Biomolecular recognition processes; the chemistry and biology of protein-saccharide interactions

A description of our research follows; a short summary can be found here.

Chemical Synthesis. Our group develops and implements synthetic methods that provide access to biologically active compounds for hypothesis-driven and discovery-driven research. This important foundation of our program offers chemically-oriented researchers new opportunities to develop and apply their synthetic skills. Biochemically- and biologically-oriented researchers benefit from access to unique biologically active ligands. Areas of current focus include:

• Target-oriented synthesis of natural products and natural product-like structures; diversity-oriented synthesis of ligands. The synthetic compounds are used to explore cell surface receptor interactions, oligosaccharide biosynthesis, carbohydrate recognition, and glycoconjugate function (1).
• Development of new strategies for peptide/protein synthesis that provide access to sequences with critical posttranslational modifications (e.g., glycosylation, tyrosine sulfation) that can be used to elucidate the biological roles of these modifications (2). For a discussion of the rationale behind these projects, see: C&ENews.
• Synthesis of multivalent ligands (compounds displaying multiple binding epitopes) that can be used to control receptor position. The synthetic ligands facilitate the investigation of processes ranging from signal transduction in chemotaxis to disarming proteins that mediate inflammatory responses (3).

Biological Recognition Processes. We use our synthetic ligands along with methods derived from fields ranging from biochemistry to molecular and cell biology to biophysics to investigate biological recognition processes and their consequences.

We are especially interested in extracellular receptor-ligand interactions and how they elicit/inhibit cellular responses. In addition to exploring clustering cell-surface recognition events, we are investigating protein aggregation processes, such as those that occur in Alzheimer’s Disease. A sampling of our current interests is listed below.

• New strategies for modulating carbohydrate – protein interactions in inflammation. The selectins are a family of 3 cell surface proteins (E-, P- and L-selectin) that participate in the recruitment of white blood cells to sites of inflammation. Selectin function depends on their interaction with carbohydrate epitopes. We have generated a variety of synthetic ligands for L-selectin and used them to explore selectin recognition and as templates for inhibitor design. We created synthetic multivalent displays that bind to cell-surface L-selectin and promote its proteolytic release from the cell (4 and 5). Our current focus is on understanding the mechanism of L-selectin downregulation. In addition, we are using diversity-oriented synthesis to generate natural product-derived glycomimetics that inhibit the selectins.
• We have synthesized ligands that can selectively facilitate cell – cell interactions. Such compounds may serve as new types of targeting agents for the clearance and/or destruction of pathogenic cells (6).
• In collaboration with Regina Murphy’s group, we have discovered agents that interfere with fibril formation of the beta-amyloid protein, the major protein component found in the plaques that occur in the brains of Alzheimer’s Disease (AD) patients. We are developing new methods to identify agents that bind to beta-amyloid peptide. Our longterm goals are to develop strategies to inhibit protein aggregation and to generate agents that can be used to explore these deleterious events.

Biological Mechanisms. The molecules on the cell surface, including membrane-associated proteins, lipids, and carbohydrates, act as a remarkable conduit of information. The resident cell-surface compounds have the critical role of reporting to the interior on extracellular conditions (e.g., presence of nutrients or toxins), so that the cell can respond appropriately. By understanding how natural signaling processes are manipulated, we can design synthetic ligands that can promote specific cellular responses.

• Current estimates indicate that 1 in 5 people are infected with tuberculosis (TB), and resistance to current treatments is growing. Because mycobacteria (including those that cause tuberculosis or TB) require galactofuranose residues to build their cell walls, compounds that inhibit galactofuranose incorporation would serve as outstanding leads for antimicrobial agents. We are investigating key enzymes involved that mediate the incorporation of galactofuranose residues into cell walls. The UDP-galactopyranose mutase is an intriguing enzyme involved in generating UDP-galactofuranose, the requisite biosynthetic precursor.
  • The galactopyranose mutase enzyme is fascinating because it requires a reduced flavin co-factor to catalyze the interconversion of UDP galactopyranose and UDP-galactofuranose (7). We anticipate that the mechanism by which it catalyzes this isomerization will be unprecedented. We have developed an expression system that produces large quantities of this intriguing enzyme for biochemical studies.
  • We have developed an efficient synthesis of the UDP-galactofuranose and its analogs. These compounds provide us with key reagents to study the process of galactofuranose incorporation.
• Different cellular responses can be elicited through a single receptor; thus, it is not enough to identify a ligand that binds to or activates a receptor to control its function. Evidence indicates that the position of a receptor within the cell membrane can influence its function. We are generating synthetic ligands that use non-covalent interactions to organize receptors into different patterns on the cell surface (8). We have found these ligands can reveal unique aspects of signaling mechanisms, and we have focused initially on those that lead to bacterial chemotaxis. We are especially interested in how bacteria can respond to attractants with such high sensitivity (i.e., ca. 5% change in concentration at 10 nM) over such a broad concentration range (i.e., 5 orders of magnitude).

Bacterial Chemotaxis:

• Our results indicate that the cellular location of bacterial and archael chemoreceptors is conserved.
• We have used molecular modeling to design synthetic chemoattractants (9).
• We have found that the chemotactic responses of E. coli can be modulated using ligands that cluster the chemoreceptors to different extents (10). Our studies suggest that bacteria can amplify and modulate their signals by controlling the clustering of cell surface receptors. Movies of bacterial movement in response to our compounds can be seen by clicking on the quicktime movie icons below.
• Our findings have implications for regulating signaling processes in bacteria, and we are pursuing this avenue for the design of new antimicrobial agents.

We are applying what we have learned from bacterial chemotaxis to investigate the signaling processes that lead to antibody production through the B cell receptor.

More detailed information can be found in our publications.

Quicktime is required to view the following movies, you may download a free version here.
No attractant	1mM Glactose	1mM Monomer	100 mM 25mer