Our long term objective is to understand the initiation of endotoxic shock, a frequent, lethal complication of bums and trauma. Research from our laboratory has begun to clarify the events involved. Lipopolysaccharides (LPS) from gram-negative bacteria enter the bloodstream and bind to LPS binding protein (LBP), a 60 kD serum glycoprotein discovered and characterized by the applicant. LPS-LBP complexes then bind to CD14, a monocyte (MO) specific, 55 kD plasma membrane receptor, leading to MO activation. LPS alone is much less efficient at activating MO. Depending on the extent of MO activation, the monokines which are induced either initiate the bactericidal activities of phagocytic cells or initiate endotoxic shock. Thus LBP occupies a central role in regulating MO responses to LPS and the consequent balance of monokine induced responses. It follows that understanding the structural basis for LPS binding to LBP and for LPS-LBP complex binding to CD14 is critical to understanding and perhaps manipulating the activation of MO by LPS-LBP complexes. We propose three approaches to identifying the structural bases for these interactions of LBP. One will be to cross-link LPS to LBP at the LPS binding site and determine which amino acids of LBP are in proximity to LPS. The second approach will be to create antibodies to defined portions of the structure of LBP and determine their effects on the binding of LPS to LBP and of LPS-LBP complexes to CD14. The third approach will be to use recombinant DNA technology to construct LBP mutants, altering regions of the molecule indicated to be important for binding of LPS and CD14 by the antibody and cross-linking data. The functional abilities of the mutations should pinpoint the LPS and CD14 binding sites. In addition to their utility in understanding the structural bases for LPS and CD14 binding, some of the reagents we will develop can be tested for potential therapeutic utility.
Showing the most recent 10 out of 31 publications
