully functional hardware CDU is available, and where a few hours may cost many thousands of dollars. The FMC trainer provides a fully implemented hardware CDU simulation including maintenance menus, and enables crews and maintenance personnel full exposure to a type-specific CDU in an affordable part-task trainer package. Currently available for 737NG aircraft, future FMC trainer versions will include Airbus A320 and Boeing 747, with others to follow based on demand.
"Flight training centers are often overcrowded and simulated practice flights are at the mercy of when the center's next Level D flight simulator becomes available," says ASTi Training Specialist Scott Moore. "With the combined solution of Airliner1's FMC trainer and ASTi's SERA, flight training centers will be able to provide trainees with a method of flying a simulated route and practicing realistic ATC communications on a platform that costs less than 1 percent of a Level D flight simulation training device."


text:
Hlongwane sings same tune as other witnesses
By Corruption Watch 15 Dec 2014, 12:39
By Lee-Ann Alfreds
He came, he spoke, he left. That was the sum of Fana Hlongwane's eagerly-awaited appearance at the Arms Procurement Commission.
In one of the shortest appearances on the stand, the middleman accused of pocketing hundreds of millions from the arms deal:
* first tried to have the media banned from taking photographs and video recordings while he testified;
* then questioned why he had been called as "times had changed and a black man from Soweto could also be progressive";
* before denying paying anyone involved in the arms deal to influence the awarding of contract.
A one-time advisor to former defence minister Joe Modise, Hlongwane is accused by arms critics of being a key intermediary who – on behalf of arms deal company BAE – pushed for South Africa to buy Hawk trainer jets and Gripen fighter aircraft from BAE-Saab, which formed the bulk of the package. The City Press revealed BAE had forked out over R200-milion in commission to Hlongwane for assisting them in winning arms deal contracts and managing their offset projects in South Africa.
The Arms Procurement Commission is investigating allegations of fraud and corruption in the arms deal which is estimated to have cost South Africa more than R70-billion.
Eventful appearance
Despite its brevity, the appearance of Hlongwane – whose testimony was postponed last month after his legal team indicated they were not ready to proceed – was eventful.
First, his advocate Jaap Celliers applied to bar the media from showing his picture because, according to his lawyer, this would infringe on his client's safety, privacy and the security of his family, and negatively affect his business, Sapa reported. "Up until now, there has never been any publication of him in the press and he's able to move around freely," said Celliers, highlighting the fact that his client was a "very private" person.
"If published, everyone in South Africa will recognise him and this will affect his family," he said, adding that there were previous threats made against Hlongwane.
Celliers requested commission chairman Willie Seriti order that no pictures be taken of Hlongwane during the proceedings and even in the foyer during adjournments.
Seriti had indicated that he had no authority outside the chambers before Hlongwane abandoned his application.
Taking the stand, Hlongwane then questioned why he had been called to testify, Sapa reported. "Why am I here?" he asked the commission.
He said he did not understand why there was an issue over the payments made to him when he was Modise's consultant because it was up to two entities to decide on payment. "You cannot criminalise a businessman simply because of quantum," he said.
Lashing out at critics who had claimed he was involved in corrupt activities which resulted in the massive payouts, Hlongwane said they did this because they failed to understand that times had changed and a black man from Soweto could also be progressive.
"I do not support an inferiority mindset," said Hlongwane.
He claimed that when white people funds pocketed funds, these were regarded as bonuses which were above board.
More denials
Hlongwane said none of the witnesses who had testified had provided any evidence supporting claims of corruption in the deal.
He said former president Thabo Mbeki and various cabinet ministers who had testified had provided clear and unequivocal evidence.
"They rejected any suggestion of improper influence relating to the award or conclusion of any of the SDPP [strategic defence procurement package] contracts," said Hlongwane. "Apart from the fact that their evidence was clear on this issue, their evidence was never disputed."
Hlongwane denied allegations that he or his company had had any influence on the arms deal.
"I can further categorically state that I did not pay any gratification to anybody who was involved in the procurement process in order to influence such person relating to the award or conclusion of any of the contracts awarded and concluded in the SDPP," he said.
Hlongwane, the last witness to testify, had been scheduled to deliver his testimony and cross-examination over two days. But he concluded his testimony in less than one without any questions being posed from the panel and legal representatives.
The term of the commission has been extended to April 30 next year.
He came, he spoke, he left. That was the sum of Fana Hlongwane's eagerly-awaited appearance at the Arms Procurement Commission. Former defence advisor Hlongwane questioned why he was even asked to appear, and like others before him, denied any knowledge of irregular goings-on.
hlongwane-text.jpg
Tags: arms deal, Corruption Watch, corruption, bribery, official site, Seriti Commission, arms procurement commission, Fana Hlongwane
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redpajama_set_name: RedPajamaCommonCrawl


BRAMPTON, ON (August 2, 2018) – West Nile virus has once again been detected in mosquitos collected in the Region of Peel. So far this summer, three mosquito pools have tested positive for the virus. The Region reminds residents that it is not uncommon to find West Nile virus in local mosquitos.
West Nile virus became established across much of North America in the late 1990s and public health units, including Region of Peel – Public Health, have run annual programs to help monitor and control the population of mosquitos that carry the virus.
"Public Health maintains and monitors 33 mosquito traps that are set up across the cities of Mississauga and Brampton and the Town of Caledon. These traps are tested weekly from June to September and with this recent confirmation of West Nile virus activity, we are increasing our efforts in the area surrounding the positive traps by looking for breeding sites and re-treating catch basins with larvicide," says Paul Callanan, Director of Health Protection at the Region of Peel.
The Region of Peel encourages residents to take extra precautions during dusk and dawn when mosquito activity is higher. Wear light coloured, long sleeved shirts and pants and use mosquito repellent containing DEET if older than six months of age. Children under six months can be protected with insect netting.
Turn over old tires or wheelbarrows; empty out pails and plastic wading pools; change the water in birdbaths often.
Report standing water if present longer than a week on public property at peelregion.ca/health/vbd/rep-stwater or by calling Region of Peel - Public Health at 905-799-7700.
The Region of Peel has had no confirmed human cases of West Nile virus this season.
For more information, visit peelregion.ca/westnile or call Region of Peel - Public Health at 905-799-7700 from 8:30 a.m. to 4:30 p.m., Monday to Friday. Caledon residents can call toll free at 905-584-2216.


text:
Posted on October 21, 2015 by Community Common
Piketon's Legg signs with Rio Grande
JACKSON, Ohio – University of Rio Grande head volleyball coach Billina Donaldson has announced the recent signing of Piketon High School senior Emily Legg for the 2016 season.
Legg, a 5-foot-11 middle blocker/outside hitter, has been sidelined of late by injury for head coach Allison Robbins's RedStreaks, who take a 11-9 record into play on Tuesday night.
"I'm beyond excited to have the opportunity to sign with Rio Grande," said Legg, whose brother, Evan, completed a four-year career with the Rio men's basketball team last season. "I'm looking forward to my future at Rio and I know my best volleyball is ahead of me.
This season, Legg totaled 86 kills, 51 digs, 21 blocks (20 solos) and 12 service aces. As a junior, she had 64 kills, 35 digs, 31 blocks and eight aces.
Off the court, Legg is a High Honors student who ranks third in her graduating class. She's also a member of the National Honor Society.
"Coach Donaldson really believes in me and has confidence that I will fit well into the team," Legg said. "Knowing that my former teammate – and now teammate again – Aleah Pelphrey, has had so much success at Rio influenced my decision. The village of Rio Grande is similar to Piketon – a very small, tight-knit community which has a family atmosphere that I feel most comfortable in."
Donaldson said that Legg should provide immediate depth, not to mention competition, along the net.
"Emily will make a fine addition to our front row," Donaldson said. "We're looking forward to having another tall girl who can challenge our hitters for playing time. She does a lot of things well and we can't wait to see how she develops in the coming years."
Legg, who plans to major in Middle Childhood Education (English and History), was familiar with Rio Grande even before her brother chose to play for the RedStorm, attending a number of basketball camps as a youngster.
Legg said she knows she needs to become a better hitter, but added that it's a process that she's ready to get started on.
"My best assets as a player are my ability to block, my hitting, my court knowledge and leadership," she said. "I need to be more versatile and have more 'power' behind my hit – to take a better approach."
And what does Legg hope her freshman campaign produces?
"Personally, I want to contribute to my team's success and earn quality minutes," she said. "Overall, I want to make the national tournament and win."
Legg is the daughter of Scott and Tracy Legg of Piketon.
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text:
McSherrystown gives Conewago ultimatum
The negotiations over McSherrystown Borough and Conewago Township's plan to merge police departments came to a crest Monday
McSherrystown gives Conewago ultimatum The negotiations over McSherrystown Borough and Conewago Township's plan to merge police departments came to a crest Monday Check out this story on eveningsun.com: http://evesun.co/2ewxmGr
Lillian Reed, lreed@eveningsun.com Published 3:22 p.m. ET Oct. 13, 2016 | Updated 4:11 p.m. ET Oct. 13, 2016
File Photo(Photo: Davin Jurgensen - The Evening Su)
Conewago Township and McSherrystown Borough have been in talks to merge police departments for more than a year.
Conewago officials indicated this week that they wish for their newly hired police chief to also be chief of the regional police force when it is created.
McSherrystown officials gave Conewago an ultimatum that they must agree to allow the borough's chief to be second-in-command or risk the merger falling apart.
The tumultuous negotiations over McSherrystown Borough and Conewago Township's plan to merge police departments came to a crest Wednesday, with borough officials issuing the township an ultimatum.
If Conewago officials want their newly hired police chief, Curtis McCoy, to be the chief of a larger, regional force, officials must agree to allow McSherrystown's chief, Michael Woods, to be second-in-command.
Those were the latest terms discussed at the McSherrystown Borough Council meeting Wednesday. No members of the public were present.
Council member Douglas Duvall told fellow officials Wednesday that Conewago expects its new chief to become the chief of the regional force.
McSherrystown Mayor Anthony Weaver initially wasn't comfortable striking such a bargain without considering Woods for the job as well, he said.
"When I talked to (McCoy), he seemed to be a great guy and very knowledgeable," Weaver said. "But we've got a chief who's just as knowledgeable."
Duvall countered that the McSherrystown chief had an opportunity to apply for the Conewago police chief position himself, but didn't. Woods, who was present at the meeting, quickly interjected that the qualifications listed in the advertisement for the position barred him from consideration.
After several minutes of discussion, council members agreed that if Conewago wanted them to accept McCoy as the regional police chief, they would need an assurance that Woods would be second-in-command.
Council members did not ask Woods if he wanted the position. President Pat McKim-Bortner told Woods he could decide for himself later if he was interested.
Woods declined to comment Thursday when asked how he felt about the position but did state he felt loyal to the department and wanted the regionalization to work so long as it was done correctly.
Officials from both municipalities have debated the logistics of merging police departments for more than a year with little progress.
The two municipalities, along with New Oxford Borough, began looking into a merger during the summer of 2015 as a way to save money for taxpayers and increase the level of police service for residents. New Oxford later withdrew when it became clear the deal would not be completed by 2017.
The plan loomed near failure in February when Weaver, the man responsible for hatching the project, vowed to do whatever it took to stop it.
Weaver, who is a retired McSherrystown police officer, explained at the time that he believed the municipalities' plan was leaning on a deeply flawed feasibility study. He was also upset that another municipal official outside the borough had stated he should not be involved in the project.
McKim-Bortner tried in May to strike a fresh start with Conewago by removing councilman David Bolton from the regionalization committee. Weaver also stepped down from the committee in protest.
Bolton, who objected to his removal, was replaced on the committee by council members Duvall and Joseph VonSas.
Duvall and VonSas will present the borough's condition to Conewago before council's next meeting in November.
If Conewago officials do not agree to McSherrystown's terms, the plan may fail to move forward.
"I can live with that," Weaver said of the borough's decision. "If they come back and say 'No, we're not going to agree to that,' then I don't think we have to be in talks anymore."
"We won't be," Duvall said.
Read or Share this story: http://evesun.co/2ewxmGr


text:
Jan Hubertus (* 5. Februar 1920 in Nijmegen; † 9. Oktober 1995 in Baden; heimatberechtigt in Wettingen) war ein Schweizer Maler.

Leben und Werk
Jan Hubertus wurde auf hoher See zwischen Holland und Indonesien geboren und wuchs in Java unter dem Einfluss von buddhistischen Mönchen auf. 1931 kehrte er mit seinen Eltern nach Geertruidenberg zurück. Seine künstlerische Ausbildung erhielt er an der Kunstakademie Antwerpen, die er mit neunzehn Jahren abschloss. Es folgten Studienaufenthalte in Paris und London. An der Universität von Leuven besuchte Hubertus freie Kurse in Archäologie und Kunstgeschichte.

Nach den Kriegsjahren, die er als Soldat und im niederländischen Widerstand verbrachte, reiste er durch Europa und Afrika. Seine Malerei entwickelte sich in diesen Jahren unter dem Einfluss der Künstlergruppe CoBrA hin zum abstrakten Expressionismus. 1956 wurde Hubertus der zweite Sekretär des holländischen Künstlerverbandes und hatte dadurch regen Austausch mit ausländischen Kunstschaffenden. Ab 1960 zog er sich aus dem Kunstbetrieb zurück und setzte sich intensiv mit dem östlichen Gedankengut auseinander.

1965 lernte Hubertus seine Frau Maria kennen und übersiedelte in die Schweiz, wo in den nächsten 30 Jahren sein Hauptwerk entstand. Er lebte zurückgezogen und malte nur nachts. Es folgten weitere Studienaufenthalte im Tibet-Institut Rikon und Reisen nach Asien. Seine Kenntnisse in der Meditation gab er auch anderen Menschen weiter.

In den sechziger und siebziger Jahren entstanden, ausgehend von meditativen Übungen, Bilder mit kalligrafischen Strukturen. In den achtziger Jahren kamen Objekte dazu, oft in Verbindung von Holz, Stein und beschriftetem Papier. 1982 wurden Teile von Hubertus' Werken bei einem Brand zerstört.

Jan Hubertus verstarb während der Vorbereitungen zu seiner Ausstellung im Aargauer Kunsthaus, die im Frühjahr 1996 stattfand.

Weblinks

Einzelnachweise

Maler (Schweiz)
Maler (Niederlande)
Schweizer
Geboren 1920
Gestorben 1995
Mann

redpajama_set_name: RedPajamaWikipedia


1 Preheat oven to 425 degrees.
2 Place cupcake liners in muffin tin.
3 In a large bowl, combine all ingredients until well mixed.
4 Fill liner of cups 2/3 full.
5 Bake for 15 to 20 minutes or until light golden brown.
6 Top cupcakes with frosting, sprinkle on chopped bacon and drizzle with syrup.
7 Enjoy one (or three) pancake cupcakes with a glass of cold milk.


text:
Q: 2 Async WCF services I have an asyncronous WCF service, that uses a client proxy to call an 2nd SOAP WCF service. I have no control of the SOAP Java service, but i can set the configuration on the service reference to run Asyncronously.
How would i get a result from the 2nd Async Service, to pass values back to the 1st down to the Client??
public class AddService : IAddService
{
    // SOAP Java service reference

    ResultServiceClient proxy = new ResultServiceClient();

    public int AddNumbers(int x, int y)
    {

        // Am i on the right track here to use BeginXXX, EndXXX?
        proxy.BeginGetResult(x, y, new AsyncCallback(OnEndAdd), null);

        /// how to return a result here.??????
        return result;
    }

    void OnEndAdd(IAsyncResult result)
    {
        int result = proxy.EndGetResult(result);
    }
}

A:    [ServiceBehavior(InstanceContextMode = InstanceContextMode.PerCall)]
    public class AddService : IAddService
    {
        // SOAP Java service reference
        ManualReseEvent _resetEvent=new ManualResetEvent(false);
        ResultServiceClient proxy = new ResultServiceClient();
        private int _result=-1;

        public int AddNumbers(int x, int y)
        {

            // Am i on the right track here to use BeginXXX, EndXXX?
            proxy.BeginGetResult(x, y, new AsyncCallback(OnEndAdd), null);
            _resetEvent.WaitOne(TimeSpan.FromSeconds(10);
            return _result;
        }

        void OnEndAdd(IAsyncResult result)
        {
            _result = proxy.EndGetResult(result);
            _resetEvent.Set();
        }
    }

What's going on here?
We make the call and then we use the ManualResetEvent to pause the thread and wait for the callback to be made.  The ManualResetEvent is told to wait 10 seconds and then continue executing.
ManualResetEvent has multiple overloads for the Wait method, from infinite - Wait() - to the one I'm using, which takes a TimeSpan.  _result is now a member variable so we can access the value from the initial calling method.  I've also marked the service with a "PerCall" service behaviour.  This means that a new instance of this object is instantiated per call to it.  I do this because we've got an up-to 10 second delay while we call the Java service and we don't want to block other calls to this service by other users.

redpajama_set_name: RedPajamaStackExchange


text:
Q: How to add a Metadata String to an LLVM module with the C++ API? I'm trying to add a metadata string to my LLVM module. The stripped down version of what I'm trying is
#include <llvm/LLVMContext.h>
#include <llvm/Module.h>
#include <llvm/Metadata.h>

using namespace llvm;

int main() {
    Module* module = new Module("test", getGlobalContext());
    MDString::get(module->getContext(), "test");
    module->dump();
}

I can compile and run it:
Desktop% g++ llvm.cc -o llvm `llvm-config --cppflags --ldflags --libs all`
Desktop% ./llvm
; ModuleID = 'test'

But as one can see, the metadata does not show up.
Can I somehow add the string to the module? The module itself only seems to offer access to named metadata. Now I don't know where else I could look. Any suggestions?
Supplement: I got the feeling that you can't just have a metadata string "floating around" in your module, it seems like you have to add it to a named metadata node. Is that right?

A: Try this:
#include <llvm/LLVMContext.h>
#include <llvm/Module.h>
#include <llvm/Metadata.h>

using namespace llvm;

int main() {
  Module* module = new Module("test", getGlobalContext());

  Value *Elts[] = {
    MDString::get(module->getContext(), "test1")
  };
  MDNode *Node = MDNode::get(getGlobalContext(), Elts);

  NamedMDNode *NMD = module->getOrInsertNamedMetadata("test2");
  NMD->addOperand(Node);

  module->dump();
}

I am not sure if you are able to have metadata "floating around" as you say.  If it's not attached to any part of your program then what good is it doing?  I've been looking into MD a bit lately...  I found similar code in lib/Analysis/DIBuilder.cpp.  Good luck.