When most people talk about sugars, they think of the molecules we consume and use for metabolism. While carbohydrates are obviously an important part of our diet, sugar molecules are actually used for a number of different functional roles in biology like cell signaling and protein folding. Sugars that serve functional roles in the cell are called glycans. As a glycoscience group, the Godula lab is interested in exploring the chemistry and biology of glycans in cells.
Our understanding of glycans lags significantly behind major biomolecules, like proteins and nucleic acids. In our interdisciplinary field of glycoscience, we apply chemistry, cell biology, and engineering to bridge this gap. Glycans play essential roles in host-pathogen interactions, development, and hold great potential for regenerative medicine. The overlapping project areas in our lab integrate these disciplines to answer complex questions about the roles of glycans in development, signaling, and pathogensis.
Fluorogenic probes for glycomics
Chemical Tools for glycomics
The tools of molecular biology enabled the study of nucleic acids and proteins and sparked a revolution in biomedical research. Having a general toolset to manipulate and understand glycans would be similarly transformative. With this goal in mind, we have developed a method for introducing glycans into macromolecular scaffolds without the need for carbohydrate synthesis or prefunctionalization (JACS).
The cellular glycocalyx is essential for conveying biochemical signals from the outside environment to cells during differentiation. For instance, glycosaminoglycans (GAGs), highly sulfated polysaccharides, recruit and present growth factors to their receptors, thus triggering downstream signaling and gene transcription. We are interested in recapitulating the unique functions of GAGs in synthetic materials, which can be delivered to the surface of living cells using a membrane engineering strategy. In this JACS report, we show the glycocalyx of embryonic stem cells can be edited to promote neural differentiation.
Synthetic neoPGs, with affinity for specific growth factors (GFs), can be introduced into the plasma membranes of embryonic stem cells (ESCs) deficient in heparan sulfate biosynthesis. There, the neoPGs assume the function of native PGs, thereby rescuing GF-mediated signaling, and promoting neural specification.
NeoPGs promote neural differentiation in ESCs, similar to highly sulfated, soluble heparin
Remodeling the glycocalyx with synthetic glycopolymers
Neural rosette differentiation of glycan-remodeled embryonic stem cells
Macromolecular remodeling: 3-D cell culture
Extracellular glycans orchestrate the formation of gradients of signaling factors in tissues during development. Initally, we have developed a method for remodeling the glycocaylx of mutant Ext1–/– mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells. See our ACS ChemBio paper for additional information!
Describe what you offer here. add a few choice words and a stunning pic to tantalize your audience and leave them hungry for more.
Glycocalyx remodeling promotes differentiation in embryoid bodies
Taking the next step in our approach to remodel the glycocaylx of mESC embryoid bodies, the tunability of 3-D modeling was by establishing gradients of growth factors by spatial patterning of glycan mimetics on EBs.
We have capitalized on the ability of amphiphilic lipid-functionalized glycopolymers with affinity for FGF2 to assem-ble into nanoscale vesicles with tunable dimensions and extracellular matrix penetrance. Upon size-dependent diffusion into EBs, the vesicles fused with the plasma membranes of stem cells, giving rise to concentric gradients of cells with enhanced FGF2-binding. View the full report here.
Ext1-/- embryoid bodies do not naturally bind FGF2, but when remodeled with different size glycopolymers, the binding of FGF2 is restored and spatially tunable.
Ext1-/- embryoid bodies do not naturally bind FGF2, but when remodeled with different size glycopolymers, the binding of FGF2 is restored and spatially tunable
Electron microscopy images of Embryoid bodies
Small molecule tools for glycoscience
Small molecules are potent modulators of the glycocalyx. In contrast for genetic or enzymatic methods to control or abolish glycan interactions, small molecules are abundant, cheap, and accessibly. In our Stem Cells paper, we use the small molecule glycan antagonist, surfen, to reversibly control the onset of stem cell differentiation.
Surfen, small molecule GAG antagonist
Additionally, we are developing small molecule probes for activity of glycan modifying proteins. Fluorogenic probes will enable facile measurement of activity directly.
The addition of surfen arrests stem cells in a pluripotent state, which can then be triggered to differentiate by surfen removal.
Untreated (left) vs. Fluorogenic probe (right)
DNA-hybrid tools for glycoscience
DNA is nature's way to store information, but DNA aptamers can also be used as a potent targeting tool for therapeutics. An expanding area of our lab focuses on development of nucleic acid conjugate glycomaterials for the precision editing and engineering of glycans at the cell surface. This will impart control over cell signaling events in the context of development!
Characterization of DNA-glycan materials
Glycans in biological problems
Glycans in host-pathogen interactions
Mucins are linear heavily glycosylated proteins forming a protective mucous layer on epithelial surfaces lining many of our organs. Many pathogens have evolved to exploit glycans in the mucosal barrier to gain entry into host cells. While the specificities of pathogens, such as the Influenza A virus toward individual host glycan structures are coming into focus, very little is known about how the three dimensional presentation of glycans in mucosal barriers dictate successful infection events. We are integrating nanoscale glycomaterials with high-throughput glycomics platforms (see our ChemComm paper!) to catalog how parameters, such as glycan valency, density, and organization define host glycan-pathogen interactions.
We are also developing nanomaterials-based assays for in-field detection of emerging Influenza A pandemic strains. By combining high-affinity probes with viral-enzyme amplification detection methods, we have been able to capture and profile glycan binding phenotypes of viruses in primary samples collected from mallard ducks in Northern California (in collaboration with the Boyce lab at UC Davis, reported in Virology). More broadly, we are focusing on the development of a suite of probes for antibody-free detection of various glycan-binding pathogens for rapid response during the onset of pandemics.
Glycan bead arrays enable detection of low-abundant virus
Complementary to our materials-based glyco-engineering program are our efforts to modulate glycan interactions within the glycocalyx using small molecules. We have reported in ACS Central Science on a new drug screening assay to identify inhibitors of viral neuraminidase enzymes that can slow the escape of Influenza A from mucosal barriers en route to host cells and prevent infection by reinforcing the natural protective functions of mucosal glycans.
Synthetic mucus-like nanobarrier
Synthesis of glycopolymer scaffolds for array-based interrogation of virus-glycan interactions
Glycans as key modulators of biology:
physical and biochemical properties
In the mucosal epithelium, glycosylated mucins can extend for hundreds of nanometers. While glycans are usually assed on the cell surface in the context of binding affinity, the physical bulk of the glycocalyx affects binding events at the crowded cellular boundary. Using a synthetic "spectator glycocalyx" we seek to better understand the effect of a bulky glycocalyx on cell morphology and binding events with lectins. Read more about the steric effects of the glycocaylx here!
Turkey Red Blood Cells without remodeled glycocalyx display thin, edges, convex morphology
Turkey Red Blood Cells remodeled with bulky glycocaylx show effects on cell morphology and lectin binding!
Glycocalyx-engineering can also be used to control complex multi-cellular events, such as the formation of the neuromuscular synapse, which may enable the bottom-up assembly of artificial tissues in the future. We envision that advances in precision glycan editing will provide new methods for therapeutic intervention after traumatic CNS or PNS injury or to reverse the effects of neurodegeneration. Read our ACS chem. neuroscience report to see more about how remodeling membranes with our synthetic materials affects development of the neuromuscular junction.
Acetylcholine receptor clustering for neuromusclar junction formation
Glycans also play critical roles in the extracellular environment. When the extracellular space is modified with heparinoid conjugates, growth factor sequestration affects cellular proliferation and signaling. Checkout this Bioconjugate Chemistry report to see more!