Research Accomplishments:? Progress was made primarily in the area of RAGE (Receptor for advanced glycation end products). RAGE is expressed on macrophages, SMC, vascular endothelium and cardiac myocytes. Diabetes-associated vascular dysfunction can be prevented by in vivo blockade of RAGE. Upon interaction with glycated proteins, RAGE elicites a proinflammatory cascade that includes hyperpermiability of blood vessels, monocyte adhesion and generation of reactive oxygen species. RAGE is also a signal transduction receptor for proinflammatory S100/calgranulins, HMGB1/amphoterin and the integrin Mac-I. ? In an attempt to optimize the crystallization of RAGE for higher resolution diffraction, several more soluble fragments of human RAGE including fusion proteins were designed and successfully expressed in E coli. These included further shortening the linker with the N-terminal maltose-binding protein (MBP) fusion, altering the length of the C-terminus in the second domain, removing the second domain altogether and including Rat Rage in addition to human RAGE. ? Preliminary data from our lab has shown for the first time that RAGE recognizes both DNA and RNA as ligands with submicromolar affinity. We pursued this further using gel filtration and surface plasmon resonance (SPR, Biacore) experiments. Only dsDNA and dsRNA bound to RAGE; ssDNA did not. The curved scatchard plot and subsequent mathematical modeling (using Origin software) revealed that the binding is most likely follows a one-dimensional lattice model in which RAGE binds with no sequence specificity to dsDNA. This type of binding results in an initial high binding affinity of RAGE to DNA that is proportional to the length of DNA. However, as more ligand (e.g. RAGE) molecules bind to the DNA, and available sites are reduced, the affinity also decreases in a manner that resembles negative cooperativity. An analysis of several Biacore experiments and subsequent modeling revealed a binding footprint of approximately 8 nucleotides and an intrinsic dissociation constant (for one binding footprint) of 10-20 nanomolar with no positive cooperativity. This suggests that RAGE may be a sensor of some sort for naked DNA or RNA in various extracellular spaces where RAGE is expressed. Such a function may play a role in detecting necrotic or damaged cells, signaling the potential presence of a pathogen. Potential future experiments include crystallizing RAGE with dsDNA or dsRNA fragments and functional studies with RAGE in cell cultures to more fully explore the potential functional roles of RAGE in pathogen recognition and signaling.
| Park, HaJeung; Adsit, Floyd G; Boyington, Jeffrey C (2005) The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1. J Biol Chem 280:13593-9 |
