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  • Identification of key amino acid residues determining ligand binding specificity, homodimerization and cellular distribution of human Galectin-10
    Glycobiology (IF 3.664) Pub Date : 2018-09-20
    Su J, Song C, Si Y, et al.

    Charcot-Leyden crystal protein/Gal-10, abundantly expressed in eosinophils and basophils, is related to several immune diseases. Recently, crystallographic and biochemical studies showed that Gal-10 cannot bind lactose, because a glutamate residue (Glu33) from another monomer blocks the binding site. Moreover, Gal-10 actually forms a novel dimeric structure compared to other galectins. To investigate the role that Glu33 plays in inhibiting lactose binding, we mutated this residue to glutamine, aspartate, and alanine. The structure of E33A shows that Gal-10 can now bind lactose. In the hemagglutination assay, lactose could inhibit E33A from inducing chicken erythrocyte agglutination. Furthermore, we identified a tryptophan residue (Trp127) at the interface of homodimer that is crucial for Gal-10 dimerization. The variant W127A, which exists as a monomer, exhibited higher hemagglutination activity than wild type Gal-10. The solid phase assay also showed that W127A could bind to lactose-modified sepharose-6B, whereas wild type Gal-10 could not. This indicates that the open carbohydrate binding site of the W127A monomer can bind to lactose. In addition, the distribution of EGFP-tagged Gal-10 and its variants in HeLa cells was investigated. Because Trp72 is the highly conserved in the ligand binding sites of galectins, we used EGFP-tagged W72A to show that Gal-10 could not be transported into the nucleus, indicating that Trp72 is crucial for Gal-10 transport into that organelle.

  • Unraveling synthesis of the cryptococcal cell wall and capsule
    Glycobiology (IF 3.664) Pub Date : 2018-04-10
    Wang Z, Li L, Doering T.

    Fungal pathogens cause devastating infections in millions of individuals each year, representing a huge but underappreciated burden on human health. One of these, the opportunistic fungus Cryptococcus neoformans, kills hundreds of thousands of patients annually, disproportionately affecting people in resource-limited areas. This yeast is distinguished from other pathogenic fungi by a polysaccharide capsule that is displayed on the cell surface. The capsule consists of two complex polysaccharide polymers: a mannan substituted with xylose and glucuronic acid, and a galactan with galactomannan side chains that bear variable amounts of glucuronic acid and xylose. The cell wall, with which the capsule is associated, is a matrix of alpha and beta glucans, chitin, chitosan, and mannoproteins. In this review, we focus on synthesis of the wall and capsule, both of which are critical for the ability of this microbe to cause disease and are distinct from structures found in either model yeasts or the mammals afflicted by this infection. Significant research effort over the last few decades has been applied to defining the synthetic machinery of these two structures, including nucleotide sugar metabolism and transport, glycosyltransferase activities, polysaccharide export, and assembly and association of structural elements. Discoveries in this area have elucidated fundamental biology and may lead to novel targets for antifungal therapy. In this review, we summarize the progress made in this challenging and fascinating area, and outline future research questions.

  • Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways
    Glycobiology (IF 3.664) Pub Date : 2018-07-17
    Gonzalez-Gil A, Porell R, Fernandes S, et al.

    Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells—primarily eosinophils and mast cells—where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose–acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

  • Analysis of protein landscapes around N-glycosylation sites from the PDB repository for understanding the structural basis of N-glycoprotein processing and maturation
    Glycobiology (IF 3.664) Pub Date : 2018-07-18
    Suga A, Nagae M, Yamaguchi Y.

    Asparagine-linked glycans (N-glycans) are attached onto nascent glycoproteins in the endoplasmic reticulum (ER) and subsequently processed by a set of processing enzymes in the ER and Golgi apparatus. Accumulating evidence has shown that not all N-glycans on glycoproteins are uniformly processed into mature forms (hybrid and complex types in mammals) through the ER and Golgi apparatus, and a certain set of glycans remains unprocessed as an “immature” form (high-mannose type in mammals). Much attention has been paid to environmental factors regulating N-glycoprotein maturation, such as the expression levels of glycosyltransferases/glycosidases. On the other hand, the influence of the 3D structure of the carrier glycoprotein on N-glycan maturation has been investigated mostly using individual model glycoproteins. To obtain more insights into N-glycoprotein maturation, we herein analyze glycoprotein structures extracted from the Protein Data Bank. We confirm that site-specific N-glycan processing is largely explained by the solvent accessibility of the glycosylated Asn residue and of the conjugated N-glycan. Potential bias of protein structural features toward immature or mature forms was explored within a range of concentric circles of fully folded glycoproteins. There does appear to be bias in amino acid composition and secondary structure. Most notably, γ-branched amino acid residues (Asn+Asp+Leu) occur more frequently on unstructured loop regions of immature glycoproteins. Structural features of the protein surface around the N-glycosylated site do seem to affect N-glycan processing and maturation.

  • Structural and functional analysis of Alg1 beta-1,4 mannosyltransferase reveals the physiological importance of its membrane topology
    Glycobiology (IF 3.664) Pub Date : 2018-07-18
    Xu X, Li S, Wang N, et al.

    In eukaryotes, the biosynthesis of a highly conserved dolichol-linked oligosaccharide (DLO) precursor Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) begins on the cytoplasmic face of the endoplasmic reticulum (ER) and ends within the lumen. Two functionally distinguished heteromeric glycosyltransferase (GTase) complexes are responsible for the cytosolic DLO assembly. Alg1, a β-1, 4 mannosyltransferase (MTase) physically interacts with Alg2 and Alg11 proteins to form the multienzyme complex which catalyzes the addition of all five mannose to generate the Man5GlcNAc2-PP-Dol intermediate. Despite the fact that Alg1 plays a central role in the formation of the multi-MTase has been confirmed, the topological information of Alg1 including the molecular mechanism of membrane association are still poorly understood. Using a combination of bioinformatics and biological approaches, we have undertaken a structural and functional study on Alg1 protein, in which the enzymatic activities of Alg1 and its variants were monitored by a complementation assay using the GALpr-ALG1 yeast strain, and further confirmed by a liquid chromatography–mass spectrometry-based in vitro quantitative assay. Computational and experimental evidence confirmed Alg1 shares structure similarity with Alg13/14 complex, which has been defined as a membrane-associated GT-B GTase. Particularly, we provide clear evidence that the N-terminal transmembrane domain including the following positively charged amino acids and an N-terminal amphiphilic-like α helix domain exposed on the protein surface strictly coordinate the Alg1 orientation on the ER membrane. This work provides detailed membrane topology of Alg1 and further reveals its biological importance at the spatial aspect in coordination of cytosolic DLO biosynthesis.

  • EpsN from Bacillus subtilis 168 has UDP-2,6-dideoxy 2-acetamido 4-keto glucose aminotransferase activity in vitro
    Glycobiology (IF 3.664) Pub Date : 2018-07-27
    Kaundinya C, Savithri H, Rao K, et al.

    The gene epsN of Bacillus subtilis 168 was cloned and overexpressed in Escherichiacoli. Purified recombinant EpsN is shown to be a pyridoxal 5′-phosphate (PLP)-dependent aminotransferase by absorption spectroscopy, l-cycloserine inhibition and reverse phase HPLC studies. EpsN catalyzes the conversion of UDP-2,6-dideoxy 2-acetamido 4-keto glucose to UDP-2,6-dideoxy 2-acetamido 4-amino glucose. Lys190 was found by sequence comparison and site-directed mutagenesis to form Schiff base with PLP. Mutagenesis studies showed that, in addition to Lys190, Ser185, Glu164, Gly58 and Thr59 are essential for aminotransferase activity.

  • Biophysical analysis of sialic acid recognition by the complement regulator Factor H
    Glycobiology (IF 3.664) Pub Date : 2018-07-27
    Schmidt C, Hipgrave Ederveen A, Harder M, et al.

    Complement factor H (FH), an elongated and substantially glycosylated 20-domain protein, is a soluble regulator of the complement alternative pathway (AP). It contains several glycan binding sites which mediate recognition of α2-3-linked sialic acid (FH domain 20) and glycosaminoglycans (domains 6–8 and 19–20). FH also binds the complement C3-activation product C3b, a powerful opsonin and focal point for the formation of C3-convertases of the AP feedback loop. In freely circulating FH the C3b binding site in domains 19–20 is occluded, a phenomenon that is not fully understood and could be mediated by an intramolecular interaction between FH’s intrinsic sialylated glycosylation and its own sialic acid binding site. In order to assess this possibility, we characterized FH’s sialylation with respect to glycosidic linkage type and searched for further potential, not yet characterized sialic acid binding sites in FH and its seven-domain spanning splice variant and fellow complement regulator FH like-1 (FHL-1). We also probed FH binding to the sialic acid variant Neu5Gc which is not expressed in humans but on heterologous erythrocytes that restrict the human AP and in FH transgenic mice. We find that FH contains mostly α2-6-linked sialic acid, making an intramolecular interaction with its α2-3-sialic acid specific binding site and an associated self-lock mechanism unlikely, substantiate that there is only a single sialic acid binding site in FH and none in FHL-1, and demonstrate direct binding of FH to the nonhuman sialic acid Neu5Gc, supporting the use of FH transgenic mouse models for studies of complement-related diseases.

  • Plasma contact activation by a fucosylated chondroitin sulfate and its structure–activity relationship study
    Glycobiology (IF 3.664) Pub Date : 2018-08-17
    Lin L, Xu L, Xiao C, et al.

    Plasma contact system is the initial part of both the intrinsic coagulation pathway and kallikrein–kinin pathway, which mainly involves three proteins: coagulation factor XII (FXII), prekallikrein (PK) and high-molecular weight kininogen. Fucosylated chondroitin sulfate (FCS) is a unique sulfated glycosaminoglycan (GAG) composed of a chondroitin sulfate-like backbone and sulfated fucose branches. The native FCS was preliminary found to cause undesired activation of the plasma contact system. How this unusual GAG functions in this process remains to be clarified. Herein, the relationship between its structure, plasma contact activation and its effects on the PK–FXII reciprocal activation loop were studied. The recalcification time assay indicated that the FCS at high concentration could be procoagulant which may be attributed to its contact activation activity. The structure–activity relationship study indicated that its high molecular weight and distinct fucose side chains are required for contact activation by FCS, although the sulfate substitution types of its side chains have less impact. In human plasma, the native FCSs potently induced FXII-dependent contact activation. However, in purified systems FCS did not significantly activate FXII per se or induce its autoactivation, whereas FCS significantly promoted the activation of PK by factor XIIa. Polysaccharide–protein interaction assays showed that FCS bound to PK with higher affinity than other contact system proteins. These data suggested that potent contact activation by FCS requires the positive feedback loop between PK and FXII. These findings contribute to better understanding of contact activation by complex GAG.

  • Structural and conformational studies of the heparan sulfate mimetic PI-88
    Glycobiology (IF 3.664) Pub Date : 2018-08-17
    Elli S, Stancanelli E, Handley P, et al.

    The heparan sulfate mimetic PI-88 is a complex mixture of sulfated oligosaccharides with anti-metastatic and anti-angiogenic activity due to its potent inhibition of heparanase and heparan sulfate-dependent angiogenic growth factors. It was recently in Phase III clinical trials for postresection hepatocellular carcinoma. The major oligosaccharide constituents of PI-88 were prepared for the first time by sulfonation of individually purified phosphorylated oligosaccharides isolated from the PI-88 precursor. PI-88 and its components were subjected to detailed 1D and 2D NMR spectroscopic analysis. The spectra of the individual components greatly assisted the assignment of minor resonances in the 1H NMR spectrum of PI-88. The data also showed that the majority of the oligosaccharides in PI-88 are fully sulfated and that undersulfated species present are largely due to anomeric desulfation. The solution conformation of the phosphomannopentaose sulfate (major component) of PI-88 was then determined by a combination of molecular dynamics simulations and NOE measurements which may provide insights into its binding interactions with target proteins.

  • A pipeline to translate glycosaminoglycan sequences into 3D models. Application to the exploration of glycosaminoglycan conformational space
    Glycobiology (IF 3.664) Pub Date : 2018-09-18
    Clerc O, Mariethoz J, Rivet A, et al.

    Mammalian glycosaminoglycans are linear complex polysaccharides comprising heparan sulfate, heparin, dermatan sulfate, chondroitin sulfate, keratan sulfate and hyaluronic acid. They bind to numerous proteins and these interactions mediate their biological activities. GAG–protein interaction data reported in the literature are curated mostly in MatrixDB database (http://matrixdb.univ-lyon1.fr/). However, a standard nomenclature and a machine-readable format of GAGs together with bioinformatics tools for mining these interaction data are lacking. We report here the building of an automated pipeline to (i) standardize the format of GAG sequences interacting with proteins manually curated from the literature, (ii) translate them into the machine-readable GlycoCT format and into SNFG (Symbol Nomenclature For Glycan) images and (iii) convert their sequences into a format processed by a builder generating three-dimensional structures of polysaccharides based on a repertoire of conformations experimentally validated by data extracted from crystallized GAG–protein complexes. We have developed for this purpose a converter (the CT23D converter) to automatically translate the GlycoCT code of a GAG sequence into the input file required to construct a three-dimensional model.

  • Identity and role of the non-Conserved Acid/Base Catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata
    Glycobiology (IF 3.664) Pub Date : 2018-09-10
    Perrella N, Withers S, Lopes A.

    α-L-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-L-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-L-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-L-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolysed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterised. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organisation reinforce the necessity of structural studies of new fucosidases.

  • Nature-inspired engineering of an F-type lectin for increased binding strength
    Glycobiology (IF 3.664) Pub Date : 2018-09-10
    Mahajan S, Ramya T.

    Individual lectin-carbohydrate interactions are usually of low affinity. However, high avidity is frequently attained by the multivalent presentation of glycans on biological surfaces coupled with the occurrence of high order lectin oligomers or tandem repeats of lectin domains in the polypeptide. F-type lectins are L-fucose binding lectins with a typical sequence motif, HX(26)RXDX(4)R/K, whose residues participate in L-fucose binding. We previously reported the presence of a few eukaryotic F-type lectin domains with partial sequence duplication that results in the presence of two L-fucose-binding sequence motifs. We hypothesized that such partial sequence duplication would result in greater avidity of lectin-ligand interactions. Inspired by this example from Nature, we attempted to engineer a bacterial F-type lectin domain from Streptosporangium roseum to attain avid binding by mimicking partial duplication. The engineered lectin demonstrated 12-fold greater binding strength than the wild-type lectin to multivalent fucosylated glycoconjugates. However, the affinity to the monosaccharide L-fucose in solution was similar and partial sequence duplication did not result in an additional functional L-fucose binding site. We also cloned, expressed and purified a Branchiostoma floridae F-type lectin domain with naturally occurring partial sequence duplication and confirmed that the duplicated region with the F-type lectin sequence motif did not participate in L-fucose binding. We found that the greater binding strength of the engineered lectin from S. roseum was instead due to increased oligomerization. We believe that this Nature-inspired strategy might be useful for engineering lectins to improve binding strength in various applications.

  • Galectin-3 binds selectively to the terminal, non-reducing end of β(1→4)-galactans, with overall affinity increasing with chain length
    Glycobiology (IF 3.664) Pub Date : 2018-09-11
    Miller M, Zheng Y, Zhou Y, et al.

    Galactans are linear polysaccharides of β(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in β-strands 4, 5 and 6 on the canonical, β-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.

  • Selectins in cancer immunity
    Glycobiology (IF 3.664) Pub Date : 2018-01-17
    Borsig L.

    Selectins are vascular adhesion molecules that mediate physiological responses such as inflammation, immunity and hemostasis. During cancer progression, selectins promote various steps enabling the interactions between tumor cells and the blood constituents, including platelets, endothelial cells and leukocytes. Selectins are carbohydrate-binding molecules that bind to sialylated, fucosylated glycan structures. The increased selectin ligand expression on tumor cells correlates with enhanced metastasis and poor prognosis for cancer patients. While, many studies focused on the role of selectin as a mediator of tumor cell adhesion and extravasation during metastasis, there is evidence for selectins to activate signaling cascade that regulates immune responses within a tumor microenvironment. L-Selectin binding induces activation of leukocytes, which can be further modulated by selectin-mediated interactions with platelets and endothelial cells. Selectin ligand on leukocytes, PSGL-1, triggers intracellular signaling in leukocytes that are induced through platelet’s P-selectin or endothelial E-selectin binding. In this review, I summarize the evidence for selectin-induced immune modulation in cancer progression that represents a possible target for controlling tumor immunity.

  • Targeting sialic acid–Siglec interactions to reverse immune suppression in cancer
    Glycobiology (IF 3.664) Pub Date : 2018-01-27
    Adams O, Stanczak M, von Gunten S, et al.

    Changes in sialic acids in cancer have been observed for many years. In particular, the increase of sialoglycan density or hypersialylation in tumors has been described. Recent studies have identified mechanisms for immune evasion based on sialoglycan interactions with immunoregulatory Siglec receptors that are exploited by tumor cells and microorganisms alike. Siglecs are mostly inhibitory receptors similar to known immune checkpoints including PD-1 or CTLA-4 that are successfully targeted with blocking antibodies for cancer immunotherapy. Here, we summarize the known changes of sialic acids in cancer and the role Siglec receptors play in cancer immunity. We also focus on potential ways to target these Siglec receptors or sialoglycans in order to improve anti-cancer immunity.

  • Glycan-directed CAR-T cells
    Glycobiology (IF 3.664) Pub Date : 2018-02-17
    Steentoft C, Migliorini D, King T, et al.

    Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy.

  • Cancer glycan epitopes: biosynthesis, structure and function
    Glycobiology (IF 3.664) Pub Date : 2018-03-28
    Pearce O.

    Aberrant glycan epitopes are a classic hallmark of malignant transformation, yet their full clinical potential in cancer diagnostics and therapeutics is yet to be realized. This is partly because our understanding of how these epitopes are regulated remains poorly understood. In this review cancer glycan epitopes for the major glycan classes are summarized with a focus on their biosynthesis, structure and role in cancer progression. Their application as cancer biomarkers, in particular the more recent work on cancer glycoforms, and the advantages these offer over the glycan or protein alone are discussed. Finally, emerging concepts which expand on the current view of the cancer glycan epitope beyond the single structure, to patterns and the whole glycocalyx, are described. These new approaches that consider the cancer glycan epitope as a glycoform, or as a pattern of many epitope structures, are providing new targets both for cancer biomarkers and therapeutics currently in development at the bench and the clinic.

  • Through the barricades: overcoming the barriers to effective antibody-based cancer therapeutics
    Glycobiology (IF 3.664) Pub Date : 2018-06-12
    Dalziel M, Beers S, Cragg M, et al.

    Since the turn of the century, cancer therapy has undergone a transformation in terms of new treatment modalities and renewed optimism in achieving long-lived tumor control and even cure. This is, in large part, thanks to the widespread incorporation of monoclonal antibodies (mAbs) into standard treatment regimens. These new therapies have, across many settings, significantly contributed to improved clinical responses, patient quality of life and survival. Moreover, the flexibility of the antibody platform has led to the development of a wide range of innovative and combinatorial therapies that continue to augment the clinician’s armory. Despite these successes, there is a growing awareness that in many cases mAb therapy remains suboptimal, primarily due to inherent limitations imposed by the immune system’s own homeostatic controls and the immunosuppressive tumor microenvironment. Here, we discuss the principal barriers that act to constrain the tumor-killing activity of antibody-based therapeutics, particularly those involving antibody glycans, using illustrative examples from both pre-clinical and market approved mAbs. We also discuss strategies that have been, or are in development to overcome these obstacles. Finally, we outline how the growing understanding of the biological terrain in which mAbs function is shaping innovation and regulation in cancer therapeutics.

  • Sensitized genetic backgrounds reveal differential roles for EGF repeat xylosyltransferases in Drosophila Notch signaling
    Glycobiology (IF 3.664) Pub Date : 2018-08-30
    Ashutosh Pandey, David Li-Kroeger, Maya K Sethi, Tom V Lee, Falk F R Buettner, Hans Bakker, Hamed Jafar-Nejad

    In multicellular organisms, glycosylation regulates various developmental signaling pathways including the Notch pathway. One of the O-linked glycans added to epidermal growth factor-like (EGF) repeats in animal proteins including the Notch receptors is the xylose-xylose-glucose-O oligosaccharide. Drosophila glucoside xylosyltransferase (Gxylt) Shams negatively regulates Notch signaling in specific contexts. Since Shams adds the first xylose residue to O-glucose, its loss-of-function phenotype could be due to the loss of the first xylose, the second xylose or both. To examine the contribution of the second xylose residues to Drosophila Notch signaling, we have performed biochemical and genetic analysis on CG11388, which is the Drosophila homolog of human xyloside xylosyltransferase 1 (XXYLT1). Experiments in S2 cells indicated that similar to human XXYLT1, CG11388 can add the second xylose to xylose-glucose-O glycans. Flies lacking both copies of CG11388 (Xxylt) are viable and fertile and do not show gross phenotypes indicative of altered Notch signaling. However, genetic interaction experiments show that in sensitized genetic backgrounds with decreased or increased Notch pathway components, loss of Xxylt promotes Delta-mediated activation of Notch. Unexpectedly, we find that in such sensitized backgrounds, even loss of one copy of the fly Gxylt shams enhances Delta-mediated Notch activation. Taken together, these data indicate that while the first xylose plays a key role in tuning the Delta-mediated Notch signaling in Drosophila, the second xylose has a fine-tuning role only revealed in sensitized genetic backgrounds.

  • An F-type lectin domain directs the activity of Streptosporangium roseum alpha-L-fucosidase
    Glycobiology (IF 3.664) Pub Date : 2018-08-30
    Ritika Bishnoi, Sonal Mahajan, T N C Ramya

    F-type lectins are phylogenetically widespread but selectively distributed fucose-binding lectins with fucose- and calcium-binding sequence motifs and an F-type lectin fold. Bacterial F-type lectin domains frequently occur in tandem with various protein domains in diverse architectures, indicating a possible role in directing enzyme activities or other biological functions to distinct fucosylated niches. Here, we report the biochemical characterization of a Streptosporangium roseum protein containing an F-type lectin domain in tandem with an NPCBM-associated domain and a family GH 29A alpha-L-fucosidase domain. We show that the F-type lectin domain of this protein recognizes fucosylated glycans in both α and β linkages but has high affinity for a Fuc-α-1,2-Gal motif and that the alpha-L-fucosidase domain displays hydrolytic activity on glycan substrates with α1-2 and α1-4 linked fucose. We also show that the F-type lectin domain does not have any effect on the activity of the cis-positioned alpha-L-fucosidase domain with the synthetic substrate, 4-Methylumbelliferyl-alpha-L-fucopyranoside or on inhibition of this activity by L-fucose or Deoxyfuconojirimycin hydrochloride. However, the F-type lectin domain together with the NPCBM-associated domain enhances the activity of the cis-positioned alpha-L-fucosidase domain for soluble fucosylated oligosaccharide substrates. While there are many reports of glycoside hydrolase activity towards insoluble and soluble polysaccharides being enhanced by cis-positioned carbohydrate binding modules on the polypeptide, this is the first report, to our knowledge, of enhancement of activity towards aqueous, freely diffusible, small oligosaccharides. We propose a model involving structural stabilization and a bind-and-jump action mediated by the F-type lectin domain to rationalize our findings.

  • The effect of streptozotocin-induced hyperglycemia on N-and O-linked protein glycosylation in mouse ovary
    Glycobiology (IF 3.664) Pub Date : 2018-08-28
    Abdulrahman M Shathili, Hannah M Brown, Arun V Everest-Dass, Tiffany C Y Tan, Lindsay M Parker, Jeremy G Thompson, Nicolle H Packer

    Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography–ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans; with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.

  • A widely compatible expression system for the production of highly O-GlcNAcylated recombinant protein in E. coli
    Glycobiology (IF 3.664) Pub Date : 2018-08-23
    Hong Gao, Minghui Shi, Ruihong Wang, Chaojie Wang, Changlun Shao, Yuchao Gu, Wengong Yu

    O-GlcNAcylation is a ubiquitous and dynamic post-translational modification on serine/threonine residues of nucleocytoplasmic proteins in metazoan, which plays a critical role in numerous physiological and pathological processes. But the O-GlcNAcylation on most proteins is often substoichiometric, which hinders the functional study of the O-GlcNAcylation. This study aimed to improve the production of highly O-GlcNAcylated recombinant proteins in Escherichia coli (E. coli). To achieve this goal, we constructed a bacterial artificial chromosome-based chloramphenicol-resistant expression vector co-expressing OGT and key enzymes (GlmM and GlmU) of the UDP-GlcNAc synthesis pathway in E. coli, which can effectively increase the O-GlcNAcylation of the OGT target protein expressed by another vector. The results revealed that the expression of GlmM and GlmU increase the cellular concentration of UDP-GlcNAc in E. coli, which markedly enhanced the activity of the co-expressed OGT to its target proteins, such as H2B, p53 and TAB1. Altogether, we established a widely compatible E. coli expression system for producing highly O-GlcNAcylated protein, which could be used for modifying OGT target proteins expressed by almost any commercial expression vectors in E. coli. This new expression system provides possibility for investigating the roles of O-GlcNAcylation in the enzymatic activity, protein-protein interaction and structure of OGT target proteins.

  • Active site complementation and hexameric arrangement in the GH family 29; a structure-function study of α-l-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus
    Glycobiology (IF 3.664) Pub Date : 2018-08-23
    Terézia Kovaľová, Tomáš Kovaľ, Eva Benešová, Patricie Vodičková, Vojtěch Spiwok, Petra Lipovová, Jan Dohnálek

    α-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from non-reducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild type enzyme was active on most of the tested naturally-occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule, however the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.

  • Glycosylation profiling of dog serum reveals differences compared to human serum
    Glycobiology (IF 3.664) Pub Date : 2018-08-20
    Anna-Janina Behrens, Rebecca M Duke, Laudine M C Petralia, David J Harvey, Sylvain Lehoux, Paula E Magnelli, Christopher H Taron, Jeremy M Foster

    Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG – the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.

  • Cancer Immunotherapy
    Glycobiology (IF 3.664) Pub Date : 2018-07-10
    Oliver M T Pearce, Heinz Läubli

    Advancements in our understanding of how tumor cells evade immune control have led to new therapeutic approaches, and the introduction of cancer immunotherapies into daily oncological practice. Inhibitors—most of the time blocking antibodies—of inhibitory receptors called immune checkpoints, including PD-1 and CTLA-4, can induce remissions even in patients with advanced disease (Chen and Mellman 2017). For example, patients with metastatic melanoma had a median survival of less than 12 months only 10 years ago, but treatment with blocking antibodies that target PD-1 and CTLA-4 results in 58% of patients with a 3-year survival (Wolchok et al. 2017). The success of these immune checkpoint inhibitors has spurred the development...

  • Acinetobacter baumannii K20 and K21 capsular polysaccharide structures establish roles for UDP-glucose dehydrogenase Ugd2, pyruvyl transferase Ptr2 and two glycosyltransferases
    Glycobiology (IF 3.664) Pub Date : 2018-08-09
    Anastasiya A Kasimova, Johanna J Kenyon, Nikolay P Arbatsky, Alexander S Shashkov, Anastasiya V Popova, Mikhail M Shneider, Yuriy A Knirel, Ruth M Hall

    Infections caused by Acinetobacter baumannii isolates from the major global clones, GC1 and GC2, are difficult to treat with antibiotics, and phage therapy, which requires extensive knowledge of the variation in the surface polysaccharides, is an option under consideration. The gene clusters directing synthesis of capsular polysaccharide (CPS) in A. baumannii GC1 isolate A388 and GC2 isolate G21 differ by a single glycosyltransferase (gtr) gene. They include genes encoding a novel UDP-glucose dehydrogenase (Ugd2) and a putative pyruvyltransferase (Ptr2). The composition and structures of the linear K20 and K21 tetrasaccharide repeats (K units) of the CPSs isolated from A338 and G21, respectively, were established by sugar analyses and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The K20 and K21 CPSs are the first known to include GlcpA produced by Ugd2 and d-galactose with an (R)-configured 4,6-pyruvic acid acetal added by Prt2. The first sugar in the tetrasaccharide K units is 2-acetamido-4-amino-2,4,6-trideoxy-d-glucose (D-QuipNAc4N) that carries a 4-N-[(S)-3-hydroxybutanoyl] group in some K units and a 4-N-acetyl group in the others. Accordingly, K unit polymerases WzyK20 and WzyK21 form a β-D-QuipNAc4NR-(1→2)-D-Galp bond. The K20 and K21 units differ only in the configuration of the glycosidic linkages of d-GlcpNAc allowing the unique inverting glycosyltransferases Gtr43 and the retaining glycosyltransferase Gtr45 to be assigned to formation of the β-D-GlcpNAc-(1→4)-D-GlcpA and α-D-GlcpNAc-(1→4)-D-GlcpA linkages, respectively.

  • Members of the GalNAc-T family of enzymes utilize distinct Golgi localization mechanisms
    Glycobiology (IF 3.664) Pub Date : 2018-08-06
    Jessica L Becker, Duy T Tran, Lawrence A Tabak

    Mucin-type O-glycosylation is an evolutionarily conserved and essential post-translational protein modification that is initiated in the Golgi apparatus by a family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). GalNAc-Ts are type II membrane proteins which contain short N-terminal tails located in the cytoplasm, a transmembrane domain that crosses the Golgi membrane, to which is connected a stem region that tethers the C-terminal catalytic and lectin domains that reside in the Golgi lumen. Although mucin-type O-glycans have been shown to play critical roles in numerous biological processes, little is known about how the GalNAc-Ts are targeted to their site of action within the Golgi complex. Here, we investigate the essential protein domains required for Golgi localization of four representative members of the GalNAc-T family of enzymes. We find that GalNAc-T1 and -T2 require their cytoplasmic tail and transmembrane domains for proper Golgi localization, while GalNAc-T10 requires its transmembrane and luminal stem domains. GalNAc-T7 can use either its cytoplasmic tail or its luminal stem, in combination with its transmembrane domain, to localize to the Golgi. We determined that a single glutamic acid in the GalNAc-T10 cytoplasmic tail inhibits its ability to localize to the Golgi via a cytoplasmic tail-dependent mechanism. We therefore demonstrate that despite their similarity, different members of this enzyme family are directed to the Golgi by more than one set of targeting signals.

  • Ligand binding and retention in snake gourd seed lectin (SGSL). A crystallographic, thermodynamic and molecular dynamics study
    Glycobiology (IF 3.664) Pub Date : 2018-08-06
    Thyageshwar Chandran, Nukathoti Sivaji, Avadhesha Surolia, Mamannamana Vijayan

    Snake gourd seed lectin (SGSL) is a non-toxic homologue of type II ribosome inactivating proteins (RIPs) which contain a catalytic domain and a lectin domain. Isothermal titration calorimetry (ITC) measurements of the interactions of the protein with LacNAc, Lac, Gal, Me-α-Gal were carried out and the crystal structures of the native protein and its complex with Lac were determined. The crystal structure of the Me-α-Gal complex has already been determined. While the crystal structure showed the presence of two-sugar binding sites, one on each of the two domains of the lectin chain, ITC measurements indicated the presence of only one binding site. In order to resolve this anomaly, Molecular Dynamics (MD) simulations were carried out on the native protein and on its complexes with Me-α-Gal and Lac. Simulations were also performed on the protein after reducing the inter-chain disulphide bridge between the two chains. The crystal structures and the simulations confirmed the robustness of the protein structure, irrespective of the presence or absence of the disulphide bridge. The simulations indicated that although two sites can bind sugar, only the ligand at one site is retained in a dynamic situation. The studies thus bring out the subtle relationship between binding and retention of the ligand.

  • 更新日期:2018-08-02
  • Meeting Report of the International Life Science Integration Workshop 2018
    Glycobiology (IF 3.664) Pub Date : 2018-07-10

    The first International Life Science Integration Workshop was held on 5–9 March 2018 in Tokyo, Japan. This workshop was sponsored by the Glycoinformatics Consortium (GLIC), Japan Science and Technology Agency (JST) and the National Bioscience Database Center of Japan (NBDC). The first 2 days of this workshop consisted of a symposium with invited talks from experimentalists and bioinformatics experts in the life sciences. The last 3 days were devoted to a Hackathon to further develop communication between the different software projects and support the integration of different life science domains. This was one of the first meetings where glycomics software developers met with leading proteomics and lipidomics resource experimentalists, and the hackathon sessions provided a forum to discuss strategies for data integration and improved data interoperability in the life science domain. The results of the workshop are presented here.

  • Functional crosstalk among oxidative stress and O-GlcNAc signaling pathways
    Glycobiology (IF 3.664) Pub Date : 2018-04-03
    Po-Han Chen, Jen-Tsan Chi, Michael Boyce

    In metazoans, thousands of intracellular proteins are modified with O-linked β-N-acetylglucosamine (O-GlcNAc) in response to a wide range of stimuli and stresses. In particular, a complex and evolutionarily conserved interplay between O-GlcNAcylation and oxidative stress has emerged in recent years. Here, we review the current literature on the connections between O-GlcNAc and oxidative stress, with a particular emphasis on major signaling pathways, such as KEAP1/NRF2, FOXO, NFκB, p53 and cell metabolism. Taken together, this work sheds important light on the signaling functions of protein glycosylation and the mechanisms of stress responses alike and illuminates how the two are integrated in animal cell physiology.

  • A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches
    Glycobiology (IF 3.664) Pub Date : 2018-06-08
    Paulo A G Soares, Kátia A Ribeiro, Ana P Valente, Nina V Capillé, Stephan-Nicollas M C G Oliveira, Ana M F Tovar, Mariana S Pereira, Eduardo Vilanova, Paulo A S Mourão

    Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in β-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the β-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

  • Quiescin sulfhydryl oxidase 1 (QSOX1) glycosite mutation perturbs secretion but not Golgi localization
    Glycobiology (IF 3.664) Pub Date : 2018-05-25
    Ben Horowitz, Gabriel Javitt, Tal Ilani, Yair Gat, David Morgenstern, Frederic A Bard, Deborah Fass

    Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that dictate QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

  • Identification of serum glycoprotein ligands for the immunomodulatory receptor blood dendritic cell antigen 2
    Glycobiology (IF 3.664) Pub Date : 2018-06-11
    Jong-won Kim, James Budzak, Yu Liu, Sabine A F Jégouzo, Kurt Drickamer, Maureen E Taylor

    Blood dendritic cell antigen 2 (BDCA-2) is a C-type lectin found on the surface of plasmacytoid dendritic cells. It functions as a glycan-binding receptor that downregulates the production of type I interferons and thus plays a role in oligosaccharide-mediated immunomodulation. The carbohydrate recognition domain in BDCA-2 binds selectively to galactose-terminated bi-antennary glycans. Because the plasmacytoid dendritic cells function in a plasma environment rich in glycoproteins, experiments have been undertaken to identify endogenous ligands for blood dendritic cell antigen 2. A combination of blotting, affinity chromatography and proteomic analysis reveals that serum glycoprotein ligands for BDCA-2 include IgG, IgA and IgM. Compared to binding of IgG, which was previously described, IgA and IgM bind with higher affinity. The association constants for the different subclasses of immunoglobulins are below and roughly proportional to the serum concentrations of these glycoprotein ligands. Binding to the other main serum glycoprotein ligand, α2-macroglobulin, is independent of whether this protease inhibitor is activated. Binding to all of these glycoprotein ligands is mediated predominantly by bi-antennary glycans in which each branch bears a terminal galactose residue. The different affinities of the glycoprotein ligands reflect the different numbers of these galactose-terminated glycans and their degree of exposure on the native glycoproteins. The results suggest that normal serum levels of immunoglobulins could downmodulate interferon stimulation of further antibody production.

  • Streptococcal Siglec-like adhesins recognize different subsets of human plasma glycoproteins: implications for infective endocarditis
    Glycobiology (IF 3.664) Pub Date : 2018-06-14
    Barbara A Bensing, Qiongyu Li, Dayoung Park, Carlito B Lebrilla, Paul M Sullam

    Streptococcus gordonii and Streptococcus sanguinis are typically found among the normal oral microbiota but can also cause infective endocarditis. These organisms express cell surface serine-rich repeat adhesins containing “Siglec-like” binding regions (SLBRs) that mediate attachment to α2-3-linked sialic acids on human glycoproteins. Two known receptors for the Siglec-like adhesins are the salivary mucin MG2/MUC7 and platelet GPIbα, and the interaction of streptococci with these targets may contribute to oral colonization and endocarditis, respectively. The SLBRs display a surprising diversity of preferences for defined glycans, ranging from highly selective to broader specificity. In this report, we characterize the glycoproteins in human plasma recognized by four SLBRs that prefer different α2-3 sialoglycan structures. We found that the SLBRs recognize a surprisingly small subset of plasma proteins that are extensively O-glycosylated. The preferred plasma protein ligands for a sialyl-T antigen-selective SLBR are proteoglycan 4 (lubricin) and inter-alpha-trypsin inhibitor heavy chain H4. Conversely, the preferred ligand for a 3′sialyllactosamine-selective SLBR is glycocalicin (the extracellular portion of platelet GPIbα). All four SLBRs recognize C1 inhibitor but detect distinctly different glycoforms of this key regulator of the complement and kallikrein protease cascades. The four plasma ligands have potential roles in thrombosis and inflammation, and each has been cited as a biomarker for one or more vascular or other diseases. The combined results suggest that the interaction of Siglec-like adhesins with different subsets of plasma glycoproteins could have a significant impact on the propensity of streptococci to establish endocardial infections.

  • β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain
    Glycobiology (IF 3.664) Pub Date : 2018-05-11
    Hari P Dulal, Yoshiyuki Adachi, Naohito Ohno, Yoshiki Yamaguchi

    Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1–3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1–3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223 and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein–protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling.

  • Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases
    Glycobiology (IF 3.664) Pub Date : 2018-06-20
    Susannah M L Gagnon, Max S G Legg, Robert Polakowski, James A Letts, Mattias Persson, Shuangjun Lin, Ruixiang Blake Zheng, Brian Rempel, Brock Schuman, Omid Haji-Ghassemi, Svetlana N Borisova, Monica M Palcic, Stephen V Evans

    Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

  • Prognostic role of the sialyltransferase ST6GAL1 in ovarian cancer
    Glycobiology (IF 3.664) Pub Date : 2018-07-16
    Beatrice Wichert, Karin Milde-Langosch, Vladimir Galatenko, Barbara Schmalfeldt, Leticia Oliveira-Ferrer

    Aberrant sialylation of glycoproteins has been detected in many tumours, and up-regulation of the beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6GAL1) has been implicated with tumour aggressiveness and chemoresistance in experimental models. In our present study we aimed to study the prognostic or predictive role of ST6GAL1 in ovarian carcinoma, using two independent ovarian cancer cohorts. ST6GAL1 mRNA levels were retrieved from a publicly available database (n=517), and ST6GAL1 protein levels were analysed by western blot analysis in a cohort of 204 ovarian tumour samples. The results were correlated with clinical and histological tumour parameters and follow-up information. High ST6GAL1 mRNA levels significantly correlated with lymphovascular invasion and shorter survival, whereas high ST6GAL1 protein expression was associated with advanced stage, distant metastasis and shorter recurrence-free intervals. In both cohorts the prognostic role was most pronounced in tumours without macroscopically visible residual tumour after surgery. In these cases, ST6GAL1 expression levels might help to identify cases with a higher risk of chemoresistance and metastatic relapse that might require an adapted therapeutic regime.

  • Demystifying O-GlcNAcylation: hints from peptide substrates
    Glycobiology (IF 3.664) Pub Date : 2018-03-22
    Jie Shi, Rob Ruijtenbeek, Roland J Pieters

    O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

  • KIAA1199 expression and hyaluronan degradation colocalize in multiple sclerosis lesions
    Glycobiology (IF 3.664) Pub Date : 2018-07-07
    Mathieu Marella, Laurence Jadin, Gilbert A Keller, Barry J Sugarman, Gregory I Frost, H Michael Shepard

    Modification of hyaluronan (HA) accumulation has been shown to play a key role in regulating inflammatory processes linked to the progression of multiple sclerosis (MS). The aim of this study was to characterize the enzymatic activity involved in HA degradation observed within focal demyelinating lesions in the experimental autoimmune encephalomyelitis (EAE) animal model.

  • Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy
    Glycobiology (IF 3.664) Pub Date : 2018-06-19
    Laurino Carmen, Vadala Maria, Julio Cesar Morales-Medina, Annamaria Vallelunga, Beniamino Palmieri, Tommaso Iannitti

    Duchenne Muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterised by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix of mammalian cells. These macromolecules interact with numerous ligands, including extracellular matrix constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarising the state of the art of GAG dysregulation within the extracellular matrix in DMD and discussing future therapeutic perspectives.

  • Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate–carbohydrate interactions
    Glycobiology (IF 3.664) Pub Date : 2018-04-03
    Marit Sletmoen, Thomas A Gerken, Bjørn T Stokke, Joy Burchell, C Fred Brewer

    The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate–carbohydrate interactions, CCIs) of Tn (Tn–Tn) and STn (STn–STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, which may participate in oncogenesis.

  • Global aspects of viral glycosylation
    Glycobiology (IF 3.664) Pub Date : 2018-03-21
    Ieva Bagdonaite, Hans H Wandall

    Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.

  • TCA cycle-powered synthesis of fucosylated oligosaccharides
    Glycobiology (IF 3.664) Pub Date : 2018-05-24
    Ningzi Guan, Hyun-Dong Shin, Lingfeng Long, Parastoo Azadi, Rachel Chen

    Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progress, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, given the stunning diversity and many varied applications of glycans, broadening the range of glycans accessible via microbial synthesis is of paramount importance. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular “power house,” the TCA cycle, to provide the cellular energy for synthesis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis by-products. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding and pH control in the synthesis stage. The system could achieve several grams per liter (3–4 g/L) within a 24-h period in shaker flask cultivation for two targets, fucosyllactose and fucosyllactulose, demonstrating efficiency of the biocatalyst developed and its applicability to both natural and non-natural targets. To the best of our knowledge, this is the first use of TCA cycle intermediates as the energy source for oligosaccharide synthesis and the first description of successful synthesis of fucosyllactulose with titers in several grams per liter.

  • The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans
    Glycobiology (IF 3.664) Pub Date : 2018-05-11
    Jorick Vanbeselaere, Shi Yan, Anja Joachim, Katharina Paschinger, Iain BH Wilson

    O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

  • Generation and characterization of a site-specific antibody for SIRT1 O-GlcNAcylated at serine 549
    Glycobiology (IF 3.664) Pub Date : 2018-04-24
    Hui Shan, Jiahui Sun, Minghui Shi, Xue Liu, Zhu Shi, Wengong Yu, Yuchao Gu

    O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies thousands of proteins. However, the roles and mechanisms of O-GlcNAcylation have been clarified in only a few proteins, and one of the main reasons for this is the lack of site-specific anti-O-GlcNAc antibodies. Recently, we found that SIRT1, which is an NAD+-dependent deacetylase, is O-GlcNAcylated at the serine 549 site (S549) and plays a cytoprotective role under stress. However, the mechanism underlying the roles of SIRT1 O-GlcNAcylation remains unclear. Here, we describe a site-specific antibody for SIRT1 O-GlcNAcylated at S549, named SIRT1-549-O. This antibody can be used for immunoprecipitation and western blotting assays, and it can be used to recognize the endogenous levels of both human and mouse SIRT1 O-GlcNAcylation. Therefore, this antibody not only provides an effective method to further understand the roles of SIRT1 O-GlcNAcylation but also makes it possible to discover the genetic and pharmacological factors that could regulate SIRT1 activity by modulating its O-GlcNAcylation.

  • Quantifying the binding stoichiometry and affinity of histo-blood group antigen oligosaccharides for human noroviruses
    Glycobiology (IF 3.664) Pub Date : 2018-03-19
    Ling Han, Ruixiang Zheng, Michele R Richards, Ming Tan, Elena N Kitova, Xi Jiang, John S Klassen

    Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis. Many HuNoVs recognize histo-blood group antigens (HBGAs) as cellular receptors or attachment factors for infection. It was recently proposed that HuNoV recognition of HBGAs involves a cooperative, multistep binding mechanism that exploits both known and previously unknown glycan binding sites. In this study, binding measurements, implemented using electrospray ionization mass spectrometry (ESI-MS) were performed on homodimers of the protruding domain (P dimers) of the capsid protein of three HuNoV strains [Saga (GII.4), Vietnam 026 (GII.10) and VA387 (GII.4)] with the ethyl glycoside of the B trisaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-OC2H5) and free B type 1 tetrasaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-(1→3)-d-GlcNAc) in an effort to confirm the existence of new HBGA binding sites. After correcting the mass spectra for nonspecific interactions that form in ESI droplets as they evaporate to dryness, all three P dimers were found to bind a maximum of two B trisaccharides at the highest concentrations investigated. The apparent affinities measured for stepwise binding of B trisaccharide suggest positive cooperativity. Similar results were obtained for B type 1 tetrasaccharide binding to Saga P dimer. Based on these results, it is proposed that HuNoV P dimers possess only two HBGA binding sites. It is also shown that nonspecific binding corrections applied to mass spectra acquired using energetic ion source conditions that promote in-source dissociation can lead to apparent HuNoV–HBGA oligosaccharide binding stoichiometries and affinities that are artificially high. Finally, evidence that high concentrations of oligosaccharide can induce conformational changes in HuNoV P dimers is presented.

  • Fine-tuning the structure of glycosaminoglycans in living cells using xylosides
    Glycobiology (IF 3.664) Pub Date : 2018-05-24
    Andrea Persson, Ulf Ellervik, Katrin Mani

    Xylosides can induce the formation and secretion of xyloside-primed glycosaminoglycans when administered to living cells; however, their impact on the detailed glycosaminoglycan structure remains unknown. Here, we have systematically investigated how the xyloside concentration and the type of xyloside, as well as the cell type, influenced the structure of xyloside-primed glycosaminoglycans in terms of the heparan sulfate and chondroitin/dermatan sulfate proportion and disaccharide composition. We found that although greatest influence was exerted by the cell type, both the xyloside concentration and type of xyloside impacted the proportion of heparan sulfate and the complexity of chondroitin/dermatan sulfate. The disaccharide composition of the chondroitin/dermatan sulfate was influenced by the xyloside concentration and type of xyloside to a higher extent than that of the heparan sulfate; the proportion of 4S-sulfated disaccharides in the chondroitin/dermatan sulfate decreased and the proportions of 6S-sulfated and/or nonsulfated disaccharides increased both with increasing concentrations of xyloside and with increasing xyloside hydrophobicity, whereas the proportion of nonsulfated disaccharides was primarily altered in the heparan sulfate with increasing concentrations of xyloside. Our results indicate that it is feasible to not only produce large amounts of glycosaminoglycans in living cells but also to fine-tune their structures by using xylosides of different types and at different concentrations.

  • Novel thioglycoside analogs of α-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells
    Glycobiology (IF 3.664) Pub Date : 2018-05-06
    Ashanty M Melo, Lei Zhang, Éilis F Dockry, Andreea Petrasca, Yasmeen G Ghnewa, Eamon P Breen, Maria E Morrissey, Ciara O’Reilly, Robyn Bruen, Andrew O’Meara, Joanne Lysaght, Xiangming Zhu, Derek G Doherty

    Invariant natural killer T (iNKT) cells recognize glycolipid antigens bound to CD1d molecules on antigen-presenting cells. Therapeutic activation of iNKT cells with the xenogeneic glycolipid α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in murine models, but clinical trials using α-GalCer-stimulated human iNKT cells have shown limited efficacy. We synthesized a series of thioglycoside analogs of α-GalCer with different substituents to the galactose residue and found that two of these compounds, XZ7 and XZ11, bound to CD1d-transfected HeLa cells and activated lines of expanded human iNKT cells. Both compounds stimulated cytolytic degranulation by iNKT cells and while XZ7 preferentially stimulated the production of the antitumor cytokine interferon-γ (IFN-γ), XZ11 preferentially stimulated interleukin-4 (IL-4) production. This biased T helper type 1 effector profile of XZ7 was also evident when iNKT were stimulated with dendritic cells presenting this glycolipid. Separate analysis of the responses of CD4+, CD8α+ and CD4−CD8− iNKT cells indicated that XZ7 preferentially activated CD8α+ iNKT cells, and to a lesser degree, CD4−CD8− iNKT cells. The partial agonist effect of glycolipid XZ7, inducing cytotoxicity and IFN-γ production but not IL-4 production, indicates that specific protumour activities of iNKT cells can be abolished, while preserving their antitumor activities, by introducing structural modifications to α-GalCer. Since XZ7 was much less potent than α-GalCer as an iNKT cell agonist, it is unlikely to be superior to α-GalCer as a therapeutic agent for cancer, but may serve as a parent compound for developing more potent structural analogs.

  • Distinct amino acid residues confer one of three UDP-sugar substrate specificities in Acinetobacter baumannii PglC phosphoglycosyltransferases
    Glycobiology (IF 3.664) Pub Date : 2018-04-13
    Christian M Harding, M Florencia Haurat, Evgeny Vinogradov, Mario F Feldman

    Acinetobacter baumannii is an opportunistic human pathogen with the highest reported rates of multidrug resistance among Gram-negative pathogens. The capsular polysaccharide of A. baumannii is considered one of its most significant virulence factors providing resistance against complemented-mediated killing. Capsule synthesis in A. baumannii is usually initiated by the phosphoglycosyltransferase PglC. PglC transfers a phosphosugar from a nucleotide diphosphate-sugar to a polyprenol phosphate generating a polyprenol diphosphate-linked monosaccharide. Traditionally, PglC was thought to have stringent specificity towards UDP-N-N′-diacetylbacillosamine (UDP-diNAcBac). In this work we demonstrate that A. baumannii PglC has the ability to utilize three different UDP-sugar substrates: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) or UDP-diNAcBac. Using phylogenetic analyses, we first demonstrate that A. baumannii PglC orthologs separate into three distinct clades. Moreover, all members within a clade are predicted to have the same preference for one of the three possible sugar substrates. To experimentally determine the substrate specificity of each clade, we utilized in vivo complementation models and NMR analysis. We demonstrate that UDP-diNAcBac is accommodated by all PglC orthologs, but some orthologs evolved to utilize UDP-GlcNAc or UDP-GalNAc in a clade-dependent manner. Furthermore, we show that a single point mutation can modify the sugar specificity of a PglC ortholog specific for UDP-diNAcBac and that introduction of a non-native PglC ortholog into A. baumannii can generate a new capsule serotype. Collectively, these studies begin to explain why A. baumannii strains have such highly diverse glycan repertoires.

  • Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia
    Glycobiology (IF 3.664) Pub Date : 2018-04-28
    Ngoc Thy Nguyen, Romain R Vivès, Magali Torres, Vincent Delauzun, Els Saesen, Véronique Roig-Zamboni, Hugues Lortat-Jacob, Pascal Rihet, Yves Bourne

    The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.

  • Glycoengineering design options for IgG1 in CHO cells using precise gene editing
    Glycobiology (IF 3.664) Pub Date : 2018-03-27
    Morten A Schulz, Weihua Tian, Yang Mao, Julie Van Coillie, Lingbo Sun, Joachim S Larsen, Yen-Hsi Chen, Claus Kristensen, Sergey Y Vakhrushev, Henrik Clausen, Zhang Yang

    Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.

  • Structural basis of oligosaccharide processing by glycosaminoglycan sulfotransferases
    Glycobiology (IF 3.664) Pub Date : 2018-06-06
    Tarsis F Gesteira, Vivien J Coulson-Thomas

    Heparan sulfate (HS) is a sulfated polysaccharide that plays a key role in morphogenesis, physiology and pathogenesis. The biosynthesis of HS takes place in the Golgi apparatus by a group of enzymes that polymerize, epimerize and sulfate the sugar chain. This biosynthetic process introduces varying degrees of sulfate substitution, which are tightly regulated and directly dictate binding specificity to different cytokines, morphogens and growth factors. Here we report the use of molecular dynamics simulations to investigate the dynamics of substrate recognition of two glycosaminoglycan (GAG) sulfotransferases, N-deacetylase-N-sulfotransferase and 2-O-sulfotransferase to the HS chain during the biosynthetic process. We performed multiple simulations of the binding of the sulfotransferase domains to both the HS oligosaccharide substrate and sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPs). Analysis of extended simulations provide detailed and useful insights into the atomic interactions that are at work during oligosaccharide processing. The Fast Information Matching method was used to detect the enzyme global dynamics and to predict the pairwise contact of residues responsible for GAG-enzyme binding and unbinding. The correlation between HS displacement and the location of the modified GAG chain were calculated, indicating a possible route for HS and heparin during sulfotransferase processing. Our data also show sulfotransferases contain a conserved interspaced positively charged amino acid residues that form a patch which controls the protein-GAG binding equilibrium. Together, our findings provide further understanding on the fine-tuned complex mechanism of GAG biosynthesis. Our findings can also be extrapolated to other systems for calculating rates of protein-GAG binding.

  • Addicted to sugar: roles of glycans in the order Mononegavirales
    Glycobiology (IF 3.664) Pub Date : 2018-06-06
    Victoria Ortega, Jacquelyn A Stone, Erik M Contreras, Ronald M Iorio, Hector C Aguilar

    Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell-cell signaling, protein folding, and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, Rabies, Measles, and Nipah viruses. We discuss glycobiological findings by genera in alphabetical order within each of eight Mononegavirales families, namely the bornaviruses, filoviruses, mymonaviruses, nyamiviruses, paramyxoviruses, pneumoviruses, rhabdoviruses, and sunviruses.

  • Cytosolic galectin-3 and -8 regulate antibacterial autophagy through differential recognition of host glycans on damaged phagosomes
    Glycobiology (IF 3.664) Pub Date : 2018-02-23
    I-Chun Weng, Hung-Lin Chen, Tzu-Han Lo, Wei-Han Lin, Huan-Yuan Chen, Daniel K Hsu, Fu-Tong Liu

    While glycans are generally displayed on the cell surface or confined within the lumen of organelles, they can become exposed to the cytosolic milieu upon disruption of organelle membrane by various stresses or pathogens. Galectins are a family of β-galactoside-binding animal lectins synthesized and predominantly localized in the cytosol. Recent research indicates that some galectins may act as “danger signal sensors” by detecting unusual exposure of glycans to the cytosol. Galectin-8 was shown to promote antibacterial autophagy by recognizing host glycans on ruptured vacuolar membranes and interacting with the autophagy adaptor protein NDP52. Galectin-3 also accumulates at damaged phagosomes containing bacteria; however, its functional consequence remains obscure. By studying mouse macrophages infected with Listeria monocytogenes (LM), we showed that endogenous galectin-3 protects intracellular LM by suppressing the autophagic response through a host N-glycan-dependent mechanism. Knock out of the galectin-3 gene resulted in enhanced LC3 recruitment to LM and decreased bacterial replication, a phenotype recapitulated when Galectin-8-deficient macrophages were depleted of N-glycans. Moreover, we explored the concept that alterations in cell surface glycosylation by extracellular factors can be deciphered by cytosolic galectins during the process of phagocytosis/endocytosis, followed by rupture of phagosomal/endosomal membrane. Notably, treatment of cells with sialidase, which removes sialic acid from glycans, resulted in increased galectin-3 accumulation and decreased galectin-8 recruitment at damaged phagosomes, and led to a stronger anti-autophagic response. Our findings demonstrate that cytosolic galectins may sense changes in glycosylation at the cell surface and modulate cellular response through differential recognition of glycans on ruptured phagosomal membranes.

  • Airway glycomic and allergic inflammatory consequences resulting from keratan sulfate galactose 6-O-sulfotransferase (CHST1) deficiency
    Glycobiology (IF 3.664) Pub Date : 2018-04-06
    Tadahiro Kumagai, Takumi Kiwamoto, Mary E Brummet, Fan Wu, Kazuhiro Aoki, Zhou Zhu, Bruce S Bochner, Michael Tiemeyer

    Siglec-F is a pro-apoptotic receptor on mouse eosinophils that recognizes 6′-sulfated sialyl Lewis X and 6′-sulfated sialyl N-acetyl-lactosamine as well as multivalent sialyl N-acetyl-lactosamine structures on glycan arrays. We hypothesized that attenuation of the carbohydrate sulfotransferase 1 (CHST1) gene encoding keratan sulfate galactose 6-O-sulfotransferase, an enzyme likely required for 6′-sulfation of some of these putative Siglec-F glycan ligands, would result in decreased Siglec-F lung ligand levels and enhanced allergic eosinophilic airway inflammation. Tissue analysis detected CHST1 expression predominantly not only in parenchymal cells but not in airway epithelium, the latter being a location where Siglec-F ligands are located. Western blotting of lung extracts with Siglec-F–Fc fusion proteins detected ≈500 kDa and ≈200 kDa candidate Siglec-F ligands that were not appreciably altered in CHST1−/− lungs compared with normal mouse lungs. Characterization of the O-linked glycans of lung tissue and bronchoalveolar lavage fluid detected altered sialylation but minimal change in sulfation. Eosinophilic airway inflammation was induced in wild-type (WT) and CHST1−/− mice via sensitization to ovalbumin (OVA) and repeated airway challenge. After OVA sensitization and challenge, Siglec-F ligands on airway cells, and numbers of eosinophils and neutrophils accumulating in the airways, both increased to a similar degree in WT and CHST1−/− mouse lungs, while macrophages and lymphocytes increased significantly more in CHST1−/− mouse airway compared with normal mouse lungs. Therefore, keratan sulfate galactose 6-O-sulfotransferase does not contribute to the synthesis of glycan ligands for Siglec-F in the airways, although its absence results in exaggerated accumulation of airway macrophages and lymphocytes.

  • Crystal structures of an archaeal chitinase ChiD and its ligand complexes
    Glycobiology (IF 3.664) Pub Date : 2018-03-21
    Yuichi Nishitani, Ayumi Horiuchi, Mehwish Aslam, Tamotsu Kanai, Haruyuki Atomi, Kunio Miki

    Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

  • Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis
    Glycobiology (IF 3.664) Pub Date : 2018-03-07
    Felipe C O B Teixeira, Eliene Oliveira Kozlowski, Kayene Vitória de A Micheli, Ana Cristina E S Vilela-Silva, Lubor Borsig, Mauro S G Pavão

    Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell–platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet–tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure–function relationship or even for future treatments.

  • Recent Approaches for Directly Profiling Cell Surface Sialoform
    Glycobiology (IF 3.664) Pub Date : 2018-05-24
    Xiaoqing Zhang, Huan Nie, Joshua Whited, Dan Wang, Yu Li, Xue-Long Sun

    Sialic acids (SAs) are nine-carbon monosaccharides existing at the terminal location of glycan structures on the cell surface and secreted glycoconjugates. The expression levels and linkages of SAs on cells and tissues, collectively known as sialoform, present the hallmark of the cells and tissues of different systems and conditions. Accordingly, detecting or profiling cell surface sialoforms is very critical for understanding the function of cell surface glycans and glycoconjugates and even the molecular mechanisms of their underlying biological processes. Further, it may provide therapeutic and diagnostic applications for different diseases. In the past decades, several SA-specific binding molecules have been developed for detecting and profiling specific sialoforms of cells and tissues; the experimental materials have expanded from frozen tissue to living cells; and the analytical technologies have advanced from histochemistry to fluorescent imaging, flow cytometry and microarrays. This review summarizes the recent bio-affinity approaches for directly detecting and profiling specific SAs or sialylglycans, and their modifications of different cells and tissues.

  • 更新日期:2018-05-04
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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