AN AMINO-TERMINAL FRAGMENT OF HUMAN LIPOPOLYSACCHARIDE-BINDING PROTEIN RETAINS LIPID-A BINDING BUT NOT CD14-STIMULATORY ACTIVITY

LPS-binding protein (LBP) mediates the pro-inflammatory effects of bac terial LPS by enhancing LPS-induced cytokine production by monocytic c ells. LBP binds specifically to LPS to generate a complex that interac ts with the CD14 receptor on the surface of responsive cells. To ident ify the biologically active regions of the protein responsible for med iating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP(2 5)).s of LBP bound to LPS with the same affinity, and similarly inhibi ted LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP(25) were compared for their ability to mediate CD14-dependent LPS effects on c ells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP(25) had n o significant activity in these assays. Similarly, rLBP was able to me diate LPS-induced TNF production by human PBMC whereas rLBP(25) was es sentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably locat ed in the C-terminal region of the protein. Thus, the LPS-binding acti vity of LBP can be separated from the CD14-stimulatory activity, sugge sting these activities are mediated by structural elements residing in different regions of the protein.