Purification and antimicrobial properties of three defensins from rat neutrophils

Abstract

Three cysteine-rich cationic peptides, designated RatNP-1, RatNP-3, and RatNP-4, were purified from an acid extract of rat polymorphonuclear neutrophils, sequenced, and tested for antimicrobial activity. The peptides ranged from 29 to 32 amino acids in length (Mrs 3,252 to 3,825), and each contained all eight invariantly conserved "framework" residues that are characteristic of defensins. Each of the peptides killed Escherichia coli ML-35, Acinetobacter calcoaceticus HON-1, Staphylococcus aureus 502A, and Candida albicans 820 in vitro. RatNP-1, the most cationic rat defensin, was also the most potent. With this report, a total of 13 distinct defensins have been characterized in the polymorphonuclear leukocytes of four mammalian species. The existence of the defensin system in rats should facilitate investigations of the in vivo role of defensins in experimental infections.

Defensins promote fusion and lysis of negatively charged membranes

“…29-34 amino acid residues long (M, = 3,500-4,000) and have been found in rats (Eisenhauer et al, 1989), rabbits , and guinea pigs (Selsted & Harwig, 1987), as well as humans (Ganz et al, 1985;Selsted et al, 1985b;Wilde et al, 1989). The amino acid sequences of the defensins have been determined and when they are aligned (Table 1) to emphasize the positions of the conserved residues, a family of cationic peptides is revealed whose net charge varies from +2 for defensin HNP-3 to +9 for defensin NP-1.…”

“…bSources: 'human ; 'human (Wilde et al, 1989); 3guinea pig (Selsted & Harwig, 1987); 4rabbit ; 5rat (Eisenhauer et al, 1989). At pH 7.4, assuming the charge on histidine is neutral.…”

Antimicrobial activity of chicken and turkey heterophil peptides CHP1, CHP2, THP1, and THP3

“…Heterophils were collected as previously described from sterile peritoneal exudates induced in 6-week-old broiler chickens and turkey poults (n = 80) (Sabet et al, 1977;Harmon et al, 1992). The granules were separated from the heterophils and peptides were extracted in acetic acid then purified by size exclusion column chromatography and high pressure liquid chromatography as previously described (Eisenhauer et al, 1989;Yamashita and Saito, 1989;Selsted et al, 1993). Four avian peptides, CHP 1 and 2, and THP 1 and 3 (Evans et al, 1994), were used for the antimicrobial assays described in this study.…”

Defensins are mitogenic for epithelial cells and fibroblasts

“…Defensins are cationic antimicrobial peptides that are stored in cytoplasmic granules of neutrophils and some macrophages (reviewed in Lehrer et al, 1991). These unique molecules, first demonstrated by Zeya and Spitznagel (1963), have been isolated from rabbit macrophages and neutrophils (Selsted et al, 1984;Selsted et al, 1985a,b), and human Selsted 1985c;Wilde et al, 19891, guinea pig (Selsted and Harwig, 1987), and rat (Eisenhauer et al, 1989) neutrophils. Sequence analysis of defensins from these species has revealed that they range in size from 29-34 amino acids, eight of which are invariant.…”