The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to intracellular serine and threonine residues is a ubiquitous post-translational modification (PTM) found in all higher eukaryotes. Like other PTMs, it is finely regulated in response to stimuli and dysregulated in multiple diseases. However, unlike other PTMs, methods to detect and profile the dynamics of O-GlcNAc glycosylation are still in their infancy. Herein, we discuss the background, development, and application of new chemical tools that have allowed for some of the first systems-level investigations of O-GlcNAcylation in different cells, organ systems, and disease states. We also significantly advance established techniques for the detection and monitoring of O-GlcNAc on proteins of interest. Using these new techniques, we first uncover a novel O-GlcNAcylation site on Cdk5 and show that this site can dynamically regulate Cdk5 activity in the context of neurodegenerative disease. Next, we apply novel chemical, mass spectrometric, and computational tools to, for the first time, uncover cellular networks engaged by O-GlcNAcylation in vivo. Finally, we undertake the systematic optimization of mass spectrometry based O-GlcNAcomics and use these new insights to significantly advance our understanding of O-GlcNAcylation dynamics in metabolic diseases of the liver. Overall, the techniques developed and data generated herein are closing the methodological and intellectual gaps between the study of O-GlcNAc glycosylation and that of other PTMs.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9JD4TZ9, author = {Jensen, Elizabeth Hwang}, title = {Elucidating the Role of O-GlcNAc Glycosylation in Neurobiology and Neurodegeneration}, school = {California Institute of Technology}, year = {2018}, doi = {10.7907/Z9JD4TZ9}, url = {https://resolver.caltech.edu/CaltechTHESIS:12192017-155744407}, abstract = {O-GlcNAc glycosylation is a dynamic, inducible post-translational modification (PTM) essential for neuronal homeostasis and found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Intracellularly, O-GlcNAc modification is cycled by two enzymes in mammalian cells: O-GlcNAc transferase (OGT) appends O-GlcNAc to serine or threonine residues and O-GlcNAcase (OGA) removes O-GlcNAc. OGT modifies over 1000 different proteins, but the lack of a well-defined consensus sequence or substrate structural constraints has hampered efforts to predict sites a priori. Furthermore, the identification of O-GlcNAc modification sites has been obstructed by the difficulty of enriching and detecting O-GlcNAc using traditional biochemical methods. Here, we established and employed biological and chemical tools to illuminate the role of O-GlcNAc in neuronal function.
In Chapter 2, we sought to determine the role of O-GlcNAc in learning, memory, and neurodegeneration. Deletion of the OGT gene causes early postnatal lethality in mice, complicating efforts to study O-GlcNAc glycosylation in mature neuronal function and dysfunction. We demonstrated that the loss of OGT in the forebrain of adult mice (OGT cKO) leads to progressive neurodegeneration, including neuronal death, neuroinflammation, hyperphosphorylated tau, amyloidogenic Aβ-peptides, and memory deficits. In the hippocampus, we showed that OGT ablation lead to the upregulation of neuroinflammatory genes and the downregulation of cholesterol biosynthetic genes. Additionally, a gene network analysis (WGCNA), qPCR, and immunohistochemistry (IHC) revealed that loss of O-GlcNAc perturbed cell cycle progression in the hippocampal neurons. In the hippocampus, we identified increased neuroinflammatory gene transcription in OGT cKO mice and both tau neurofibrillary tangle (NFT)-forming and amyloid-forming Alzheimer’s disease (AD) mouse models. However, only OGT cKO and NFT-forming mice displayed decreased synaptic gene expression, suggesting that NFT formation and OGT cKO compromise hippocampal synaptic transcription. These studies indicate that O-GlcNAcylation regulates pathways vital for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.
In order to understand the critical O-GlcNAc-mediated neuronal functions that underlie OGT cKO dysfunction, we next developed and utilized novel biological and chemical tools in order to identify key OGT interactors and substrates in the brain in Chapter 3. Due to the lack of a well-defined OGT substrate sequence and structural constraints, OGT is believed to obtain its substrate specificity through its interactome where specific interactors target OGT to specific substrates. In order to identify these interactors, we used CRISPR/Cas9 to generate a novel mouse with a minimally tagged OGT in order to identify the endogenous OGT brain interactome using tandem affinity purification and MS methods. The preliminary OGT brain interactome consisted of previously identified OGT interactors and substrates as well as novel interactors. The identified OGT interactors were enriched for ribosomal and cytoskeletal proteins in addition to axonal, dendritic, and neuronal cell body proteins, implicating OGT as a pivotal mediator of neuronal structure and function.
In addition to the OGT interactome, we sought to uncover OGT’s substrates or the O-GlcNAcome. We developed an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labeling followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) installed a new MS-compatible linker designed for facile purification and release of O-GlcNAcylated proteins for downstream MS analysis. We validated the approach by identifying several established O-GlcNAc sites on the proteins α-crystallin and OGT as well as discovering new, previously unreported sites on both proteins. Notably, these novel sites on OGT lie in key functional domains of OGT, underscoring how this site identification method can reveal important biological insights into protein activity and regulation.
Finally, in Chapters 4 and 5, we focus on the post-translational modification (PTM) code on a specific transcription factor (TF), CREB (cAMP response element binding protein). CREB regulates memory formation through its transcriptional control of neuronal metabolism, activity, differentiation, development, and survival. CREB phosphorylation at serine 133 has been previously shown to enhance CREB-mediated transcription while CREB glycosylation at serine 40 has been shown to decrease CREB-mediated transcription. However, the exact gene networks modulated by and potential interplay between CREB glycosylation and phosphorylation have not been explored. Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we showed that CREB O-GlcNAcylation is important for neuronal activity and excitability while phosphorylation at serine 133 regulated the expression of genes involved in neuronal differentiation. Using WGCNA, we demonstrated that CREB O-GlcNAcylation at serine 40 and phosphorylation at serine 133 mediate mutually exclusive gene networks. The glycosylation-deficient mutant enhanced neuronal activity- and excitotoxicity-related gene networks while the phosphorylation-deficient mutant perturbed neuronal differentiation and amino and fatty acid metabolism-related gene networks. Our work sheds light on the regulation of CREB through PTMs to modulate neuronal function and delineate the roles of O-GlcNAcylation and phosphorylation in modulating neuronal excitability and neuronal development and metabolism respectively. Altogether, these studies demonstrate that O-GlcNAc modification is a critical mediator of neuronal homeostasis and neurodegeneration.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9RJ4GPT, author = {Miller, Gregory Martin}, title = {Eph Receptor Clustering by Chondroitin Sulfate Inhibits Axon Regeneration}, school = {California Institute of Technology}, year = {2018}, doi = {10.7907/Z9RJ4GPT}, url = {https://resolver.caltech.edu/CaltechTHESIS:02132018-133402404}, abstract = {Chondroitin sulfate proteoglycans (CSPGs) play important roles in the developing and mature nervous system, where they guide axons, maintain stable connections, restrict synaptic plasticity, and prevent axon regeneration following CNS injury. The chondroitin sulfate glycosaminoglycan (CS GAG) chains that decorate CSPGs are essential for their functions. Through these sugar chains, CSPGs are able to bind and regulate the activity of a diverse range of proteins and through these interactions can regulate neuronal growth. These CS-protein interactions depend on specific sulfation patterns within the CS GAG chains, and accordingly, particular CS sulfation motifs are upregulated during development, in the mature nervous system, and in response to CNS injury. Thus, spatiotemporal regulation of CS GAG biosynthesis may provide an important mechanism to control the functions of CSPGs and modulate intracellular signaling pathways. Here, we will discuss these sulfation-dependent processes and highlight how the CS sugars on CSPGs contribute to neuronal growth, axon guidance, and plasticity in the nervous system.
Chondroitin sulfate proteoglycans (CSPGs) are a major barrier to regenerating axons in the central nervous system (CNS), exerting their inhibitory effect through their polysaccharide side chains. Chondroitin sulfate (CS) potently inhibits axon regeneration through modulation of inhibitory signaling pathways induced by carbohydrate binding to protein ligands and receptors. Here, we identify a novel carbohydrate-protein interaction between CS and EphA4 that inhibits axon regrowth. We characterize the mechanism of activation and demonstrate how carbohydrate binding induces phosphorylation of the intracellular kinase domain through clustering of cell surface EphA4. Collectively, our studies present a novel mechanism of EphA4 activation by CS independent of the canonical ephrin ligands and uncover the role of this interaction in inhibition of neurite regrowth after injury. Our results underscore a mechanism of action by which carbohydrates can function as direct, activating ligands for protein receptors and provide mechanistic insights into the inhibition of axon growth by CS following injury to the CNS.
Chondroitin sulfate proteoglycans (CSPGs) regulate neuronal plasticity, as well as axon regeneration and guidance through their ability to bind protein ligands and cell surface receptors. In this way, extracellular CSPGs can modulate the activity of intracellular signaling pathways. Here, a computational analysis of EphA4-CS interactions is performed to characterize the importance of key arginine and lysine residues towards CS binding, and to identify structural differences in CS-A, CS-C, CS-D, and CS-E docking to EphA4. Carbohydrate-induced Eph receptor clustering could be a general mechanism of Eph receptor activation. To identify additional Eph receptors that interact with CS, CS-E was docked to all EphA and EphB family members to predict relative binding affinities. The relative strengths of the predicted binding energies are: EphB4 > EphA8 > EphA1 > EphA3 > EphB1 > EphB3 > EphA7 > EphA5 > EphA4 > EphA6 > EphB2 > EphB6 > EphA2. In addition, the arginine and lysine residues that mediate CS binding are identified for each Eph receptor. These computational predictions provide mechanistic insights into Eph receptor activation by chondroitin sulfate and have implications for inhibition of axon regeneration following injury to the nervous system and axon guidance during development.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/TPD7-FB87, author = {Yang, Kuang-Wei}, title = {Synthesis, NMR Solution Structure, and Neuritogenic Activity of Chondroitin Sulfate D and E}, school = {California Institute of Technology}, year = {2018}, doi = {10.7907/TPD7-FB87}, url = {https://resolver.caltech.edu/CaltechTHESIS:05292018-232454800}, abstract = {Chondroitin sulfate are ubiquitously expressed linear, sulfated polysaccharides that play critical roles in neuronal development and regeneration growth factor signaling, morphogenesis, and virus invasion. The diverse sulfation patterns presented by chondroitin sulfate has been suggested to regulate its activity, but the structural complexity and heterogeneity have hampered the understanding of structure-activity relationship. Therefore, we envisioned that chemically synthesized chondroitin sulfate oligosaccharide may provide a unique opportunity to specifically study the functions of sulfation patterns.
Here, we report the synthesis of a CS-D and CS-E tetrasaccharide in a step-efficient manner. By generating a disaccharide precursor from hydrolysis of polysaccharides, we were able to streamline the synthesis and reduce the number of steps by one-third comparing to the traditional synthesis without losing versatility of the synthetic route and functionality of the final product. With the structurally defined molecules, we were able to determine the NMR solution structure of CS-D and CS-E. In this work, we accomplished the first structural study of CS-D tetrasaccharide and the most thorough study of CS-E to date. Furthermore, we also discovered the existence of a second conformer in CS-D, which is the first time for such behavior to be observed experimentally in chondroitin sulfate. The electrostatic potential surface constructed based on the NMR structure presented unique structural features that may allow proteins to interact specifically.
The CS-D and CS-E tetrasaccharide, along with a CS-D disaccharide, was investigated for their neuritogenic activity. We discovered that the CS-D tetrasaccharide specifically stimulates dendritic growth whereas the CS-E tetrasaccharide preferentially promoted axonal growth, revealing the potential critical role chondroitin sulfate with specific sulfation patterns may play in the nervous system. The lack of activity of the CS-D disaccharide suggested that the minimum motif required for activity of CS-D is a tetrasaccharide.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9610XCH, author = {Griffin, Matthew Everett}, title = {Discovering Biological Roles of Glycosaminoglycans and Protein O-GlcNAcylation Using Chemical Tools}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9610XCH}, url = {https://resolver.caltech.edu/CaltechTHESIS:06042017-183300784}, abstract = {Carbohydrates surround nearly every cell in the human body. Glycosaminoglycans like chondroitin sulfate and heparan sulfate on the cell surface regulate protein ligand engagement and receptor activation to control a variety of biological processes including development, angiogenesis, and neuronal growth. These polysaccharides exert activity through protein binding to their diverse chemical structures. Therefore, the development of methods to tailor glycosaminoglycan populations at the cell surface with defined structures could provide novel approaches to control biological activity. Herein, two new methods to engineer the cell surface glycocalyx with known glycosaminoglycans are reported. Together, these methods provide complementary short- and long-term approaches to change carbohydrate structures at the cell surface and guide neuronal growth and stem cell differentiation. It is also critical to identify unknown protein-carbohydrate interactions that underlie biological phenomena. Studies delineating novel GAG interactions with an orphan receptor and related soluble ligands are reported herein as well as work towards understanding the biological functions of these newly discovered interactions. These results showcase the utility of chemical biology and biochemical tools to discover and modulate various GAG-protein interactions in diverse biological systems.
Within the cell, thousands of proteins are modified by O-GlcNAc glycosylation, a process that is uniquely catalyzed by a single transferase and hydrolase pair unlike many other post-translational modifications. O-GlcNAcylation functions in many biological contexts including transcription, translation, proteostasis, and metabolism. Key to understanding its effects on these physiological phenomena is the discovery of O-GlcNAc modification sites. However, due to a number of technical challenges, O-GlcNAc proteomics has not progressed nearly as quickly as phosphoproteomics. Thus, developing new methods to enrich O-GlcNAcylated substrates and map modification sites is critical to unravel the myriad functions of O-GlcNAc. Herein, a labeling approach using a chemically cleavable tag is reported as an improved method to capture and release O-GlcNAcylated substrates. Unlike other methods, the cleavable Dde tag is quantitatively removed under mild, neutral conditions and leaves a minimal residual tag on the O-GlcNAcylated peptide to be analyzed. Moreover, the Dde linker outcompetes a previously used UV-cleavable tag both at the protein and peptide enrichment levels. Together, these results highlight the potential usefulness of this method to illuminate novel roles of O-GlcNAcylation in diverse systems.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9PR7T0K, author = {Cheung, Sheldon Ting Fong}, title = {Discovery and Development of Small-Molecule Modulators for the Sulfation of Glycosaminoglycans and Studying the Role of O-GlcNAc on CREB through Semisynthesis}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z9PR7T0K}, url = {https://resolver.caltech.edu/CaltechTHESIS:10032016-103410952}, abstract = {Glycosaminoglycans (GAGs) are sulfated polysaccharides that play key roles in many cellular processes, ranging from viral invasion and cancer metastasis to neuronal development. Their diverse biological activities stem from their complex sulfation patterns, which are tightly regulated in vivo. For instance, the GAG chondroitin sulfate (CS) has been shown to undergo regiochemical sulfation during development and after spinal cord injury. However, few tools exist to modulate specific GAG sulfation patterns and study their importance in different biological contexts. Here, we identified the first cell-permeable small molecule that can selectively inhibit GAG sulfotransferases and modify the fine structure of GAGs. We demonstrate that the inhibitor reduces GAG sulfation in vitro and in cells and reverses CS-E-mediated inhibition of neuronal outgrowth. This small molecule may serve as a useful lead compound or chemical tool for studying the importance of CS and other GAGs in normal biology and disease.
The β-N-acetyl-D-glucosamine (O-GlcNAc) post-translational modification plays a major role in many diseases such as cancer, diabetes, and neurodegenerative disorders, but much is still unknown about its molecular-level influence on protein structure and function. Although post-translational modifications have been known to induce important structural changes in proteins, notably, no structures of O-GlcNAcylated proteins exist. The challenge of obtaining homogeneous glycoproteins bearing the GlcNAc sugar at defined sites has hindered the structural and biochemical studies of this modification. Here we have utilized a semisynthetic approach to generate a homogeneously O-GlcNAcylated form of cyclic-AMP response element binding protein (CREB) for structural and functional studies.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9K35RN1, author = {Chaubard, Jean-Luc}, title = {Development of Chemoenzymatic Labeling Approaches for the Detection of Fucosylated Biomarkers}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z9K35RN1}, url = {https://resolver.caltech.edu/CaltechTHESIS:05052016-123724002}, abstract = {Protein fucosylation regulates a diverse set of physiological functions such as memory and learning, development, and disease pathogenesis. However, our current understanding of these processes is far behind that of other post-translational modifications, such as phosphorylation. This is, in part, due to the lack of tools available for the study of this important protein modification. To address this need, I have developed novel chemoenzymatic methods that enable the labeling and detection of unique forms of fucosylation, specifically fucose-α(1-2)-galactose (Fucα(1-2)Gal) and core fucose. Additionally, novel glycosyltransferase assays were developed in-house to aid in the future development of both new and existing chemoenzymatic approaches.
I have demonstrated that the approach to detect Fucα(1-2)Gal is highly selective for this disaccharide motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of this motif on live cells, discriminating malignant from normal cells. I have also shown that the chemoenzymatic detection of core fucose exhibits superior specificity towards this glycan on a variety of complex N-glycans and when compared to current fucose-specific lectins. Further, the approach is amenable to detection of core fucosylated glycans from multiple biological settings, can be exploited as an antibody-conjugation method, and can be integrated into a diagnostic platform for the profiling of protein specific core fucosylation levels. These approaches represent new potential strategies for biomarker identification and expand the technologies available for understanding the role of these important fucosylated glycans in physiology and disease.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z97W695K, author = {Wang, Andrew Chih-Kae}, title = {Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z97W695K}, url = {https://resolver.caltech.edu/CaltechTHESIS:06032015-135024245}, abstract = {O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer’s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.
Given the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/JQ2Z-EN67, author = {Sheng, Gloria J.}, title = {Tunable Heparan Sulfate Glycomimetics for Modulating Chemokine Activity}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/JQ2Z-EN67}, url = {https://resolver.caltech.edu/CaltechTHESIS:05272014-224131184}, abstract = {Heparan sulfate (HS) glycosaminoglycans participate in critical biological processes by modulating the activity of a diverse set of protein binding partners. Such proteins include all known members of the chemokine superfamily, which are thought to guide the migration of distinct subsets of immune cells through their interactions with HS proteoglycans on endothelial cell surfaces. Animal-derived heparin polysaccharides have been shown to reduce inflammation levels through the inhibition of HS-chemokine interactions; however, the clinical usage of heparin as an anti-inflammatory drug is hampered by its anticoagulant activity and potential risk for side effects, such as heparin-induced thrombocytopenia (HIT).
Here, we describe an expedient, divergent synthesis to prepare defined glycomimetics of HS that recapitulate the macromolecular structure and biological activity of natural HS glycosaminoglycans. Our synthetic approach uses a core disaccharide precursor to generate a library of four differentially sulfated polymers. We show that a trisulfated glycopolymer antagonizes the chemotactic activities of pro-inflammatory chemokine RANTES with similar potency as heparin polysaccharide, without potentiating the anticoagulant activities of antithrombin III. The same glycopolymer also inhibited the homeostatic chemokine SDF-1 with significantly more efficacy than heparin. Our work offers a general strategy for modulating chemokines and dissecting the pleiotropic functions of HS/heparin through the presentation of defined sulfation motifs within multivalent polymeric scaffolds.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, month = {July}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z90G3H45, author = {Brown, Joshua Micah}, title = {Investigation of Receptors for the Modulation of Neuronal Growth by Chondroitin Sulfate}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/Z90G3H45}, url = {https://resolver.caltech.edu/CaltechTHESIS:09162013-140043647}, abstract = {A major obstacle to neural regeneration after injury in the central nervous system (CNS) is the environment encountered by injured axons. This environment is inhibitory due to proteins expressed by the CNS myelin as well as molecules present in the glial scar. Experimental results have implicated chondroitin sulfate proteoglycans (CSPGs) as major inhibitors of axonal regeneration after CNS injury, but until recently, the mechanisms of this inhibition were not well understood. Furthermore, the complex nature of the chondroitin sulfate (CS) chains made it difficult to study their contribution to CSPG function. This thesis describes a specific carbohydrate epitope, CS-E, that is primarily responsible for the inhibition of CNS axonal regrowth in the presence of CSPGs. We show that removal or blocking of the CS-E motif via genetic elimination of the enzyme responsible for generating CS-E or a monoclonal antibody that binds specifically to the CS-E motif significantly reduces the inhibitory activity of CSPGs on axon growth. Furthermore, we show that CS-E functions as a protein recognition element to engage receptors, including the transmembrane protein tyrosine phosphatase PTPσ, which had been previously established to be a receptor for CSPGs. Finally, we show that the protein tyrosine kinase receptor EphA4 is a novel receptor for the CS-E motif, and as with PTPσ, neurons deficient in EphA4 exhibit reduced inhibition by CS-E. Our results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides directs important physiological processes, and establish the importance of the chemical structure of CS chains in modulating the activity of CSPGs in vivo. The identification of receptors that mediate the inhibitory effect of CS-E advances our understanding of the mechanisms of axon regeneration following injury to the CNS when CS-E expression is upregulated. These findings provide us with the opportunity to develop therapies for the recovery of axonal outgrowth after damage to the nervous system, which in conjunction with blocking approaches targeting the CS motif, can provide a powerful strategy for allowing recovery after injury to the CNS.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z99021SG, author = {Krishnamurthy, Chithra}, title = {Chemical Probes to Study Fucosylated Glycans}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/Z99021SG}, url = {https://resolver.caltech.edu/CaltechTHESIS:06052013-123658997}, abstract = {Fucosylated glycans have many critical biological roles, from leukocyte adhesion to host-microbe interactions. However, a molecular level understanding of these sugars has been lacking, in part due to the chemical and structural diversity of glycans that make them challenging to study. In order to gain a deeper understanding of fucosylated glycans, we have explored the use of chemical probes to study these structures. In Chapters 1 and 2, we apply a metabolic labeling technique for the investigation of fucosylated glycans in neurons, where they have been implicated in learning and memory processes. However, the molecular mechanisms by which these sugars influence neuronal processes are not well understood, and only a handful of fucosylated glycoproteins have been identified. In order to facilitate our understanding of these processes, we exploit non-natural fucose analogs to identify the fucose proteome in rat cortical neurons, identifying proteins involved in cell adhesion, neuronal signaling, and synaptic transmission. Moreover, we track fucosylated glycoproteins in hippocampal neurons, and show that fucosylated glycoproteins localize to the Golgi, axons, and dendrites, and are enriched in synapses. In Chapter 4, we report a new chemoenzymatic strategy for the sensitive detection of the Fucα(1-2)Gal epitope, which has been implicated in tumorigenesis as a potential biomarker of cancer progression. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. These approaches represent new potential applications and strategies for the investigation of fucosylated glycans, and expand the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/Z9N58JCD, author = {Rogers, Claude Joseph}, title = {Discovery of New Roles for Chondroitin Sulfate in Neurotrophin Signaling and Retinotopic Development}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/Z9N58JCD}, url = {https://resolver.caltech.edu/CaltechTHESIS:01272013-150337805}, abstract = {Chondroitin sulfate (CS), a member of the glycosaminoglycan family of linear polysaccharides, is involved in the formation and maintenance of neuronal networks. CS has dual roles in regulating neuronal morphology: promoting or inhibiting neuronal outgrowth, depending on the context. A single sulfated epitope, CS-E, is capable of inducing both types of activity.
Members of the neurotrophin (NT) family of growth factors are required for CS- E-induced neurite outgrowth in hippocampal neurons. Here, we demonstrate that CS is capable of forming ternary complexes with NTs and their receptors. These complexes were discovered using a novel, carbohydrate microarray-based approach that allows for the rapid screening of such interactions. To support these findings, we computationally determined the CS-E-binding site of the complexes, suggesting a structural basis for the interaction. In addition, we showed that CS-E is capable of attenuating NT signaling in cells, consistent with our computational and microarray data. This is the first demonstration that CS-E is involved in NT signaling and that CS is capable of supporting multimeric signaling complexes.
In addition to stimulating growth factor signaling, CS has been known to repulsively guide retinal ganglion cell (RGC) axons for over twenty years. However, its function in vivo is unknown. RGCs are the only neuron type that transmits visual information to the brain, and their guidance, which maps a topographic projection of the retina to the superior colliculus (SC), is tightly regulated. Here, we show that CS-E is required for the proper formation of this topographic order. CS-E, but not the other major sulfation patterns, is a repellent guidance cue for RGC axons, with a graded activity profile from low to high along the dorsal-ventral axis of the retina, congruent with EphB3 expression. EphB3 binds specifically to CS-E with physiologically relevant affinity, and is required for CS-E-mediated guidance. CS-E-null mice have defects in topographic mapping in which ventral axons form ectopic termina- tions medial to their correct location in the SC. These results indicate that CS is a repulsive guidance cue required to map the dorsal-ventral axis of the retina along the lateral-medial axis of the SC. This is the first report of a non-protein topographical guidance cue.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/ETC4-N802, author = {Wibowo, Arif}, title = {Identification of Fucose-α(1-2)-Galactose Binding Proteins in the Mammalian Brain}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/ETC4-N802}, url = {https://resolver.caltech.edu/CaltechTHESIS:06012013-114632816}, abstract = {Fucose-α(1-2)-galactose (Fucα(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fucα(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fucα(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/E16S-6T52, author = {Oh, Young In}, title = {Synthesis and Biological Activity of Anticoagulant Heparan Sulfate Glycopolymers}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/E16S-6T52}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022013-204012683}, abstract = {Heparin has been used as an anticoagulant drug for more than 70 years. The global distribution of contaminated heparin in 2007, which resulted in adverse clinical effects and over 100 deaths, emphasizes the necessity for safer alternatives to animal-sourced heparin. The structural complexity and heterogeneity of animal-sourced heparin not only impedes safe access to these biologically active molecules, but also hinders investigations on the significance of structural constituents at a molecular level. Efficient methods for preparing new synthetic heparins with targeted biological activity are necessary not only to ensure clinical safety, but to optimize derivative design to minimize potential side effects. Low molecular weight heparins have become a reliable alternative to heparin, due to their predictable dosages, long half-lives, and reduced side effects. However, heparin oligosaccharide synthesis is a challenging endeavor due to the necessity for complex protecting group manipulation and stereoselective glycosidic linkage chemistry, which often result in lengthy synthetic routes and low yields. Recently, chemoenzymatic syntheses have produced targeted ultralow molecular weight heparins with high-efficiency, but continue to be restricted by the substrate specificities of enzymes.
To address the need for access to homogeneous, complex glycosaminoglycan structures, we have synthesized novel heparan sulfate glycopolymers with well-defined carbohydrate structures and tunable chain length through ring-opening metathesis polymerization chemistry. These polymers recapitulate the key features of anticoagulant heparan sulfate by displaying the sulfation pattern responsible for heparin’s anticoagulant activity. The use of polymerization chemistry greatly simplifies the synthesis of complex glycosaminoglycan structures, providing a facile method to generate homogeneous macromolecules with tunable biological and chemical properties. Through the use of in vitro chromogenic substrate assays and ex vivo clotting assays, we found that the HS glycopolymers exhibited anticoagulant activity in a sulfation pattern and length-dependent manner. Compared to heparin standards, our short polymers did not display any activity. However, our longer polymers were able to incorporate in vitro and ex vivo characteristics of both low-molecular-weight heparin derivatives and heparin, displaying hybrid anticoagulant properties. These studies emphasize the significance of sulfation pattern specificity in specific carbohydrate-protein interactions, and demonstrate the effectiveness of multivalent molecules in recapitulating the activity of natural polysaccharides.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/FTJS-8M82, author = {Clark, Peter Michael}, title = {New Tools for Studying O-G1cNAc Glycosylation and Chondroitin Sulfate Proteoglycans and Studies on the Roles of O-G1cNAc Glycosylation on the Transcription Factor CREB}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/FTJS-8M82}, url = {https://resolver.caltech.edu/CaltechTHESIS:01132011-095304695}, abstract = {The addition and removal of the monosaccharide N-acetyl-D-glucosamine (GlcNAc) to serine and threonine residues of proteins has emerged as a critical regulator of cellular processes. However, studies of O-GlcNAc in such complex systems as the brain have been limited, in part due to the lack of tools. Here we report the development of new tools for studying O-GlcNAc, and the application of these and other tools for studying the roles of O-GlcNAc in the brain.
Working from a previously established chemoenzymatic method, we designed an isotopic labeling strategy for probing the dynamics of O-GlcNAc glycosylation using quantitative proteomics. With this tool, we show that O-GlcNAc is dynamically modulated on specific proteins by excitatory stimulation of the brain in vivo. Separately, we improved this chemoenzymatic strategy by integrating [3+2] azide-alkyne cycloaddition chemistry to attach biotin and fluorescent tags to O-GlcNAc residues. These tags allow for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc modified proteins. With this strategy, we identified over 146 novel glycoproteins from the mammalian brain.
The transcription factor cAMP-response element binding protein (CREB) is critical for numerous functions in the brain, including neuronal survival, neuronal development, synaptic plasticity, and long-term memory. We show that CREB is highly glycosylated in the brain and discover new glycosylation sites on CREB in neurons. One of these sites is dynamically modulated and is important for regulating CREB. Removal of this glycosylation site alters CREB-mediated functions in vitro and in vivo. These studies are the first demonstration that O-glycosylation at a specific site on a specific protein is critical for neuronal function and behavior.
Chondroitin sulfates (CS) are sulfated linear polysaccharides important in neuronal development and viral invasion. Depending on their sulfation patterns, CS molecules differ dramatically in their functions. We developed a computational method to model the structure and function of CS. Using this approach, we show that different CS tetrasaccharides have distinct solution structures. We also modeled the CS binding site on a variety of proteins and discovered that CS may be important in modulating protein-protein interactions.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/QG38-9P18, author = {Murrey, Heather Elizabeth}, title = {Identification and Characterization of the Plasticity-Relevant Fucose-α(1-2) Galactose Glycoproteome from Mouse Brain}, school = {California Institute of Technology}, year = {2009}, doi = {10.7907/QG38-9P18}, url = {https://resolver.caltech.edu/CaltechETD:etd-12182008-145714}, abstract = {Fucα(1-2)Gal carbohydrates have been implicated in cognitive processes such as learning and memory. However, a molecular level understanding of their functions has been lacking. This thesis describes multiple chemical and biological approaches that we have undertaken to elucidate the molecular mechanisms by which fucosyl sugars mediate neuronal communication. We demonstrate that Fucα(1-2)Gal carbohydrates play an important role in the regulation of synaptic proteins and neuronal morphology. We identify synapsins Ia and Ib as prominent Fucα(1-2)Gal glycoproteins in rat hippocampus, and fucosylation protects synapsin I from proteolytic degradation by the calcium-activated protease calpain. Synapsin fucosylation has important consequences on neuronal growth and morphology, with defucosylation leading to stunted neurites and delayed synapse formation. In addition, we identify the Fucα(1-2)Gal proteome from mouse olfactory bulb using lectin affinity chromatography. We discover four major classes of Fucα(1-2)Gal glycoproteins, including the immunoglobulin superfamily of cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, and synaptic vesicle-associated proteins. Protein fucosylation is regulated by FUT1 in mouse olfactory bulb, and olfactory bulb development is impaired in FUT1-deficient mice. In particular, FUT1 KO animals exhibit defects in the olfactory nerve and glomerular layers of olfactory sensory neurons expressing the fucosylated cell adhesion molecules NCAM and OCAM. Lastly, we explore the molecular mechanisms of protein fucosylation by metabolic labeling with alkynyl- and azido-fucose derivatives. We demonstrate that fucosylated glycoconjugates are present along both axons and dendrites of developing neuronal cultures, as well as in the Golgi body. We identify the fucosylated proteome from cultured cortical neurons, and demonstrate that proteins such as NCAM, the MARCKS family of proteins, and the inositol 1,4,5 triphosphate receptor are fucosylated. In addition, we can label fucosylated glycans in vivo, which will have important consequences for studies on the dynamics of protein fucosylation in living animals. Cumulatively, our studies suggest important functional roles for fucosyl-carbohydrates in the nervous system, and implicate an extended role for fucose in the molecular mechanisms that may underlie synaptic plasticity and neuronal development.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/CDGH-MJ49, author = {Gama, Cristal Ivette}, title = {Understanding the Chemical Basis of Neuronal Development and Communication: I. The Role of Fucose α(1-2) Galactose Carbohydrates in Neuronal Growth. II. Structure-Function Analysis of Chondroitin Sulfate in the Brain}, school = {California Institute of Technology}, year = {2009}, doi = {10.7907/CDGH-MJ49}, url = {https://resolver.caltech.edu/CaltechETD:etd-10302008-123855}, abstract = {Although carbohydrates are known to participate in many processes including inflammation and cancer metastasis, their functional roles are only beginning to be understood on a molecular level. Unlike DNA and proteins, carbohydrate structures are not template-encoded and are challenging to detect in vivo and manipulate for structure-function analyses. New tools are needed to complement biochemical and genetic approaches to advance our understanding of carbohydrates and their physiological roles. We seek to understand the roles of carbohydrates in regulating the structure and function of proteins in the brain. Our focus is on two modifications that are important in neuronal communication and development: fucosylation (Part I) and chondroitin sulfate modifications (Part II).
In Part I, we describe our progress in elucidating the molecular mechanisms by which fucosyl saccharides regulate neuronal communication. Previous studies have shown that preventing formation of fucoseα(1-2)galactose saccharides causes reversible amnesia in animals, suggesting that these sugars play essential roles in learning and memory. However, proteins expressing the fucoseα(1-2)galactose epitope or proteins binding this epitope have not been identified. Using chemical probes, we established that fucoseα(1-2)galactose associated proteins participate in a novel carbohydrate-mediated pathway for regulating neuronal growth. Additionally, we found that fucoseα(1-2)galactose glycoproteins are prevalent in developing brain and that synapsin Ia/Ib are the major fucoseα(1-2)galactose glycoproteins in adult brain. In our attempts to identify Fucα(1-2)Gal lectins, we have established that multivalent polymers enhance our ability to capture and characterize such proteins.
In Part II, we describe our efforts toward understanding the role of chondroitin sulfate glycosaminoglycans in neuronal development. Chondroitin sulfate glycosaminoglycans are structurally complex and heterogeneous in nature, thus hampering efforts to understand their precise biological roles. It is thought that chondroitin sulfate activity is dictated by a sulfation code, where distinct sulfation sequences are spatially and temporally regulated. We have developed a chemical approach to evaluate the structure-activity relationship of chondroitin sulfate as it effects neuronal growth. We generated the first synthetic library of well-defined chondroitin sulfate oligosaccharides containing various sulfation sequences and have demonstrated that the chondroitin sulfate-E sequence is a stimulatory motif that promotes the growth of several neuron types. Moreover, we determined that chondroitin sulfate-E stimulation was facilitated through activation of the midkine/PTPζ and BDNF/TrkB pathways.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/HWFJ-P813, author = {Saliba, Katie Rose}, title = {Methodologies for the Rapid Synthesis of Hexoses and Their Application Towards a Differentially-Protected Chondroitin Sulfate Tetrasaccharide}, school = {California Institute of Technology}, year = {2008}, doi = {10.7907/HWFJ-P813}, url = {https://resolver.caltech.edu/CaltechETD:etd-05292008-230310}, abstract = {Carbohydrates play many roles in biology, but their study has been hindered by the paucity of methods available to rapidly access hexoses. In 2004, the MacMillan laboratory published a two-step aldol methodology that allows access to the erythrohexoses allose, glucose, and mannose. Described herein is the development of two methodologies to access hexoses. First, the two-step aldol methodology for accessing the erythrohexoses was expanded to allow access to a differentially-protected mannosamine and gulose. Also described is the discovery of a one-step aldol methodology for accessing hexoses, which has allowed access to a protected allose and gulose.
This methodology was applied to the synthesis of a differentially-protected chondroitin sulfate di- and tetrasaccharide. Chondroitin sulfate is a complex linear polysaccharide composed of alternating glucuronic acid and galactosamine residues that are heterogeneously sulfated. Combining the aldol methodology with a Cerny epoxide methodology developed in the Hsieh-Wilson laboratory, a core disaccharide was accessed. Model studies confirmed each position could be accessed selectively. Elaboration of this disaccharide to the protected tetrasaccharide was hindered by an unfavorable rearrangement during the tetrasaccharide coupling, so a second core disaccharide was synthesized. This core disaccharide was elaborated to a common intermediate to confirm that it should still allow selective access to each position, and then the disaccharide was elaborated towards the desired protected tetrasaccharide.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {MacMillan, David W. C. and Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/MT8P-JB95, author = {Khidekel, Nelly}, title = {A Chemoenzymatic Strategy toward Understanding O-GlcNAc Glycosylation in the Brain}, school = {California Institute of Technology}, year = {2007}, doi = {10.7907/MT8P-JB95}, url = {https://resolver.caltech.edu/CaltechETD:etd-03022007-083916}, abstract = {Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O-GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification’s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain.
Our approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically ‘tag’ O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. Here, we have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/ZEXB-6889, author = {Lamarre-Vincent, Nathan}, title = {Identification and Functional Analysis of O-G1cNAc Glycosylation on the Transcription Factor cAMP-Response Element Binding Protein}, school = {California Institute of Technology}, year = {2007}, doi = {10.7907/ZEXB-6889}, url = {https://resolver.caltech.edu/CaltechETD:etd-05292007-183523}, abstract = {The survival and development of organisms requires the ability of cells to communicate with the environment and with surrounding cells. This demand has led to the evolution of a number of methods used for communication. Chief among these is the ability to modify protein function with post-translational modifications (PTMs). PTMs allow cells to use a single protein for a variety of tasks and link protein activity with a specific environmental or cellular cue. Modification of transcription factors has arisen as a key model for the study of PTMs and their effects on cell processes. PTMs modulate transcriptional activity required for key processes such as development, differentiation and cell survival.
The eukaryotic transcription factor cAMP-response element binding protein (CREB) is a transcription factor that confers dynamic control of a number of cellular processes including neuronal and pancreatic cell survival, gluconeogenesis and neuronal long-term potentiation. CREB is activated by phosphorylation of single serine residue. The observation that a number of kinase signaling cascades converge on CREB has led to the question of how cells deal with the apparent loss of signal identity that occurs as a result of this convergence. In this thesis I describe the identification, characterization and functional analysis of a novel PTM of CREB, O-GlcNAc glycosylation, that provides an additional level of control of CREB activity. CREB glycosylation moderates phosphorylation-dependent CREB activity and reduces CREB-dependent gene expression in pancreatic [beta]-cells, and as a result promotes [beta]-cell death, as observed in type II diabetes. CREB glycosylation offers us an example of how cells use multiple PTMs to control protein function and how dysfunction in the regulation of these modifications may contribute to disease states.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, } @phdthesis{10.7907/c6pj-np35, author = {Tully, Sarah Erin}, title = {Synthesis and Biological Activity of Chondroitin Sulfate Biopolymers}, school = {California Institute of Technology}, year = {2007}, doi = {10.7907/c6pj-np35}, url = {https://resolver.caltech.edu/CaltechETD:etd-12132006-122200}, abstract = {Chondroitin sulfate glycosaminoglycans are ubiquitously expressed linear, sulfated polysaccharides involved in cell growth, neuronal development and spinal cord injury. The different sulfation motifs presented by chondroitin sulfate may regulate its activity, but efforts to understand the precise biological roles of this glycosaminoglycan have been hampered by its complexity and heterogeneity. Here, we report the synthesis of well-defined chondroitin sulfate oligosaccharides through a convergent approach that permits installation of sulfate groups at precise positions along the carbohydrate backbone, biological evaluation of the synthetic molecules, and generation of antibodies that recognize the distinct sulfation motifs.
Using the chondroitin sulfate oligosaccharide library, we demonstrate that specific sulfation patterns act as molecular recognition elements for growth factors, and modulate neuronal growth. We identified a chondroitin sulfate tetrasaccharide, CS-E, which stimulates the growth and differentiation of multiple neuron types. Through use of carbohydrate microarrays, we found that the CS-E tetrasaccharide binds to a variety of proteins involved in promoting neurite outgrowth. A CS-E disaccharide, an unsulfated tetrasaccharide, and three other sulfated tetrasaccharides, CS-A, CS-C, and CS-R, were also investigated, and showed little effect on neurite outgrowth and reduced growth factor binding compared to the CS-E tetrasaccharide. These studies represent the first, direct investigations into the structure-activity relationships of chondroitin sulfate using homogeneous synthetic molecules, define a tetrasaccharide as a minimal motif required for function, and reveal the importance of sulfation in chondroitin sulfate bioactivity.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Hsieh-Wilson, Linda C.}, }