Purification of the 11- and 5-kDa antibacterial polypeptides from guinea pig neutrophils

Abstract

It has been known that neutrophils contain various antimicrobial components in the granules, which contribute to the oxygen-independent host defense mechanism. In this study, we have isolated the two antimicrobial polypeptides from guinea pig neutrophil granules. Urea-SDS-PAGE analysis revealed that the molecular masses of the polypeptides were 11 and 5 kDa under nonreducing conditions. Under reducing conditions, the molecular mass of the 5-kDa polypeptide did not change, whereas the molecular mass of the 11-kDa polypeptide changed to about 5 kDa, suggesting that the 11-kDa polypeptide is a dimer composed of 5-kDa subunits joined with a disulfide bond. The amino acid composition and sequence data indicated that the 5-kDa subunit of the 11-kDa polypeptide contained 9 lysine, 8 arginine, and 1 cysteine residues and that the 11-kDa polypeptide was a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 (4599 Da) joined with one disulfide bond. Amino acid sequence of the 11-kDa polypeptide showed partial homology (19-30%) to the active peptides of rabbit and human cationic antimicrobial proteins of 18 kDa (CAP18), suggesting the 11-kDa polypeptide might be a homologue of CAP18. In contrast, the amino acid analysis of the 5-kDa antibacterial polypeptide revealed that the polypeptide was composed of 41 amino acids (5007 Da) containing 7 lysine, 10 arginine, and 2 cystine residues. However, sequence analysis indicated that the N-terminus of the 5-kDa polypeptide was likely blocked. The 11- and 5-kDa polypeptides showed almost the same antibacterial activities; ED50 values were 30-35 nM against Escherichia coli and 90-120 nM against Staphylococcus aureus, which were 4- to 20-fold lower than those of defensins. Furthermore, the 11- and 5-kDa polypeptide retained the antibacterial activities even at the physiological concentration of NaCl (0.15 M), although the antibacterial activity of defensin was completely lost in the presence of NaCl.