Claude J. Rogers

Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here, we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation, or expensive radiolabels. In addition, the glycosylation state (e.g., mono-, di-, tri-) of proteins is established, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation, and discover an unexpected reverse yin-yang relationship on the transcriptional repressor MeCP2, which was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

A sulfated carbohydrate epitope inhibits axon regeneration after injury

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury.

Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes.

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.

Microarray method for the rapid detection of glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) perform numerous vital functions within the body. As major components of the extracellular matrix, these polysaccharides participate in a diverse array of cell-signaling events. We have developed a simple microarray assay for the evaluation of protein binding to various GAG subclasses. In a single experiment, the binding to all members of the GAG family can be rapidly determined, giving insight into the relative specificity of the interactions and the importance of specific sulfation motifs. The arrays are facile to prepare from commercially available materials.

Predicting glycosaminoglycan surface protein interactions and implications for studying axonal growth

Significance Glycans and proteins are important partners in the regulation of fundamental biological processes such as the immune response, migration, differentiation, morphogenesis, angiogenesis, axon guidance, and response to CNS injury. Understanding glycan–protein interactions is critical to mapping the biological functions of glycans and developing new therapies for diseases such as cancer, autoimmune disorders, and neurodegenerative disorders. It is difficult to extract information about molecular-level interactions from experiments, and theory/computation has not been able to provide definite information to aid the interpretation of experiments. Our glycosaminoglycan (GAG)-Dock methodology addresses this challenge, furthering understanding of GAG–protein interactions by predicting the binding structures and how they depend on glycan structure and predicting precise effects of mutations that can be used to validate and interpret the interactions. Cell-surface carbohydrates play important roles in numerous biological processes through their interactions with various protein-binding partners. These interactions are made possible by the vast structural diversity of carbohydrates and the diverse array of carbohydrate presentations on the cell surface. Among the most complex and important carbohydrates are glycosaminoglycans (GAGs), which display varied stereochemistry, chain lengths, and patterns of sulfation. GAG–protein interactions participate in neuronal development, angiogenesis, spinal cord injury, viral invasion, and immune response. Unfortunately, little structural information is available for these complexes; indeed, for the highly sulfated chondroitin sulfate motifs, CS-E and CS-D, there are no structural data. We describe here the development and validation of the GAG-Dock computational method to predict accurately the binding poses of protein-bound GAGs. We validate that GAG-Dock reproduces accurately (<1-Å rmsd) the crystal structure poses for four known heparin–protein structures. Further, we predict the pose of heparin and chondroitin sulfate derivatives bound to the axon guidance proteins, protein tyrosine phosphatase σ (RPTPσ), and Nogo receptors 1–3 (NgR1-3). Such predictions should be useful in understanding and interpreting the role of GAGs in neural development and axonal regeneration after CNS injury.

Unexpected Acetylcholinesterase Activity of Cocaine Esterases

A series of proteins with cocaine esterase ability have been shown to hydrolyze acetylcholine with similar rate enhancements. Docking studies revealed that acetylcholine binds in a similar orientation as cocaine. These results suggest that acetylcholinesterase activity may be inherent to cocaine esterases.

Discovering Additional Chemical and Biological Functions for 3-Oxo-N-Acylhomoserine Lactones

Introduction The term quorum sensing has been coined to describe the ability of a population of unicellular bacteria to act as a multicellular organism in a cell-density-dependent manner, that is, a way to sense “how many are out there” [1]. Bacteria use small diffusible molecules to exchange information amongst themselves. An important class of autoinducers is the family of N-acylhomoserine lactones (AHLs) used by Gram negative bacteria. Variation in N-acyl chain length and oxidation state of AHLs provide for bacterial strain specificity in the signaling process and subsequent synchronization of gene expression. Upon reaching a critical threshold concentration, they bind to their cognate receptor proteins, triggering the expression of target genes. Pseudomonas aeruginosa is a common environmental microorganism that has acquired the ability to take advantage of weaknesses in the host immune system to become an opportunistic pathogen in humans [2]. Over the last ten years, significant progress has been made in elucidating the molecular mechanisms underlying P. aeruginosa pathogenicity. Two different AHLs, N-(3-oxododecanoyl) homoserine lactone 1 and N-butyrylhomoserine lactone, have been identified as the main quorum sensing signaling molecules in P. aeruginosa [3]. Importantly, genes regulated by this mechanism control the expression of virulence factors as well as the formation of structures known as biofilms [4]. Recently, we have assigned new roles for these compounds through the demonstration that 1 performs a previously unrecognized role; the autoinducer itself and a corresponding degradation product derived from an unusual Claisen-like condensation reaction function as innate bactericidal agents [5].