Structure and functions of endotoxin-binding peptides derived from CAP18.

CAP18 (cationic antimicrobial protein, 18kDa) is a 142 amino acid protein originally isolated from rabbit granulocytes using agglutination of LPS-coated erythrocytes as an assay. CAP-18 is composed of an N-terminal domain of unknown function (CAP181-105) and a C-terminal LPS-binding domain (CAP18106-142). Synthetic CAP18106-142 and CAP18106-137, a 32-amino acid peptide resulting from the truncation of 5 amino acids from the C-terminus of CAP18106-142, inhibited LPS-induced tissue factor generation, nitric oxide production and TNF release by macrophages. Mice treated with CAP18106-142 or CAP18106-137 were significantly protected from LPS lethality. Although CAP18106-142 and CAP18106-137 were highly active, other fragments of CAP18106-142, including CAP18110-142 with a truncated N-terminus, did not exhibit LPS-binding and LPS-neutralizing activities. Both peptides had broad anti-microbial activity against both Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa (IC50; 40-100 nM) and Gram-positive bacteria such as Staphylococcus aureus(Methicillin sensitive and resistant strains) and Streptococcus pneumoniae (IC50; 100-200nM). We cloned a CAP18 family protein from human granulocytes. The cloned cDNA encoded 140 amino acid residues. Human CAP18 (CAP181-140) was highly homologous to that of rabbit. A 32- amino-acid C-terminal fragment (CAP18104-135) was shown to bind LPS, inhibit LPS-induced tissue factor generation by murine macrophages, and protect mice from LPS lethality. This peptide exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria. We hypothesize that CAP18 and the derived peptides bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.