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 G1LRKKFRKTRKRIQKLGRKIGKTGRKVWK AWREYGQIPYPCRI43(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; ED50values were 30–35 nMagainstEscherichia coliand 90–120 nMagainstStaphylococcus 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 (0.15M), although the antibacterial activity of defensin was completely lost in the presence of NaCl.