No description available.

Yes / The disulfide bond plays an important role in the formation and stabilisation of higher order structures of peptides and proteins, while in recent years interest in this functional group has been extended to carbohydrate chemistry. Rarely found in nature, glycosyl disulfides have attracted significant attention as glycomimetics, with wide biological applications including lectin binding, as key components of dynamic libraries to study carbohydrate structures, the study of metabolic and enzymatic studies, and even as potential drug molecules. This interest has been accompanied and fuelled by the continuous development of new methods to construct the disulfide bond at the anomeric centre. Glycosyl disulfides have also been exploited as versatile intermediates in carbohydrate synthesis, particularly as glycosyl donors. This review focuses on the importance of the disulfide bond in glycobiology and in chemistry, evaluating the different methods available to synthesise glycosyl disulfides. Furthermore, we review the role of glycosyl disulfides as intermediates and/or glycosyl donors for the synthesis of neoglycoproteins and oligosaccharides, before finally considering examples of how this important class of carbohydrates have made an impact in biological and therapeutic contexts. / The authors thank the Institute of Cancer Therapeutics (University of Bradford) Doctoral Training Centre for financial support.

Les conversions enzymatiques des antigènes du système des groupes sanguins ABO et de l'antigène majeur de xénotransplantation α3Gal représentent des alternatives thérapeutiques afin d'améliorer l'approvisionnement des hôpitaux en sang de groupe O et la transplantation d'organes. La famille GH110 des glycoside hydrolases comprend des enzymes actives sur les antigène B et α3Gal à pH neutre et fonctionnant avec un mécanisme catalytique par inversion. La sous‐famille GH110_A regroupe des membres actifs exclusivement sur l'antigène B, tandis que la sous‐famille GH110_B regroupe des membres actifs sur les antigènes B et α3Gal. Le travail présenté dans ce manuscrit décrit les études structurales et fonctionnelles de deux α‐galactosidases, BtGal110A (GH110_A) et BtGal110B (GH110_B), de la famille GH110 provenant de la bactérie commensale Bacteroides thetaiotaomicron et représentatives des chacune des ces deux sous‐familes. La spécificité de substrat et la grande efficacité catalytique de ces enzymes à pH neutre ont été confirmées grâce à des oligosaccharides mimant l'extrémité des antigènes B et α3Gal. BtGal110A et BtGal110B se replient en hélice β droite et se distinguent d'autres protéines au repliement similaire par la présence de deux domaines additionnels qui adoptent un repliement en demi tonneau β et participent à l'architecture du site actif. La machinerie catalytique de BtGal110A et BtGal110B est constituée de trois acides aspartiques, semblable à celle des enzymes des familles GH28 et GH49, qui partagent la même repliement en hélice β. / Enzymatic conversions of the ABO blood group antigens and of the major xenotransplantation α3Gal antigen represent therapeutic alternatives intended to improve supply of hospitals with requested blood type O and organs. The family GH110 of glycoside hydrolases includes enzymes active towards B and α3Gal antigen within neutral pH and employs an inverting catalytic mechanism. The GH110_A subfamily gathers enzymes with exquisite substrate specificity towards the B antigen, while the GH110_B subfamily gathers enzymes active towards the B and the α3Gal antigens. The present manuscript describes the functional and structural studies of two α‐galactosidases BtGal110A (GH110_A) and BtGal110B (GH110_B) from the commensal bacteria Bacteroides thetaiotaomicron, two representative members of each of these two subfamilies. The substrate specificity and high catalytic efficiency of these enzymes at neutral pH were confirmed using oligosaccharides corresponding to the terminal carbohydrate sequences of the B and α3Gal antigens. BtGal110A and BtGal110B contain a right‐handed β‐helix, but he presence of two additional domains tfolded as half β‐barrels, which participate to the architecture of the active site distinguishes BtGal110A and BtGal110B fro other related glycoside hydrolases. The catalytic machinery consists of three aspartic acids, reminiscent of that of enzymes from the GH28 and GH49 families, which share a similar β‐helix fold. Our observations support the membership of family GH110 to the clan‐N of glycoside hydrolases.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações relevantes sobre o papel desempenhado pela sinalização de insulina e IGF-1 durante a progressão de carcinomas. / Colorectal cancer is a major cause of cancer-related morbidity and mortality. In Brazil, it is the third most common cancer. Many studies have been developed to clarify several molecular features which regulate phenotypic changes exhibited by cells that constitute colorectal cancer, however, comparatively, there are few studies dedicated to investigate co- and post-translational modifications of proteins in this process. Glycosylation is the most common post-translational modification of proteins. Approximately fifty percent of all proteins are glycosylated. During malignant transformation, changes in the expression profile of glycans (carbohydrates covalently bound to proteins or lipids) may be involved in a variety of events, including the loss of cellcell and cellmatrix adhesion, migration, invasion, and evasion of apoptosis. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors, swainsonine and tunicamycin, in cells derived from colorectal cancer (Caco-2, HCT-116 e HT-29). Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in HCT-116 colon cancer cells. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment. Moreover, we confirmthe existence of an interplay between stimulation with insulin and IGF-1 and bisecting GlcNAc N-glycans expression in MDA-MB435 cancer cells, providing new insights into the role that Insulin/IGF-I signaling play during carcinoma progression.

Dendritic cells (DCs) are regulators of the immune system and express a class of pattern recognition receptors known as C-type lectin receptors (CLRs) to recognize and respond to carbohydrates (glycans). Dendritic cells are hypothesized to be key mediators in the immune response to implanted materials and ligation of CLRs has been shown to have diverse effects on DC phenotype ranging from tolerogenic to pro-inflammatory. Thus, designing future biomaterials and combination products that harness the potential of CLR ligation on DCs has great promise. Additionally, many of the proteins which adsorb to biomaterials when implanted are glycosylated and thus understanding this interaction would provide further insight into the host response to currently implanted materials. However, DC responses to glycans presented from non-phagocytosable surfaces has not been well characterized and optimal factors for DC phenotype modulation by surface presented glycans are unknown. Additionally, studies relating DC response to glycan structures from soluble and phagocytosable displays to that of non-phagocytosable display have not been performed. This is of critical importance to the field because of the extremely limited supply of complex glycan structures that are able to be obtained. Because of this limited supply of glycans the trend in glycomics has been toward creation of glycan microarrays to assess initial candidates of interest for further study. However, the assumption that cell response to these glycoconjugate microarrays is equivalent to soluble or phagocytosable conjugates has not been validated. Therefore, the purpose of this study was to 1) determine the optimal molecular contextual variables of glycoconjugate presentation from a non-phagocytosable surface, namely, charge, density, and glycan structure for modulating DC phenotype; and 2) determine if modality of glycoconjugate presentation, i.e. soluble, phagocytosable, or non-phagocytosable will modulate DC phenotype differentially. To determine the effect of the molecular contextual variables primary human immature DCs (iDCs) were exposed to a range of adsorbed glycoconjugates in a 384 well plate and their subsequent phenotype assessed via a novel in house produced high throughput (HTP) assay. Bovine serum albumin (BSA) was modified to have a range of glycan densities and isoelectric points to determine which of these were optimal for DC phenotype modulation. Next, several poly-mannose structures were presented to DCs to determine if DC response was structure specific. Finally, contextual variables were modeled in a multivariate general linear model to determine underlying trends in DC behavior and optimal factors for glycan presentation from non-phagocytosable surfaces. To determine the effect of the modality of glycoconjugate display on DCs, optimized glycoconjugates from 1) were adsorbed to the wells of a 384 flat well plate, delivered at varying soluble concentrations, or adsorbed to phagocytosable 1 µm beads and subsequent DC phenotype assessed via the HTP assay. The cell response to the glycoconjugates was then validated to be CLR mediated and the DC response to glycan modality was modeled in another general linear model. Results from these studies show that highly cationized high density glycoconjugates presented from non-phagocytosable flat well display modulate DC phenotype toward a pro-inflammatory phenotype to the greatest extent. Additionally, significant impacts on DC phenotype in response to adsorbed conjugates can be seen when grouping glycan structure by terminal glycan motif. Finally, DC response to glycoconjugates were found to be CLR mediated and that each modality of glycan display is significantly different, in terms of DC phenotype, from the others. These results provide indications for the future design of glycan microarray systems, biomaterials and combination products. Furthermore, this work indicates that different mechanisms are involved in binding and processing of surface bound versus soluble glycoconjugates. With further study these differences could be harnessed for use in the next generation of biomaterials.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O câncer colorretal representa uma das maiores causas de morbidade e mortalidade relacionadas ao câncer. No Brasil, é o terceiro tipo de câncer mais frequente em homens e mulheres. Muitos estudos estão sendo desenvolvidos no sentido de esclarecer os diversos aspectos moleculares que regulam as alterações fenotípicas exibidas pelas células que constituem o câncer colorretal, no entanto, comparativamente, ainda são poucos os que são dedicados a investigar o papel de modificações co- e pós-traducionais neste processo. Entre os vários tipos destas modificações que ocorrem em proteínas, a glicosilação é a mais comum. Cogita-se que aproximadamente cinquenta por cento de todas as proteínas são glicosiladas. Durante a transformação maligna, mudanças no perfil de expressão de glicanos (carboidratos covalentemente ligados a proteínas ou lipídios) estão envolvidas em uma variedade de mecanismos celulares, tais como: perda da adesão célula-célula e célula matriz, migração, invasão e evasão da apoptose. Neste estudo, foi investigada a atividade antitumoral de inibidores da biossíntese de N-glicanos, swainsonina e tunicamicina, em células derivadas de câncer colorretal (Caco-2, HCT-116 e HT-29). Os resultados obtidos mostram que o tratamento das células HCT-116 com tunicamicina inibe mecanismos celulares relacionados ao fenótipo maligno, como formação de colônia dependente e independente de ancoragem, migração e invasão. Estes resultados sugerem que modulação da biossíntese de N-glicanos parece ser uma potencial ferramenta terapêutica para o tratamento do câncer colorretal. Em outra etapa do trabalho, foram avaliados também o impacto da estimulação com insulina e IGF-1 na expressão N-glicanos bissectados em células tumorais MDA-MB-435. Os resultados obtidos confirmaram também a existência de uma relação entre a estimulação dos receptores de insulina e IGF-1 e a regulação da expressão de N-glicanos bissectados em células tumorais MDA-MB-435, fornecendo assim informações relevantes sobre o papel desempenhado pela sinalização de insulina e IGF-1 durante a progressão de carcinomas. / Colorectal cancer is a major cause of cancer-related morbidity and mortality. In Brazil, it is the third most common cancer. Many studies have been developed to clarify several molecular features which regulate phenotypic changes exhibited by cells that constitute colorectal cancer, however, comparatively, there are few studies dedicated to investigate co- and post-translational modifications of proteins in this process. Glycosylation is the most common post-translational modification of proteins. Approximately fifty percent of all proteins are glycosylated. During malignant transformation, changes in the expression profile of glycans (carbohydrates covalently bound to proteins or lipids) may be involved in a variety of events, including the loss of cellcell and cellmatrix adhesion, migration, invasion, and evasion of apoptosis. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors, swainsonine and tunicamycin, in cells derived from colorectal cancer (Caco-2, HCT-116 e HT-29). Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in HCT-116 colon cancer cells. Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for CRC treatment. Moreover, we confirmthe existence of an interplay between stimulation with insulin and IGF-1 and bisecting GlcNAc N-glycans expression in MDA-MB435 cancer cells, providing new insights into the role that Insulin/IGF-I signaling play during carcinoma progression.

No description available.

No description available.

Yes / Protein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development. / Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant.

The conserved N-linked glycosylation site on the Fc domain of IgG1 antibodies is essential for maintaining a functionally active conformation of the antibody. Different glycoforms of the Fc exhibit widely different effector functions. Similarly, therapeutic antibodies, with engineered glycosylation, exhibit altered binding to cellular Fc receptors (FcRs). Here, X-ray crystallographic structures were obtained for biosynthetic intermediate glycoforms of human IgG1 Fc bearing: unprocessed oligomannose-type, intermediate hybrid-type, and mature complex-type glycans. The fully processed Fc protein crystallised in an “open” conformation with glycans forming canonical stabilising interactions on the protein surface. Analysis of the biosynthetic intermediates revealed that these stabilising hydrophobic protein-glycan interactions are formed only after processing by Golgi -mannosidase II. Mutagenesis of hydrophobic residues on Fc disrupted crucial protein-glycan interactions resulting in the selective destabilization of the 3-arm of the glycan chain with the 6-arm closely matching that seen for the native structure. However, carbohydrate analysis of released glycans shows increased processing on both arms indicating a more accessible and flexible glycan in the mutant structure suggesting that the crystallographic structure of these antibody glycans represents a minor low-energy conformation. The importance of Fc glycosylation is highlighted by endoglycosidases which eliminate Fc effector function. The crystallographic structure of enzymatically deglycosylated IgG Fc revealed a significant collapse of the of Cγ2 domains resulting in a ‘closed’ quaternary conformation, incompatible with Fc receptor binding. This provides a structural explanation for immune deactivating properties of endoglycosidases including those under preclinical development for the treatment of antibody-mediated immune pathology. One such bacterial endoglycosidase, Endo S, was studied further and revealed a specificity for complex-type glycans of the type found on IgG but no hydrolytic activity towards an engineered IgG Fc with oligomannose-type glycans. Introduction of both the engineered monoclonal IgG and endoglycosidase in serum led to a dramatic increase in FcR binding as the competitive binding of serum IgG for FcRs was selectively eliminated. This approach is a general technique for boosting the effector signal of therapeutic antibodies.