LPS (lipopolysaccharide) is the endotoxin of Gram-negative bacteria and the most important inducer of innate immune responses in human. It is the principal component of the outer membrane of Gram-negative bacteria. Humans have evolved to detect low levels of LPS to alarm the immune system and prepare for further infection. Monocytes orchestrate the innate immunity response to LPS by expressing a variety of inflammatory cytokines that include tumor necrosis factor-alpha (TNF-∝). However, over-reaction to the presence of LPS can lead to sepsis, septic shock, or systemic inflammatory response syndrome. Septic shock or systemic inflammatory syndrome is considered the most important cause of death in the intensive care units and its mortality reaches 50%. Despite much effort there is no good therapy for this problem. Therefore, studies on the molecular basis of LPS recognition by monocytes and the regulation of inflammatory genes are required to identify novel therapeutic approaches.
LPS is initially recognized by LBP (lipopolysaccharide binding protein) in serum. Then, LPS is transferred from LBP to CD14 located on the membrane of the immune cells. Ultimately CD14 transfers the bound LPS to TLR-4 and MD-2 complex that initiates intracellular signaling and secretion of various cytokines.
To understand LPS binding and transfer mechanisms in atomic detail, I undertook crystallographic studies of MD-2. For this purpose, I overproduced MD-2 in Hi5 insect cells using recombinant baculoviruses and purified the protein by ion-exchange chromatography and gel filtration. Through in vitro binding experiments, I confirmed the interactions between MD-2 and LPS. In addition, I found that the purified MD-2 interact with E5564, a LPS antagonist. MD-2 protein was crystallized by vapor diffusion technique. Structure determination is in progress and the crystal structure of MD-2 and TLR4 complex will be used as a template for the design of novel drugs against sepsis and other LPS-related diseases.
