Endotoxin binding protein : Novel therapeutic strategy of endotoxin shock.

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We purified 18kDa cationic anti-microbial protein (CAP18) with lipopolysaccharide (LPS)-neutralizing activities from rabbit granulocytes. We also cloned a CAP18 family protein from a human bone marrow library. The cloned DNA encoded 140 amino acid residues (CAP181-140). Like the rabbit protein this molecule is comprised of two domains, a highly conserved N-terminal domain of unknown function and a less conserved C-terminal anti-microbial and LPS-neutralizing domain. In the present study, we synthesized new human peptides to identify in more detail the active domain within C-terminal fragment. Synthetic peptide (27 mer, F12-V38) of C-terminal fragment binds to LPS,inhibits LPS-induced activation of Limulus amebocyte lysate, LPS-induced reactive nitrogen release by macrophages and protect mice from LPS lethality. Treatment with the 27 mer peptide also blocked the increase in TNF-alpha levels induced by LPS injection. this peptide also showed antimicrobial activity versus both gram-negative bacteria such as Escerichia coli O157 : H7, salmonella typhimiurium, Klebsiella pneumoniae, Pseudomonas aeruginosa and gram-positive bacteria such as Staphylo-coccus aureus and Streptococcus pneumoniae. Truncation of hydrophobic amino acids from this 27 mer peptide caused a significant decrease in all activities indicating that this 27-mer fragment is the minimal antimicrobial and LPS-neutralizing domain of CAP18.The amphipathic and alpha-helical structure of this peptide may play a major role in the expression of these activities. The importance of C-terminus hydophobic residues in CAP18 was also suggested. CAP18 and derived peptides may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.