Michael Tiemeyer - US grants

Cell-cell recognition mediated by cellular contact contributes to the formation of specific synaptic connectivity during neural development. Localization to the cell surface, inherent structural diversity and regulat expression are properties ascribed to the carbohydrate moieties of glycoconjugates that are also to be expected of cellular recognition molecules. For cell surface carbohydrates to mediate, cell-cell interaction however,, endogenous protein receptors (lectins) should exist that recognize and discriminate between oligosaccharide ligands. A novel Drosophila melanogaster protein, called "gliolectin", is such a lectin. Identified through adhesion-based expression cloning, gliolectin is expressed by a subset of the glial cells at the midline of the embryonic Drosophila nervous system. Gliolectin's spatial and temporal expression pattern positions it to mediate the glia/neural or glia/glial interactions at guide formation of embryonic axonal commissures. Alterations in gliolectin expression (partial loss-of-function mutations) disrupt axonal outgrowth early in development. To define gliolectin's role in axonal guidance, this proposal takes advantage of the potential for genetic and molecular analysi of function offered by studying development in Drosophila. Phenotypes associated with null mutations in the gliolectin gene, with ectopic- expression of gliolectin protein and with double mutants that combine gliolectin mutations with mutations in other genes active at the Drosophila midline will be characterized. Immuno-electron microscopic examination of gliolectin's normal expression pattern will indicate whether gliolectin lie at the interface between midline glial cells or between glia and axon, a distinction important for interpreting mutant phenotypes. Also, gliolectin' carbohydrate binding will be further characterized in vitro and in situ to define the degree to which binding specificity controls specificity in cellular recognition. These complementary genetic, ultrastructural and biochemical approaches will define gliolectin's function and place it in th context of other molecular activities suggested to guide axon outgrowth across the Drosophila midline. Conserved molecular expression and developmental function have suggested that the Drosophila ventral midline is analogous to the floorplate of the vertebrate neural tube. Therefore, th identification of a vertebrate gliolectin homologue will place a lectin at crucial crossing point for vertebrate commissural axon pathfinding. Elucidation of cellular recognition mechanisms in neural tissue, including carbohydrate-mediated interactions, is essential for realization of full functional regeneration following CNS injury.

[unreadable] DESCRIPTION (provided by applicant): The surfaces of all eukaryotic cells are richly endowed with complex glycoconjugates. Cell surface glycans, together with the protein and lipid backbones to which they are linked, form the interfaces at which cell-cell interactions occur. Consistent with their subcellular location and immense structural diversity, specific glycans function as cell surface tags that allow cells to appropriately interact with each other and with their local environment. Therefore, mechanisms that control glycan expression also control developmental, immunological, and pathological cellular behaviors. Using genetic and molecular techniques, we discovered that a Drosophila Toll-like receptor (TLR), called Tollo/Toll-8, is a key component of a mechanism that leads to tissue-specific glycan expression. Tollo activity induces the neural-specific expression of a set of N-linked oligosaccharides known as the HRP-epitope. Tollo is not expressed on neurons but is found on ectodermal cells that contact differentiating neurons. This new function for a TLR provides the means to understand how cell-cell interactions influence tissue-specific glycosylation. The molecular pathway through which the Tollo signal is propagated will be identified and characterized by generating new mutations that affect expression of the HRP-epitope. In addition to our mutagenesis screen, we will determine whether previously identified genes, that are components of TLR signaling pathways in other contexts, are also active in regulating glycan expression. To place new genes in context and to determine the cellular mechanism of Tollo function, the ability of altered forms of Tollo to induce neural-specific glycosylation will be investigated. Finally, the specificity of Tollo induced glycan expression will be assessed by determining the diversity of HRP-epitopes in the embryo and by characterizing the full extent of glycan changes caused by loss of Tollo function. The importance of specific glycosylation for normal tissue function indicates that mechanisms controlling glycan expression should ultimately provide new targets for therapeutic intervention in a broad range of human pathologies, including CNS injury, inflammatory/immune disorders, and tumor cell metastasis. [unreadable] [unreadable] [unreadable]

The specific aims of this proposal test the central hypothesis that expression profiles of cellular glycans can serve as unique and sensitive fingerprints, capable of defining specific cell types or reporting cell status during culture. The development of markers and reagents for purifying specific sub-populations of cells is a high priority for advancing the therapeutic and investigational uses of hESCs. Despite their predominant localization to the cell surface, glycoconjugates have been under targeted for such uses. This deficiency reflects the lack of broadly accessible techniques or expertise for characterizing these complex macromolecules. We have developed a sophisticated suite of tools for quantitatively analyzing, with unprecedented depth, the glycans, glycoproteins, and glycolipids isolated from small amounts of material. We have successfully applied these tools to assess glycan expression changes associated with the differentiation of mouse ESCs and now propose to apply our developed glycomic and glycoproteomic techniques to questions of fundamental importance for human ESC biology. Our tools will allow us to screen for changes in glycosylation that occur globally or on specific molecules as a result of aneuploidy, cell culture conditions, passage number, cell pedigree, and differentiation. In Specific Aim 1, we will define the glycomic fingerprints of hESC lines, and hESCs differentiated toward definitive endoderm, mesoderm, and neural precursor populations. The impact of aneuploidy and cell culture conditions will also be assessed. In Specific Aim 2, we will map identified glycan markers, which define hESCs or differentiated cell fates, to specific sites on proteins and lipid cores. In Specific Aim 3, we will generate tools for detecting and enriching sub-populations of hESCs and differentiated cells based on the expression of specific glycomic and/or glycoproteomic markers. Completion of these aims will not only provide novel approaches for characterizing, defining, and enriching specific cell types but will also provide a basis for exploring the role of glycosylation in hESC self renewal and differentiation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Using several technologies developed in the Resource Center, we have investigated in detail the relative expression patterns of glycoprotein and glycolipid glycans in both human and mouse embryonic stem cell lineages. The emphasis is on identifying significant changes when cells are differentiated into other cell types. In addition, we have compared the glycans in several different stem cell lines, as well as in iPS cells. We are also developing tools to enhance high-throughput glycomic analysis and automated annotation of MS data.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Technology developed in support of the aims of the NCRR Biomedical Glycomics grant is applicable to many different systems. The non-biased methods for glycan characterization are sufficiently sensitive to allow detection of minor glycans from small amount of tissue. As a challenge to these methods, we have undertaken the detection of sialylated glycan processing intermediates in wild-type and mutant strains of Drosophila melanogaster, an organism that produces very small amounts of sialylated glycans. The Panin lab has amassed mutants in enzymes necessary for the production, transport, and utilization of CMP-sialic acid, as well as in enzymes that make glycan structures that are acceptors for the Drosophila sialyltransferase. The Tiemeyer lab has analyzed glycan profiles for mutant tissues provided by the Panin lab.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Technology developed in support of the aims of the NCRR Biomedical Glycomics grant is applicable to many different systems. The non-biased methods for glycan characterization are sufficiently sensitive to allow detection of minor glycans. The Doering lab has previously generated Cryptococcus mutants that present altered capsule morphology as a result of altered glycan synthesis. The glycan products of these mutant genes are not always known. We have used our glycomic technology to identify a novel phosphodiester linkage between Xyl and Man as a product of a specific xylosylphosphotransferase.

Infiltration and activation of inflammatory leukocytes drive the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD), two lung inflammatory diseases (LIDs) that produce signiflcant human suffering and consume considerable medical resources. Eosinophils and neutrophils express different members of the siglec family of glycan binding proteins, and siglec activation on either cell population suppresses lung inflammation by inducing granulocyte apoptosis. In humans, eosinophils express Siglec-8 and neutrophils express Siglec-9. Candidate ligands that bind these siglecs were identified through in vitro glycan array screening, predicting key structural features to be expected of potent, endogenous siglec counter-receptors. However, endogenous glycan counter-receptors for Siglec-8 and -9, as well as the proteins or lipids to which they are attached remain to be determined. Furthermore, the mechanisms that control the expression of siglec counter-receptors are unknown, but, if understood, could be invoked to facilitate granulocyte apoptosis through enhanced counter-receptor synthesis. HYPOTHESIS: The expression of endogenous siglec counter-receptors is regulated by innate signaling mechanisms that coordinate the presentation of potent pro- and anti-infiammatory glycans. AIMS: The total glycome of human lung tissue and isolated cell types will be characterized by mass spectrometry and orthogonal analytic approaches to define the full diversity of potential siglec counter-receptor configurations. In collaboration with Proiect 3 (Schnaar), proteomic analysis of affinity-purified materials extracted from human lung tissue will identify protein carriers that present siglec counter-receptors. Emerging evidence reveals crosstalk between siglec and toll-like receptor signaling, indicating the existence of innate regulatory networks for controlling glycan expression. Therefore, dynamic changes in cell-specific glycan expression will be assessed following cytokine or Toll-like receptor agonist administration to isolated and co-cultured lung cell types. The aims are designed to identify the diversity of endogenous counter-receptors for siglecs and to deconvolute the signaling pathways that regulate counter-receptor expression in order to enhance the development of potent therapeutics. RELEVANCE (See instructions): This project will identify glycan structures that possess anti-inflammatory activity in lung inflammatory diseases (LID) such as asthma and chronic obstructive pulmonary disease. The mechanism by which lung tissue and leukocytes normally control the synthesis of these anti-inflammatory glycans will also be studied in order to enhance their production and thereby reduce LID severity.

DESCRIPTION (provided by applicant): The glycan (carbohydrate) components of the glycoproteins and glycolipids that adorn the surfaces of all eukaryotic cells, as well as the secreted glycoproteins produced by these cells, interact with glycan binding proteins to mediate cellular processes that include differentiation, activation and migration of immune cells, quality control in protein folding, and regulation of protein composition at the cell surface. Despite the importance of these processes, molecular level descriptions of the regulation of glycan synthesis, the influence of glycans on glycoprotein structure, and the functional responses of cells to protein-glycan interactions remain at their infancy. In part this reflects the significant challenges presented by glycan structural diversity and by the complexity of cellular responses to the varied glycan presentations found at diverse biological interfaces. A key to overcoming these challenges is the cooperation of researchers with skills of sufficient diversity to allow the formulation of comprehensive solutions. This proposal requests funding for a series of workshops (two per year), one general meeting, and a networking infrastructure, that will promote the interaction of researchers with these diverse skills in order to solve challenging problems in glycoscience. The communication network and workshop structure proposed here is built on the successful experience of the Consortium for Functional Glycomics (CFG), an entity funded by a Glue Grant for the last ten years, and its pool of now more than 500 participating investigators. The proposed workshops will broaden the scope of glycoscience investigations and facilitate the formation of new research teams to address major issues of importance to human health.

PROJECT SUMMARY/ABSTRACT Although ongoing technical advances are accelerating the pace and sophistication of data acquisition in glycoscience, the transformation of these data to glycobiology knowledge, insight, and understanding is slowed by the limited number of tools that facilitate their integration with biological knowledge from genetics, proteomics, pathology, and other disciplines. Our grant application describes the development of an integrated, extendable, and cross-disciplinary resource providing tools and data to address specific scientific questions that can currently be answered only by extensive literature-based research and manual collection of data from disparate databases and websites. Using insight gained during our planning grant activities, including a workshop focused on evaluating existing resources and community needs, we propose to develop a broadly relevant and sustainable glycoinformatics resource to connect glycoscience with the explosion of data that is revolutionizing biology. We identified critical gaps that need to be filled and challenges that must be overcome to create an enduring and sustainable glycoinformatics resource that goes beyond mapping glycan data to genes and proteins to identify and integrate diverse multidisciplinary knowledge from EMBL-EBI, NCBI, UniProt, UniCarbKB, CAZy, Gene Ontology and other sources. To maximize synergy among these resources, we propose a new glycan array data repository and enhanced ontologies to facilitate integration of glycan and glycoconjugate expression and interaction data with other information. Evaluating these data in the context of knowledge about genetic mutations, gene expression, protein function and other phenomena will provide new opportunities for systems-level understanding of the roles of glycosylation in disease and development. This comprehensive data integration framework will provide unprecedented support for complex queries spanning diverse data types relevant to glycobiology. Technical advances required to implement this framework include evidence tagging of data, ontology and standards development, and new interfaces that enable data mining, sharing, and dissemination. Community engagement, especially with scientists who do not specialize in glycobiology, will be emphasized to maximize the relevance of our resource. We will develop a portal to make all this information publicly available in standard formats supported by NCBI and EMBL-EBI and in new formats we develop, promoting sharing of data and their ultimate integration into these widely used informatics resources.  

ABSTRACT With significance in biotechnology, biomedicine, and basic research, glycobiology?s applications are widespread. Technological advancements in the field of glycobiology have expanded in parallel with the influx of an array of data within the glycosciences community. The broad range of experimental approaches, disparate nature of available datasets, and the seemingly piecemeal strategies required to construct comprehensive interpretations create inherent barriers for glycoscience researchers to utilize all available information. The mission of GlyGen has been to target and mitigate such challenges by developing procedures and a platform which integrates or builds upon glycoconjugate structure-function data from different resources. GlyGen, a NIH-funded international effort, captures and integrates over 90% of available glycoconjugate data, harmonizing and managing diverse outputs such as glycans, proteins, and genes integrated with genomics, pathway, and disease information. Since its inception, the GlyGen team has built a user-friendly platform complete with analytical tools and comprehensive, exportable data sets to ease the burden for researchers. Within the Swiss Institute of Bioinformatics (SIB), the Proteome Informatics Group (PIG) has worked extensively to develop the glycoinformatics resource GlyConnect, which focuses on the molecular characterization of protein glycosylation through an integrated, expertly- curated platform, specializing in structure analysis and producing novel data sets, such as site-specific glycan data. Despite each resource?s efforts to mitigate challenges, difficulties in amassing the amalgam of data required to fully examine microheterogeneity within glycobiology still persist. By utilizing their distinct strengths, the proposed collaborative research between GlyGen and GlyConnect will focus on further integrating site-specific protein-glycan data to generate more comprehensive data sets, where increasing the data availability in GlyGen is expected to accelerate basic and translational research. Currently, the major resources for site-specific protein-glycan data are UniCarbKB and UniProtKB, though the amount of available data from these databases, or other similar resources, is not substantial. To address this limitation, GlyConnect and GlyGen will develop an advanced, scalable, and site-specific protein-glycan annotation pipeline. This pipeline will be constructed using existing data in GlyConnect, in addition to roughly 100 publications identified and prioritized through current literature mining efforts in GlyGen. Moreover, front and back-end software developments will be implemented on the GlyGen platform, allowing glycoscience researchers to submit site-specific glycan data through a validated submission system. The proposed research will create a standardized methodology for more efficient data submission efforts, expand on the available site-specific protein-glycan data for the glycobiology community, and facilitate data sharing amongst glycoscience researchers. 1

ABSTRACT Recent years have highlighted the significant impact of glycans in biomedical research especially in aspects of immunology, role in biological functions, impact in diseases, and more. Technical advancements in recent years have allowed researchers to perform glycomics and glycoproteomics analysis more accurately and efficiently, resulting in rapid growth of the volume of data in the literature and a great need for robust evidence-based archival glycan record maintenance. This need can be at least partially met by a repository that allows authors to submit and update data while database developers provide mechanisms for data archiving without any editing. We propose to develop a prototype glycan archive (a primary database) to store registered glycan structures and allow users to deposit new glycans which will be shared with existing resources. It is expected that this effort will lead to the development of an internationally managed glycan archive collaboration, parallel in concept to the International Nucleotide Sequence Database Collaboration (INSDC) that harmonizes and maintains databases like GenBank, DDBJ, ENA for nucleotides. The glycan archive prototype will be developed by GlyGen under subdomain gsa.glygen.org. and it will house all glycans sourced from existing public databases like GlyTouCan, PubChem, ChEBI, KEGG glycan, and others. Robust QC procedures will enhance the value of the submitted records and all glycan records will be associated with an unique ID that will be cross-referenced to other databases. Novel informatic-based structural validation will flag submissions that contain unexpected features, allowing additional evaluation before or after incorporation into the archive or secondary databases. We anticipate that the proposed mechanism will be highly valuable for researchers, data curators, bioinformatics databases, and also provide a historical record of when a particular glycan was published.