当前期刊: Glycobiology Go to current issue    加入关注   
显示样式:        排序: 导出
我的关注
我的收藏
您暂时未登录!
登录
  • Application of a gut-immune co-culture system for the study of N-glycan-dependent host–pathogen interactions of Campylobacter jejuni
    Glycobiology (IF 4.194) Pub Date : 2020-01-15
    Zamora C, Ward E, Kester J, et al.

    An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host–pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

    更新日期:2020-01-23
  • Crystal structures of a β-trefoil lectin from Entamoeba histolytica in monomeric and a novel disulphide bond-mediated dimeric forms
    Glycobiology (IF 4.194) Pub Date : 2020-01-21
    Khan F, Kurre D, Suguna K.

    β-trefoil lectins are galactose/N-acetyl galactosamine specific lectins, which are widely distributed across all kingdoms of life and are known to perform several important functions. However, there is no report available on the characterization of these lectins from protozoans. We have performed structural and biophysical studies on a β-trefoil lectin from Entamoeba histolytica (EntTref) which exists as a mixture of monomers and dimers in solution. Further, we have determined the affinities of EntTref for rhamnose, galactose and different galactose-linked sugars. We obtained the crystal structure of EntTref in a sugar-free form (EntTref_apo) and a rhamnose-bound form (EntTref_rham). A novel Cys residue-mediated dimerization was revealed in the crystal structure of EntTref_apo while the structure of EntTref_rham provided the structural basis for the recognition of rhamnose by a β-trefoil lectin for the first time. To the best of our knowledge, this is the only report of the structural, functional and biophysical characterization of a β-trefoil lectin from a protozoan source and the first report of Cys-mediated dimerization in this class of lectins.

    更新日期:2020-01-23
  • Allotype-specific processing of the CD16a N45-glycan from primary human natural killer cells and monocytes
    Glycobiology (IF 4.194) Pub Date : 2020-01-21
    Patel K, Roberts J, Barb A.

    Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in NK cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human natural killer (NK) cells, but structures at N45 were relatively homogeneous. In this study we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. An ESI-MS/MS analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from a data collected on donors expressing only predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors.

    更新日期:2020-01-23
  • 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.

    更新日期:2020-01-14
  • Are there specific antibodies against Neu5Gc epitopes in the blood of healthy individuals?
    Glycobiology (IF 4.194) Pub Date : 2020-01-02
    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.

    更新日期:2020-01-04
  • The Sialyltransferase ST6GAL1 Protects Against Radiation-Induced Gastrointestinal Damage
    Glycobiology (IF 4.194) Pub Date : 2020-01-02
    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.

    更新日期:2020-01-04
  • Differential Distribution of N- and O-Glycans and Variable Expression of Sialyl-T Antigen on HeLa Cells—Revealed by Direct Fluorescent Glycan Imaging
    Glycobiology (IF 4.194) Pub Date : 2020-01-02
    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.

    更新日期:2020-01-04
  • Characterization of disease-specific chondroitin sulfate non-reducing end accumulation in mucopolysaccharidosis IVA
    Glycobiology (IF 4.194) Pub Date : 2020-01-02
    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.

    更新日期:2020-01-04
  • Structural and functional analysis of four family 84 glycoside hydrolases from the opportunistic pathogen Clostridium perfringens
    Glycobiology (IF 4.194) Pub Date : 2019-09-06
    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.

    更新日期:2019-12-22
  • Intracellular galectins control cellular responses commensurate with cell surface carbohydrate composition
    Glycobiology (IF 4.194) Pub Date : 2019-09-25
    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.

    更新日期:2019-12-22
  • 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
    Glycobiology (IF 4.194) Pub Date : 2019-09-16
    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.

    更新日期:2019-12-22
  • ragp: Pipeline for mining of plant hydroxyproline-rich glycoproteins with implementation in R
    Glycobiology (IF 4.194) Pub Date : 2019-09-11
    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.

    更新日期:2019-12-22
  • Advanced glycation end products (AGEs), protein aggregation and their cross talk: new insight in tumorigenesis
    Glycobiology (IF 4.194) Pub Date : 2019-09-11
    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.

    更新日期:2019-12-22
  • Expanding the capillary electrophoresis based glucose unit database of the GUcal app
    Glycobiology (IF 4.194) Pub Date : 2019-12-12
    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.

    更新日期:2019-12-13
  • An atomistic perspective on ADCC quenching by core-fucosylation of IgG1 Fc N-glycans from enhanced sampling molecular dynamics
    Glycobiology (IF 4.194) Pub Date : 2019-12-12
    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.

    更新日期:2019-12-13
  • Heparan sulfate inhibits transforming growth factor β signaling and functions in cis and in trans to regulate prostate stem/progenitor cell activities
    Glycobiology (IF 4.194) Pub Date : 2019-12-12
    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.

    更新日期:2019-12-13
  • Microarray Analyses of Closely Related Glycoforms Reveal Different Accessibilities of Glycan Determinants on N-Glycan Branches
    Glycobiology (IF 4.194) Pub Date : 2019-12-06
    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.

    更新日期:2019-12-06
  • The O-GalNAcylating enzyme GALNT5 mediates carcinogenesis and progression of cholangiocarcinoma via activation of AKT/ERK signaling
    Glycobiology (IF 4.194) Pub Date : 2019-12-04
    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.

    更新日期:2019-12-05
  • Directed Evolution of a Remarkably Efficient Kdnase from a Bacterial Neuraminidase
    Glycobiology (IF 4.194) Pub Date : 2019-12-04
    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.

    更新日期:2019-12-05
  • Fucoidan suppresses the gastric cancer cell malignant phenotype and production of TGF-β1 via CLEC-2
    Glycobiology (IF 4.194) Pub Date : 2019-11-19
    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.

    更新日期:2019-11-19
  • Modulation of hepatocyte sialylation drives spontaneous fatty liver disease and inflammation
    Glycobiology (IF 4.194) Pub Date : 2019-11-19
    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.

    更新日期:2019-11-19
  • Identification of Tn Antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies
    Glycobiology (IF 4.194) Pub Date : 2019-11-19
    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].

    更新日期:2019-11-19
  • Glycoxidative profile of cancer patient serum: A clinical result to associate glycation to cancer
    Glycobiology (IF 4.194) Pub Date : 2019-11-19
    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.

    更新日期:2019-11-19
  • Cosmc is required for T cell persistence in the periphery
    Glycobiology (IF 4.194) Pub Date : 2019-07-18
    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.

    更新日期:2019-11-07
  • Direct fluorescent glycan labeling with recombinant sialyltransferases
    Glycobiology (IF 4.194) Pub Date : 2019-07-30
    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.

    更新日期:2019-11-07
  • Bottom-up analysis using liquid chromatography–Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers
    Glycobiology (IF 4.194) Pub Date : 2019-07-30
    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.

    更新日期:2019-11-07
  • PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells
    Glycobiology (IF 4.194) Pub Date : 2019-07-30
    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.

    更新日期:2019-11-07
  • Semirational design and engineering of grapevine glucosyltransferases for enhanced activity and modified product selectivity
    Glycobiology (IF 4.194) Pub Date : 2019-07-30
    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.

    更新日期:2019-11-07
  • GAG positioning on IL-1RI; A mechanism regulated by dual effect of glycosylation
    Glycobiology (IF 4.194) Pub Date : 2019-07-18
    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.

    更新日期:2019-11-07
  • Carbo-loading in Coccidioides spp.: a quantitative analysis of CAZyme abundance and resulting glycan populations
    Glycobiology (IF 4.194) Pub Date : 2019-11-06
    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.

    更新日期:2019-11-06
  • Real- time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells
    Glycobiology (IF 4.194) Pub Date : 2019-11-06
    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.

    更新日期:2019-11-06
  • Society for Glycobiology Awards-2019.
    Glycobiology (IF 4.194) Pub Date : 2019-09-19

    更新日期:2019-11-01
  • 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

    更新日期:2019-11-01
  • The GlySpace Alliance: towards a collaborative global glycoinformatics community.
    Glycobiology (IF 4.194) Pub Date : null
    Kiyoko F Aoki-Kinoshita,Frederique Lisacek,Raja Mazumder,William S York,Nicolle H Packer

    更新日期:2019-11-01
  • Analysis of differential expression of glycosyltransferases in healing corneas by glycogene microarrays.
    Glycobiology (IF 4.194) Pub Date : 2018-12-07
    Chandrassegar Saravanan,Zhiyi Cao,Steven R Head,Noorjahan Panjwani

    更新日期:2019-11-01
  • Tunicamycin-induced ER stress in breast cancer cells neither expresses GRP78 on the surface nor secretes it into the media.
    Glycobiology (IF 4.194) Pub Date : 2019-04-28
    Jesús E Serrano-Negrón,Zhenbo Zhang,Andrea P Rivera-Ruiz,Aditi Banerjee,Eva C Romero-Nutz,Neysharie Sánchez-Torres,Krishna Baksi,Dipak K Banerjee

    更新日期:2019-11-01
  • Identification and characterization of a Drosophila melanogaster ortholog of human beta1,4-galactosyltransferase VII.
    Glycobiology (IF 4.194) Pub Date : 2002-09-24
    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.

    更新日期:2019-11-01
  • Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase.
    Glycobiology (IF 4.194) Pub Date : 2000-04-15
    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.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Molecular models for mouse sperm-oocyte binding.
    Glycobiology (IF 4.194) Pub Date : 2010-12-29
    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.

    更新日期:2019-11-01
  • Cholera toxin B subunit binding does not correlate with GM1 expression: a study using mouse embryonic neural precursor cells.
    Glycobiology (IF 4.194) Pub Date : 2006-09-12
    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.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Asparagine linked glycosylation in Giardia.
    Glycobiology (IF 4.194) Pub Date : 2005-06-07
    Phillips W Robbins,John Samuelson

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • 更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Shooting HARE.
    Glycobiology (IF 4.194) Pub Date : 2003-05-20
    Bård Smedsrød,Staffan Johansson,Sergij Goerdt

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Expression of histo-blood group A antigen increases resistance to apoptosis and facilitates escape from immune control of rat colon carcinoma cells.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • An investigation of the interactions of E-selectin with fuco-oligosaccharides of the blood group family.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • Identification, cloning, purification, and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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+).

    更新日期:2019-11-01
  • Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • A complete alpha1,3-galactosyltransferase gene is present in the human genome and partially transcribed.
    Glycobiology (IF 4.194) Pub Date : 2002-12-25
    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.

    更新日期:2019-11-01
  • Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids.
    Glycobiology (IF 4.194) Pub Date : 2002-12-04
    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.

    更新日期:2019-11-01
  • O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase.
    Glycobiology (IF 4.194) Pub Date : 2002-12-04
    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.

    更新日期:2019-11-01
  • 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.
    Glycobiology (IF 4.194) Pub Date : 2002-12-04
    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.

    更新日期:2019-11-01
Contents have been reproduced by permission of the publishers.
导出
全部期刊列表>>
2020新春特辑
限时免费阅读临床医学内容
ACS材料视界
科学报告最新纳米科学与技术研究
清华大学化学系段昊泓
自然科研论文编辑服务
加州大学洛杉矶分校
上海纽约大学William Glover
南开大学化学院周其林
课题组网站
X-MOL
北京大学分子工程苏南研究院
华东师范大学分子机器及功能材料
中山大学化学工程与技术学院
试剂库存
天合科研
down
wechat
bug