RL-37, an alpha-helical antimicrobial peptide of the rhesus monkey

Abstract

Rhesus monkey bone marrow expresses a cathelicidin whose C-terminal domain comprises a 37-residue alpha-helical peptide (RL-37) that resembles human LL-37. Like its human counterpart, RL-37 rapidly permeabilized the membranes of Escherichia coli ML-35p and lysed liposomes that simulated bacterial membranes. When tested in media whose NaCl concentrations approximated those of extracellular fluids, RL-37 was considerably more active than LL-37 against staphylococci. Whereas human LL-37 contains five acidic residues and has a net charge of +6, rhesus RL-37 has only two acidic residues and a net charge of +8. Speculating that the multiple acidic residues of human LL-37 reduced its efficacy against staphylococci, we made a peptide (LL-37 pentamide) in which each aspartic acid of LL-37 was replaced by an asparagine and each glutamic acid was replaced by a glutamine. LL-37 pentamide's antistaphylococcal activity was substantially greater than that of LL-37. Thus, although the precursor of LL-37 is induced in human skin keratinocytes by injury or inflammation, its insufficiently cationic antimicrobial domain may contribute to the success of staphylococci in colonizing and infecting human skin.

FIG. 1

cDNA sequence of the rhesus monkey cathelicidin, RL-37. The precursor has 170 residues, a mass of 18,861 Da, and a pI of 10.06. The predicted signal sequence (33 residues) is underlined, and the expected mature peptide is shown in bold face type. The stop codon is indicated by asterisks, and the polyadenylation site is indicated by italics.

FIG. 2

Comparison of rhesus and human cathelicidin peptides. Both signal peptides (underlined) have 30 residues, of which 28 (93.3%) are identical. Both cathelin domains have 101 residues, of which 93 (92%) are identical. The mature domains (dotted underlined) of RL-37 and LL-37 both contain 37 residues, of which 25 (67.6%) are identical. Mature rhesus RL-37 has a mass of 4,100.9 Da, a theoretical pI of 11.20, and a net charge of +8. Mature human LL-37 has a mass of 4527.34, a pI of 10.61, and a net charge of +6. A vertical line connects identical residues, and a plus sign identifies conservative substitutions. Residues that differ in the human and rhesus peptides are shown in boldface type.

FIG. 3

Activity against E. coli, P. aeruginosa, and L. monocytogenes. Radial diffusion assays were performed in underlay gels that contained different amounts of NaCl (0 mM, 100 mM, or 200 mM), in addition to their common basic ingredients (10 mM sodium phosphate buffer, pH 7.4; Trypticase soy broth powder, 0.3 mg/ml; and 1% [wt/vol] agarose).

FIG. 4

Membrane permeabilization. Stationary-phase E. coli ML-35P (2.5 × 10⁷ CFU/ml) was suspended in 10 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl, 0.3 mg of Trypticase soy broth powder per ml, and fluorogenic substrates for β-lactamase and β-galactosidase. Fluorescence monitoring began immediately after the addition of 2.5 μg of RL-37. Controls were incubated under identical conditions but in the absence of the peptide.

FIG. 5

Liposome lysis. A cationic thoadicarbocyanine dye was encapsulated in liposomes formulated to resemble gram-positive and gram-negative membranes. Fluorescence measurements were made 10 min after the addition of peptides.

FIG. 6

LPS binding. We used a chromogenic Limulus amoebocyte assay to obtain binding isotherms for E. coli 0111:B4 lipopolysaccharide. The peptides examined included polymyxin B, LL-37, LL-37 pentamide, RL-37, and rabbit CAP-18 (another LPS binding cathelicidin). The EC₅₀ (i.e., the peptide concentrations that bound 50% of the LPS) are shown and provide an approximate binding constant.

FIG. 7

CD. Spectra labeled A, B, and C are shown. Spectrum A was obtained in 10 mM sodium phosphate buffer, pH 7.4. Spectrum B was obtained in liposomes that contained POPE-POPG-cardiolipin in a 3:1:0.44 molar ratio that simulated gram-negative membranes. These liposomes were dispersed in 10 mM sodium phosphate buffer, pH 7.4. Spectrum C was obtained in a dispersion of diphosphoryl lipid A from E. coli F583 (Sigma) in 10 mM sodium phosphate buffer. In each experiment, the peptide concentration was 120 μM and the lipid-to-peptide molar ratio was 20:1.