Chemical Probes to Study Fucosylated Glycans

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

Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb

Fucose-α(1−2)-galactose [Fucα(1−2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucα(1−2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes

End-functionalized glycopolymers as mimetics of chondroitin sulfate proteoglycans

Glycosaminoglycans are sulfated polysaccharides that play important roles in fundamental biological processes, such as cell division, viral invasion, cancer and neuroregeneration. The multivalent presentation of multiple glycosaminoglycan chains on proteoglycan scaffolds may profoundly influence their interactions with proteins and subsequent biological activity. However, the importance of this multivalent architecture remains largely unexplored, and few synthetic mimics exist for probing and manipulating glycosaminoglycan activity. Here, we describe a new class of end-functionalized ring-opening metathesis polymerization (ROMP) polymers that mimic the native-like, multivalent architecture found on chondroitin sulfate (CS) proteoglycans. We demonstrate that these glycopolymers can be readily integrated with microarray and surface plasmon resonance technology platforms, where they retain the ability to interact selectively with proteins. ROMP-based glycopolymers are part of a growing arsenal of chemical tools for probing the functions of glycosaminoglycans and for studying their interactions with proteins

Chemoenzymatic Probes for Detecting and Imaging Fucose-α(1-2)-galactose Glycan Biomarkers

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα(1-2)Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα(1-2)Gal glycans. 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. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease

Chemoenzymatic approaches to the imaging and detection of cancer relevant fucosylated glycoconjugates

A major focus in the field of glycomics has been the development of new strategies for the detection and quantification of glycans and glycoconjugates. With alterations in glycoconjugate structure being a hallmark of various cancers, these strategies can discover new cancer biomarkers and be developed into new clin. diagnostic tools. Here we report chemoenzymic strategies for the rapid, sensitive detection of cancer-relevant fucosylated glycoconjugates. Our methods exploit non-mammalian glycosyltransferases that accept non-natural donor substrates. We then use "Click" chem. to append reporter tags for the detection of these glycans. We have developed methods for the detection of glycans contg. fucoseα(1-2)galactose (Fucα(1-2) Gal), a motif implicated cancer pathogenesis, as well as core fucosylated glycans, a carbohydrate modification that is upregulated in various cancer states and mediates cell signaling events. We demonstrate the specificity and utility of these methods for the detection of cancerous compared to healthy cells and tissues

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2‑nitro-N-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p

Chemoenzymatic Probes for Detecting and Imaging Fucose-α(1-2)-galactose Glycan Biomarkers

The disaccharide motif fucose-α­(1-2)-galactose (Fucα­(1-2)­Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα­(1-2)­Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα­(1-2)­Gal glycans. 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. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease

Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb

Fucose-α(1−2)-galactose [Fucα(1−2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucα(1−2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes