MIT Chemistry Directory

Laura L Kiessling

Visiting Professor
All cells on Earth possess a carbohydrate coat. Emerging evidence indicates that this coat serves as a critical conduit of information, but little is known at the molecular level. Elucidating how carbohydrates are assembled, how they are recognized, and how they function are issues at the scientific frontier. Using ideas and approaches that range from synthetic chemistry to cell biology, our research group is addressing the critical issues at this frontier. A summary of the key elements of our research program along with relevant references can be found below.
 
Chemistry to Explore and Exploit Multivalent Protein–Carbohydrate Interactions
 
The position of carbohydrates on the cell surface renders them uniquely poised to engage with proteins on the surfaces of other cells or pathogens. Indeed, protein–carbohydrate interactions have been implicated in physiological processes ranging from fertilization to development to immune system function. Given these important functions, it seems surprising that individual protein-carbohydrate interactions are so weak: Their association constants are typically 1000- to 1,000,000-fold poorer than those of protein–protein interactions. To compensate for their low affinity, most protein–carbohydrate interactions are multivalent. Multivalent interactions occur when multiple binding groups (e.g., carbohydrates) on one cell bind to multiple copies of a receptor (e.g., a protein) on another cell. By combining energetic contributions of multiple individual complexes, multivalent interactions are kinetically labile yet exhibit high apparent affinity. These attributes of multivalent interactions complicate the study of protein–carbohydrate interactions. It is challenging to determine if a protein–carbohydrate interaction is relevant, to assess the molecular mechanisms that contribute to formation and stabilization of protein–carbohydrate complexes, and to design potent inhibitors of protein–carbohydrate interactions. To address all of these issues, we developed novel and general synthetic routes to multivalent carbohydrate displays (Fig. 1) 

Fig 1.jpg


Fig 1. The ring-opening polymerization can be used to generate defined carbohydrate-substituted polymers. B: Multivalent carbohydrate derivatives can block cell – cell interactions.
 
We reasoned polymer chemistry would be a powerful approach to assemble multivalent carbohydrate derivatives. We sought to devise a method to synthesize multivalent ligands with control over length and valency. Most polymerization reactions are incompatible with the polar functional groups of carbohydrates and give rise to mixtures of polymers that vary widely in valency and length. We surmised that the ring opening metathesis polymerization (ROMP) could be ideal for generated defined multivalent carbohydrate displays. ROMP can be a living polymerization, a reaction in which chain elongation occurs more readily than termination. As a result, we can use ROMP to assemble carbohydrate-substituted polymers with defined lengths, valencies, and arrangements of functional groups (Fig. 2). Our first indication of the power of ROMP for making bioactive polymers was our finding that ROMP can be used to generate carbohydrate-substituted polymers that block cell–cell interactions. Our subsequent studies underscore the versatility of this synthetic strategy for generating multivalent carbohydrate derivatives with tailored biological activities.
 

fig 2.jpg


Fig 2. Schematic depiction of ligands that can be generated using “living” polymerization reactions. Different colors represent different binding epitopes. Polymers can also be uniquely end-labeled (green) with a reporter (e.g., a fluorophore).
 
We used ROMP to create polymers that varied in chemical structure to probe the mechanisms underlying multivalent binding. Multivalent interactions had been thought to be non-selective, but our data challenge this paradigm. We found that multivalent ligands can by highly specific. Moreover, we used our synthetic expertise to carry out the first comparison of how the architecture of multivalent ligand influences its function. One finding to emerge from this investigation is that multivalent ligands generated by ROMP are especially effective at clustering proteins. We subsequently synthesized multivalent carbohydrate displays that inhibit a target carbohydrate-binding protein (L-selectin) by a novel mechanism: they cluster the carbohydrate-binding protein and induce it proteolytic release from the cell surface. This method of altering protein function had been unprecedented and it prompted us to consider using multivalent ligands to explore signaling pathways.
 
Multivalent Carbohydrates As New and Powerful Probes of Signal Transduction
 
The ability of polymers generated by ROMP to cluster proteins prompted us to examine their utility for activating signal transduction. Multiprotein complexes are the critical mediators of signal transduction. We created multifunctional ligands to direct the formation of multiprotein complexes (Fig. 3). Such ligands, which can be used like small molecules for temporal control, can illuminate how protein assembly regulates cellular responses. For example, we used carbohydrate-based multivalent attractants to reveal how bacteria sense attractants and repellents. We found that a single receptor type is not sufficient to sense a specific attractant – an array of receptors is required for proper sensing and movement toward attractants. In effect, the clustered receptors act as a type of sensory organ (nose) that allow bacteria to respond sensitively and appropriately to stimuli in the environment. We have recently created multifunctional compounds that can co-cluster multiple copies of a carbohydrate-binding protein with copies of another signaling receptor (Fig 3). We synthesized compounds that promote the formation of specific multiprotein complexes to down-regulate signaling on immune cells. Thus, we can use our knowledge of synthetic chemistry and the cellular building blocks to assemble complexes that augment immune responses (for vaccine development) or attenuate them (for treatment of autoimmune diseases).
 
 
Fig 3. Schematic depiction of how multivalent ligands can cluster multiple types of proteins. Left: Multivalent compounds with only one group (blue) can cluster the B cell receptor (BCR, blue) and activate B cells of the immune system to lead to antibody production. Right: Multivalent compounds (blue and red) that can co-cluster two receptors (CD22 and the BCR) downregulate immune responses. Compounds like this could help treat autoimmune diseases.
 
Carbohydrate Polymer Biosynthesis: A New Target for Anti-Tuberculosis Agents
 
In addition to elucidating how carbohydrates function, the Kiessling group also seeks to understand how they are assembled. The biosynthesis of polysaccharides is of special interest. An understanding of how cells generate these polysaccharides is lacking, yet polysaccharides are the most abundant organic compounds on Earth. Impetus for understanding how these molecules are built has implications from energy and sustainability to human health. We envision gains in harvesting the energy from cellulose, the creation of polysaccharide-based biodegradable materials, and the identification of polysaccharide synthesis inhibitors as new antibiotics. Our results to date have been aimed at the latter objective. We focused on a polysaccharide composed of galactofuranose residues, which is present in the cell wall of the mycobacteria that cause tuberculosis (TB). Galactofuranose residues are not present in humans, so the enzymes that mediate galactofuranose incorporation are potential drug targets. The need for such targets is urgent, because many cases of TB are antibiotic resistant and the disease ranks 2nd (behind HIV) in causing death worldwide. We elucidated the chemical mechanism of a critical enzyme responsible for galactofuranose incorporation. This enzyme functions via an unexpected chemical mechanism. Our data indicate that the flavin (vitamin B2) cofactor is used as a covalent catalyst. Though the chemistry of vitamin B2 has been studied for over 70 years, our findings have added to the chemical transformations possible for vitamin B2. With an understanding of this enzyme, we used our expertise in molecular interactions and chemical synthesis to generate small molecule inhibitors (Fig. 4). The resulting compounds block mycobacterial cell growth. Our investigations identify a new drug target for treatment of mycobacterial diseases, including tuberculosis.
 

fig 4.gif


Fig 4. Compound that blocks galactofuranose incorporation into the mycobacterial cell wall.
 
 
Carbohydrate Polymer Biosynthesis: Controlling Length
 
The biological function of a polysaccharide depends on its length. Unlike nucleic acids and proteins, carbohydrate polymer biosynthesis occurs without a template. For template-independent polymerization reactions, little is known about how length is controlled. To address this issue, we focused on the carbohydrate polymerase GlfT2, which builds the galactofuranose-containing polysaccharide in the cell wall of mycobacteria (including Mycobacterium tuberculosis). We devised a mass spectrometry assay and used it to reveal that GlfT2 is a processive polymerase with an intrinsic ability to control polymer length. Our results prompted us to propose a model for polysaccharide length control. The carbohydrate polymerase GlfT2 binds to both ends of the growing polysaccharide. When the polysaccharide gets too long, the enzyme cannot maintain multipoint binding so the complex dissociates. In this way, the length of the polysaccharide depends on the multipoint binding. This mode of tethering is a general strategy that other carbohydrate polymerases could use to regulate polysaccharide length.
 
Carbohydrate Polymers and Human Embryonic Stem Cell Pluripotency
 
Our experience in activating signaling with multivalent carbohydrate derivatives led us to become interested in using surfaces to deliver signals that direct cellular decisions. Like polymers, surfaces can display multiple copies of binding epitopes and therefore cluster proteins that activate signaling. This mode of directing and controlling cellular responses is largely unexplored. To test the feasibility of the strategy, we developed an array strategy to pattern surfaces, such that materials that alter cell adhesion and/or cell behavior can be identified (Fig. 5). The surfaces that we have begun to study display peptides, which can bind to cell surface receptors or cell surface carbohydrates.
 
Fig. 5. Strategy for synthesis of arrays for identifying surfaces that support cell attachment or elicit cellular responses. Alkane thiols functionalized with peptides or small molecules are immobilized on a gold surface to generate array elements. The immobilized alkane thiols assemble to form a well-ordered array.
 
We used our arrays to identify surfaces that would support the growth of human embryonic stem (hES) cells. The ability to grow human cells in culture is the basis for remarkable advances in fields ranging from cell biology to medicine. Human ES cells are remarkable because they can be cultured indefinitely and can differentiate into every cell type in the body. The ability to propagate hES cells provides new opportunities to test drug candidates on human cells and to generate cells for therapies. Still, to exploit the full potential of hES cells for regenerative medicine, developmental biology, and drug discovery, chemically defined culture conditions are needed. Reproducible cellular responses depend on having a fully defined environment. In addition, contamination of animal cells and/or animal-derived components typically used for hES cell culture remains a safety concern. We have addressed the limitations of standard methods. Using our surface array strategy, we identified synthetic surfaces that can support the long-term culture of hES cells. A surprising result of this screen is the simplicity of the identified surfaces. They do not present complex mixtures of proteins, but rather display a single glycosaminoglycan-binding peptide. This finding was especially exciting to us because glycosaminoglycans are polysaccharides present on the surface of all mammalian cells. While others have examined surfaces that can interact with cell-surface proteins, our data indicate that carbohydrate polymers are important in pluripotency. The surfaces we devised highlight the critical role of cell surface polysaccharides in regulating cell fate decisions. They also offer a chemically defined environment for propagating hES cells. The simplicity and efficiency of our results led the Director of NIGMS to highlight them to Congress last year as an example of how basic science can yield economic and scientific benefits.
 
 

 

Contact Information

t: 6082620541
e: KIESSLING@CHEM.WISC.EDU