EXPRESSION CLONING COMPONENTS OF THE LPS RECEPTORS

Scripps Research Institute, La Jolla CA

Investigators

Abstract

Systemic Inflammatory Response Syndrome (SIRS) or septic shock from bacterial infection, trauma or burn injury can be fatal. Over one-half of septic shock cases are a result of gram-negative bacterial infection. Structure/function studies of gram-negative lipopolysaccharide (LPS) have revealed a group of compounds that act as LPS antagonists. Lipid Iva is an LPS precursor molecule that has a unique species-specific bioactivity. Lipid Iva is an LPS antagonist in human cells and exhibits full LPS agonist activity in murine cells. This species-specific difference in bioactivity is believed to be a result of the differences in structure between the murine and human LPS signaling receptor. We used the species-specific difference to isolate, by expression cloning, a molecule that may be involved in LPS signaling. Our preliminary results describe the isolation and DNA sequence analysis of a partial murine cDNA (G21) that enables human cells to respond to lipid Iva. In addition we demonstrate that a stable human cell line containing the murine cDNA responds to lipid Iva stimulation in a CD14-dependent manner. Therefore this murine cDNA may be a part of the LPS recognition complex. Our goal for the two years of this proposal is to isolate and characterize both the murine and human full-length cDNAs encoding this receptor. These cDNAs will be used to construct stable cell lines to confirm lipid Iva and LPS dependent induction of inflammatory mediators (TNFalpha,ICAM-1). In vitro mutagenesis studies will be employed to identify the areas(s) involved in ligand recognition. If this receptor is part of the innate immune response to bacteria, we will have a new target for the design of drugs to fight septic shock.