Ronald Schnaar - US grants

Complex carbohydrates and complementary carbohydrate receptors on opposing cells may mediate neural cell interactions. The current application focuses on gangliosides, a major class of cell surface glycoconjugates in the central nervous system (CNS), as potential neural recognition molecules. High-affinity ganglioside conjugates were synthesized and used to identify a novel brain ganglioside receptor which may by involved in neural cell interactions. Purification and molecular characterization of this and related receptors will provide unprecedented molecular and immunological tools with which to probe the functions of gangliosides and related glycosphingolipids. Myelin-associated ganglioside receptor. A high-affinity, ganglioside- specific binding activity was recently identified on CNS myelin membranes. Its saccharide specificity and membrane distribution suggest that it may be involved in oligodendroglial-axonal recognition. The primary goal of the next grant period will be the isolation and molecular characterization of this binding activity. Various synthetic ganglioside conjugates have been prepared for use as high-affinity radioligands, affinity chromatography matrices, and photoaffinity probes. Molecular biological techniques will be used to determine the sequence of the myelin ganglioside receptor and to generate molecular and immunological tools with which to probe its distribution, developmental expression, and function. Synaptosomal ganglioside receptor. A second ganglioside-specific binding activity was found enriched on rat brain synaptosomes. Saccharide specificity and tissue/subcellular distribution studies of this second brain ganglioside receptor will be performed, leading to its purification and molecular characterization. Sulfoglucuronyl glycosphingolipids in peripheral nervous system (PNS) cell recognition. Schwann cells, the myelin forming cells of the PNS, were found to adhere specifically to purified sulfoglucuronyl glycolipids, anionic glycosphingolipids implicated in certain peripheral neuropathies. Receptors complementary to these lipids were detected on PNS membranes. They will be characterized for saccharide and tissue specificity, with the goal of purification, molecular characterization, and functional studies.

The eucaryotic cell surface has been implicated in the control of cell adhesion, growth, motility, morphology and differentiation. Presumably, this is accomplished via specific cell surface receptors which "sense" the cell's molecular and cellular environment and allow the cell to respond accordingly. These control mechanisms are central in the process of orderly morphogenesis and in neoplastic transformation and metastasis. The molecular mechanisms for cell-cell interactions are unknown, and their elucidation remains a major challenge. This proposal investigates the role of a major class of cell surface molecules--complex carbohydrates--in the control of cell surface interactions through the use of "cell surface analogs": synthetic or natural cell surface glycoconjugates covalently immobilized on otherwise inert plastic surfaces. Primary hepatocytes and macrophages as well as normal and transformed fibroblast cell lines in culture will be used in our studies. Two complementary experimental approaches are proposed: 1. Cell Responses to Carbohydrate-derivatized Surfaces. Hepatocytes and macrophages adhere to carbohydrate-derivatized surfaces in a sugar-specific manner. Carbohydrate-directed adhesion is followed, as in cell-cell adhesion, by distinct responses which alter the nature of the adhesive bonds. Thus, hepatocytes appear to covalently modify extracellular carbohydrates subsequent to initial adhesion, perhaps via phosphorylation. This proposal details experiments which will identify the biochemistry of the extracellular carbohydrate modification(s) and their relationship to cell adhesion. These studies may reveal new information on the biochemical mechanisms underlying the control of cell behavior by intercellular interactions. 2. Cell Surface Components in Growth Regulation. The control of cellular growth is important to orderly morphogenesis and is lost upon neoplastic transformation. Evidence suggests that cell-cell contact regulates the rate of cell division via cell surface molecules which bind to complementary receptors on apposing cells. We will use fibroblasts in cell culture to test the ability of partially purified membrane glycoproteins, immobilized on "cell surface analogs", to control cell division. This approach may help identify the active cell surface component(s).

Inflammation is marked by extensive granulocyte extravasation from the blood into tissues at a site of infection or immunologic challenge. Adhesion molecules and their complementary ligands on granulocytes and on the blood vessel wall endothelium control the extent and identity of granulocytes entering the tissue at a site of injury or insult. E- selectin, which is expressed on activated endothelial cells, is a member of the selectin adhesion protein family which mediates cell-cell adhesion events early in this immune response by binding to cell surface glycoconjugates. The identification of normal human granulocyte glycoconjugates responsible for E-selectin-mediated cell adhesion will be important to our understanding of inflammation in its many guises, and may offer novel avenues for inflammation control. Although carbohydrate structures which bind to selectins have been identified, the natural ligands for E-selectin on normal human granulocytes have yet to be fully elucidated. The principal investigators have obtained data indicating that one or more glycosphingolipids on normal human neutrophils and eosinophils act as E-selectin ligands. The current application proposes to combine proven expertise in granulocyte cell adhesion, flow cytometry, and glycosphingolipid glycobiology to identify and characterize normal human granulocyte glycosphingolipid ligands for E-selectin, and to evaluate their role(s) in granulocyte function. Aim 1 will directly test the hypothesis that glycosphingolipids constitute major ligands for E-selectin on intact human neutrophils and eosinophils using in vitro cell adhesion assays in conjunction with highly specific enzymes, antibodies, and glycoconjugate biosynthesis inhibitors. Aim 2 will use techniques for glycosphingolipid isolation, resolution and functional analysis to identify and isolate glycosphingolipid(s) from normal human neutrophils and eosinophils capable of specific binding to E- selectin. Aim 3 will use microanalytical techniques, antibodies, and specific enzymes, to determine the structure of glycosphingolipid ligand(s) identified and isolated in Aim 2. Aim 4 will test the glycosphingolipids on intact cells and individual glycosphingolipids purified and characterized in Aims 2 and 3 for their role(s) in granulocyte cell adhesion under static conditions and physiological flow. The knowledge gained from these studies may be applicable to the design of improved anti-inflammatory drugs, to understanding certain immune dysfunctions, and may provide insight into the physiological roles of glycosphingolipids.

DESCRIPTION (provided by applicant): Myelination is essential to the stability, structure and function of myelinated axons. Myelin enhances axon survival, regulates the axon cytoskeleton, impacts nodes of Ranvier, and inhibits axon regeneration. In each case, molecules on myelin engage complementary molecules on axons to initiate cell-to-cell signaling. Knowledge of the receptors and ligands responsible may provide insight into dysmyelinating disorders and help enhance axon regeneration. Myelin associated glycoprotein (MAG) participates in all of these effects of myelin on axons. Mice lacking MAG express myelin, but display axon degeneration, altered axon cytoskeleton, and altered nodes of Ranvier. MAG also inhibits axon regeneration. MAG binds to multiple ligands on the axon. Among these, gangliosides are implicated in several of MAG's effects. Mice lacking complex gangliosides display axon degeneration, altered axon cytoskeleton, and altered nodes of Ranvier. Gangliosides are also ligands for MAG-mediated inhibition of axon regeneration in vitro. Aim 1. The major MAG-binding gangliosides in nerve tissue, GDla and GT1b, are synthesized by addition of a sialic acid to the terminal galactose of non-MAG binding gangliosides GM1 and GD1b respectively. The sialyltransferases responsible have not been established, hi collaboration with Dr. Jamey Marth (UCSD), we propose a program to identify the genes responsible and construct a mouse strain lacking GDla and GTlb. This mouse will provide a definitive test of the role of these MAG ligands in vivo and cellular substrates for reconstitution of MAG function in vitro. Aim 2. Independent studies implicate gangliosides and the Nogo66 receptor, NgR, as independent MAG ligands. We will test whether different neurons use different functional MAG ligands, and whether they act independently or interactively, using cerebellar granule neurons, dorsal root ganglion neurons, and embryonic stem cell derived motoneurons in vitro. Aim 3. Clustering gangliosides is sufficient to initiate neuronal signaling, including RhoA activation and inhibition of axon outgrowth. Since gangliosides populate the outer leaflet of the plasma membrane, they must recruit other molecules to generate transmembrane signals. We will use cell surface ganglioside engineering in situ to identify ganglioside-associated proteins that may serve as proximal molecules in ganglioside-mediated signaling.

DESCRIPTION (provided by applicant): The adult mammalian central nervous system (CNS) is a profoundly inhibitory environment for axon regeneration. This is due, in significant measure, to multiple axon regeneration inhibitors (ARI's) in the milieu of a CNS injury. These include Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp) and chondroitin sulfate proteoglycans (CSPG). Each ARI binds to complementary ligands on the nerve or axon surface, halting axon regeneration. Rapidly emerging knowledge of ARI's and their ligands provides previously unanticipated opportunities to block their actions and potentially enhance axon regeneration. The relative contributions of the different known ARl's in blocking axon regeneration in vivo are unknown. This R21 Exploratory/Developmental Grant application describes a highly directed, short-term program to block the known ARI's, individually and in combination, in a well characterized in vivo spinal cord injury model. The proposed studies take advantage of the latest findings regarding the molecular nature of ARI's. The results may direct future studies, in that they will provide comparative data on the effects of blocking the different inhibitory systems side by side in the same lesion model. Depending on the outcome, they may encourage future efforts to focus on multi inhibitor blocking rather than on blocking single systems. The "exploratory/developmental" aspects of the proposed approach include (i) multi-inhibitor blocking in a single lesion model; and (ii) the use of the tools of glycobiology as experimental therapeutics. Each ARI and/or its nerve cell surface ligand(s) are glycosylated. Moreover, glycosylation plays key structural and functional roles in inhibiting axon regeneration. The nerve cell ligand for Nogo, NgR, is a glycosylphosphatidylinositol (GPI)-Iinked glycoprotein, as is OMgp. MAG is a sialic acid binding protein that binds to nerve cell surface gangliosides GDla and GTlb, as well as to NgR. Finally, the sugar chains of CSPG mediate its inhibition of axon regeneration. The tools of glycobiology, in concert with novel biochemical tools, provide feasible means to block ARI's and enhance axon regeneration in vivo. We will use the following glyco-enzymes for this purpose: phosphatidylinositol-specific phospholipase C (PI-PLC), to cleave the GPI anchors of NgR and OMgp, neuraminidase to cleave GDla and GTlb, and chondroitinaseABC to cleave CSPG. The biochemical efficacy of each enzyme will be monitored immunohistochemically, and their effects on regeneration will be determined after spinal cord lesion (dorsal hemisection) in rats. ARI-blocking saccharides, peptides, and antibodies will also be used to further enhance the value of these studies. The resulting data may provide insight on the relative contributions of the different ARI's in an in vivo model, and may support evaluation of new approaches to enhance axon regeneration after CNS injury.

DESCRIPTION (provided by applicant): Spinal cord injury typically results in life-long loss of nerve function accompanied by profound morbidity and mortality. The current project will use a well-established animal model for human spinal cord injury - spinal cord contusion in the rat - to investigate novel ways to enhance recovery. Our approach is based on our recent discovery that delivery of the enzyme sialidase to the site of experimental spinal cord injuries results in significant enhancements in spinal axon outgrowth, locomotor recovery, and cardiovascular reflex recovery. We now propose to quantify a battery of behavioral, neurophysiological and neuroanatomical outcomes to explore the potential of sialidase, alone and in combination with other treatments, to enhance recovery after spinal cord injury. Our proposal is based on a wealth of data indicating that central nervous system axons have the capacity to regenerate, but are inhibited from doing so by endogenous axon regeneration inhibitors (ARI's), including myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein on residual myelin and chondroitin sulfate proteoglycan (CSPG) on the glial scar. Each ARI binds to complementary receptors on axons, halting axon outgrowth. Knowledge of ARI's and ARI receptors provides new opportunities to block ARI actions and enhance recovery. For example, the enzyme sialidase destroys sialoglycans, a class of ARI receptors for MAG, and the enzyme chondroitinase ABC (ChABC) destroys CSPG. Anti-ARI therapies, individually or in combination, may enhance axon regeneration and improve functional recovery after spinal cord injury. We now propose to: (i) Test the hypothesis that sialidase delivery to the site of a spinal cord contusion injury in the rat will enhance axon plasticity and/or regeneration, resulting in significant functional recovery; (ii) Test the hypothesis that combining independent anti-ARI therapies, such as sialidase and ChABC, will result in additive or synergistic enhancements of recovery after spinal cord contusion injury, and (iii) Use our knowledge of sialoglycans and sialidases to identify the molecular target(s) of therapeutic sialidase and discover the best sialidase(s) for preclinical studies. PUBLIC HEALTH RELEVANCE: The mature central nervous system, including the spinal cord, is overwhelmingly inhibitory for axon regeneration, severely limiting recovery after traumatic injury and resulting in life-long loss of function. Remarkably, axons have the ability to regenerate, but are inhibited from doing so by molecules that accumulate at injury sites. Destroying or blocking these molecules may permit axons to regenerate, greatly enhancing functional recovery.

The Lung Inflammatory Disease Program of Excellence in Glycosciences (LID-PEG) has Projects and Cores at three institutions and a breadth of capabilities ranging from chemistry to preclinical therapeutic models. With this diversity, communications will be a key to LID-PEG progress and success. A major role in effective communication will be provided by Core A, the Administrative Core. Core A will take responsibility for inter-program communications both through web-based video conferencing and face-to-face meetings. The Core will coordinate and oversee the regular video-conference meetings of the Executive Committee, organize the annual Program Group Meeting (which will rotate among the three sites), organize meetings of the LID-PEG with its Internal and External advisory boards and support LID-PEG participation in the annual Investigator's meeting in Bethesda. In addition to travel planning and reimbursement for LID-PEG meeting participants. Core A will coordinate communications with NHLBI and the Administrative Center, manage budgets and fulfill NIH reporting requirements.

DESCRIPTION (provided by applicant): Cell surface glycans and complementary glycan binding proteins are intimately involved in inflammatory processes. Members of this Program team and others discovered that certain members of the siglec family of glycan binding proteins are inhibitors of inflammation. This Lung Inflammatory Disease Program of Excellence in Glycosciences (LID-PEG) focuses on the anti-inflammatory functions of siglecs and their glycan counter-receptors (ligands) in moderating ongoing inflammation in the lung. THEME: Specific glycans expressed on lung tissues engage complementary glycan binding proteins (Siglec-8, Siglec-9, Siglec-1) on inflammatory cells to limit lung inflammation. Knowledge of the glycan structures and glycan binding proteins involved, the control of their expression, and the mechanisms responsible for translating glycan engagement into regulation of the inflammatory response will provide new insights into the progression of inflammatory lung diseases. Synthetic glycan-decorated nanoparticles and antibodies that target glycan binding proteins on inflammatory cells will limit inflammatory damage. The insights gained may lead to novel diagnostic tools and therapeutic compositions that treat inflammatory diseases of the lung and other tissues relevant to the goals of the NHLBI. Four closely integrated Projects and two Cores at three major glycobiology centers will coordinate efforts to reach the Project goals: Project 1, Treating lung inflammation by targeting siglecs (B. Bochner, Johns Hopkins); Project 2, Siglec-targeted nanoparticles for lung and cardiovascular disease (J. Paulson, Scripps); Project 3, Human lung counter-receptors for Siglec-8 and Siglec-9 (R. Schnaar, Johns Hopkins); Project 4, Regulated expression of siglec counter-receptors (M. Tiemeyer, CCRC/U. Georgia); Core C, Shared Resources Core: Carbohydrate Synthesis (J. Paulson, Scripps); and Core D, Inflammatory Animal Models Core (Z. Zhu, Johns Hopkins). The Program is supported by an Administrative Core (Core A) and a Skills Development Core (Core B) that provides trainees with diverse experiences at the three centers. This Program will provide novel insights into the glycosciences of lung inflammatory diseases. RELEVANCE: Asthma and Chronic Obstructive Pulmonary Disease (COPD), lung diseases that cause extensive illness and death, involve infiltration of damaging inflammatory cells. Normally, sugar molecules in the lung engage complementary sugar binding molecules on inflammatory cells, signaling them to halt and limiting tissue damage. This project defines anti-inflammatory sugar molecules and uses them to develop new treatments. (End of Abstract)

Glycosciences Skills Development is a key component of the Lung Inflammatory Disease Program of Excellence in Glycoscience (LID-PEG). Four postdoctoral fellows will be designated as Skills Development Trainees with terms of 3-4 years. At any time, one Skills Development Trainee will be associated with (and supported by) each Project. Training will consist of three components: (1) Site-specific courses in glycobiology - With the assumption that glycobiology training is often modest at the doctoral and pre-doctoral levels, Skills Development Trainees will enroll in site-specific courses in the fundamentals of glycobiology. These opportunities will include Essentials of Glycobiology (at UCSD for trainees at Scripps), Advanced Topics in Glycobiology (at University of Georgia for trainees at the CCRC) and a newly created graduate-level course on Glycobiology directed by Dr. Schnaar (for trainees at Johns Hopkins). (2) Annual Skills Development Retreats - Each year all Skills Development Trainees will travel to one of the three Program sites (in rotation) for hands-on training. This will provide specialized experiences in glycoconjugate purification, analysis, chemistry and function. In addition, it will provide opportunities for every trainee to visit and network with glycoscientists at three of the major glycobiology centers in the US. (3) Enrichment and networking opportunities. Each site provides exanded opportunities for informal glycoscience training through specialized glycobiology seminar series and local or regional glycobiology interest groups. In addition, all trainees will attend the annual Society for Glycobiology meeting, the Glycobiology Gordon Conference, or other international or specialized meetings in the glycosciences.

Asthma and chronic obstructive pulmonary disease (COPD) involve accumulation and activation of inflammatory cells in the lung. Inflammation in asthma is marked by the influx of eosinophils, mast cells, and CD4+ T-cells whereas COPD is marked by the influx of neutrophils, macrophages and CD8+ T-cells. Members of the Siglec family of glycan binding proteins (sialic acid-binding immunoglobulin-like lectins) are expressed selectively on subsets of inflammatory cells: Siglec-8 on allergic inflammatory cells (eosinophils, mast cells and basophils) and Siglec-9 on monocytes, neutrophils, and some T-cells. Both Siglec-8 and Siglec-9 suppress inflammation. Crosslinking siglecs on inflammatory cells inhibits release of proinflammatory mediators, enhances release of anti-inflammatory mediators, or induces apoptosis/death, depending on the inflammatory cell type. HYPOTHESIS: Glycoconjugate ligands (siglec counter-receptors) in the lung, consisting of endogenous glycoproteins and/or glycolipids, engage Siglec-8 on incoming allergic inflammatory cells and Siglec 9 on COPD inflammatory cells as a feedback mechanism to limit ongoing inflammatory responses. AIMS: This project will isolate, identify and characterize the human lung counter- receptors for Siglec-8 and Siglec-9. Glycoconjugates (glycoproteins and/or glycolipids) will be extracted from human lung and the major siglec-binding entities will be isolated by conventional, lectin, and Siglec affinity chromatography. The resulting siglec-interacting molecules will be analyzed by mass spectrometry in collaboration with Project 4 of this program. The expression of counter-receptors will be characterized on cultured human lung epithelial and/or endothelial cells and the enzymes responsible for their biosynthesis will be determined by RNAi knockdown. Siglec counter-receptor expression will be compared on normal and diseased human nasal epithelium. The properties of counter-receptors on mouse lung epithelium will be compared with those from human lung. Knowledge of the endogenous human lung counter-receptors for Siglec-8 and Siglec-9 may provide insights useful in understanding the progression of lung inflammatory diseases and may provide improved lead structures for anti-inflammatory therapeutics. RELEVANCE (See instructions): Asthma and COPD, lung diseases that cause extensive illness and death, involve infiltration of damaging inflammatory cells. Normally, sugar molecules in the lung engage complementary molecules on inflammatory cells, signaling them to halt and limiting tissue damage. This project will define those anti-inflammatory sugar molecules in an effort to better understand lung inflammatory diseases and to find new ways to treat them.

DESCRIPTION (provided by applicant): Recent studies suggest that the majority of HIV infected patients develop mild or asymptomatic neurocognitive dysfunction despite being aviremic on antiretroviral therapy. Coupled with the observation that the viral burden in the brain may be much larger than previously anticipated, the possibility that small amounts of viral products over extended periods of time may directly cause neuronal dysfunction needs to be considered. One of these viral proteins, Tat is particularly attractive since its production and release from HIV-infected cells is not impacted by currently available antiretroviral agents. Substantial amount of data from our laboratory over the last 15 years clearly shows that Tat interacts directly with the NMDA receptors to cause neuronal injury. Over the previous five years of funding we further characterized the mechanisms of Tat-mediated neuronal injury and discovered that the mechanism of excitation of the NMDA receptor by Tat is unlike any other known agonist. To further characterize these interactions we will use a combination of in vitro and in vivo models to determine the role of Tat-NMDA receptor interactions in mediating synaptodendritic injury and using molecular studies including crystallography we will determine the exact molecular basis of these protein-protein interactions. We anticipate that these studies will not only have important implications for understanding the pathophysiology of HIV associated neurocognitive disorders but will also advance our understanding of the physiological and pathological states in which the NMDA receptor plays a role. The grant application will examine three specific aims: Aim 1: To characterize the post-translational modifications and essential residues of Tat that mediates its neurotoxic properties. Aim 2: To determine the role of Tat-NMDA receptor interaction in mediating synaptodendritic injury Aim 3: To determine the structural basis of the interactions between Tat and the NMDA receptor.

? DESCRIPTION (provided by applicant): Regulation of excitatory synapses is central to learning and memory, and their dysregulation leads to human cognitive diseases including intellectual disability. Glutamate is the major excitatory neurotransmitter and AMPA-type glutamate receptors (AMPARs) are the predominant mediators of excitatory neurotransmission. Accessory molecules exquisitely regulate AMPAR expression at the synapse to drive synaptic plasticity, learning and memory. We discovered unanticipated AMPAR regulatory molecules, gangliosides and the transmembrane protein Nicalin, the study of which are the basis for this project. Gangliosides, sialylated glycosphingolipids, are prominent structures on the surfaces of neurons, with four structures - GM1, GD1a, GD1b, and GT1b - comprising >95% of all gangliosides in the brains of mice and humans alike. Human disorders of ganglioside biosynthesis invariably result in cognitive disability, often accompanied by seizures, outcomes phenocopied in mutant mice. The link between gangliosides, learning, and excitatory neurotransmission had been unclear. Using an unbiased molecular screen we found that ganglioside GT1b associates with a molecular complex responsible for endocytosis of AMPARs, especially those containing the GluR2 subunit known to regulate neuronal excitability. We found that GluR2 binds to a different major brain ganglioside, GM1. We hypothesize that different gangliosides on nerve cells segregate GluR2-containing AMPARs from their endocytosis complex, enhancing their stability. Since GluR2-containing AMPARs limit seizures and are essential for long-term potentiation (LTP) and long-term depression (LTD), molecular correlates of learning and memory, our findings may mechanistically link ganglioside expression to learning, memory, and seizures. Individuals with altered ganglioside expression may suffer pathological changes in the steady state levels of GluR2-containing AMPARs resulting in seizures and diminished learning. This project tests the functional consequences of the interactions between brain gangliosides, AMPARs, and AMPAR endocytosis complexes. Pharmacological, enzymatic and genetic tools will be used to predictably alter the expression of gangliosides and the ganglioside-associated trafficking protein Nicalin on nerve cells and tissues. Specific alterations in gangliosides and Nicalin will be quantified analytically and immunocytochemically, then altered ganglioside and Nicalin expression on cultured hippocampal neurons will be correlated with changes in AMPAR trafficking at baseline and in response to chemically-induced LTP and LTD. Acute hippocampal slices from ganglioside-altered mutant mice will be used to test the role of gangliosides in stimulus-evoked LTP and LTD. Finally, reconstitution with exogenous gangliosides will be used to test the ability of specific ganglioside structures and ganglioside mimetics to modulate AMPAR trafficking, LTP and LTD. If successful, this exploratory project will open new areas of research on the functions of gangliosides and ganglioside binding proteins in regulating neurotransmitter receptor expression and function.

The program directors and faculty from three of the six prior NHLBI Program of Excellence in Glycoscience (PEG) centers have joined forces with top quality physician scientists at the Cleveland Clinic and at Johns Hopkins to create a unique career development program that will train the next generation of leaders in biomedical research involving glycans and human disease. Scholars will be selected by either an advertised national search or by selecting individuals who were already nationally recruited by highly competitive searches to local fellowship programs in cardiology, pulmonology or biomedical engineering. Five scholars will be selected during the first cycle of the Program. Five additional Scholars will be selected in the second cycle of the Program. Each Scholar will have at-least two co-mentors, one a leading expert in glycoscience and the other(s) leading experts in clinical/translational research on a disease of high priority to NHLBI. Selection of Scholars will favor individuals with the M.D. or M.D.-Ph.D. degree. The objectives of our career development program are to provide immersion in state-of-the art glycosciences relevant to human disease for both Scholars and their clinical co-mentors. The career development program will consist of a multi-faceted immersion in disease-related glycosciences. Research experiences will be supplemented by a) advanced topic lectures; b) translational glycoscience examples & applications; c) thematic literature reviews; d) glycoscience seminar series; and e) research in progress. This training will be simulcast at the Cleveland Clinic or Johns Hopkins University (JHU) as needed. f) Scholars will also take a hands-on laboratory course, where they will learn major skills required to do research in glycosciences. Biomedical glycoscience career development will be further supported by professional development opportunities, focused on grant writing, presentation skills, and networking. Attendance at all Program activities by Scholars will be mandatory. Participation and progress of Scholars will be collectively overseen by the internal oversight committee, comprised of all of the Co-P.I.s. A formal electronic system for evaluating both the Scholars and mentors will be established. The overall Program will be evaluated annually by an outside review panel, comprised of leading glycoscientists. We will also monitor publications, presentations and career trajectories of present and former Scholars to evaluate the overall success of the Program. Our Program will create a cadre of physician scientists who not only will become leaders in disease-related research, but also will be well versed in the glycosciences required to understand the molecular basis of disease. True success of the Program will occur as these Scholars are now able to pass on their knowledge of the importance of glycans to their own Fellows when they become independent physician scientists.

Gangliosides, sialylated glycosphingolipids found on all vertebrate cells and tissues, play well-established roles in diverse molecular signaling pathways that impact human diseases including diabetes, cancer, neuro- degenerative proteinopathies, intellectual disability, and many others. The major ganglioside structures are well-defined, finite, and shared across vertebrate species. Their glycans regulate cell signaling independent of a protein carrier. Most gangliosides reside on the cell surface with their ceramide lipids embedded in the plasma membrane and their glycans extending outward. They regulate cell physiology in two modes, cis and trans. As cis regulators, they associate laterally via glycan binding to transmembrane proteins in the same cell membrane to regulate their function. As trans recognition molecules they engage proteins in the extracellular milieu or on apposing cells, mediating cell-cell interactions. Both cis and trans interactions are specific for ganglioside glycan structures and essential for human health. Most ganglioside functions and ganglioside-protein interactions remain poorly understood due to lack of broadly accessible, adaptable and validated tools and optimized methods for their use. It is the goal of this project to address this need using chemical biology technologies to synthesize a defined set of major gangliosides carrying minimally disruptive bifunctional photoreactive and alkyne (click chemistry) tags. Optimized and validated protocols will be generated to deliver bifunctionally tagged gangliosides to the outer leaflet of the plasma membrane of cells, where most gangliosides reside and function. Our goal is to synthesize the ganglioside probe toolkit, validate appropriate cell delivery of the probes, validate their use to identify glycan-specific ganglioside binding proteins, and transfer the reagents and protocols to other biomedical research laboratories within the term of this project. The deliverables, ganglioside probes and validated methods for their use, will be distributed broadly to biomedical researchers to discover the identities, specificities, distributions and functions of ganglioside binding proteins relevant to a variety of human cells, tissues, and diseases.