Representing glycophenotypes: semantic unification of glycobiology resources for disease discovery.

While abnormalities related to carbohydrates (glycans) are frequent for patients with rare and undiagnosed diseases as well as in many common diseases, these glycan-related phenotypes (glycophenotypes) are not well represented in knowledge bases (KBs). If glycan-related diseases were more robustly represented and curated with glycophenotypes, these could be used for molecular phenotyping to help to realize the goals of precision medicine. Diagnosis of rare diseases by computational cross-species comparison of genotype-phenotype data has been facilitated by leveraging ontological representations of clinical phenotypes, using Human Phenotype Ontology (HPO), and model organism ontologies such as Mammalian Phenotype Ontology (MP) in the context of the Monarch Initiative. In this article, we discuss the importance and complexity of glycobiology and review the structure of glycan-related content from existing KBs and biological ontologies. We show how semantically structuring knowledge about the annotation of glycophenotypes could enhance disease diagnosis, and propose a solution to integrate glycophenotypes and related diseases into the Unified Phenotype Ontology (uPheno), HPO, Monarch and other KBs. We encourage the community to practice good identifier hygiene for glycans in support of semantic analysis, and clinicians to add glycomics to their diagnostic analyses of rare diseases

Genetics of Golgi apparatus regulation in mammalian cells

Ph.DDOCTOR OF PHILOSOPH

Elucidating the role of the family of GalNAc-Transferases in aberrant protein O-glycosylation in the progression of epithelial ovarian cancer

Le cancer épithélial de l’ovaire (CEO) est la forme de cancer gynécologique la plus létale. Ainsi, la compréhension des changements moléculaires associés à ce cancer métastatique ovarien peut mener à l’identification de nouvelles cibles thérapeutiques essentielles. La glycosylation, une modification post-traductionnelle, joue un rôle important dans de nombreuses fonctions cellulaires. Cette glycosylation participe à des événements physiopathologiques majeurs durant la progression tumorale. De plus, il a été prouvé que l’expression aberrante des structures glycanes interfère avec des mécanismes cellulaires comme l’adhésion, la migration et la prolifération des cellules. Dans ce contexte, notre laboratoire a récemment montré que le gène codant pour la protéine N-acétylgalactosaminyltransférase 3 (GALNT3), membre de la famille des GalNAcTransférases (GalNAc-Ts), est hypométhylé et que la protéine GALNT3 est plus fortement exprimée dans les tumeurs CEO dont la sévérité est de grade élevé (“high-grade (HG) serous”), en comparaison avec des tumeurs à potentiel malin faible (“low malignant potential (LMP) ”) et des tissus ovariens normaux. Ces observations indiquent un fort potentiel oncogénique pour le gène GALNT3 dans les stades avancés du CEO. Ces premières constatations suggèrent également que la surexpression de GALNT3 peut jouer un rôle important dans la tumorigenèse du CEO en augmentant sa dissémination via une O-glycosylation de type mucine aberrante. Ces glycosylations anormales peuvent donc être impliquées dans la carcinogenèse ovarienne et nécessitent une étude approfondie. Dans ce projet de recherche, nous proposons d’approfondir les observations déjà obtenues in vitro en utilisant un modèle in vivo chez la souris, afin d’élucider le rôle fonctionnel de la GALNT3 et d’autres membres de cette famille dans la progression du CEO. A partir d’une étude de glycoprotéomique indépendante de la masse, qui a permis d’identifier des glycopeptides intacts ou métaboliquement marqués, ce projet de recherche a rendu possible la définition précise du rôle de GALNT3 dans la O-glycosylation des cibles de type mucine au sein des cellules CEO. Ainsi, via une recherche ciblée dans la base de données « SwissProt » du protéome humain, nous avons trouvé plusieurs centaines de glycoprotéines et glycopeptides uniques, différemment exprimés dans les clones cellulaires dépourvus en iv GALNT3 KD. Par la suite, nous avons identifié les gènes codant pour ces glycoprotéines et glycopeptides. Nous avons notamment trouvé, parmi la liste, un groupe de gènes impliqués dans le métabolisme cellulaire dont les modifications post-traductionnelles sont, de manière intéressante, principalement supprimées dans les clones GALNT3 KD. De plus, nous nous sommes intéressés aux autres membres de la famille des GalNAc-Ts dans le CEO et nous avons montré que de multiples membres et pas uniquement GALNT3 peuvent jouer un rôle important dans la dissémination et la progression du CEO. De plus, une découverte très intéressante fut la redondance possible des rôles joués par certains membres de la famille des GalNAc-Ts dans le CEO. Ainsi, nous avons identifié GALNT6 qui serait, à l’image de GALNT3, impliquée dans la dissémination et la progression du CEO. Cette implication du GALNT6 est supportée par le fait que cette protéine a les mêmes fonctions que GALNT3, suggérant un effet compensatoire de GALNT6 en absence de GALNT3. Pour tester cette hypothèse, nous avons abolie l’expression des deux protéines GALNT3 et GALNT6, in vivo, et nous avons observé une effet significatif sur la formation des tumeurs et la survie des animaux. Pour la suite de ce projet, nous proposons d’analyser la structure glycane des différentes glycoprotéines identifiées dans les cellules cancéreuses, afin de déterminer les altérations des modifications O-glycanes suite à la perte d’expression de GALNT3 et d’autres membres de la famille des GalNAc-Ts. En conclusion, notre étude contribue à comprendre la participation du glycoprotéome dans la tumorigenèse du CEO et à identifier d’autres cibles de type mucine ou des O-glycoprotéines dont l’expression aberrante serait modulée dans le CEO. Ainsi, pris dans son ensemble, ce projet de recherche montre la possibilité de discriminer entre des cellules cancéreuses et des cellules contrôles via les glycosylations de leurs protéines et permet d’entrevoir la glycobiologie comme une voie prometteuse pour l’identifier de nouveaux biomarqueurs pour le diagnostic du CEO.Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, thus understanding the molecular changes associated with ovarian cancer metastasis could lead to the identification of essential therapeutic targets. Glycosylation is a post-translational modification (PTM) of proteins playing a major role in various cell properties. Glycosylation participates in major pathophysiology events during tumor progressions, and the aberrant expression of glycan structures was shown to interfere with cell properties such as cell adhesion, migration, and proliferation. The lab has previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-Transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade (HG) serous EOC tumors, compared to low malignant potential (LMP) EOC tumors and normal ovarian tissues. Taken together, the data obtained were indicative of a strong oncogenic potential of the GALNT3 gene in advanced EOC and suggest that GALNT3 overexpression might contribute to EOC dissemination through aberrant mucin O-glycosylation, thus specifying some of the putative mechanisms of abnormal glycosylation implicated in ovarian carcinogenesis, which warrant further investigation. The current research project focused on expanding the in vitro observations obtained by using animal models to investigate in vivo the functional significance of GALNT3 and other close members of the GalNAc-Ts gene family in serous EOC progression. Moreover, by applying a mass-independent chemical glycoproteomics platform to characterize intact, metabolically labeled glycopeptides, this project more profoundly characterized the role of GALNT3 in aberrant O-glycosylation of mucin-like targets in EOC cells. Isotopically recorded ions were searched against the Swiss-Prot human proteome; and data obtained were indicative of hundreds of unique glycoproteins and glycopeptides that were differentially expressed upon GALNT3 KD. Related gene groups were identified, and interestingly, genes implicated in mechanisms of cellular metabolic functions, and PTMs were found to be predominantly suppressed in GALNT3 KD clones. In accordance, we also investigated the role of other members of the GalNAc-T family in EOC and we showed that multiple members and not only GALNT3 can play an important role in EOC cancer dissemination and progression. One very interesting finding was the redundant role some members of the GalNAc-T family members play in EOC. We investigated the compensatory functions of GALNT3 and GALNT6, and we were able to demonstrate these two genes can impose that synthetic backup. Furthermore, we found that and their ablation can affect animal survival and tumor formation as observed both in vivo and in vitro. In continuation of this work, this project will focus on analyzing the glycan structures of those differentially expressed glycoproteins, to further examine the specific O-glycans alterations associated with the GALNT3 and other members of the GalNAc-Ts upon gene knockout (KO). Fully elaborated glycopeptides can reveal structural details of the glycoproteome, thus our results could give important information on the glycome in EOC cells, and the identification of other O-glycoproteins/mucin-like targets whose aberrant expression may be modulated by these in EOC. Taken together, the ability to mark differences in the glycosylation of proteins between cancer cells and control cells can emphasize glycobiology as a promising field for potential biomarker identification

Method Development for the Identification of Alternatively Sialylated Glycoproteins in Non-Small Cell Lung Cancer

Non-small cell lung cancer represents 85% of all lung cancers with an average 5 year life expectancy of 15-20%. A wealth of data suggests that altered glycosylation contributes to the progression of these tumors and efforts to improve the specificity of biomarkers have logically shifted towards glycoproteomic investigations. For this purpose, recent innovations in experimental strategies and analytical techniques could be combined to provide a more detailed characterization of glycans than previously achievable. These new methods have not yet been assessed for feasibility in experimental procedures useful for discovery phase efforts. Therefore, we aimed to investigate the utility of novel glycoproteomic and glycomic approaches for defining alterations in glycosylation which may accompany disease progression. To this end, we implemented an azido-sugar metabolic labeling, alkyne-agarose bead enrichment, and liquid chromatography-tandem mass spectrometry for profiling glycoproteins in a cell model of lung cancer induced to express transforming growth factor beta ligand-1. This approach identified putative changes in the sialylation of glycoproteins related to metabolic, cell adhesion, glycan biosynthesis, and extracellular matrix-related proteins. The application of a secondary digest to glycopeptide-bound beads, using peptide-N-glycosidase-F, was useful for verifying N-glycan sites as well as exposing previously undetected glycoproteins. A stable isotope labeling of amino acids in cell culture approach was used to gauge if altered protein expression was contributing to differential capture, however this method provided limited information due to a low overlap of proteins identified from enriched vs. unenriched fractions. In another set of experiments, we applied a novel derivatization strategy combined with high resolution/high mass accuracy mass spectrometry for discerning glycan structures in human lung cancer proximal fluids. This procedure effectively defined the sialic acid anomeric configuration of several prevalent species and identified preliminary trends in the expression of oligomannose and complex glycans in clinically-relevant materials. Finally, matrix-assisted laser desorption ionization imaging mass spectrometry was used to spatially resolve the distribution of N-glycans in lung tissues matched to the proximal fluids. Histological assessment of these tissues facilitated cross-reference of acquired glycan species to regions of interest and provided a direct means for assessing how these trends correlated in the proximal fluids

Personalized Medicine in the Field of Inflammatory Skin Disorders

Skin inflammation is associated with a wide range of conditions which represent major health issues worldwide. Skin and mucosal surfaces represent the primary interface between the human body and the environment, susceptible to numerous factors whose action results in diseases produced by chemical substances, mechanical trauma, microbial agents, radiation, etc. Inflammation, a complex network of interactions between soluble molecules and cells, represents the main modality of the skin’s response to injuries. Numerous studies have revealed close links between chronic inflammation, oxidative stress, and carcinogenesis. Chronic inflammation induces the activation of various cell types and an increase in the production of reactive oxygen species, promoting the initiation of a malignant process. Identifying specific biomarkers is essential for understanding molecular mechanisms and developing therapies appropriate to the patient’s characteristics.Personalized medicine is an emerging field of medicine that has the potential to predict which therapy will be safe and efficacious for specific patients using an individual’s genetic profile to guide decisions regarding the diagnosis, treatment, as well as prevention of disease. This book gathers articles that present recent advancements in research involving the mechanisms that underlie the development of inflammatory skin disorders, skin and mucosal inflammation in general

Molecular Biology of Selenium in Health and Disease

The trace mineral selenium is still regarded as one of the most interesting and health-beneficial elements. In addition to the Editorial containing a dedication to Dr. Leopold Flohé, this Special Issue contains 13 research articles and 8 reviews, with over 120 different contributors covering many of the most important subjects concerning the study of selenium. The articles address both selenium as well as selenoproteins and their molecular roles, providing important considerations regarding this trace element’s impact on human and animal health and disease